Volume 3, Issue 3 (2016) Tropical Plant Research

TROPICAL PLANT RESEARCH An International Journal

© Society for tropical Plant Research® ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265

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https://doi.org/10.22271/tpr.2016.v3.i3

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Contents An inoculum of endophytic fungi for improved growth of a traditional ricevariety in Sri Lanka W.A.D.K. Wijesooriya and N. Deshappriya* Morphological and molecular characterization of Colletotrichum capsici causing leaf-spot of soybean Rajyasri Ghosh*, Sreetama Bhadra and Maumita Bandyopadhyay Structural study of Gilbertiodendron dewevrei mono-dominant forest based on mature individuals in the Masako forest reserve (Tshopo province, Democratic Republic of the Congo) Francine K. Botelanyele, Patience K. Kahola, Jean-Leon K. Kambale, Nicole S. Assani, Esther I. Yokana, Prosper S.Yangayobo, Honorine N. Habimana, Monizi Mawunu and Koto-te-Nyiwa Ngbolua* First report on three new diatom species from the Hooghly District, West Bengal Nilu Halder Effect of various dormancy breaking treatments on seed germination, seedling growth and seed vigour of medicinal plants Ashwani Kumar Bhardwaj, Sahil Kapoor, Avilekh Naryal, Pushpender Bhardwaj, Ashish Rambhau Warghat, Bhuvnesh Kumar and Om Prakash Chaurasia* Some ethnomedicinal plants used against high blood pressure in Bargarh district in Western Odisha (India) S. K. Sen* and L. M. Behera Uncultivated fodder grass for cattle R. Prameela* and M. Venkaiah Ecological studies of mangroves species in Gulf of Khambhat, Gujarat Vandna Devi and Bhawana Pathak* Floristic assessment of different habitats of Parvati Aranga wildlife sanctuary and adjacent Tikri forest area, Gonda, Uttar Pradesh, India Vineet Singh*, S. K. Srivastava and L. M. Tewari Intra-specific variation in response of Neem (Azadirachta indica A. Juss) to elevated CO2 levels and biochemical characterization of differently responding plants C. Buvaneswaran*, K. Arivoli, T. Sivaranjani, E. Menason, K. Vinothkumar, S. Padmini and S. Senthilkumar* Morphological characters of Chaetoceros lorenzianus (Bacillariophyceae) isolated from North Arabian Sea after Tasman Spirit oil spill Asma Tabassum*, Hina Baig and Aliya Rehman Diversity and distribution of Litsea in Chikkamagaluru, Karnataka S. G. Srinivas and Y. L. Krishnamurthy* Alternaria polypodiicola, a new foliicolous fungus discovered on Microsorum punctatum from Uttar Pradesh, India Shambhu Kumar* and Raghvendra Singh Floristic composition and biological spectrum of weeds in agro-climatic zone of Nalbari district, Assam, India D. K. Bhattacharjya* and S. K. Sarma

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Contents Differential responses of pea seedlings to salicylic acid under UV-B stress Chanda Bano, N. B. Singh* and Sunaina Exotic species invasion threats to forests: A case study from the Betla national park, Palamu, Jharkhand, India Preeti Kumari and A. K. Choudhary* Disease progression in potato germplasm from different reaction groups against potato virus Y in relation to environmental factors Ata-ul-Haq, Yasir Iftikhar, Muhammad Irfan Ullah, Mustansar Mubeen*, Qaiser Shakeel, Faheema Bakhtawar and Iram Bilqees A note on precociouspollen germination in Woodfordia fruticosa (L.) Kurz. Kanak Sahai*, Krishna Kumar Rawat and Dayanidhi Gupta New species and new records of Graphis (Ostropales: Graphidaceae) from Eastern Ghats, India Satish Mohabe, Anjali Devi B., Sanjeeva Nayaka and A. Madhusudhana Reddy* Seed priming with spermine ameliorates salinity stress in the germinated seedlings of two rice cultivars differing in their level of salt tolerance Saikat Paul and Aryadeep Roychoudhury* Isolation and characterization of lectin from the leaves of Euphorbia tithymaloides (L.) Aruna A. Jawade, Shubhangi K. Pingle*, Rajani G. Tumane, Anvita S. Sharma, Archana S. Ramteke and Ruchika K. Jain Nutritional composition and fungi deterioration of canned tomato products collected from Ibadan, South-western Nigeria S. G. Jonathan, B. J. Babalola, O. J. Olawuyi, J. A. Odebode* and A. O. Ajayi Taxonomic account of an Indian endemic, monotypic genus Adenoon Dalzell with a note on lectotypification of Adenoon indicum Dalzell. Bandana Bhattacharjee*, Sobhan Kumar Mukherjee and P. Lakshminarasimhan Floristic composition and vegetation analysis of Hulikal Ghat region, central Western Ghats, Karnataka Vinayaka K. S.* and Krishnamurthy Y. L. Role of dicot angiosperms in the livelihood of Mishing community in Sonitpur district, Assam, India Jintu Sarma and Ashalata Devi* Finger millet (Eleusine coracana L.) grain yield and yield components as influenced by phosphorus application and variety in Western Kenya Wekha N. Wafula*, Korir K. Nicholas, Ojulong F. Henry, Moses Siambi and Joseph P. Gweyi-Onyango Bidens bachulkarii (Asteraceae-Heliantheae): A new species from Western Ghats, India D. G. Jagtap* and M. Y. Cholekar A checklist of succulent plants of Ahmedabad, Gujarat, India Ruchi M. Patel, Umerfaruq M. Qureshimatva*, Rupesh R. Maurya and Hitesh A. Solanki Lichens in 50 ha permanent plot of Mudumalai Wildlife Sanctuary, Tamil Nadu, India Komal K. Ingle, Sanjeeva Nayaka* and H. S. Suresh Development and characterization of microsatellite markers for Osyris lanceolata Hochst. & Steud., an endangered African sandalwood tree species John O. Otieno*, Stephen F. Omondi, Annika Perry, David W. Odee, Emmanuel T. Makatiani, Oliver Kiplagat and Stephen Cavers Rice false smut [Ustilaginoidea virens (Cooke) Takah.] in Paraguay Lidia Quintana*, Susana Gutiérrez, Marco Maidana and Karina Morinigo

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 470–480, 2016 DOI: 10.22271/tpr.2016.v3.i3.063 Research article

An inoculum of endophytic fungi for improved growth of a traditional ricevariety in Sri Lanka W.A.D.K. Wijesooriya and N. Deshappriya* Department of Botany, Faculty of Science, University of Kelaniya, Kelaniya, Sri Lanka *Corresponding Author: [email protected] [Accepted: 12 September 2016] Abstract: Use of chemical fertilizers and pesticides in rice cultivation has incurred many environmental and health problems in Sri Lanka. Therefore, there is renewed interest in cultivating traditional rice varieties as they are more amenable to organic farming practices. However, as the yield of these varieties is comparatively low, strategies to enhance their performance should be investigated. As endophytes of plants are reported to promote growth and yield of a number of crop varieties, this study was aimed at studying the endophytic fungal assemblage present in the traditional rice variety Kuruluthuda with a view to evaluate their capacity to enhance plant growth and yield. Twenty seven endophytic fungal species were isolated from different parts of Kuruluthuda rice plants collected from a paddy field cultivated using organic fertilizers in the Gampaha district, Sri Lanka. Two frequently isolated endophytic fungal spp. i.e. Acremonium and Arthrobotrys (frequencies of isolation 60% and 38.6% respectively) were introduced separately and in combination torice seedlings using spore suspension and plate methods to determine their effect on growth and yield under green house and field conditions. All endophyte inoculated plants showed a significant difference (P ≤ 0.05) in plant growth (height, fresh weight and dry weight), number of tillers and yield when compared withnon-inoculated plant sunder both green house and field conditions. The effect of Acremonium and Arthrobotrys when introduced in combination showed a significant difference (P ≤ 0.05) in the fresh weight, dry weight, tiller number and yield (weight of seeds harvested) when compared to their individual effects under field conditions which indicates that the two endophytes in combination can be used as a better inoculum to improve biomass and yield of the plants of rice variety tested. This is the first report of the endophytic mycoflora of the rice variety Kuruluthuda and their potential use for growth promotion and yield enhancement. Keywords: Fungal endophytes - Acremonium - Arthrobotrys - Kuruluthuda - Growth enhancement. [Cite as: Wijesooriya WADK & Deshappriya N (2016) An inoculum of endophytic fungi for improved growth of a traditional ricevariety in Sri Lanka. Tropical Plant Research 3(3): 470–480] INTRODUCTION Rice (Oryza sativa L.) is the staple food of a large part of the world population (FAO 1994). Improvement of rice grain yield has been dependent on the introduction of new high yielding varieties that are heavily dependent on the use of chemical fertilizers and pesticides. The need for long term, indiscriminate application of chemical fertilizers has led to hazardous influences on the environment as well as on human and animal health. Traditional rice varieties are highly amenable to organic farming practices that cause minimum damage to the environment and sustain soil health (Rathnabharathi 2009). Therefore, there is renewed interest in cultivating traditional rice varieties combined with organic farming practices. However, these varieties are known to be low producers of yield and thus their performance has to be improved to meet the demand of the highly expanding population. Recent studies aimed at improving the productivity of plants using alternative cultivation practices have focused on the possibility of using microorganisms to increase root growth and nutrient uptake of plants, fix nitrogen as well as decrease plant stress and disease incidence (Montesinos 2003). Endophytes are microorganisms that can colonize internal plant tissues without causing apparent harm to the host (Petrini 1991). They are symbionts in plants and benefit the plant by enhancing growth directly by releasing www.tropicalplantresearch.com

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Received: 11 May 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.063

Wijesooriya & Deshappriya (2016) 3(3): 470–480 . phyto-hormones (i.e. auxins, giberallin and cytokinin) which are biosynthesized through their own pathways (Contreras-Cornejo 2009, You et al. 2012). These additional concentrations of hormones improve root and shoot biomass and reinforce the root system of plants which facilitates the acquisition of mineral nutrients from the soil (Feng et al. 2000) and enable plant roots to increase nitrogen and phosphorus uptake (Ryan et al. 2008). Indirectly, they increase the availability of the nutrients in the rhizosphere by mobilization of phosphate from organic phosphate sources (Tarafdar & Claassen 1998) and producelow molecular weight metabolites (i.e. mycotoxins, altechromones and flavonoids) which are required for the normal functioning of plants under adverse conditions (Strobel 2003). Diazotrophic bacterial and actinomycete populations of rice are reported to promote plant growth promotion and to possess the potential for nitrogen fixation and disease resistance (Barraquio et al. 1997, Tian et al. 2004). Fungal endophytes also have been detected in cultivated rice and they have shown to possess antagonistic or plant growth stimulating properties (Yuan et al. 2009, Naik et al. 2009). Previous studies in Sri Lanka have shown that some endophytes present in the traditional rice varieties Suwandel, Kaluheenati and Herath Banda have the ability to increase the growth of plants significantly (Atugala & Deshappriya 2015, Ponnawila & Deshappriya 2014). However, the endophytic fungal assemblage of the rice variety Kuruluthuda and their effect on plant growth and yield has not been reported in Sri Lankapreviously. Therefore, the present study was carried out with the objectives:  To isolate the endophytic fungal assemblage present in different plant parts of the rice variety Kuruluthuda.  To develop method(s) of introducing endophytic fungi into seedlings.  To evaluate the effect of the most frequently isolated fungal species on the performance of plants under green house and field conditions with a view to develop an inoculum for improved plant growth and productivity. MATERIALS AND METHODS Sample collection and microscopic observation of roots Nine week old healthy, intact plants of the traditional rice variety Kuruluthuda were randomly collected from a field treated with organic fertilizers at Eldeniya, Sri Lankaduring the Maha season (September to February) and transported to the laboratory in clean polythene bags. Roots were cut into 7 cm long pieces and washed under running tap water for 20 minutes. Squashed preparations of root segments were observed under the light microscope and the presence of root inhabiting endophytic fungi was confirmed by trypan blue staining method (Yuan et al. 2009). Isolation and identification of endophytic fungi of therice variety Kuruluthuda Leaf pieces stem pieces, root segments and seeds of Kuruluthuda were used to isolate endophytic fungi. Each plant part was cleaned separately by rinsing under running tap water for 10-15min and the cleaned plant parts were surface sterilized using the sterilization protocols reported previously (Atugala & Deshappriya 2015). After treatments with surface sterilizing agents, each plant part and seeds were washed with three consecutive changes of sterilized distilled water (SDW). The edges of the surface sterilized leaf, stem and root segments were trimmed and twenty five pieces of each of the plant parts and seeds were transferred aseptically into petri plates containing Malt Extract Agar (MEA) supplemented with tetracycline (50 mg.l-1). After 10 days incubation at room temperature (RT) (30˚C), endophytic fungal speciesthat grew out were subcultured onto fresh Potato Dextrose Agar (PDA) plates supplemented with tetracycline (50 mg.l-1). Pure cultures of each isolated colony was obtained by hyphal tip method (Tutte 1969) and stored at 4˚C. The fungal cultures were identified based on morphological characters (i.e. features of hyphae and sporulating structures, colony morphology, texture and color) to genus level using standard keys of identification (Domschet al. 1993). Isolation frequencies and percentages of dominant fungi were calculated as following (Fisher & Petrini 1991, Goveas et al. 2011): Isolation frequency = (Total number of isolates yielded in a given trial/ Total number of sample segments in the same trial) × 100 Percentage dominance = (Number of isolates collected from the samples/ Total number of leaf/stem/root/seed samples) × 100 www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . Inoculation of endophytic fungal species into seedlings The effect of most frequently isolated fungal endophytes i.e. Acremonium and Arthrobotrys on rice plant growth and yield was determined by inoculating each fungus separately and together to three day old seedlings of the rice variety Kuruluthuda. Seed germination Seeds were surface sterilized as described in 2.2 above. The surface sterilized seeds were soaked overnight in SDW and allowed to germinate by wrapping with a wet cloth for three days at RT (30˚C). Inoculation of seedlings Inoculation of seedlings was carried out using two methods, (a) spore suspension method and (b) plate method. (a) Spore suspension method: 10 day old cultures of Acremonium and Arthrobotrys grown on PDA plates were used to prepare the spore suspensions. 5 ml of SDW was added to each culture and the spores were dislodged using a sterilized glass spreader. The resulting spore suspensions were transferred to sterilized petri dishes and spore concentrations adjusted to 1×106 spores/ml. Inoculation of the seedlings was done in the following manner: (i) Immersion of seedlings in 40 ml of each quantified fungal spore suspension separately in a petri plate. (ii) Immersion of seedlings in a mixture of 20 ml each of spore suspensions (40 ml in total) in a petri plate. (iii) Immersion of seedlings in 40 ml of SDW in a petri plate (control). Ten seedlings were used for each treatment and they were immersed in the spore suspensions/SDW overnight at RT (30˚C).There were ten replicates for each treatment. (b) Plate method: 10 day old Acremonium and Arthrobotrys cultures grown on PDA were used for the plate method and the inoculation was carried out as follows; (i) Seedlings were placed on each fungal culture plate separately and incubated for 2–3 days at RT (30˚C). (ii) Seedlings were placed on Acremonium culture plates for 2 consecutive days and subsequently on Arthrobotrys culture platesfor 2 consecutive daysat RT (30˚C). (iii) Seedlings transferred onto PDA+tetracycline plates without the fungus served as the controls. Ten seedlings were used for each treatment and there were ten replicates for each treatment. Confirmation of entry and establishment of inocula To test whether the inoculations were successful, randomly selected 15 seedlings subjected to each treatment of both methods including controls were surface sterilized (75% ethanol for 30 s, 1% NaOCl for 10 min and 70% ethanol for 30 s) and transferred onto PDA plates supplemented with tetracycline (50 mg.l-1). Determination of the effect on plant growth and yield The effect of the endophytes was evaluated under greenhouse conditionas pot experiment and in the field using seedlings inoculated with the fungal species separately and together by both methods of inoculation. Pot experiments in greenhouse Fifty seedlings selected randomly from the five replicates of each treatment were washed with SDW and planted in polythene bags (31 cm of diameter and 24.4 cm of height) filled with soil treated with organic fertilizers transported from a paddy field, which had been autoclaved for 20 min (121°C and 15 lb.in-2). Overall eighty bags were prepared at a rate of five seedlings per bag. Plants were watered regularly and maintained under a temperature range 30˚C (day) and 20˚C (night) in the greenhouse for 3 months. Field trials Autoclaved (at 121˚C and 15 lb.in-2 for 20 min) coconut coir was layered on plastic trays (31 cm × 20 cm) and seedlings subjected to each treatment were planted in the trays (150 seedlings per tray). Plants were allowed to grow under greenhouse conditions for 2 weeks with regular watering of the coconut coir. 14 day old plants were introduced into a pre-leveled field prepared for planting and they were hand-planted separating the groups of plants subjected to different treatments according to randomized block design (Fig. 1). Plants were allowed to grow in the field for 4 months until maturation under controlled conditions:the water depth was maintained evenly at 5 inches with 2 weekly additions of a liquid organic fertilizer mixture supplemented with dolomite (green manure, cow dung and dolomite, 7:2:1). www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 .

Figure 1. Lay out of the field trials.

Parameters Evaluated Height, fresh and dry weights of the plants under greenhouse and field conditions were measured for each treatment separately at 2 weeks, 4 weeks and 10 weeks after planting. The length of the crown to the end point of the flag leaf was measured as plant height. Dry weight of the plant was measured by oven drying at 60˚C until a constant weight was reached. Number of tillers per plant of the plants grown under field conditions was measured at the panicle initiation stage (10 weeks after planting). Weight of the seeds per plant was measured at harvesting stage. The results were analyzed statistically using ANOVA and pair wise comparisons of treatments were done using T-test. RESULTS Microscopic observations of endophytic fungi in Kuruluthuda

Figure 2. Endophytic fungal structures in Kuruluthuda roots: A–B, Trypan blue stained hyphae of endophytic fungal species (10×40×2); C–D, Sclerotium-like structures of colonized endophytic fungi (10×40×3).

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . Root segments stained withtrypan blue were examined for the presence of endophytic fungal structures. Intra and intercellular hyphae were seen in the root cortex parallel to the longitudinal axis of the roots (Fig. 2A & B), and intra-cellular, sclerotium-like structures were observed in roots (Fig. 2C & D). Table 1. Occurrence of endophytic fungi in different parts of Kuruluthuda plants.

Total number of Number of fungal Percentage frequency of samples species isolated isolation Root 25 13 52 Stem 25 6 24 Leaf 25 5 24 seed 25 3 12 Total 125 27 21.6 Note: Fungi isolated from surface sterilized plant parts placed on MEA plates. There were 25 replicates for each plant part. Plant part

Isolation and identification of endophytic fungi from Kuruluthuda rice variety A total of 27 endophytic fungi were isolated from 125 samples of root, stem, leaf and seeds of plants of Kuruluthuda assessed. Endophytic fungi were isolated at a higher frequency from root segments (Table 1). Acremonium, Arthrobotrys, Colletotrichum and Humicola showed ubiquitous colonization as they were isolated at least from 3 different plant parts and Acremonium was the most dominant endophytic fungus present in Kuruluthuda (Table 2). Table 2. Dominant endophytic fungal percentage and their colonized plant part of Kuruluthuda.

Endophytic fungi Acremonium Arthrobotrys Aspergillus sp1 Aspergillus sp2 Aureobasidium Chaetomium Colletotrichum Curvularia Fusarium Humicola Penicillium sp1 Penicillium sp2 Phoma sp1 Phoma sp2 Rhizoctonia Rhizopus sp1 Trichoderma sp1 Trichoderma sp2 Unidentified genus 1 Unidentified genus 2 Sterile Mycelia (SM) 1 SM 2 SM 3 SM 4 SM 5 SM 6

Colonized plant part seed , stem, leaf root, stem, leaf root root root root stem, leaf, seed root seed root, stem, leaf root stem root stem leaf stem root root root root stem root root leaf root, leaf seed

Percentage dominance 60.0 38.6 10.4 20.8 0.8 10.4 26.4 13.6 3.2 33.6 6.4 8.0 8.0 4.8 0.8 4.0 5.6 1.6 13.6 8.8 4.0 0.8 2.4 4.8 9.6 0.8

Effectiveness of the methods used for endophyte inoculation to rice seedlings Two of the most frequently isolated fungal endophytes i.e. Acremonium and Arthrobotrys were inoculated to seedlings separately and in combination to test their effect on the growth and productivity of the rice variety www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . Kuruluthuda using spore suspension and plate methods. To test for successful entry and colonization of seedlings, re-isolations were carried out. Fungal species identical to those that were inoculated were re-isolated from the seedlings inoculated using both methods. The inoculated fungal species were not isolated from the noninoculated seedlings treated with SDW (controls). These results confirmed that both methods of inoculation of the endophytes into seedlings were successful.

Figure 3. Variation in shoot length after 10 weeks: A–D, Kuruluthuda plants grown under field conditions; E–H, Kuruluthuda plants grown under greenhouse conditions; A,E, Acremonium inoculated plants; B,F, Arthrobotrys inoculated plants; C,G, Acremonium&Arthrobotrys inoculated plats; D,H, non-inoculated plants. Plants inoculated with both endophytic fungi show a significant increase of plant height (arrowed).

Effect of Acremonium and Arthrobotrys on growth and yield of Kuruluthuda A significant difference (P ≤ 0.05) in plant height as well as fresh and dry weights was observed for the plants subjected to all 3 treatments (i. Acremonium inoculation, ii. Arthrobotrys inoculation, iii. Acremonium and Arthrobotrys combined inoculation) when compared with non-treated plants under both field and greenhouse conditions after two, six and ten weeks (Table 3,4 & 5) (Fig 3). Plant height, fresh weight and dry weight of all treated and non-treated plants increased with time up to 10 weeks and finally became constant at panicle initiation stage. Table 3. Effect of endophyte inoculation on plant height 2, 6 and 10 weeks after planting.

Treatment

After 2 weeks Field Greenhouse 18.7 ± 0.28a 17.9 ± 0.37a 18.5 ± 0.38a 19.1 ± 0.50a b 19.2 ± 0.37a 20.0 ± 0.45b

Mean plant height (cm) ± SE After 6 weeks After 10 weeks Field Greenhouse Field Greenhouse 71.8 ± 0.98a 64.9 ± 1.44a 106.0 ± 1.21a 93.6 ± 2.03a 79.5 ± 0.91b 78.5 ± 1.64a b 113.7 ± 0.74b 108.5 ± 1.93b b b b 81.6 ± 1.01 84.0 ± 0.90 117.1 ± 0.84 110.2 ± 1.88b

Acremonium inoculation Arthrobotrys inoculation Acremonium & Arthrobotrys inoculation Non inoculation 14.5 ± 0.32b 14.3 ± 0.41c 58.3 ± 0.93c 51.0 ± 1.16c 98.3 ± 0.84c 73.6 ± 1.24c Note: n=20; mean ± SE; Mean values sharing common letters in each row are not significantly different p ≤ 0.05. Plants inoculated with Arthrobotrys showed a significant difference(P ≤ 0.05) in plant height, fresh weight, dry weight, tiller number and yield compared to the plants inoculated with Acremonium (Table 3,4,5,6 & 7) (Fig 3 A,B & E,F). There was a significant difference (P ≤ 0.05) in fresh and dry weights in plants inoculated with both fungal species separately and in combination in both greenhouse and field trials as compared with noninoculated plants. This was shown as early as 6 weeks (Table 4 & 5). There was no significant difference (P ≤ 0.05) in plant height and in tiller number between Arthrobotrys single inoculation and combined inoculation of both fungal species. However it was significantly different (P ≤ 0.05) when compared to Acremonium www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . inoculated plants after 6 and 10 weeks of planting (Table 3 & 6). Combined effect of two endophytic fungi showed an increase of the fresh weight and dry weight than their individual effects after 6 and 10 weeks of planting (Table 4 & 5). Table 4. Effect of endophyte inoculation on fresh weight 2, 6 and 10 weeks after planting.

Mean fresh weight (g) ± SE After 2 weeks After 6 weeks After 10 weeks Field Greenhouse Field Greenhouse Field Greenhouse 0.28 ± 0.01a 0.18± 0.02a 3.41 ± 0.98a 1.71 ± 0.38a 7.25 ± 0.22a 5.20 ± 019a 0.39 ± 0.02b 0.20 ± 0.02 a 3.85 ± 0.78b 1.82 ± 0.51b 7.86 ± 0.20b 5.25 ± 022a b 0.40 ± 0.03 0.21 ± 0.02a 4.21 ± 0.53c 2.15 ± 0.48c 9.31 ± 0.19c 5.74 ± 013b

Treatment

Acremonium inoculated Arthrobotrys inoculated Acremonium & Arthrobotrys inoculated Non inoculated 0.13 ± 0.01c 0.08 ± 0.01b 2.24 ± 0.37d 1.14 ± 0.36d 6.24 ± 0.12d 3.87 ± 0.17c Note: n=20; mean ± SE; Mean values sharing common letters in each row are not significantly different p ≤ 0.05. Table 5. Effect of endophyte inoculation on dry weight 2, 6 and 10 weeks after planting.

Treatment

Mean dry weight (g) ± SE After 2 weeks After 6 weeks Field Greenhouse Field Greenhouse 0.049 ± 0.002a 0.034± 0.002a 1.095 ± 0.23a 0.478 ± 0.02a a b b 0.050± 0.003 0.050 ± 0.02 1.214 ± 0.16 0.584± 0.035a b a 0.052 ± 0.003 0.055 ± 0.02b 1.586 ± 0.12c 0.748 ± 0.11c

After 10 weeks Field Greenhouse 2.478 ± 0.05a 1.115 ± 0.05a b 2.707 ± 0.021 1.244 ± 0.036b 3.421 ± 0.002c 1.586 ± 0.01c

Acremonium inoculated Arthrobotrys inoculated Acremonium & Arthrobotris inoculated Non inoculated 0.030 ± 0.001b 0.014 ± 0.001c 0.894 ± 0.08d 0.361 ± 0.013d 1.987 ± 0.04d 0.758 ± 0.02d Note: n=20; mean ± SE; Mean values sharing common letters in each row are not significantly different p ≤ 0.05. A significant difference (P ≤ 0.05) in tiller number and yield per plant was observed in the plants subjected to all 3 treatments compared with non-treated plants under field conditions (Table 6 & 7). Combined inoculation of Acremonium and Arthrobotrys showed an increase of yield than the plants subjected to other two treatments (Table 7). However data indicated that the overall effect of Arthrobotrys was higher than that of Acremonium. There was no significant difference in plant height between plants grown under greenhouse and field condition but the fresh weight and dry weight increased in all treated and non-treated plants under field condition when compared to greenhouse condition (Table 3). Table 6. Mean number of tillers at panicle initiation stage. (After10 weeks of planting)

Treatment Mean number of tillers± SE Acremonium inoculated 5.5 ± 0.14a Arthrobotrys inoculated 6.0 ± 0.14b Acremonium & Arthrobotrys inoculated 7.3 ± 0.16b Non inoculated 4.6 ± 0.11c Note: n=20; mean of tillers ± SE; mean values that do not share a letter are significantly different p ≤ 0.05. Table 7. Mean yield/plant (g) of Kuruluthuda variety. (After 12 weeks)

Treatment Mean yield per plant (g)± SE Acremonium inoculated 5.99 ± 0.12a Arthrobotrys inoculated 7.57 ± 0.12b Acremonium & Arthrobotrys inoculated 9.65 ± 0.23c Non inoculated 4.54 ± 0.17d Note: n=20; mean of yield/plant ± SE; mean values that do not share a letter are significantly different p ≤ 0.05. The mean weight of seeds per plant is considered as yield. DISCUSSION There is renewed interest in the cultivation of traditional rice varieties using organic farming practicesin Sri Lanka due to numerous health problems such as the Chronic Kidney Disease of Unknown Etiology (CKDU) which is spreading fast amongst farmers in the country as well as various environmental problems. The traditional rice varieties, whilst responding well to organic cultivation practices, produce low yields and thus, finding means of yield improvementis important. Research based on the development of effective organic fertilizers consisting of applicable microorganism preparations for increased yield is a viable solution to the problem. Endophytic fungi that grow as symbiontsin plants have been reported to enhance growth in a number www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . of crops as well as in organic rice cultivation (Angelard et al. 2010).The ability of endophytic fungi to improve plant growth and reduce rice blast incidence in traditional rice varieties of Sri Lanka has also been reported. (Atugala & Deshappriya 2015, Ponnawila & Deshappriya 2014). Therefore, the present study was carried out to test whether fungal endophytes of the Sri Lankan traditional rice variety Kuruluthuda would show a similar capacity for growth and yield enhancement of rice plants under green house and field conditions. Determination of the endophytic association was initialized by isolation of the endophyticfungal assemblage present in different parts of plants of the variety Kuruluthuda. As the use of fresh plant material is a critical requirement for successful isolation, fresh plant samples collected from the field were used for fungal isolations within 48 hours. Direct observation of trypan blue stained squashed root preparations indicated the co-existence of endophytic fungi inside roots without causing apparent damage to plant tissue. Since the endophytes are a group of microorganisms thatcan spend a part of their life cycle in different parts of the same plant (Petrini et al. 1992), most parts of the plant i.e. roots, stem, leaves and seeds were used for the isolations. A suitable surface sterilization regime is essential to eliminate epiphytic microbes present on the plant surface whilst maintaining the viability of the endophytes. Therefore, the most effective surface sterilizing regimes developed in a previous study (Atugala & Deshappriya 2015) were used for the effective surface sterilization of the plant parts used for isolations. Once surface sterilized,the plant parts were plated on MEA medium supplemented with tetracycline (50 mg/L) as soil and endophytic fungi are reported to grow more effectively on MEA resultingin a higher fungal yield and species richness (Bills & Polishook 1993). Tetracycline added to the medium restrained bacterial growth until emergence of fungal colonies from the plant segment. Isolation of endophytic fungi from healthy Oryza sativa plants has been reported previously to yield a few fungal genera such as Fusarium, Aspergillus, Curvularia, Penicillium, Gilmaniella and Arthrobotrys foliicola at a high frequency (Zakaria et al. 2010). Most fungal genera isolated from Sri Lankan traditional rice varieties Suwandel and Kaluheenati belonged to the class ascomycetes and they have been identified as being members of commonly observed genera of soil fungi, e.g. Fusarium, Penicillium, Rhizopus, Rhizoctonia, Absidia, Aspergillus and Paecilomyces (Atugala & Deshappriya 2015). In a comparison of endophytic assemblages between traditional and newly improved rice varieties in Sri Lanka, higher fungal colonization and species richness have been shownby the traditional rice varieties Suwandel and Herath Banda compared to the newly improved variety Bg 352. Fusarium and Arthrobotrys have been identified as the most frequently colonized endophytic fungi in Herath Banda traditional variety (Ponnawila & Deshappriya 2014). In the present study, 27 different fungal genera wereisolated from the variety Kuruluthuda. However, unlike in the previous studies, the most frequently isolated genera were Acremonium, Arthrobotrys, Colletotrichum and Humicola. Acremonium was mostly isolated from leaf and stem, Arthrobotrys from roots, Colletotrichum from stem and Humicola from leaves. Colonization and isolation frequencies of isolates in each plant part revealed that Kuruluthuda has a rich endophytic fungal diversity that is ubiquitous within the whole plant. Nevertheless, most isolates were restricted to specified areas of the plants. A surprisingly rich endophytic community was associated with roots but Fusarium and sterile mycelia species were restricted only to seeds. The combined and individual effects of the two most frequently isolated fungal endophytes Acremonium and Arthrobotryson growth and production of variety Kuruluthuda was tested under field and greenhouse conditions. Previous studies have shown that fungal inoculation into immature roots of seedlings was successful when they were in direct contact with fungal cultures (Fischer 1962, Herre et al. 2007) or immersed in a spore suspension (Champion et al. 1973, Abdel-Motaal et al. 2010). In this study, both methods were used and the ability to reisolate the same fungal species from the seedlingsas that of the inoculated species confirmed the success of both spore suspension and plate methods in introducing the endophytes to plant seedlings. This is significant as successful and easy introduction of inoculum into plants is an essential aspect of development of a biofertilizer. In the present study,endophyte inoculated plants showed a significant increase of plant height, fresh weight and dry weight (P ≤ 0.05) when compared with non-inoculated plants during a 10 week period under both field and greenhouse conditions. These results are in agreement with previous studies carried out in Sri Lanka (Ponnawila & Deshappriya 2014, Atugala & Deshappriya 2015) and indicate that rice plant growth can be enhanced by introducing their endophyticmyco flora. Mechanisms of growth enhancement were not studied in the present study. However evidence from previous studies have indicated that fungal endophytes promote plant growth and performance in a number of ways www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . including the production of siderophores, supplying biologically fixed nitrogen and by secretion of plant growth regulators (Herre et al. 2007, Kedar et al. 2014). The endophytic fungi of Potentilla fulgensis is reported to have the metabolic machinery to secrete plant growth hormones such as cytokinins, auxins and gibberellins which promote seed germination and growth (Bhagobaty & Joshi 2009). Khan et al. (2015) reported that two fungal endophytes, Fusarium and Alternaria isolated from the leaves of Solanumnigrum, increased the chlorophyll content, root-shoot length, and biomass production of Dongjin rice plantswhen inoculated at the seedling stage. Gas chromatography/ mass spectrometry analysis for the culture filtrates of each fungal endophytic isolate revealed that both Fusarium and Alternaria could produce 54 and 30 µ.gm.L-1 of indole acetic acid, respectively. In addition, Kedar et al. (2014) revealed a significant effect on shoot height, root length, fresh and dry weights of shoots and roots of experimental maize plants inoculated with the endophytic fungus Phoma compared to non-inoculated plants. It was also found that the roots of germinated seeds treated with the Phoma extract showed development of additional root hairs allowing the absorption of more nutrients which subsequently resulted in increased growth and biomass of plants. Based on these reports, it can be concluded that endophytes have a number of mechanisms to enhance plant growth and the similar trend of growth enhancement observed in the present study may also be attributed to one or more of such mechanisms. In most previous studies, individual effects of two endophytic fungi on host plant performances have been tested. Waqas et al. (2012) reported that the endophytic fungi Phomaglomerata and Penicillium sp. significantly promoted the shoot growth of rice as well as increased plant biomass and related growth parameters of cucumber plants compared to non-inoculated plants. However, the combined effect of two endophytes has not been evaluated in these studies. In the present study, the combined effect of two endophytic fungal species showed an increase of the fresh and dry weights when compared with their individual effects on Kuruluthuda plants under both field and greenhouse conditions. It can be concluded that Acremonium and Arthrobotrys may have a synergistic effect that results inimproved plant weight. A significant difference of plant height could not be observed between Arthrobotrys single inoculation and combined inoculation with both fungal species. However, there was a significant increase in the height of plants inoculated with Arthrobotrys when compared with those inoculated with Acremonium indicating that Arthrobotrys might have the ability to improve plant height than Acremonium. Although tillers of the Kuruluthuda variety were formed 4 weeks after planting, the number of tillers was counted at the panicle initiation stage to obtain an accurate count of tillers per one bush. An increased number of tillers could be observed for the plants inoculated with both fungi. It is reported that endophytic fungi associated with many agricultural crops improve crop production significantly (Yuan et al. 2009). In this study also endophytes inoculated Kuruluthuda plants yielded a significantly higher amount of seeds when compared with non-inoculated plants. Plants inoculated with both Acremonium and Arthrobotrys gave a higher yield as well as improved fresh and dry weights. Therefore it can be concluded that the combination of two endophytic fungi can result in increased biomass and rice yield. These results were evident as early as six weeks after planting and remained to be so uptothe harvesting stage (12 weeks). There was no difference in plant height between plants grown under greenhouse and field conditions but increased fresh weight and dry weight could be observed under field conditions when compared to greenhouse condition. The enhanced effect of endophyte inoculation shown by plants grown under field conditions when compared with those grown under greenhouse conditions was both an interesting and encouraging result. Based on these very promising results, an inoculum consisting of the two fungal endophytes i.e. Acremonium and Arthrobotrys could be utilized to enhance the biomass and yield of the rice variety Kuruluthuda. Thisis the first report on the endophytic fungal assemblage present in the varietyKuruluthuda and their potential to be used for improved productivity of the rice variety under field conditions. The possibility of developing such an inoculum as a biofertiliser for other rice varieties after more stringent testscould be the solution to the many problems associated with the use of chemical fertilizers in rice cultivation in Sri Lanka and worldwide. CONCLUSIONS  Kuruluthuda traditional rice variety consists of a high diversity of fungal endophytesin most parts of the plant i.e. root, stem, leaf, and seeds. www.tropicalplantresearch.com 478

Wijesooriya & Deshappriya (2016) 3(3): 470–480 .  Spore suspension method and plate method can be used for successful inoculation of fungal endophytes into seedling roots.  Acremonium and Arthrobotrys can improve the growth of rice plants of the variety Kuruluthuda and yield under field conditions.  Combined effect of the two endophytes enhances the fresh weight, dry weight, number of tillers and yield of rice plants significantly as compared to their effect when inoculated separately. REFERENCES Abdel-Motaal F, Nassar MSM, El-Zayat SA, El-Sayed MA & Ito S (2010) Antifungal activity of endophytic fungi isolated from Egyptian Henbane (Hyoscyamus muticus L.). Pakistan Journals of Botany 42: 2883– 2894. Angelard C, Colard A, Niculita-Hizel H, Croll D & Sanders IR (2010) Segregation in a Mycorrhizal fungus alters rice growth and symbiosis-specific gene transcription. Current biology 20: 1261–1221. Atugala DM & Deshappriya N (2015) Effect of endophytic fungi on plant growth and blast diseaseincidence of two traditional rice varieties. Journal of National Science Foundation Sri Lanka 43: 173–187. Barraquio WL, Revilla L & Lodha JK (1997) Isolation of endophytic diazotrophic bacteria from wetland rice. Plant and Soil 194: 15–24. Bhagobaty RK & Joshi SR (2009) Promotion of seed germination of Green gram and Chick pea by Penicillium verruculosum RS7PF, a root endophytic fungus of Potentilla fulgens L. Advanced Biotech 8(7): 16–18. Bills GF & Polishook JD (1993) Abundance and diversity of microfungi in leaf litter of a lowland rain forest in Costa Rica. Mycologia 86: 187–198. Champion MR, Brunet D, Mauduit ML & Ilami R (1973) Methods for testing the resistance of bean varieties to anthracnose (Collectotrichum lindemuthianum. And Magn.) Briosi and Cav.P.C.R.Seances acad. Agricultural Food Reaschers 59: 951. Contreras-Cornejo HA (2009) Trichoderma virens, a plant beneficial fungus enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiology 149: 1579–1592. Domsch KH, Dams W & Anderson PL (1993) Compendium of soil fungi, IHW- Verlag press, Berlin, Germany. FAO (1994) Rice Production. Food and Agriculture Organization of the United Nations Year Book 47: 70–71. Feng QL, Chen GH, Cui FZ, Kim TN & Kim JO (2000) A mechanistic study of the antibacterial effect of silver ion on E. coli and S. aureus. Journals of Biomedical Materials research 52: 662–668. Fischer GW (1962) Induced by hybridization and point of inoculation on the symptomatology of barley covered smut. Phytopathology 1962: 52. Fisher PJ & Petrini O (1991) Fungal saprobes and pathogens as endophytes of rice (Oryza sativa L.). New Phytology 120: 137–143. Goveas SW, Madtha R, Nivas SK & D’Souza L (2011) Isolation of endophytic fungi from Coscinium fenestratum- a red listed endangered medicinal plant. EurAsian journal of BioSciences 5: 48–53. Herre EA, Mejia LC, Kyllo DA, Rojas E, Maynard Z, Butler L & Van bael SA (2007) Ecological implications of anti-pathogen effects of tropical fungal endophytes and mycorrhizae. Ecology 88 (3): 550–558. Kedar A, Rathod D, Yadav A, Agarkar G & Rai M (2014) Endophytic Phoma sp. isolated from medicinal plants promotes the growth of Zea mays. Bioscience 6: 132–139. Khan AR, Ullah I, Waqas M, Shahzad R, Hong S, Park G, Jung BK, Lee I & Shin J (2015) Plant growthpromoting potential of endophytic fungi isolated from Solanum nigrum leaves. World Journal of Microbiology and Biotechnology 31(9): 1461–1466. Montesinos E (2003) Plant-associated microorganisms: a view from the scope of microbiology. International Microbiology 6: 221–223. Naik BS, Shashikala J & Krishnamurthy YL (2009) Study on the diversity of endophytic communities from rice (Oryza sativa L.) and their antagonistic activities in vitro. Microbiological Research 164: 290–296. Petrini O (1991) Evaluation of fungal endophytes in aerial roots of Ficus benghalensis. Fungal Ecology 17: 175–187. Petrini O, Sieber TN, Toti L & Viret O (1992) Ecology, metabolite production and substrate utilization in endophytic fungi. Natural Toxins 1: 185–196. www.tropicalplantresearch.com

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Wijesooriya & Deshappriya (2016) 3(3): 470–480 . Ponnawila PVAR & Deshappriya N (2014) Investigation of fungal endophytes present in rice varieties Bg 352, Suwandel and Herath Banda. Proceedings 34th annual sessions of Institute of Biology Sri Lanka 34 (2): 45. Rathnabharathi W (2009) Role of traditional paddy in adaptation to climate change impact. In: Proceedings of the Role of Community in Adaptation to the Climate Change Crisis Workshop, pp. 67–80. Ryan R.P, Germaine K, Franks A, Ryan DJ & Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiology Letters278: 1–9. Strobel G (2003) Endophytes as sources of bioactive products. Microbes and Infection 5: 535–544. Tarafdar JC & Claassen N (1998) Organic phosphorous compounds as a phosphorous source of higher plants through the activity of phosphatase produced by plant roots and microorganisms. Biology and Fertility of soils 5: 308–312. Tian XL, Cao LX, Tan HM, Zeng QG, Jia YY & Han WQ (2004) Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro. World Journal of Microbiology and Biotechnology 20: 303–309. Tutte J (1969) Plant Pathologiacal Methods: Fungi and Bacteria. Burgess Publishing Company, USA, pp. 220. Waqas M, Khan AL, Kamran M,Hamayun M, Kang S, Kim Y & Lee J (2012) Endophytic Fungi Produce Gibberellins and Indole Acetic Acid and Promotes Host-Plant Growth during Stress. Molecules 17: 10754– 10773. You YH, Yoon H, Kang SM, Shin JH, Choo YS, Lee IJ, Lee JM & Kim JG (2012) Fungal diversity and plant growth promotion of endophytic fungi from six halophytes in Sun cheon Bay. Journal of Microbiology and Biotechnology 22: 1549–1556. Yuan Z, Zhang C, Lin F & Kubicek CP (2009) Identity, diversity, and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice (Oryza granulate) from a nature reserve in Yunnan, China. Applied and Environmental Microbiology 76: 1642–1652. Zakaria L, Yaakop AS, Salleh B & Zakaria M (2010) Short communication: endophytic fungi from paddy. Tropical Life Sciences Research 21: 101–107.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 481–490, 2016 DOI: 10.22271/tpr.2016.v3.i3.064 Research article

Morphological and molecular characterization of Colletotrichum capsici causing leaf-spot of soybean Rajyasri Ghosh1*, Sreetama Bhadra2 and Maumita Bandyopadhyay2 1

Department of Botany, Scottish Church College, 1 & 3 Urquhart Square, Kolkata, West Bengal, India Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, West Bengal, India *Corresponding Author: [email protected] [Accepted: 14 September 2016] 2

Abstract: In the present investigation an attempt was made to carry out morphological and molecular characterization of Colletotrichum isolate (CI) associated with leaf spot disease of soybean. Pathogenicity test was carried out with the isolate and symptoms of leaf spot were found to appear after 7 days of inoculation. The pathogen was compared with 2 isolates of Colletotrichum capsici (CII and CIII) and one isolate of Colletotrichum gloeosporoides (CIV) from chilli. On the basis of morphological and cultural characteristics the pathogen was identified as C. capsici. For molecular identification random amplified polymorphic DNA (RAPD) primers and internal transcribed spacer (ITS2) primers were tested on the genome of Colletotrichum isolates from soybean and chilli plants and the data provided characteristic genetic evidence of 100% similarity within the CI isolate from soybean and C. capsici isolates from chilli (CII and CIII). To confirm the molecular identification of the pathogen species specific primers (CcapF/CcapR) were used and it was established that the pathogen is indeed C. capsici. Results of present investigation revealed the first report of C. capsici in soybean plant. Keywords: Soybean - leaf-spot - Colletotrichum - RAPD - ITS. [Cite as: Ghosh R, Bhadra S & Bandyopadhyay M (2016) Morphological and molecular characterization of Colletotrichum capsici causing leafspot of soybean. Tropical Plant Research 3(3): 481–490] INTRODUCTION The genus Colletotrichum contains many morphologically similar taxa comprising endophytic, saprobic and plant pathogenic fungi (Photita et al. 2004, Damm et al. 2009). Anthracnose disease caused by the Colletotrichum species is a major problem worldwide. Among these species, C. gloeosporioides (Penz.) Penz. & Sacc. and C. capsici (Syd.) E.J. Butler & Bisby are most frequently cited as causal agents of anthracnose (Than et al. 2008, Gautam 2014, Ramdial & Rampersad 2015). C. capsici also has been reported to have a wide putative host range associated with symptoms of foliar blight and leaf spot diseases (Shenoy et al. 2007). Leaf spot disease caused by Colletotrichum capsici is the most important economic constraint which hamper, turmeric (Curcuma longa L.) production in major turmeric growing regions of the India, and often results in high yield losses (Adhipathi 2013, Uma Devi 2008). The occurrence of different virulent strains of Colletotrichum species has been well documented in India (Sharma et al. 2005, 2013). Numerous cases have been reported in which several Colletotrichum species or biotypes are associated with a single host (Peres et al. 2002) making their identification by morphological and physiological methods more difficult. The use of molecular marker techniques has improved the accuracy and speed of identification of Colletotrichum spp. (Cai et al. 2009). Among these molecular techniques, RAPD technique has been extensively used to investigate relationships among isolates of Colletotrichum spp. (Madhavan et al. 2010, Sangdee et al. 2011). Similarly nucleotide sequence information for the 5.8S rDNA gene and the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) have been used to design species specific primers of members of Colletotrichum for diagnostic purposes and for phylogenetic analysis (Thalhinhas et al. 2002). Soybean is an important global crop that is grown in tropical, subtropical and temperate climate. Anthracnose is a major fungal disease of soybean which is caused by Colletotrichum capsici and several related www.tropicalplantresearch.com

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Received: 21 May 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.064

Ghosh et al. (2016) 3(3): 481–490 . species. Anthracnose of soybean can cause severe symptoms and yield loss in southern Taiwan. Strains of C. truncatum and C. gloeosporioides derived from diseased pods were noted as causal agents (Chen et al.1986). Purkayastha & Banerjee (1990) reported anthracnose of soybean in India caused by C. dematium. Soybean foliage is also susceptible to a number of fungal and bacterial pathogens causing leaf spots and blights. Among leaf spot fungal diseases, brown spot disease caused by Septoria glycines and frogeye disease caused by Cercospora sojina are most frequently encountered (Hershman 2013). In the present investigation an attempt was made to carry out morphological and molecular characterization of Colletotrichum isolate associated with leaf spot disease of soybean plants. MATERIALS AND METHODS Isolation and identification of pathogens from infected plants Fungal isolate (CI) was collected from leaves of infected soybean plants grown in the field of experimental garden. The diseased leaf parts were cut at the advanced margin of lesions into small pieces (5×5 mm) and then surface disinfected with 0.1% HgCl2 for 1 min, followed by rinsing with sterile distilled water two times. The pieces were then transferred to potato dextrose agar (PDA) medium and incubated at 30±2°C for 2–3 days. Pure cultures of the fungi were obtained by single spore isolation method. The isolate was first identified to species by morphological observation under a compound microscope. Two other isolates of C. capsici (CII and CIII) and one isolate of C. gloeosporoides (CIV) from chilli were used for identification of C. capsici. Pathogenicity test In order to prove Koch’s postulate pathogenicity test was carried out. One month old soybean plants were sprayed with spore suspension of CI (106 conidia/ml) and covered with moist polythene bags for 48 hours under natural condition of light and temperature (20–30ºC). Disease intensity was measured after 5, 7, 10, 14, 21 days after inoculation following the method of Purkayastha & Banerjee (1990). Disease symptom is necrotic spots (lesion) on the leaves of plant. Six leaves from each plant were selected randomly and the number and size of the lesions on each leaf were noted. On the basis of visual observation, the necrotic lesions were graded into small, medium and large groups and a value of 0.5, 1 and 2 were given to lesions denoting their diameter of 1, 2 and more than 2 mm respectively. The number of spots in each group was multiplied by value assigned to it. The sum total values taken together for all leaves divided by the number of test plants. Cultural characters of Colletotrichum isolates Growth characteristics of fungal isolates were measured in potato dextrose agar (PDA) solid medium. The mycelial diameter and morphological nature of mycelia was recorded in solid medium. Shape and size of conidia of the fungal isolates were also observed. Fungal DNA extraction and RAPD analysis For DNA extraction, each isolate was grown in 100 ml conical flasks, containing 30 ml of potato dextrose broth, for 5 days at room temperature (28±2°C). The mycelia were harvested by filtration and stored at -20°C for 2–3 days. Freeze-dried mycelium (0.5 g) was ground to a fine powder and DNA was extracted using CTAB (hexa-decyltrimethylammonium bromide) method described by Bhadra & Bandyopadhyay (2015). RAPD analysis was performed using 25 10-mer primers initially, among which 10 were chosen for final analysis due to high reproducibility of the banding pattern generated (Table 1). Amplification was performed in 25 µl reaction volume consisting of 100 ng template DNA, 100 pmole of primer, 10 milimole dNTPs (Applied Biosystems, Carlsbad, California, USA), 1.5 U Taq DNA polymerase and 10X reaction buffer containing 15 mM MgCl2 (Merck Genei, Bangalore, India). PCR was performed using Veriti® 96-Well Thermal Cycler (Applied Biosystems, Carlsbad, California, USA) with an initial denaturation step for 5 minutes at 94 oC followed by 40 cycles of 1 minute at 94oC, 1 minute at 37oC and 2 minutes at 72oC with a final extension for 10 minutes at 72oC. 1.5 % (W/V) agarose gel was used to analyse the PCR product. Size of the amplified fragments was determined using 100bp DNA ladder (Applied Biosystems, Carlsbad, California, USA). The gel was photographed using E-Gel® Imager (Applied Biosystems, Carlsbad, California, USA). The amplified bands thus obtained were scored as present (1) or absent (0) for data analysis. Regardless of the intensity of the amplified band, each was treated as an independent entity. The Rp (resolving power) of each primer was calculated using the formula Rp= 1-[2*(0.5-p)] (where p is the proportion of taxa containing the band) (Prevost & Wilkinson 1999). The scored binary matrix was then subjected to analysis using NTSYSpc www.tropicalplantresearch.com

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Ghosh et al. (2016) 3(3): 481–490 . software, version 2.02i (Rohlf 1999). Jaccard's similarity co-efficient was calculated using SIMQUAL program using the formula NAB / (NAB+NA+NB) where NAB is the number of amplified fragments shared by samples, NA is the number of fragments in sample A, and, NB denotes those in sample B (Souframanien & Gopalakrishna 2004). Amplification and Sequencing of ITS1-5.8S-ITS2 of rDNA The four Colletotrichum isolates were further characterized by nucleotide sequence analysis after amplification of the ITS1-5.8S-ITS2 regions using the universal primers ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) and ITS5 (5’-GGA AGT AAA AGT CGT AAC AAG G-3’) (White et al. 1990), and soybean isolate CI was used as positive control. PCR reactions were performed in reaction volumes of 50 μl containing 25 ng of genomic DNA, 1×PCR buffer (Merck Genei, Bangalore, India), 0.5 mM of each dNTP (Applied Biosystems, Carlsbad, California, USA), 1 μM primers, and 1U Taq polymerase (Merck Genei, Bangalore, India). DNA amplification was performed in a Veriti® 96-Well Thermal Cycler (Applied Biosystems, Carlsbad, California, USA), and the program consisted of an initial denaturing step at 95°C for 3 min, followed by 25 cycles of 30 s at 95°C, 30 s at 54°C, and 60 s at 72°C; and a final extension step of 10 min at 72°C. PCR products, approximately 600 bp, were separated by electrophoresis in 1.5% (W/V) agarose gels and visualized by ethidium bromide staining and were photographed using the gel documentation system. Specific bands were then cut off from gel and the amplified DNA was extracted using Silica Bead DNA Gel Extraction Kit (Thermo Fisher Scientific Inc., Waltham, MA USA). DNA sequencing was performed with two primers (ITS4 and ITS5) in both directions to ensure that there was no misreading. PCR products were sequenced by ABI 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, California, USA). Alignment and edition were carried out with the CAP3 Sequence Assembly Program (Huang & Madan 1999) and visually corrected. Sequences were then compared using Mega 6.0 software program (Tamura et al. 2013). PCR using Species-Specific Primers Colletotrichum capsici specific primer pairs CcapF (5’-GTA GGC GTC CCC TAA AAA GG-3’) and CcapR (5’-CCC AAT GCG AGA CGA AAT G-3’) previously reported by Torres-Calzada et al. (2011) were used for rDNA amplification of all the samples. Amplification reactions were performed using protocol reported earlier. PCR products were visualized by electrophoresis in 1.5% (W/V) agarose gels, stained with ethidium bromide and documented using gel documentation system. RESULTS AND DISCUSSION Identification of pathogen Pathogen associated with leaf spot disease of soybean was identified as C. capsici on the basis of morphological characteristics. Pathogenicity test Healthy soybean plants were inoculated after one month of sowing and disease index was noted after 5, 7, 10, 14 and 21 days of inoculation. Symptoms were found to appear 7 days of inoculation and disease intensity was found to increase with the incubation period. Disease symptom was noted as necrotic spots (lesion) on the leaves of the plant. The results are presented in the table 1 and figures 1A & B. Table 1. Pathogenicity test of Colletotrichum capsici (CI) on healthy soybean cultivar.

Incubation period (days)a Disease index / plantb 5 0 7 3.5 10 5.75 14 7.25 21 8 Note: a - Days after inoculation; b - Average of 4 plants. Age of plant at the time of inoculation: 1 month; Inoculation potential: 106 conidia/ml Cultural characteristics The mycelial growth of Colletotrichum isolates in solid PDA medium was measured after 12 days of incubation. The CI, CII and CIII isolates showed dense white to greyish brown mycelia with ring like zonations www.tropicalplantresearch.com

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Ghosh et al. (2016) 3(3): 481–490 . in solid oat meal medium (Fig. 1C). C. gloeosporoides (CIV) produced pinkish white colony with cylindrical conidia (Table 2). Table 2. Morphological and cultural characters of strains of Colletotrichum capsici.

Cultural characters Isolate

Colony colour Margin Topography

Zonation

Grayish brown smooth Mycelium flat growth Concentric zonation CI Grayish brown smooth Mycelium flat growth Concentric zonation CII Grayish brown smooth Mycelium flat growth Concentric zonation CIII Pinkish white smooth Mycelium flat growth Concentric zonation CIV Note: Incubation period: 12 days. a - Average of three replicates.

Colony diametera 7.13±0.19 8.4±0.21 7.66±0.04 7.85 ±0.16

Morphological characters Conidia shape Falcate, fusiform Falcate, fusiform Falcate, fusiform cylindrical

Figure 1. Symptoms of leaf spot disease on infected leaves of soybean and morphological and cultural characters of the pathogen.

Morphological characters The conidia of Colletotrichum isolates from soybean and chilli (CI, CII and CIII) is falcate, fusiform with acute apices. (Fig. 1D) The size ranges from 19.65–21.00 µm in length and 3.0–3.5 µm in breadth. In C. gloeosporiodes (CIV) conidia are hyaline, one celled, cylindrical. The size ranges from 8–12 µm in length and 4–6 µm in width. www.tropicalplantresearch.com

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Ghosh et al. (2016) 3(3): 481–490 . Molecular identification of the pathogen i. RAPD-PCR analysis 25 RAPD primers were tested on the genome of CI, CII, CIII and CIV. The generated DNA fingerprints were evaluated for assessing genetic similarity among the four isolates. Data generated by 10 primers were analyzed as they reliably and reproducibly detected polymorphism among the selected isolates (Table 3). Figure 2 shows typical profile and polymorphic bands generated after amplification with the primers. Table 3. List of primers used, their sequences, and amplified products generated.

Total number of bands OPA 04 5'-AATCGGGCTG-3' 35 OPA 11 5'-CAATCGCCGT-3' 29 OPB 01 5'-GTTTCGCTCC-3' 16 OPB 07 5'-GGTGACGCAG-3' 27 OPC 09 5'-CTCACCGTCC-3' 22 OPD 04 5'-TCTGGTGAGG-3' 15 OPE 03 5'-CCAGATGCAC-3' 37 OPG 08 5'-TCACGTCCAC-3' 26 OPK 17 5'-CCCAGCTGTG-3' 32 OPL 03 5'-CCAGCAGCTT-3' 37 Primer

Sequence of primers

Number of polymorphic bands 31 24 14 23 19 14 36 23 30 36

Number of monomorphic bands 4 5 2 4 3 1 1 3 2 1

Percentage polymorphism 88.6 82.8 87.5 85.2 86.4 93.3 97.3 88.5 93.8 97.3

Resolving power (Rp) 35.5 33 16.5 31.5 23.5 15 35.5 29.5 35.5 40

Figure 2. RAPD amplification patterns of four Colletotrichum isolates using the primers OPC09 and OPA11. L: Ladder; CI, CII, CIII & CIV: Colletotrichum isolates; C: negative control.

The 10 RAPD primers produced a total of 276 amplified fragments with 250 polymorphic and 26 monomorphic bands. The average polymorphism of these primers varied from 82.8% to 97.30% with an average of 90.05%. The resolving power (Rp) of the primers varied from 15 to 40. Table 3 summarizes the above data. The dendrogram generated from the RAPD data using Jaccard's similarity coefficient showed a variation from 0.10 to 1.00 among the four isolates. While the three Colletotrichum isolates (CI, CII and CIII) separated out in a single cluster showing 0.85 similarity coefficient among them, C. gloeosporoides (CIV) showed similarity coefficient of 0.10 with them. Based on the high similarity value of 1.0 between CI and CII it may be assumed that both of them belong to the same species (Fig. 3). www.tropicalplantresearch.com

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Figure 3. Dendogram generated using Jaccard’s similarity distance obtained from RAPD primers. CI, CII, CIII & CIV: Colletotrichum isolates.

ii. Amplification and Sequencing of ITS1-5.8S-ITS2 of rDNA To further confirm the result obtained using RAPD marker-based study, PCR amplification of the ITS region of all the studied samples were also done using universal primers ITS4 and ITS5. A single band of 620 bp was obtained in all cases (Fig. 4). No nonspecific banding was found. The amplified fragments were, thus, purified and sequenced.

Figure 4. Products of PCR amplification of ITS region using primer set ITS 4and ITS 5. Lane L: Marker DNA ; CI,CII,CIII & CIV: Colletotrichum isolates; C: negative control.

The sequence data, thus obtained, were compared using Mega 6.06 software. The sequence analysis of the ITS region revealed 100% sequence similarity within the three isolates namely CI, CII and CIII. The ITS2 sequence of C. gloeosporoides (CIV), on the other hand, revealed significant difference with other three isolates (Fig. 5). The sequences were also used to generate a dendrogram revealing phylogenetic relationship among the studied taxa using UPGMA method (Fig. 6). www.tropicalplantresearch.com

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Figure 5. Alignment of ITS1-5.8S-ITS2 sequences of the rRNA gene region of all accessions used in the present investigation using ClustalW. Contig S1= CI; Contig S2=CII; Contig S3: CIII; Contig S4: C. gloeosporoides.

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Figure 6. Dendogram generated using the UPGMA method obtained from ITS sequences. CI, CII, CIII & CIV: Colletotrichum isolates.

iii. PCR using Species-Specific Primers When the primer pair CcapF/CcapR were used to amplify the genomic DNA in the present study, the two chilli isolates of C. capsici (CII and CIII) and the CI isolate from soybean tested positive with a single amplified fragment in each taxa. The size of this fragment was 394 bp in all of the C. capsici tested. The primers, however, used failed to amplify genomic DNA of C. gloeosporoides (Fig. 7).

Figure 7. Products of PCR amplification using C.capsici specific primer pairs Ccap F / Ccap R. Lane L: Marker DNA; CI, CII, CIII & CIV: Colletotrichum isolates; C: negative control.

DISCUSSION In the present investigation results of pathogenicity test indicated that Colletotrichum sp. (CI isolate) can establish leaf spot disease in susceptible soybean cultivar. Traditional identification and characterization of Colletotrichum species relies primarily on difference in morphological features such as colony, colour, size and shape of conidia and appressoria. In this study morphological characterization of Colletotrichum isolates from chilli and soybean plants were done. Results revealed that CI, CII and CIII isolates showed similar characteristics, whereas CIV is morphologically different from the other 3 isolates. This result is in agreement with a previous study by Sandgee et al. (2011) who found a morphometric overlap of conidial size among Colletotrichum species. Thangamani et al. (2011) performed morphological and physiological characterization of Colletotrichum musae, the causal organism of banana anthracnose. However morphological characterizations www.tropicalplantresearch.com

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Ghosh et al. (2016) 3(3): 481–490 . are not always adequate for reliable differentiation among species of Colletotrichum. Thus molecular methods have been employed successfully to differentiate between populations of Colletotrichum from many hosts. In the present investigation, RAPD primers were tested on genome of four Colletotrichum isolates. Analysis of data generated using 10 RAPD primers exhibited polymorphism varying from 82.76% to 97.30% with an average of 90.05%. Cluster analysis using Jaccard’s coefficient clearly separated the isolates of chilli and soybean from C. gloeosporioides. RAPD analysis was also performed by Ratancherdchai et al. (2007) on 18 isolates belonging 2 species, C. gloeosporiodes and C. capsici isolated from three varieties of chilli, i.e. chilli pepper (Capsicum annum), long cayenne pepper (C. annuum var. acuminatum) and Birds eye chilli (C. frutescens). Their study showed that a clear difference could be established between C. gloeosporiodes and C. capsici. The three C. capsici isolates analysed in the present study show similarity of 0.85–1.00 among themselves. The existence of molecular variability among isolates of C. capsici that differed in virulence was earlier established by Srinivasan et al. (2010) using RAPD markers. The result of RAPD in the present investigation was further confirmed by sequence analysis of the ITS region which revealed 100% sequence similarity between the two C. capsici isolates of chilli and CI isolate of soybean. C. gloeosporoides, on the other hand, revealed significant sequence difference with C. capsici isolates CI. Freeman (2008) used sequence analysis of ITS region to establish similarity between Colletotrichum acutatum isolates from almond and strawberry. Shi et al. (2008) had used ITS universal primer pair ITS1-F/ITS4 to characterize Colletotrichum acutatum and C. gloeosporioides isolates from flowering dogwood (Cornus florida). Katoch et al. (2016) carried out Metageographic population analysis of Colletotrichum truncatum associated with chili fruit rot and other hosts using ITS region nucleotide sequences. Molecular characterization of the soybean isolate was finally established by using species specific primer which revealed that this isolate and two other C. capsici isolates tested positive with a single amplified fragment in each taxa. C. gloeosporoides, however, failed to produce any amplified DNA fragment using these primers. The results of molecular characterization and morphological identification thus established that the CI isolate from soybean is C. capsici. C. capsici was not reported in soybean till date. Results of present investigation revealed the first report of C. capsici in soybean plant. ACKNOWLEDGEMENT Scientific assistance received from UGC, Govt. of India is gratefully acknowledged. REFERENCES Adhipathi P, Nakkeeran S & Chandrasekaran A (2013) Morphological characterization and molecular phylogeny of Colletotrichum capsici causing leaf spot disease of turmeric. The Bioscan 8: 331–337. Bhadra S & Bandyopadhyay M (2015) A fast and reliable method for DNA extraction from different plant parts of Zingiberaceae. Journal of the Botanical Society of Bengal 69(2): 91–98. Cai L, Hyde KD, Taylor PWJ, Weir BS, Waller JM, Abang MM, Zhang JZ, Yang YL, Phoulivong S, Liu ZY, Prihastuti H, Shivas RG, McKenzie EHC & Johnston PR (2009) A polyphasic approach for studying Colletotrichum. Fungal Diversity 39: 183–204. Chen LS, Chu C, Liu C, Chen RS & Tsay JG (2006) PCR-based detection and differentiation of Anthracnose pathogens, Colletotrichum gloeosporioides and C. truncatum, from vegetable soybean in Taiwan. Journal of Phytopathology 154: 654–662. Damm U, Woudenberg JHC, Cannon PF & Crous PW (2009) Colletotrichum species with curved conidia from herbaceous hosts. Fungal Diversity 39: 45–87. Freeman S (2008) Management, Survival Strategies, and Host Range of Colletotrichum acutatum on Strawberry. Hortscience 43: 66–68. Gautam AK (2014) The genera Colletotrichum: an incitant of numerous new plant diseases in India. Journal on New Biological Reports 3(1): 9–21. Hershman DE (2003) Soybean Foliar Spots and Blights. Available from: http://www.ca.uky.edu/agcollege/plant pathology/ext_files/ PPFShtml /ppfsags19.pdf (accessed: 10 Feb. 2016). Huang X & Madan A (1999 ) CAP3: A DNA sequence assembly program. Genome Research 9: 868–877. Katoch A, Prabhakar CS & Sharma PN (2016) Metageographic population analysis of Colletotrichum truncatum associated with chili fruit rot and other hosts using ITS region nucleotide sequences. Journal of Plant Biochemistry and Biotechnology 25: 64–72. www.tropicalplantresearch.com

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Ghosh et al. (2016) 3(3): 481–490 . Madhavan S, Paranidharan V & Velazhahan R (2010) RAPD and virulence analyses of Colletotrichum capsici isolates from chilli (Capsicum annuum). Plant Diseases and Protection 117: 253–257. Peres NR, Kuramae EE, Dias CM & De’souza N (2002) Identification and characterization of Colletotrichum spp. affecting fruit after harvest in Brazil. Journal of Phytopathology 150: 128–134. Photita W, Taylor PWJ, Ford R, Hyde KD & Lumyong S (2005) Morphological and molecular characterization of Colletotrichum species from herbaceous plants in Thailand. Fungal Diversity 18: 117–133. Prevost A & Wilkinson MJ (1999) A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theoretical and Applied Genetics 98: 107–112. Purkayastha RP & Banerjee R (1990) Immunoserological studies of cloxacillin-induced resistance of soybean against anthracnose. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 97: 349–359. Ramdial H & Rampersad SN (2015) Characterization of Colletotrichum spp. causing anthracnose of bell pepper (Capsicum annuum L.) in Trinidad. Phytoparasitica 43: 37–49. Ratanacherdchai K, Wang HK, Lin FC & Soytong K (2007) RAPD analysis of Colletotrichum species causing chilli anthracnose disease in Thailand. Journal of Agricultural Technology 3: 211–219. Rohlf FJ (1999) NTSYS–pc Numerical Taxonomy and Multivariate Analysis System. Version 2.02i. Exeter Software, Setauket, New York, US. Sangdee A, Sachan S & Khankhum S (2011) Morphological, pathological and molecular variability of Colletotrichum capsici causing anthracnose of chilli in the North-east of Thailand. African Journal of Microbiology Research 5: 4368–4372. Sharma G, Pinnaka AK & Shenoy BD (2013) ITS-based diversity of Colletotrichum from India. Current Research in Environmental & Applied Mycology 3: 194–220. Sharma PN, Kaur M, Sharma OP, Sharma P & Pathania A (2005) Morphological, pathological and molecular variability in Colletotrichum capsici, the cause of fruit rot of chillies in the subtropical region of northwestern India. Journal of Phytopathology 153: 232–237. Shenoy BD, Jeewon R, Lam WH, Bhat DJ, Than PP, Taylor PWJ & Hyde KD (2007) Morpho-molecular characterisation and epitypification of Colletotrichum capsici (Glomerallaceae, Sordariomycetes) the causative agent of anthracnose in chilli. Fungal Diversity 27: 197–211. Shi ABC, Kantartzi SK, Mmbaga MT, Chen P, Mrema F & Nnodu E (2008) PCR-based markers for detection of Colletotrichum acutatum and C. gloeosporioides in flowering dogwood (Cornus florida). Australasian Plant Pathology 37: 65–68. Souframanien J & Gopalakrishna T (2004) A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers. Theoretical and Applied Genetics 109: 1687–1693. Srinivasan M, Vaikuntavasan P & Rethinasamy V (2010) RAPD and virulence analyses of Colletotrichum capsici isolates from chilli (Capsicum annuum). Journal of Plant Diseases and Protection 117: 253–257. Talhinhas, P, Sreenivasaprasad S, Neves-Martins J & Oliveira H (2002) Genetic and morphological characterization of Colletotrichum acutatum causing anthracnose of lupins. Phytopathology 92: 986–996. Tamura K, Stecher G, Peterson D, Filipski A & Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. Than PP, Prihastuti H, Phoulivong S, Taylor PWJ & Hyde KD (2008) Chilli anthracnose disease caused by Colletotrichum species. Journal of Zhejiang University Science Biology 9: 764–778. Thangamani PR, Kuppusamy P, Peeran MF, Gandhi K & Thiruvendam R (2011) Morphological and physiological characterization of Colletotrichum musae the causal organism of banana anthracnose. World Journal of Agricultural Sciences 7: 743–754. Torres-Calzada C, Tapia-Tussell R, Quijano-Ramayo A, Martin-Mex R, Rojas-Herrera R, Higuera-Ciapara I & Perez-Brito D (2011) A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of Colletotrichum capsici. Molecular Biotechnology 49: 48–55. Uma Devi G (2008) Efficacy of fungicides against Colletotrichum leaf spot of turmeric. Indian Journal of Plant Protection 36: 112–113. White TJ, Bruns T, Lee S & Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH & Sninsky JJ (eds) PCR Protocols: a guide to methods and applications. Academic Press, San Diego, CA, pp. 315–322.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 491–500, 2016 DOI: 10.22271/tpr.2016.v3.i3.065 Research article

Structural study of Gilbertiodendron dewevrei mono-dominant forest based on mature individuals in the Masako forest reserve (Tshopo province, Democratic Republic of the Congo) Francine K. Botelanyele1, Patience K. Kahola1, Jean-Leon K. Kambale1, Nicole S. Assani1, Esther I. Yokana1, Prosper S.Yangayobo2, Honorine N. Habimana2, Monizi Mawunu3 and Koto-te-Nyiwa Ngbolua4* 1

University of Kisangani, Biodiversity monitoring centre, Department of Ecology and Botanical Resources Management, P.O. Box 2012 Kisangani, Democratic Republic of the Congo 2 University of Kisangani, Faculty of Science, Department of Ecology and Botanical Resources Management, P.O. Box 2012 Kisangani, Democratic Republic of the Congo 3 Department of Agronomy, College of Uige Polytechnic, University Kimpo Vita, Republic of Angola 4 University of Kinshasa, Faculty of Science, Department of Biology, Democratic Republic of the Congo *Corresponding Author: [email protected] [Accepted: 15 September 2016] Abstract: The goal of the study is to determine the vegetal composition and structure based on mature trees in the Masako Forest Reserve. A transect was installed in a mono-dominant forest with Gilbertiodendron dewevrei at 5 km from the guest house. For transect of 2,100 m, we placed a 500 m perpendicular line to the survey to a plot of 2,500 square meters in a G. dewevrei forest. An inventory was carried out in an area of 3 ha, consisting of 12 plots of 50 × 50 m. All trees with a diameter equal or greater than 30 cm were inventoried, and divided into eight size classes according to their dbh (diameter at breast height) dimension: ˂40 cm; 40 to 49.9 cm; 50 to 59.9 cm; 60 to 69.9 cm, 70 to 79.9 cm; 80 to 89.9 cm; 90 to 99.9 cm and ≥100 cm. To facilitate the inventory, the survey plots were divided in two subplots of 1,250 m2. For the entire forest, we found an overall density of 86.3 stems per hectare of which 63.3 stems/ha belonged to G. dewevrei. We also found that the basal area for an individual tree was on average 29.5 m2.ha-1 for the entire forest, and 24.2 m2.ha-1 for G. dewevrei. Keywords: Mono-dominant forest - Gilbertiodendron dewevrei - Masako Forest Reserve. [Cite as: Botelanyele FK, Kahola PK, Kambale J-LK, Assani NS, Yokana EI, Yangayobo PS, Habimana HN, Mawunu M & Ngbolua K-t-N (2016) Structural study of Gilbertiodendron dewevrei mono-dominant forest based on mature individuals in the Masako forest reserve (Tshopo province, Democratic Republic of the Congo). Tropical Plant Research 3(3): 491–500] INTRODUCTION Tropical rainforests are among to the most diverse terrestrial ecosystems of the world (Borah 2016), but they also contain zones which are dominated by a single species (mono-dominant forests). These zones pose a great enigma in tropical ecology. The mono-dominance leads to a change in the vegetal composition of the forest in which sun-loving species dominate (Fonty 2011). The high diversity of tree species, which is characteristic for tropical forests, is as well a permanent source of scientific questions as a strong constraint to improve our knowledge of the functioning of the forest ecosystem. The answers explaining the preservation of this diversity oppose the deterministic or stochastic mechanisms maintaining this high diversity (Blanc et al. 2003). However, in tropical rainforests, there are areas of low diversity too (Richards et al. 1952), where the canopy trees are dominated by one species (Richards et al. 1996). When this species reaches 50% of the relative diversity, it is considered to be a mono-dominant species (Hart et al. 1989), single-dominant Forest sensu (Connel et al. 1989).

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Received: 25 May 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.065

Botelanyele et al. (2016) 3(3): 491–500 . This is already known and documented in the tropical forests of Asia, where Drybalonopsa romatica (Dipterocarpaceae) is considered a mono-dominant species (Reitsma 1988). In the Congo basin, Gilbertiodendron dewevrei (De Wild.) J. Leonard forms extensive stands, which, in some cases, are virtually monophyletic (Whitmore 1984, Gerard 1960, Kouob 2009). Unlike heterogeneous forests, interest on the origin and preservation of mono-dominant tropical forests is recent (Toft et al. 2003). It is assumed that the massive and synchronous fruiting, low diaspores predation, tolerance to low light levels, and ecto-mycorrhizal symbiosis would be the basis for the survival of this forest type. Although they only represent a small fraction of the area of tropical forests, mono-dominant stands were described from all around the tropics. These settlements do not conform to the general pattern and it can be assumed that one or more processes controlling diversity have been altered there. Also the understanding of the mechanisms leading to these species imposing their mono-dominance can shed a (negative) light on the processes allowing the coexistence of many species (Gross et al. 2000). According to McGuire (2007), the structure of a G. dewevrei forest is the result of the impact of the port and the social temperament of this species on the light factor (Louis 1947). Therefore, we decided to approach the problems by focusing on the G. dewevrei forest, where changes are easily noticeable, not only because of its wide distribution in the tropical forest, but also because of its great vegetation diversity. The structural profiles pose a number of problems, which are sometimes difficult to solve, requiring a lot of time and resources (Mabay 1994). This is also the reason why we based our description of the morphological appearance of the forests on the vegetation composition, vertical and horizontal structure of mature individuals (dbh ≥ 30 cm) in the G. dewevrei mono-dominant forest. As such, given the extent to which inventories were made, we expected to be closer to the physionomical reality, offering one or two structural profiles. This research was motivated by the need to identify the basic data on the structural composition and plant species diversity of the G. dewevrei forest, based on mature individuals (dbh ≥ 30 cm). METERIALS AND METHODS Study site Our study was performed in the Masako Forest Reserve, which is located about 14 km northeast from the city of Kisangani (DRC) on the old road towards Buta (0° 36' 30.4" N 25° 15' 38.9" E) at an altitude of about 500 m (Sonké 1998). It covers an area of about 2,104 ha of which is occupied by primary forest (Northeast) and at least 2/3 by secondary forests (Northwest). The area was visited during three field trips of ten days (March, April and May 2009). The structure of mature individuals (dbh  30 cm) and dominant species (depending on the distribution of tree frequencies classes and the diameter structure) are aspects that have been selected to meet our goal: understanding the particular physiological nature of the primary G. dewevrei forest in Masako, as these aspects allow the easy identification of the balanced state of a plant formation. Location of the transect The transect was traced in the Masako monodominant G. dewevrei forest with the main axis directed in a NS direction and reaching 5 km from the camp site. The forest area was divided into plots and transects arranged to pass through a series of tributaries to characterize and visualize the key areas in the various topographical conditions (Mboengongo 1999). Description of the transect Along the 2,100 m main axis of the transect, we drew 500 m long perpendicular lines at 100 m intervals, giving a total of 12 lines (Fig. 1). Along these lines, random plots were selected to survey for G. dewevrei. Each of these plots measured 50 × 50 m (2,500 m2). Data collection All trees with dbh ≥ 30 cm were surveyed and their coordinates (x,y) within the plot, their diameters at a height of 1.30 m above the ground (= breast height [dbh]) were registered. Data analysis The structure diversity is defined by a set of parameters (plant diversity, density, distribution, vertical distribution, etc.) and dimensions in the plots as well as by the relationships that may exist between these www.tropicalplantresearch.com

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Botelanyele et al. (2016) 3(3): 491–500 . parameters. The latter were calculated using the model proposed by (Makana 1989, Gillet et al. 1991). Besides of these quantitative analyses, biodiversity indices were also calculated. Diversity indices 1. Margalef diversity index (DMg) This index is calculated by the following formula: DMg = (S1) / ln (N). Where, S ꞊ number of species and N = total number of individuals. 2. Menhinick diversity index (DMn) This index is calculated as follows: DMn = S /√N Where, S = number of species, N = total number of individuals. 3. Simpson diversity index (DS) This index measures the probability that two individuals, that were selected by chance, belong to the same species: DS =  pi2 with pi꞊ ni/N Where, ni = number of individuals belonging to a given species i, N = total number of individuals. 4. Shannon-Wiener diversity index According to Lejoly (1993) and Danais (1982), the Shannon-Wiener index measures the average amount of information presented by the indication of the species of an individual to the collection. H' = -(piln pi) with pi꞊ ni/N Where, N = total number of individuals (i.e. trunks), ni = number of individuals belonging to a given species i (between 0 and N), pi ranges from 0 to 1. S

N

Figure 1. Schematic representation of the sampling plots in the Masako Gilbertiodendron dewevrei mono-dominant forest.

Variation Coefficient (VC) This coefficient is used to compare two standard deviations, especially when the means are different. It also allows us to evaluate the magnitude of dispersion of data. The high value of VC indicates the larger of the dispersion around the mean (Frontier 1993, Legendre et al. 1998). VC = ᵟ× 100/M Where, ᵟ - variance, M - mean If VC ˂ 15%, the dispersion is low or less pronounced and the series is homogeneous. If VC is between 15% and 30%, the dispersion is somewhat weak and the series is relatively homogeneous, However, if VC > 30%, the dispersion is most pronounced or high and the distribution is heterogeneous. www.tropicalplantresearch.com

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Botelanyele et al. (2016) 3(3): 491–500 . Diametric and structural analysis The total diametric structure or distribution of stems per diameter class is determined by taking into account the individuals of every species (Magurran 2004). This value gives information on the stability of the stands. It can also be calculated for specific species and in that case, it represents the specific structure. The diametric structure indicates the number of stems inventoried by diameter classes. The Cartesian coordinates (x, y) of all individuals Gilbertiodendron dewevrei (with dbh ≥ 30 cm) were registered to accurately characterize the organization of the trees in each plot. The basal area is expressed in m2 (Rollet 1974) is calculated for each individual by means of the formula ST = πD² / 4 where D is the dbh. Quantitative study Quantitative determination of the data results in their structural nature consisting of a set of parameters, including the spatial distribution, species density and the relationships that interfere with these (Gounot 1969). The abundance or relative density of a species and a family is calculated as the total number of individuals of a species or family in the sample multiplied by 100: RDs = (ns / N) x 100 Where, ns is the number of individuals for a given species and N is the total number of individuals in the sample. RDf = (nf / N) x 100 Where, nf is the number of individuals for a given family and N is the total number of individuals in the sample. The relative dominance of a species or family is determined by the basal area occupied by a species or family in total basal area and is multiplied by 100 according to following formula: ∫ts∕ ∫st×100 and for the family ∫tf∕ ∫st×100 Where, ∫ts is the basal area of a species, ∫tf the basal area of a family and ∫St the total basal area in the sample Density Density is defined as the number of stems per surface unit. There are several expressions of density. However, the most commonly used is the number of stems per hectare (N/ha). Unfortunately, this expression of the density does not take into account the size of trees. RESULTS AND DISCUSSION Vegetation composition and density The floristic study in the Masako G. dewevrei mono-dominant forest resulted in an overall number of 259 individuals with dbh ≥ 30 cm, divided over 36 species, 34 genera, 19 families (Fig. 2).

Figure 2. Floristic composition of Gilbertiodendron dewevrei mono-dominant forest at the Masako Forest Reserve.

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Botelanyele et al. (2016) 3(3): 491–500 . The Gilbertiodendron dewevrei dominate the sample with 75%, followed by the Anonidium mannii and Trilepisium madagascariensis with 3% each, followed by the Funtumia africana with 2%, Klainedoxa gabonensis (1%) and other species are each represented with 16%.

A

B

C

D Figure 3. Gilbertiodendron dewevrei (De Wild.) J.Leonard: A, Seeds; B, Seedling; C, Stem with bark; D, Habitat.

Stocking densities of individuals in the Masako G. dewevrei mono-dominant forest The 3 ha sample area in the Masako G. dewevrei mono-dominant forest contained 259 trees with dbh ≥ 30 cm. On average, there were 86.3 trees per ha. Of these trees, 190 belonged to G. dewevrei, giving an average per hectare of 63.3. Quantitative analysis of the floral data The global basal areas for the Masako G. dewevrei mono-dominant forest is 29.5 m²/ha and 24.2 m²/ha for G. dewevrei individuals. The species abundance in the G. dewevrei mono-dominant forest at Masako www.tropicalplantresearch.com

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Botelanyele et al. (2016) 3(3): 491–500 . The current study indicates that the dominant species at Masako are: Gilbertiodendron dewevrei, Musanga cecropioides, Anonidium mannii, Trilepisium madagascariensis, Funtumia africana, Klainedoxa gabonensis, Petersianthus macrocarpus and Panda oleosa (Fig. 3). Vegetation structure by diametric class The diameter distribution of all species combined (total structure) is one of the stand's characteristics reflecting an equilibrium constant, which is the existence wherever moist evergreen forests are in their original state (Fournier et al. 1983). To highlight the disturbed state of forest plant community proposed a structural analysis for the species that account for the unbalanced or balanced state of the forest. Diversity indices The alpha (α) diversity was calculated using the following indices: Shannon-Wiener, Menhinick, Margalef and Simpson. This allows assessing the diversity of each group, according to the species distribution, more accurately. Margalev index gives the highest value (6.299). This shows that the Masako G. dewevrei monodominant forest is more diverse in species. It has a good evenness between the species studied. This demonstrates the dominance of the G. dewevrei forest on others in terms of species richness. The Simpson index, meanwhile, shows that the diversity of sites is not so variable because the value obtained (0.458) represents a very low diversity. On the other hand, the Menhinick index, which is based on species richness, presents a distinctly lower value (2.237) relative to Margalef index, where the number of individuals is relatively low. Comparing the Menhinick index with the one from Margalef reveals that both indices almost evolve in the same way. There is a minor difference at the species level, where the Margalef index results in higher values if there is a higher number of individuals, while that of Menhinick is low. In the current study, the Shannon index is relatively low as it represents the sum of the information given by the frequency of the various species over the 3 ha sampled surface. Statistical parameters The data for the G. dewevrei trees occurring in the twelve 50 × 50 m sample plots of the Masako Gilbertiodendron dewevrei dominated forest are presented in tables 1 and 2. Table 1. Number of Gilbertiodendron dewevrei trees with dbh ≥ 30 cm and distribution of diameter registered in the twelve 50 × 50 m sample plots.

Sample plot P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 Total Average Variance Standard deviation CV(%)

Number of trees 14 16 16 12 21 23 17 17 17 12 17 8 190 15,8 16,15 4,01 25,37

Diametric Class 30-39,9 40-49,9 50-59,9 60-69,9 70-79,9 80-89,9 90-99,9 ≥100 Total Moyenne Variance Ecart-type CV(%)

Number of trees 31 25 28 30 35 13 10 18 190 23,75 82,21 9,06 38,14

The average number of G. dewevrei trees over the sampled plots is 15.8 (Table 1). The values for the individual sample plots are more or less around this average value, with one lower value for P12 and two higher ones for P5 and P6. The coefficient variation is between 15 and 30% indicating that the dispersion is somewhat weak. The distribution of individuals in the different plots is considered relatively homogeneous. This indicates that on average, there are 23.7 G. dewevrei trees in each diametric class. However, in most of the diametric classes the number of trees differs considerably from that average. The coefficient variation exceeds 30%, www.tropicalplantresearch.com

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Botelanyele et al. (2016) 3(3): 491–500 . indicating that the dispersion is strong and that the diametric distribution of G. dewevrei should be considered heterogeneous. Over the 12 sampled plots, there were on average 21.6 trees with dbh ≥ 30 cm. The low value registered for P12 confirms the low value for G. dewevrei trees. This is also the case for the high values in P5 and P6, although these are less extreme than found for G. dewevrei. The coefficient variation is low (less than 15 %), indicating that the plots form a homogenous sample. Table 2 shows that in the G. dewevrei forest, there were on average 32.4 trees in each of the diametric classes. With the exception of classes 50 to 79.9 cm, most classes have a value which is quite different from the average value. As was already shown in figure 4 the smaller classes have a much higher number of trees, and the larger classes have a much lower number. The tree class of ≥ 100 cm contains an elevated number of trees, which might be explained as being the result of including a much wider range of diameters, with trees up to 136, 6 cm the coefficient variation exceeds 30%, confirming the heterogeneity of the sampled plots in relation to the diameter of the trees. Table 2. Number of trees with dbh ≥ 30 cm and diameter distribution registered in the twelve 50 × 50 m sample plots.

Sample plot P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 Total Average Variance Standard deviation CV(%)

Number of trees 22 23 20 23 24 26 19 21 19 21 23 18 259 21,58 5,53 2,35 10,88

Diametric Class 30-39,9 40-49,9 50-59,9 60-69,9 70-79,9 80-89,9 90-99,9 ≥100 Total Moyenne Variance Ecart-type CV(%)

Number of trees 65 40 35 33 35 18 10 23 259 32,375 124,07 11,13 34,37

Number of trees

70 60 50 40 30 20 10 0 30-39,9

40-49,9

50-59,9

60-69,9 70-79,9 Diametric classes

80-89,9

90-99,9

≥100

Figure 4. Vegetation structure by diametric class at Masako forest reserve.

The present study revealed that the sample area (3 ha) in the Masako G. dewevrei mono-dominant forest contained 259 trees with dbh ≥ 30 cm. On average, there were 86.3 trees per ha. Of these trees, 190 belonged to G. dewevrei, giving an average per hectare of 63.3. Lomba (2007), working in the G. dewevrei monodominant forest at Yoko, found 97 trees with dbh ≥ 30 cm in a 3 ha plot, resulting in an average of 32.3 threes per ha. 74 of the trees belonged to G. dewevrei (or 24.7 ha-1). These numbers indicate that at Yoko fewer mature trees were occurring, both for all species combined and for G. dewevrei separately. This is mainly due to human action as farmers harvest the average diameter (≤ 40 dbh) of species for commercial interests. www.tropicalplantresearch.com

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Botelanyele et al. (2016) 3(3): 491–500 . Quantitative analysis of the floral data revealed that the global basal areas for the Masako G. dewevrei monodominant forest is 29.5 m².ha-1 and 24.2 m².ha-1 for G. dewevrei individuals. For the Yoko forest, Masiala (2009) calculated these as 24.4 m².ha-1 and 22.7 m².ha-1, respectively. This allows us to hypothesise that the forest at Yoko is still evolving and that this is no longer the case for the forest at Masako, which also has a much more disturbed character. By comparing the species abundance in the G. dewevrei mono-dominant forest at Masako with the work of other researchers, the current study indicates that the dominant species at Masako are: Gilbertiodendron dewevrei, Musanga cecropioides, Anonidium mannii, Trilepisium madagascariensis, Funtumia africana, Klainedoxa gabonensis, Petersianthus macrocarpus and Panda oleosa. At Yoko, Masiala (2009) found the dominant species to be: Gilbertiodendron dewevrei followed by Scorodophloeus zenkeri, Julbernardia seretii, Gilbertiodendron kisantuensis and Grossera sp. In the forest of Uma, Katembo (2013) found that the dominant species are: Gilbertiodendron dewevrei (29%), followed by Cola griseiflora (8%), Diospiros sp (6%) and other species. The species composition varies from site to site. In the three sites (Masako, Yoko and Uma) the same trend in abundance of Gilbertiodendron dewevrei was encountered. However, in the two following locations, this was not the case. Indeed, in Mbiye Island, Kambale (2009) found that Coelocaryon botryoides was the most abundant species, followed by Gilbertiodendron dewevrei, Diospyros boala, Pycnanthus angolensis and Cleistanthus mildbraedii. The diameter distribution of all species combined (total structure) is one of the stand's characteristics reflecting an equilibrium constant, which is the existence wherever moist evergreen forests are in their original state (Fournier et al 1983). To highlight the disturbed state of forest plant community proposed a structural analysis for the species that account for the unbalanced or balanced state of the forest. Nshimba (2008) indicated that Gilbertiodendron dewevrei forest at Uma and the mixed forest on the Mbiye Island have the form of a "mirrored J" for the stems with dbh ≥30 cm, whereby the class size 30–39.9 cm accounts for the highest number of trees and subsequent diametric classes all have lower values. These forests have the typical diametric structure of natural forests, which is contrary to the Masako Gilbertiodendron dewevrei. Forest showing that the histogram has the form of a "mirrored J with a bulbous tail". The diametric classes between 40 and 70 cm dbh contain much higher numbers of trees, which indicate that the Masako forest is unbalanced and this is due to human action. Farmers harvest the average diameter of species for commercial interests. CONCLUSION The current study has focused on the forest structure of a Gilbertiodendron dewevrei monodominant forest based on tree species in the Masako Forest Reserve. An inventory of the mature trees (dbh ≥ 30 cm) was made on a 3 ha area, which was composed of 12 plots of 50 × 50 m each, where all trees meeting this condition were surveyed. o In the 3 ha sample area, 259 meeting the dbh condition were registered. The number of trees is 259 for the forest G. dewevrei in 3 ha. Trees belonging to the Fabaceae family were found to be the most commonly occurring group (85% of species). o The results showed that the overall tree density is 83.3 stems/ha and 63.3 stems/ha for G. dewevrei. o In the forest, the overall basal area is 29.5 m².ha-1 and for G. dewevrei this is 24.2 m².ha-1. o The vegetation was classified in eight diameter classes: 3039.9 cm; 40–49.9 cm; 50–59.9 cm; 60–69.9 cm; 70–79.9 cm; 80–89.9 cm; 90–99.9 cm and ≥100 cm. o As illustrated by the high values for the variance coefficient (exceeding 30%), the diametric distribution is heterogeneous for both the overall sample and G. dewevrei alone. Regarding the distribution of the number of trees over the sampled plots, the variation coefficient has values between 15 and 30 %, indicating that the forest is relatively homogeneous. Additional research is needed to find ways to stop the decrease and to determine measures enabling to prevent the forest overexploitation. REFERENCES Blanc L, Flores O, Molino J-F, Gourlet-Fleury S & Sabatier D (2003) Diversité spécifique et Regroupement www.tropicalplantresearch.com

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Botelanyele et al. (2016) 3(3): 491–500 . d’espèces arborescentes en forêt guyanaise. In Engref, (eds) Revue Forestière française, Nancy. Numéro spécial. connaissance et gestion de la forêt guyanaise, 131–146p. Borah N, Rabha D & Athokpam FD (2016) Tree species diversity in tropical forests of Barak valley in Assam, India. Tropical Plant Research 3(1): 1–9. Connel JH & Lowman M D (1989) Low-diversity tropical rain forests: some possible Mechanisms for their existence. The American Naturalist 134: 88–119. Danais M (1982) La diversité écologique: analyse bibliographique. Botanica Rhedonica 17: 77–104. Frontier S & Pichod-Viale D (1993) Ecosystème: structure, fonctionnement, Évolution. Collection d’écologie 21, Masson paris, 2è édit. 447 p. Fonty E (2011) Etude de l’écologie du Spirotropis longifolia DC Baill. (Leguminosae- Papilionoideae) Espèce monodominante dans les forêts de Guyane française, Ph. D. Thèsis. Université Montpellier II. Sciences Techniques, 209 p. Fournier F & Sasson A (1983) Les Ecosystèmes forestiers tropicaux d’Afrique. Paris, 473 p. Gérard Ph (1960) Etude écologique de la forêt dense à Gilbertiodendron dewevrei dans la région de l’Uélé. 159 p. Gross ND, Torti SD, Feener DH & Coley PD (2000) Monodominance in an African Rain Forest: Is Reduced Herbivory Important?. Biotropica 32(3): 430–439. Gillet F, Foucault B & Julve P (1991) La phyto-sociologie synusiale intégrée: objets et concepts. Candollea 46: 315–340. Gounot M (1969) Methodes d ‘etude quantitative de la végétation. Masson et Cie, 25 p. Hart TB, Hart JA & Murphy PG (1989) Monodominant and species-rich forests of the humid tropics: causes for their co-occurrence. The American Naturalist 133: 613–633. Katembo E (2013) Etude floristique et structurale des forêts monodominantes à Gilbertiodendron dewevrei (Dewild.) J. Léonard, sur terre ferme et sur sol hydromorphe à Uma (Province Orientale) DES/DEA. Faculté des Sciences, Université de Kisangani, 63 p. Kouob BS (2009) Organisation de la diversité végétale dans les forêts matures de terre ferme du Sud-Est Cameroun, Ph. D. Thesis. Ecole Facultaire du Bio-ingénieur. Université Libre de Bruxelles, 212 p. Kambale K (2009) Caractéristique floristique de la zone de contact entre la forêt à Gilbertiodendron dewevrei de la forêt mixte de l’ile Mbiye, Mémoire. Faculté des Sciences, Université de Kisangani, 33 p. Lomba B (2007) Contribution à l’étude de la phytodiversité de la Reserve Forestière de Yoko (Ubundu, R.D. Congo), Mémoire de DES. Faculté des Sciences, Université de Kisangani, 72 p. Louis J (1947) Contribution à l’étude des forêts équatoriales congolaises. C.R.Sem. Agr. De Yangambi, INEC. Publ., Hors-Série, pp. 902–924. Lejoly J (1993) Méthodologie ECOFAC pour les inventaires forestiers (Partie flore et végétation). Lab. Bot. Syst. Phyt. Université Libre de Bruxelles, 136 p. Legendre P & Legendre L (1998) Numerical Ecology. Developments in Environmental Modelling, Elsevier Science BV, Amsterdam, 853 p. Masiala G (2009) Analyse d’une zone de contact de la forêt à Gilbertiodendron dewevrei (De Wild.) J. Léonard avec la forêt semi-caducifoliée dans la réserve de la Yoko nord 47 (RDC). Mémoire inédit de DES, Faculté de sciences, Université de Kisangani, 103 p. Mabay K (1994) Contribution à l’étude structurale de forêts primaire et secondaire de la réserve forestière de Masako. Mémoire Faculté des Sciences, Université de Kisangani, 65 p. Makana M (1989) Contribution à l’étude floristique et écologique de la forêt à Gilbertiodendron dewevrei de la réserve forestière de Masako. Mémoire, Faculté des Sciences, Université de Kisangani, 64 p. McGuire KL (2007) Ectomycorrhizal networks may maintain monodominance in a tropical rainforest. Ecology 88(3): 567–574. Mboengongo F (1999) Contribution à l’étude écologique et systématique de champignons supérieurs (Macromycètes) de la Reserve Forestière de Masako à Kisangani (RD Congo). Mémoire, Faculté de Sciences, Université de Kisangani, 85 p. Magurran AE (2004) Measuring biological diversity. Blackwell Publishing Company, UK, 256 p. Nshimba S (2008) Etude Floristique, Ecologique et Phytosociologique des Forêts de l’ile Mbiye à Kisangani, RD Congo, Ph. D. Thesis. Faculté des Sciences, Université Libre de Bruxelles. www.tropicalplantresearch.com

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 501–507, 2016 DOI: 10.22271/tpr.2016.v3.i3.066 Research article

First report on three new diatom species from the Hooghly district, West Bengal Nilu Halder* Department of Botany, Raja Peary Mohan College, Uttarpara-712258, Hooghly, West Bengal, India *Corresponding Author: [email protected] [Accepted: 17 September 2016] Abstract: The present paper was communicated with the morpho-taxonomic description of three diatom taxa belonging to the order Pennales of the class Bacillariophyceae namely Fragilariopsis cylindrus, Achnanthidium lineare and Rhopalodia gibba var. ventricosa. Among them, the former species is rare in occurrence within freshwater ecosystem while the latter two species are found sparsely to abundant in pond, water reservoirs and other types of aquatic bodies. The limnological characteristics that supported their occurrences in water bodies were recorded and found to be congenial for their growth. The pH of water in studied aquatic bodies was observed alkaline and acceptable quantity of phosphate, nitrate-nitrogen and silica along with other physico-chemical parameters of waters were also noted. The above mentioned all three diatom taxa are new taxonomic reports from this district of West Bengal, India. Keywords: New report - Diatoms - Hooghly - West Bengal - India. [Cite as: Halder N (2016) First report on three new diatom species from the Hooghly district, West Bengal. Tropical Plant Research 3(3): 501–507] INTRODUCTION Diatoms are a major group of microscopic algae and the most common types of phytoplankton which are found in every habitat where water is present (Stoermer & Smol 1999) and their (both fossils and living forms) well preserved siliceous frustules /cell walls make them ideal tools for various applied applications. Recently, they have been using in the other fields of studies as indicators of oil and gas exploration processes. They are now successfully used in comprehensive forensic examinations (Dwivedi & Misra 2015) to detect the suspicious persons or murderers for crime investigations. It has been reported that their growth rate is faster in comparison to other phytoplanktonic taxa (Wetz & Wheeler 2007). Thus, diatoms are important algal flora among the eukaryotic algae. They are beautiful microscopic organisms with various kinds of fabulous characteristics ornamentations on their frustules or cell walls. Especially, the presence of accessory pigments like fucoxanthin and universal β-carotene give them characteristic golden coloration. Generally, they grow in single cells as unicellular forms and sometimes form chains or simple colonies of various shapes like filaments or ribbons, fans, zigzags or stars in the aquatic bodies. They are capable of growing in different trophic levels of the water bodies. As the siliceous cell walls contain hydrated silicon dioxide (SiO2) they do not decompose after death and henceforth, diatom beds or diatomite are being used as an important tool to study paleoecology, correlation analysis and to interpret or predict the phylogenetic evolutionary lineages as well as to calculate relative age dating of rocks being an important constituent of rock-forming microfossils. The diatom flora is diverse in fresh water bodies, brackish and marine ecosystems and one of the richest algal groups in India due to having wide range of climate, topology and natural habitats. Their abundant growth is controlled by the physical as well as chemical conditions of water. According to You et al. (2009) their diversity depends on gradients of water, nutrients availability, pH, temperature and altitudes of the habitats. Therefore, analysis of water has a great importance in the ecological study of diatoms. Fragilariopsis Hust. is a planktonic diatom comprising of living as well as fossil species (Lundholm & Hasle 2008). It is characteristically ribbon-shaped and valves outlines are linear with rounded apical ends. In the recent years, considerable taxonomic revisions have been made on the nomenclatural concept and generic placement of www.tropicalplantresearch.com

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Received: 28 May 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.066

Halder (2016) 3(3): 501–507 . monoraphid achnanthoid diatoms since the publication of Lange-Bertalot & Krammer (1989). They first removed the genus Achnanthes Bory from the family Achnanthaceae Kütz. and placed into two genera Achnanthidium Kütz. and Eucocconeis Cl. ex Meist. under the family Achnanthidiaceae D.G. Mann based on morphological and ecological features (Round et al. 1990, Potapova 2012). After that, a number of new genera have been created by splitting of Achnanthes Bory sensu lato. However, there are still a number of species, that have not yet been studied by LM (light microscopy) and SEM (scanning electron microscopy) or not transferred within this genus. The genus is abundant in rivers, streams and springs. They inhabit in clean and polluted waters. Their cells are small linear-lanceolate to lanceolate-elliptic with less than 30 μm long and less than 5 μm broad; consists of a concave raphe valve (RV) and a convex rapheless valve (RLV); uniseriate striae present on the RV which are comparatively denser towards the apices and the fine raphe is either straight or turned to one side (Ponader & Potapova 2007). The genus Rhopalodia O. Müll. includes 37 species of which at least 26 species are known only from the type samples from all over the world (VanLandingham 1967, Krammer 1988). It exhibited broader range of distributions, including fresh water, brackish and even marine environments. The valves are bracket-shaped in valve view with swollen in middle, indented at the central nodule, the apices are bent acutely. In girdle view, valves are lanceolate-elliptical, strongly swollen in the middle of the valve with broadly rounded apices. Raphe is excentric and chromatophore is single, laminate with irregularly margins. The present work is focused on the morpho-taxonomic investigation of the fresh water diatom flora of the class Bacillariophyceae from Hooghly district, West Bengal, India. Few taxonomic works had been reported earlier from India (Venkataraman 1939, Biswas 1949, Gonzalves & Gandhi 1952, Gandhi 1958, Sreenivasa & Duthie 1973, Anand & Kant 1976, Sarode & Kamat 1979, 1980, Barhate & Tarar 1981, Das & Santra 1982, Patel & Patel 1982, Venkateswarlu 1983, Prasad et al. 1984, Somshekar 1983, 1984, Chaturvedi 1985, Roy & Sen 1985, Pal et al. 1986, Maity et al. 1987, Shukla & Shukla 1987, Santra et al. 1989, Pal & Santra 1990, Banerjee & Santra 2001, Misra 2005, Bhakta et al. 2011, Das & Adhikary 2012, Tripathi et al. 2012, Dwivedi & Misra 2014). Except a single report (Halder & Sinha 2015) there is no work in relation to exploration of diatom flora from this locality of West Bengal. Therefore, the present work was undertaken from this area. The main objectives of the present work were to identify, explore the diversity of diatom algal flora and documentation of them in respect of ecology from this state. MATERIALS AND METHODS The diatom specimens had been collected from different places viz. Chinsurah (22°.90' N; 88°.39' E), Ganga river at Tribeni (22°.99' N; 88°.39' E) and Kuntighat (23°.01' N; 88°.41' E) of Hooghly district in West Bengal, India. The light microscopic (LM) taxonomic study of the cleaned diatom specimens was made under Olympus compound microscope with camera attachment (Model No. CH20i) and photographs were taken using Canon A480 camera. Samples were preserved in 4% formalin. The organic contents particularly calcium and irons were removed from the diatom samples by the acid digestion method in which 4 ml. of concentrated HCl (30%) and 2 ml. of saturated potassium permanganate (KMnO4) solution were added with 2 ml. of diatom sample as mentioned in materials & methods (Mitić–Kopanja et al. 2014). Cleaned diatoms were mounted with DPX mounting medium. The ecological study was carried out following the standard method described earlier by the author/s (Halder 2015a,b,c,d, Halder & Sinha 2014,2015, Halder 2016a,b). Identification of those algal species were done by following standard monographs and scientific literature viz. Hustedt (1930), Hirano (1955), Foged (1977), Lundholm & Hasle (2008), Cefarelli et al. (2010), Van De Vijver et al. (2011), Al-Hassany & Hassan (2014) etc. RESULTS AND DISCUSSION A total number of three diatom taxa belonging to the order Pennales of the class Bacillariophyceae had been morpho-taxonomically described with author citation, habitat, collection number, date of collection, significance, species abundance and ecology for the first time. Each currently accepted names had been provided with its author(s) name. All the limnological parameters except temperature and pH were expressed as mg.l-1. 1. Fragilariopsis cylindrus (Grunow) Helm. & Krieg. in Diatomeenschalen 2: 17, pl. 187–189, 1954; Hasle in Skr. Norske Vidensk Akad. I. Mat. Nat. Kl. NS. 21: 34-37, pl.12, figs. 6–12; pl. 14, figs. 1–10; pl. 17, figs. 2–4, www.tropicalplantresearch.com

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Halder (2016) 3(3): 501–507 . 1965; Lundholm & Hasle in Nov. Hedw., Beiheft 133, 237–241, figs. 1–6, 12–14, 17, 19, 20, 22–23, 2008; Cefarelli et al., in Pol. Biol. 33: 1467, 70, 81: figs. 2e–i, 7c–e, 2010. (Fig. 1A) Synonym(s): Fragilaria cylindrus Grunow; Nitzschia cylindrus (Grunow) Hasle Description: Planktonic, cells solitary or chain-forming, ribbon-shaped, attached by the valve surfaces and each cell contains two rectangular chloroplasts in the girdle view; valve shape linear and isopolar with broadly rounded ends; apical axis 14.0–23.0 µm; transapical axis 2.0–3.0 µm; transapical striae 12–16 in 10 µm; at the ends, the striae become nearly parallel to the apical axis; striae perforated and consist of 2 or rarely 3 rows of minute poroid areolae, each with 40–60 poroids in 10 μm; fibulae (continuations of costae) occur at approximately the same density as the striae; raphe canal is eccentric. Habitat: Ganga river water at Tribeni site, Hooghly district, West Bengal. Collection No: NH 804; Dated: 03.01.2011 Significance: Primary producer and a component of food chain in aquatic ecosystem. Species abundance: Rare in Hooghly district, West Bengal. 2. Achnanthidium lineare W. Sm. in Ann. & Mag. Nat. Hist. 2(15): 8, pl.1, fig. 9, 1855; Van De Vijver et al. in Algol. Stud. 136/137: 170–180, figs. 1–35, 2011. (Fig. 1B) Synonym(s): Achnanthes linearis (W. Sm.) Grunow; Achnanthes minutissima Kütz. partim sensu Krammer & Lange-Bertalot, Rossithidium lineare (W. Sm.) Round et Bukht. Description: Frustules in girdle view rectangular; valves linear or linear-lanceolate with almost parallel margins; valve apices broadly rounded, non-protracted; rapheless and raphe valves are linear; valve length 12.0– 13.5 μm, width 2.5–2.8 μm; axial area narrower, linear, weakly widening towards the central area; central area rectangular fascia; raphe filiform straight with raphe endings; striae radiate to weakly radiate throughout the entire valve; 28–32 in 10 μm; striation pattern slightly to densely spaced near apices; numbers of areolae per stria 2–3 but in this specimen striae and areolae not visible. Habitat: Pond water at Kuntighat, Hooghly district, West Bengal. Collection No: NH 509; Dated: 04.07.2009 Significance: Primary producer and a component of aquatic food chain in this pond. Species abundance: Sparsely present in Hooghly district, West Bengal.

Figure 1. A. Fragilariopsis cylindrus; B. Achnanthidium lineare; C-D. Rhopalodia gibba var. ventricosa (valve & girdle views).

3. Rhopalodia gibba var. ventricosa (Kütz.) H. Perag. & M. Perag. in Diat. Mar. France 302, pl. 77, figs. 3–5, 1900; Patrick & Reimer, The diatom of the United States, 190, pl. 28, figs. 3–4, 1975; Foged, Fresh Water Diatoms in Ireland, 106, pl. 43, fig. 7, 1977; Czarnecki & Blinn in Biblioth. Phycol. 102 , pl. 22 , fig. 12, 1978; Hadi et al. in Nov. Hedw., 534, pl. 12, fig. 217, pl. 37, fig. 3, 1984; Al-Hassany & Hassan, Asian J. Natl. & Appl. Sci. 3(1): 2, pl. 1, fig. 1, 2014. (Fig. 1C–D) Description: Planktonic, frustules bracket shaped in valve view with swollen middle, apices acutely bent and margin convex; in girdle view, valves linear-elliptical, inflated in median portion with broadly rounded poles; valves 50.5–54.5 µm long and 9.5–10.0 µm broad, having sometimes median constrictions; ventral margin www.tropicalplantresearch.com

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Halder (2016) 3(3): 501–507 . straight, arcuate or curve at the ends; dorsal margin convex; chromatophore is single in each cell, laminate with irregular margins; striae slightly radiate to parallel; costae 8 and striae 15 in 10 µm. Habitat: Water reservoir in rice field at Chinsurah, Hooghly, West Bengal. Collection No: NH 840; Dated: 18.06.2011 Significance: Primary producer in water bodies. Species abundance: Abundant in Hooghly district, West Bengal. In the recent work, three freshwater diatom taxa had been morpho-taxonomically described from this IndoGangetic plain of West Bengal, India. Fragilariopsis cylindrus sensu lato is a cold-water diatom species and was documented from polar and subpolar regions in the Arctic, Antarctic and open water as well as in ice although, a small number of species have been recorded exclusively from the higher latitudes of the Northern Hemisphere (Witkowski et al. 2000, Lundholm & Hasle 2008). It could dominate the water column, sea ice and ice edge communities (Hegseth & von Quillfeldt 2002, Cefarelli et al. 2010). Therefore, the species of the genus Fragilariopsis Hust. is abundant particularly in the sea, Arctic and Antarctic ice waters. But here, author collected the species of F. cylindrus (Grunow) Helm. & Krieg. from the lower stretch (downstream) of river Hooghly (Ganga) at Tribeni site during winter when water temperature was below the level of 20°C from West Bengal, India. It is morphologically similar to F. curta (Van Heurck) Hust. and F. kerguelensis (O’Meara) Hust. but differs it from those two species by having isopolar apical axes. The shape, measurements of valves and other identifying characteristics of this species also agreed with the type specimen and other published reports (von Quillfeldt 2001, Lundholm & Hasle 2008, Cefarelli et al. 2010). It was collected as planktonic form like Kang & Fryxell (1992). The description of species Achnanthidium lineare W. Sm. is exactly coincided with the type specimens and the valve length and width of the present specimen is matched with the European type materials especially of Scotland and France (Van De Vijver et al. 2011). The taxon Rhopalodia gibba var. ventricosa (Kütz.) H. Perag. & M. Perag. is differentiated from R. gibba (Ehr.) O. Müll. by i) its marked swelling in the middle of the valve ii) more elliptical nature of frustule in girdle view and iii) length: breadth ratio is much less than in R. gibba (Ehr.) O. Müll. Table 1. Physico-chemical characteristics of different lentic aquatic bodies during the algae sampling times (Mean±SE).

Limnological parameters Reservoir at Chinsurah Temp. (°C) 31ºC±0.18 pH 8.1±0.05 7.4±0.11 DO (mg.l-1) 4.0±0.05 BOD (mg.l-1) 90.0±5.77 COD (mg.l-1) 0.25±0.05 NO3-N (mg.l-1) -1 30.34±0.11 PO4 (mg.l ) 3.6±0.13 Silicate (mg.l-1) 6.0±0.22 SO42- (mg.l-1) -1 220.0±0.20 Total alkalinity (mg.l )

Different Sampling Sites River Ganga at Tribeni 19ºC±0.13 7.3±0.05 7.0±0.12 4.5±0.11 120.0±2.88 0.12±0.05 0.18±0.12 6.6±0.13 6.8±0.17 164.0±0.22

Pond at Kuntighat 30ºC±0.17 7.8±0.05 7.1±0.11 4.3±0.13 110±2.88 0.30±0.08 0.28±0.15 5.4±0.14 7.0±0.20 184.0±0.22

The physico-chemical characteristics of different types of water bodies during the diatom sampling times were measured and depicted in table 1. The pH of the studied aquatic ecosystems was found to be alkaline. Kamat (1965) reported that the diatoms are usually abundantly found in the alkaline water bodies. Thus, the present investigation confirmed the earlier finding. The ranges of nitrate-nitrogen and phosphate values were measured from 0.12–0.30 mg.l-1 and 0.18–0.34 mg.l-1 respectively while; silicate was recorded as 3.6–6.6 mg.l-1. This study revealed that presence of adequate amount of nitrate-nitrogen, phosphate and silicate along with other selected physico-chemical parameters favored the growth of those diatom species in the above said water bodies. The investigation also revealed that they can tolerate varying degrees of temperatures (19–31ºC), pH (pH = 7.3–8.1) and total alkalinity (164.0–220.0 mg.l-1) values. Other parameters like BOD and SO42- except COD values were found in lower amounts in the water bodies. DO was observed between 7.0 mg.l-1 and 7.4 mg.l-1 which is higher that might be due to maximum abundance of diatom and other plankton flora. Henceforth, it can be summarized that these species appear in those aquatic ecosystems which are enriched with sufficient essential nutrients. This documentation of diatom species of fresh water habitats from poorly studied region has www.tropicalplantresearch.com

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Halder (2016) 3(3): 501–507 . a great significance for future investigations on algal taxonomy and freshwater ecology. ACKNOWLEDGEMENTS The author is grateful to Dr. Sankar Narayan Sinha, Dept. of Botany, University of Kalyani, Nadia, West Bengal, India for providing opportunity to work under his guidance, co-operations and valuable suggestions. REFERENCES Al-Hassany JS & Hassan FM (2014) Taxonomic study of some epiphytic diatoms on aquatic plants from AlHawizah marshes, Southern of Iraq. Asian Journal of Natural & Applied Sciences 3(1): 1–11. Anand VK & Kant S (1976) Diatoms of Jammu Mansar lake. Geobios 3: 34–36. Banerjee A & Santra SC (2001) Phytoplankton on the rivers of Indian Sunderban mangrove estuary. Indian Biologist 33(1): 67–71. Barhate VP & Tarar JL (1981) The Algal flora of Tapti river of Bhusawal, Maharashtra. Phycological Society of India 20(1&2): 75–78. Bhakta S, Das SK, Nayak M, Jena J, Panda PK & Sukla LB (2011) Phyco-diversity assessment of Bahuda river mouth areas of east coast of Odisha, India. Recent Research in Science and Technology 22(4): 80–89. Biswas KP (1949) Common fresh water and brackish water algal flora of India and Burma. Pt. I. Records of the Botanical Survey of India 15: 1–105. Cefarelli AO, Ferrario ME, Almandoz GO, Atencio AG, Akselman R, Vernet M (2010) Diversity of the diatom genus Fragilariopsis in the Argentine sea and Antarctic waters: morphology, distribution and abundance. Polar Biology 33: 1463–1484. Chaturvedi UK (1985) Additions to algal flora of Rohilkhand division, U.P., India: IX. Diatoms from Bareilly district. Phycological Society of India 24: 163–169. Czarnecki DB & Blinn DW (1978) Diatoms of the Colorado river in Grand Canyon national park and vicinity (Diatoms of Southwestern U.S.A. II) Bibliotheca Phycologica 38: 1–181. Das PR & Santra SC (1982) Diatoms of Senchal lake, Darjeeling, West Bengal. Phycological Society of India 21: 99. Das SK & Adhikary SP (2012) Diversity of freshwater algae in Arunachal Pradesh and their distribution in different altitudes. The Journal of the Indian Botanical Society 91(1–3): 160–182. Dwivedi RK & Misra PK (2014) On the occurrence of freshwater diatoms of southern Himachal Pradesh, India. Phycological Society of India 44(1): 17–24. Dwivedi RK & Misra PK (2015) Freshwater Diatoms from Himalayan State Himachal Pradesh, India. Phycological Society of India 45(1): 30–39. Foged N (1977) Fresh water diatoms in Ireland. J. Cramer, Germany, pp. 1–220. Gandhi HP (1958) Fresh water diatoms from Kolhapur and its immediate environments. Journal of the Bombay Natural History Society 55(3): 493–511. Gonzalves EA & Gandhi HP (1952) A systematic account of the diatoms of Bombay and Salsette, Part I. The Journal of the Indian Botanical Society 31(3): 117–151. Hadi RAM, Al-Saboonchi AA & Haroon AKY (1984) Diatoms of the Shatt Al-Arab river, Iraq, Nova Hedwigia 39: 513–557. Halder N & Sinha SN (2014) New Records of Euglena acus (O.F. Müll.) Ehr. and Phacus acuminatus (A. Stokes) Huber-Pestalozzi of Euglenineae from Hooghly District, West Bengal. Journal of Academia and Industrial Research 3(7): 333–336. Halder N & Sinha SN (2015) New report of four Bacillariophycean algal species from West Bengal, India. Journal of Algal Biomass Utilization 6(2): 28–31. Halder N (2015a) Two species of Zygnemopsis (Skuja) Transeau from West Bengal, India. Tropical Plant Research 2(2): 82–84. Halder N (2015b) Morpho-taxonomy of Hydrodictyon reticulatum (L.) Lagerheim and Pediastrum tetras var. tetraodon (Corda) Hansgirg, Hooghly, West Bengal, India. Tropical Plant Research 2(3): 168–171. Halder N (2015c) Taxonomy and ecology of Coleochaete irregularis Pringsheim and Coleochaete orbicularis Pringsheim, West Bengal, India. Journal of Algal Biomass Utilization 6(4): 47– 49. Halder N (2015d) Limnological study of Dwarkeshwar river water in the downstream at Arambagh, Hooghly district, West Bengal, India. Spring 5: 10–14. www.tropicalplantresearch.com

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 508–516, 2016 DOI: 10.22271/tpr.2016.v3.i3.067 Research article

Effect of various dormancy breaking treatments on seed germination, seedling growth and seed vigour of medicinal plants Ashwani Kumar Bhardwaj, Sahil Kapoor, Avilekh Naryal, Pushpender Bhardwaj, Ashish Rambhau Warghat, Bhuvnesh Kumar and Om Prakash Chaurasia* Defence Institute of High Altitude Research, Defence Research & Development Organization, Leh-Ladakh, Jammu & Kashmir, India *Corresponding Author: [email protected] [Accepted: 20 September 2016] Abstract: Study was conducted during 2013–14 to examine the role of various dormancy breaking treatments, viz. hot water treatment, scarification, stratification, concentrated acids (H2SO4, HNO3 and HCl), gibberellic acid, potassium nitrate, alcohol, and acetone and gamma-rays irradiation on the percentage germination, seedling growth and seed vigour. Dried seeds were incubated in the plant growth chambers for 20–28 days at constant temperature of 25±2 °C under continuous light (16 hrs) photoperiod after its treatments. Maximum percent germination 97.2% was obtained in Innula racemosa followed by Rheum webbianum (95.1%), Carum carvi (93.4%), Saussurea lappa (90.01%) and Bunium persicum (81.4%) when seeds were pretreated with acid (H2SO4 for 5 minutes). According to results obtained in present study, all studied species found best germination with H2SO4 for 5 minutes in duration of 30 days. The seedlings derived from seeds exposed to the various treatments performed well when grown in a green house. Maximum length of seedlings were found in 24.3 cm in S. lappa followed by R. webbianum (23.8 cm) and C. carvi (22.2 cm) when seeds were pretreated with H2SO4 for 5 minutes, on the other side B. persicum (19.4cm) in hot water treatment at 80°C for 20 minutes and I. racemosa (17.4 cm) in 0.2 KNO3 for 10 minutes. Highest value of seed vigour index (2263) and lowest seed vigour index (390) was found in R. webbianum and B. persicum. The well developed seedlings were observed in 90 days and transplanted it for further developments. The data have implications for conservation and cultivation of the species studied. Keywords: Acid treatment - Dormancy - Gibberellic acid - Nitric acid - Seed germination. [Cite as: Bhardwaj AK, Kapoor S, Naryal A, Bhardwaj P, Warghat AR, Kumar B & Chaurasia OP (2016) Effect of various dormancy breaking treatments on seed germination, seedling growth and seed vigour of medicinal plants. Tropical Plant Research 3(3): 508–516] INTRODUCTION Seed germination is a complex physiological processes that response to environmental signals such as water potential, light and other factors. Poor seed germination is the major limiting factor of threatened medicinal plants for large scale production and cultivation under cold desert conditions. Seed germination in general can be controlled by many factors like natural germination (growth) inhibitors (Angevine & Chabot 1979). These are the derivatives of benzoic acid, cinnamic acid, coumarin, naringenin, jasmonic and abscisic acid (ABA). It has been postulated that seed coat (testa) of many plant species contains considerable amount of germination inhibitor, which prevent their seed germination (Arora & Bhojwani 1989). The first stage of germination consists of ingesting water and an awakening or activation of the germplasm. Protein components of the cells that were formed as the seed developed became inactive as it matured. Seed germination is important to know the germination pattern of a plant, more particularly the medicinal ones that might need to bring under cultivation for the primary healthcare system. The significance of the seedling in plant population ecology has long been recognized (Baskin & Baskin 1998). The germination response pattern of seeds is also regarded as a key characteristic in plant life history strategy (Baskin et al. 1993, Baskin & Baskin 1972). The variation in seed www.tropicalplantresearch.com

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Received: 19 June 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.067

Bhardwaj et al. (2016) 3(3): 508–516 . dormancy and the subsequent patterns of seedling emergence are controlled by environmental conditions. Important factors controlling the variation seed dormancy within species include the environment of the mother plant during the time of seed maturation and environmental conditions after the seeds have been released (Bewley 1997). Certain environmental conditions may be required to break dormancy, and other conditions are often required to permit germination after dormancy is broken (Bewley & Black 1994). Seeds of many species require days, weeks, or months at low temperatures to break dormancy (Bradbeer 1992), whereas others require warm temperatures for after-ripening to germinate when permissive conditions arrive (Chauhan & Johnson 2008). In some temperate species, dormancy is broken by a period of warm temperatures followed by cold stratification. This response is most often associated with morpho-physiological dormancy; however, seeds with morphophysiological dormancy have under developed embryos (Durrani et al. 1997). In order to accelerate this method, it can be combined with some treatments such as chemical applications or mechanical seed coat removal (El-Barghathi & El-Bakkosh 2005, Fenner & Thompson 2005). Many investigators have studied the effects of exogenous growth regulators on seed germination. Gibberellins eliminated the chilling requirements of peach and apple seeds and increased their germination (El-Barghathi & El-Bakkosh 2005). Recent studies have revealed that cold stratification has a direct effect on production of gibberellins (GAs) in seeds of Arabidopsis thaliana (Fernandez et al. 2002, Hartmann et al. 1997). Exogenously applied GA overcomes seed dormancy in several species (Hassan & Fardous 2003, Hradilik & Cisarova 1975) and promotes germination in some species that normally require cold stratification, light, or after-ripening (Kandari et al. 2012). Pre-chilling, scarification, and treatments with gibberellic acid (GA3) or nitric acid (KNO3) are the standard procedures used to enhance seed germination of dormant seeds. However, many attempts have been made to investigate seed germination and seedling emergence of different annual and perennial species including medicinal plants (Bewley 1997, Liebst &scheneller2008, Liza et al. 2010; Martinez-Gomez &dicenta 2001, Mayer & Poljakoff-mayber 1989, Mehanna et al. 1985). However, no study has surveyed germination patterns in medicinal plants from Ladakh region of India. MATERIALS AND METHODS Seed source Fruits of S. lappa, R. webbianum, I. racemosa, C. carvi and B. persicum were collected from their localities at an altitude of 1500–4000 m of Ladakh region of India in August 2012 (Table 1). The seeds were later air dried and stored at room temperature (25°C) before experimentation. Table 1. Brief description of plant species studied.

Name/Family Saussurea lappa C.B Clarke (Asteraceae)

Local name Habit Kuth Perennial shrub

Habitat Altitude (m) Uses Cultivated land & waste 2600–3600 Roots used as anti-arthritic, land (Lahaul valley) antiseptic, aphrodisiac, carminative and digestive agent Rheum webbianum Lachoo Perennial Moist slopes & open 3300–5200 Roots, stem and petioles used Royle (Polygonace) herb slopes (Zanskar valley) as appetizer, astringent and in the treatment of asthma, bronchitis, eye diseases, piles, etc. Inula racemosa Hook. Pushkarmool Perennial Cultivated land (Indus & 1595–2800 Roots used as anthelmintic, (Asteraceae) shrub Lahaul valley) antiseptic, anti-inflammatory and diuretic agents Carum carvi Linn. Gonyor, Biennial or Cultivated land or water 3650–3900 Fruits/seeds used as spice, (Apiaceae) jirah annual herb streams (Indus & spiti carminative, back pain, liver valley) problem and stimulant Bunium persicum Kala jirah Perennial Rocky slopes 1800–3500 Fruits/seeds used as spice, (Boiss.) B. Fedtsch. herb (Suru valley) carminative, back pain, liver (Apiaceae) problem and stimulant Seed viability assessment To ensure that the seeds used for the experiment were viable and of high quality, the sample lots were subjected to viability test using the tetrazolium technique. Three replicates (20 seeds each replicate) were www.tropicalplantresearch.com

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Bhardwaj et al. (2016) 3(3): 508–516 . subjected to 2, 3, 5, triphenyl tetrazolium chloride (TTC) test after 15, 30, 45, 60 days of storage at 4°C. In this method, seeds were longitudinally sectioned and the sections were immersed in a 0.5% aqueous solution of TTC (pH 6.5) for 24 hrs at room temperature (25°C) under controlled dark conditions. The TTC solution was drained and sections were rinsed 3 times with tap water. The topographical staining pattern of the embryos and cotyledons were studied under a dissection microscope. Seed surface sterilization and germination assessments Seeds of S. lappa, R. webbianum, I. racemosa, C. carvi and B. persicum were sterilized using 0.04% aqueous solution of mercuric chloride (HgCl2) for 15 sec. to remove any fungal infection and then rinsed with distilled water. Three replications of 30 seeds each were prepared for each treatment and control. T1, Seeds were dipped in concentrated acids, i.e. H2SO4 for 5 min; T2, gamma rays irradiation of seeds at different doses (i.e. 10–50 KR) using the 60Co gamma cell irradiator facility at the Physics Department, RTM, Nagpur University, Nagpur followed by dipping in concentrated H2SO4 for 5 min. ; T3, seeds were first pretreated in concentrated H2SO4 for 10 min. further dipped in GA3 solutions (i.e. 200 ppm) for a period of 1 hr; T4, seeds were soaked at 3 different doses of KNO3 (i.e. 0.1, 0.2 and 0.3 %) for 10 min. after presoaking in concentrated H2SO4 for 10 min; T5, scarification of seeds by P320A sandpaper (sand grain.cm-2) then dipped in GA3 solutions (i.e. 200 ppm) for a period of 1 hr; T6, seeds were stratified at -20°C for 1–30 days; T7, seeds were dipped in the hot water at 80°C for 20 minutes. and T8, seeds were first soaked in absolute alcohol and acetone for 10 minutes. All the treated seeds were placed in closed 9 cm Petridishes (Ø 9 cm) which were lined with 2 sheets of filter papers Whatman No.1 and moistened with sterilized MilliQ water. Treated seeds were placed on the moist paper for germination for 20–28 days and light was provided by philips daylight lamps (324 µmol.m-2.s-1). A clear labeled lid was placed on top of each Petri-dish denoting the treatment, temperature and replication. All these Petri-dish were then kept in plant growth chamber at 25±2 °C with relative humidity of 65% and 16 hr of light. Petri dishes were checked daily for germinated seeds and filter paper was moistened with sterilized MilliQ water as needed. Germination was determined by observing a visible radical or shoot. The number of seeds used for the germination tests were 3 replications × 90seeds/replication for each treatments. Seedling growth & vigour Seedling were incubated in plant growth chamber and monitored weekly. The growth of seedling was measured by vernier caliper in cm after 30 days of incubation. The well developed seedlings were potted in potting mixture containing Coconut + vermiculite + perlite (1:1:1) under controlled green house conditions. Initially, for 5–10 days the developed seedlings were covered with glass jars to provide sufficient moisture for growth of new shoots. During transplanting process, jars were taken off every day for 1–2 hr to acclimatize the plantlets to the external conditions. Seed vigour Index (SVI): Germination percentage × Seedling length (cm). Experiments were performed in triplicate. Data analysis The data were statistically analyzed as a factorial experiment based on completely randomized design with three replicates. Means were compared by one-way ANOVA using SPSS for windows (Version 21.0) and differences between the means were compared by Duncan’s multiple range test (DMRT). A probability of ≤0.05 was considered significant. RESULTS AND DISCUSSION Seed viability Tetrazolium chloride test showed the percentage viability of S. lappa, R. webbianum, I. racemosa, C. carvi and B. persicum as 88, 72, 78, 81 and 75 at the day of harvesting, which remains 55, 51, 55, 58 and 57 percent by 60 days of storage, showing a continuous decline with storage period (Fig. 1). A continuous decrease in seed viability was observed of different rhizomatous herbs of Himalayan region with storage period (Pupalla & Fowler 2002, Sharma et al. 2006) which supplemented the present observation. Seed germination assessments Mean and standard error comparisons of each treatment are based on Duncan’s test as presented in table 2. The effect of various durations of concentrated H2SO4 showed a positive effect of H2SO4 on seed germination, while no significant germination was observed with concentrated HCl. In case of concentrated HNO3 treatment there was no seed germination at all. Maximum germination of 97.2% was observed in I. racemosa with 5 www.tropicalplantresearch.com

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Bhardwaj et al. (2016) 3(3): 508–516 . minutes of soaking in concentrated H2SO4 followed by 95.1%, 93.4%, 90% and 81.4% in R. webbianum, C. carvi, S. lappa and B. persicum (Table 2 & Fig. 2). However, any increase or decrease in acid soaking time significantly reduced the seed germination which can be attributed to embryo damage. Poor germination or no germination in case of concentrated HCl and HNO3 respectively might be due to the inability of these treatments to break the physical dormancy. Also, the positive effect of gamma rays irradiation on seed germination is already known in many crops. Therefore, seeds were also treated with different doses of gamma rays along with concentrated H2SO4 treatment for 5 minutes. Maximum germination of 81.96% was observed when seeds of C. carvi were treated with 30 Kr gamma rays, followed by 5 min. concentrated H2SO4 treatment, followed by 77%, 76.4%, 73.04% and 45.6% in B. persicum, S. lappa, I. racemosa and R. webbianum. Obviously, any further increase or decrease dose of gamma rays or H2SO4 duration showed negative effect on the overall percent germination (Table 2 & Fig. 2).

Figure 1. Decrease in seed viability of some medicinal plants during storage at 4°C with increase in storage period.

Figure 2. Seed germination of some selected medicinal plants.

Gibberellic acid is also known to play an essential role in seed germination, stem elongation and flower development (Sharma et al. 2006). However, we have tried 100–500 ppm solution of GA3 out of which 200 ppm showed significant results. 200 ppm of GA3 treatment showed the highest germination of 69.20% in C. carvi when seed treated with 1 hr soaking in GA3 alongwith 10 min. of H2SO4 pre-soaking followed by 62%, 59.6%, www.tropicalplantresearch.com

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Bhardwaj et al. (2016) 3(3): 508–516 . 53.6% and 52.3% in R. webbianum, S. lappa, B. persicum and I. racemosa (Table 2 & Fig. 2). This means that the regulation of endogenous gibberellic levels after seed imbibitions along with specific H2SO4 presoaking time of 10 minutes is crucial factor in determining the seed germination. Lesser or longer treatment time was inhibitory in each case. A chemical treatment such as pre-treatment with H2SO4 for 15–30 minutes was found to be an effective method to increase germination. While seed treatments with KNO3 or GA3 is known to enhance the germination percentage (Silvertown & Lovett Doust 1993). When H2SO4 pre-treated seeds of I. racemosa were treated with KNO3 in different combinations the highest germination of 78.4% (Table 2 & Fig. 2) was obtained in case of pre-soaking in concentrated H2SO4 (for 10 minutes), followed by dipping in KNO3 (0.2% for 10 minutes). While, decreasingly 69%, 68.4%, 65% and 62.3% found in C. carvi, S. lappa, B. persicum and R. webbianum. Any increase or decrease in the concentration of KNO3 or soaking duration along with further increase in the presoaking time showed negative effect on the overall percent germination. Unsatisfactory germination percentage i.e. less than 20 was found in 0.3% KNO3 for 10 min. along with 10 min. H2SO4 presoaking. Scarification, stratification and hot water treatments were also found satisfactory results. Application of alcohol, acetone and HNO3 although broke the seed coat but not found better germination as compared to other treatments. Sulfuric acid treatment to remove mucilage and soaking in either of KNO3, GA3 or gamma ray treatment was found effective to allow penetration of oxygen from the surroundings to the embryos and increased germination of seeds. Germination in each case was superior over the control (10–20%). Maximum germination percentage i.e. 69.03% was found in C. carvi when seeds were stored at -20°C for 30 days followed by 67.02%, 61.23%, 59.5% and 57.23% in R. webbianum, I. racemosa, B. persicum and S. lappa. On the other hand, maximum percent germination (79.2%) found in R. webbianum when seeds treated with hot water at 80°C for 20 min. followed by 72.02%, 65.6%, 65.6% and 61.5% in S. lappa, C. carvi, B. persicum and I. racemosa while, 82.12% germination found in S. lappa when seeds treated with alcohol and acetone for 10 min. followed by 80.67%, 78%, 66.02% and 58.02% in C. carvi, I. racemosa, B. persicum and R. webbianum (Table 2 & Fig. 2). The responses of seeds to different treatments were strongly species-specific. It is obvious from the present data and similar work reported by other authors that the responses of dormant seeds of the same species to different factors are variable, depending upon the habitat of collection and duration of storage. However, some reported 70% germination in S. lappa when seeds were treated with gibberellic acid (GA) (3 μM) (Tairu et al. 2007). 89% germination was observed in R. webbianum when seeds were treated with GA3 and KNO3 (Taiz & Zeiger 2010). Similar to our observations, effectiveness of low temperature in causing dormancy removal has also been reported in other populations of C. carvi (Vleeshouwers et al. 1995) and B. persicum (Warghat et al. 2014). The low temperature requirement appeared to be replaced by GA3 in I. racemosa and C. carvi, but not in B. persicum, further signifying the species specificity in responses even of closely related species. Lowtemperature treatment of seeds could be easily adopted for the cultivation of species wherever it proved effective. According to results obtained in present study, all studied species found best germination with H 2SO4 for 5 minutes in duration of 30 days and significantly different as compared to other treatments at 5% level. Seedling growth and vigour index The effect of various durations of concentrated H2SO4 showed a positive effect of H2SO4 on seedling growth. The maximum length of seedling of S. lappa was observed 24.3 cm, when seeds were treated with H2SO4 for 5 minutes while, minimum length found 8.9 cm when seeds treated with alcohol and acetone for 10 minutes. Whereas, in R. webbianum maximum (23.8 cm) & minimum (9.7 cm), I. racemosa maximum (17.4 cm) in KNO3 & H2SO4 treatment and minimum (11.7 cm) in GA3 treatment, C. carvi maximum (22.2 cm) and minimum (10.2 cm) in H2SO4 + GA3 treatment and in B. persicum maximum (19.4 cm) in hot water treatment and minimum (5.9 cm) in alcohol + acetone for 10 minutes. Similar type of effects of treatments on seedlings growth of Q. coccifera was also found (Arora & Bhojwani 1989). There was a significant difference among the different treatments and medicinal plant seeds on seedling vigour (Table 3). R. webbianum significantly produced the best seedling vigour index 2263 which is statistically different from the other medicinal plants and treatments. However, B. persicum produced seedlings with low vigour index of 390. The bigger sized seed of R. webbianum with H2SO4 treatment for 5 min. statistically produced seedling of very high vigour index of 2263 compared to small sized seed of B. persicum with alcohol + acetone treatment for 10 min. which produced seedling that have low vigour index of 390. However, bigger sized seeds of R. webbianum and S. lappa produced the more stem girth compared to the small sized seed of B. persicum that gave less stem girth. The www.tropicalplantresearch.com

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Bhardwaj et al. (2016) 3(3): 508–516 . better seedling growth with H2SO4 treatment for 5 min. could be as a result of the early germination recorded and the maintenance of continuous growth and vigour with these treatments, during the period of observation. Table 3. Seed vigour index of medicinal plants.

Treatments T1 T2 T3 T4 T5 T6 T7 T8 Control

S. lappa 2187 1406 1168 807 575 950 900 731 143

R. webbianum 2263 757 1265 1215 809 865 1386 563 622

I. racemosa 1264 1176 654 1364 683 765 720 975 46

C. carvi 2073 844 706 1145 665 849 1207 1146 94

B. persicum 1490 1078 992 780 657 779 1273 390 483

As the seed size increases there was more food reserved in cotyledon of the seed to sustain the seedling growth than the smaller seed sizes whose food reserved could be exhausted thus affecting the seedling growth and vigour. This is agreed with the work previously done (Yamaguchi & Kamiya 2000). The significantly maximum seedling length obtained by S. lappa and R. webbianum could be that there were enough spaces within the treatments that allow growth. The maximum seedling length produced by the big sized seeds could be as results of its radicles where roots can easily attach themselves (Yamaguchi & Kamiya 2002). After the 90 th days, the well developed seedlings were transplanted to green house condition at DIHAR, Leh and placed under shade for further growth and development. However, result accomplished that 80% survival rate in green house condition and 70% in open condition of herbal garden with well developed healthy plantlets (Fig. 3). Similarly, It was found that survival rate of seedlings in the green house was high in S. lappa, R. webbianum and low in I. racemosa, C. carvi and B persicum (Yamaguchi & Kamiya 2002). Ten month old plants of these species are shown in figure 3. The transplanted seedlings of B. persicum and C. carvi did not survive beyond three month.

Figure 3. A & E, Seed-derived seedlings in petridish. B–D, Ten-month old plants growing under green house conditions.

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Bhardwaj et al. (2016) 3(3): 508–516 . CONCLUSIONS Plant propagation multiplies plants in bulk and preserves their essential genetic characteristics. Acid scarification, followed by gamma rays, addition of GA3 or KNO3 solution and hot water is a simple, efficient and cost-effective method for ensuring better seed germination and well developed seedlings. The outcomes of the present study can be gainfully utilized for multiplication of the species. The information will prove beneficial not only for conservation of species but also in boosting rural economy and the information will prove beneficial for other related species. ACKNOWLEDGEMENTS Authors are thankful to Defence Research and Development Organization (DRDO), Ministry of Defence, Government of India for providing financial assistance for the research. We are also thankful to Mr. Sandeep Singh Rawat for laboratory help. REFERENCES Angevine R & Chabot BF (1979) Seed germination syndromes in higher plants In: Solbrig OT, Jain S, Johnson GB & Raven PH (eds) Topics in Plant Population Biology. Columbia University Press, New York, pp. 188– 206. Arora R & Bhojwani SS (1989) In vitro propagation and low temperature storage of Saussurea lappa C.B. Clarke – an endangered medicinal plant. Plant Cell Report 8: 44–47. Baskin CC & Baskin JM (1998) Seeds, ecology, biogeography, and evolution of dormancy and germination. Academic Press, San Diego. Baskin CC, Chesson PL & Baskin JM (1993) Annual seed dormancy cycles in two desert winter annuals. Journal of Ecology 81: 551–556. Baskin JM & Baskin CC (1972) Ecological life cycle and physiological ecology of seed germination of Arabidopsis thaliana. Canadian Journal of Botany 50: 353–360. Bewley JD (1997) Seed germination and dormancy. Plant Cell 9: 1055–1066. Bewley JD & Black M (1994) Seeds Physiology of Development and Germination. Plenum Press, New York, USA. Bradbeer LB (1992) Vegetable production and their uses. Africana publication limited Lagos, Nigeria, pp. 67– 70. Chauhan BS & Johnson DE (2008) Influence of environmental factors on seed germination and seedling emergence of Eclipta (Eclipta prostrata) in a tropical environment. Weed Science 56: 383–388. Durrani MJ, Qadir SA, Farrulch H & Hussain F (1997) Germination ecology of Bunium persicum (Boiss) Fedtsch and Ferula oopoda (Boiss and Bulse) Boiss. Hamdard–Medicus 40, 86–90. El-Barghathi MF & El-Bakkosh A (2005) Effect of some mechanical and chemical pre-treatments on seed germination and seedling growth of Quercus coccifera (Kemes Oaks). Jerash Private University. Fenner M & Thompson K (2005) The ecology of seeds. Cambridge University Press, New York. Fernandez H, Perez C, Revilla MA & Perez–Gar-cia F (2002) The levels of GA3 and GA20 may be associated with dormancy release in Onopordum nervosum seeds. Plant Growth Regulation 38(2): 141–143. Hartmann HT, Kester DE, Davies Jr F & Genève RL (1997) Plant Propagation Principles and Practices, Sixth Edition. New Jersey, Prentice Hall. Hassan MA & Fardous Z (2003) Seed germination, pollination and phenology of Gloriosa superba L. (Liliaceae). Bangladesh Journal of Plant taxonomy 10(1): 95–97. Hradilik J & Cisarova H (1975) Studies on the dormancy of caraway (Carum carvi) achenes. Rostlinna. Vyroba 21: 351–364. Kandari LS, Rao KS, Payal KC, Maikhuri RK, Chandra A & Vanstaden JV (2012) Conservation of aromatic medicinal plant Rheum emodi Wall ex Messi. through improved seed germination. Seed Science & Technology 40: 95–101. Liebst B & Schneller JS (2008) Seed dormancy and germination behavior in two Euphrasia species (Orobanchaceae) occurring in the Swiss Alps. Botanical Journal of the Linnean Society 156: 649–656. Liza SA, Rahman MO, Uddin MZ, Hassan MA & Begum M (2010) Reproductive biology of three medicinal plants. Bangladesh Journal of Plant taxonomy 17(1): 69–78. www.tropicalplantresearch.com

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Bhardwaj et al. (2016) 3(3): 508–516 . Martinez-Gomez P & Dicenta F (2001) Mechanisms of dormancy in seeds of peach (Prunus persica (L.) Batsch) cv. GF 305. Scientia Horticulturae 91: 51–58. Mayer AM & Poljakoff-Mayber A (1989) The germination of seeds. Pergamon Press, New York, NY. Mehanna HT, Martin GC & Nishijuma C (1985) Effects of temperature, chemical treatments and endogenous hormone content on peach seed germination and subsequent seedling growth. Scientia Horticulturae 27: 63– 73. Pupalla N & Fowler JI (2002) Lesquerella seed pre-treatment to improve germination. Industrial Crops and Products 17: 61–9. Sharma RK, Sharma S & Sharma SS (2006) Seed germination behavior of some medicinal plants of Lahaul and Spiti cold desert (Himachal Pradesh): implications for conservation and cultivation. Current Science 90(8): 1113–1118. Silvertown JW & Lovett Doust J (1993) Introduction to plant population biology, 3rd edition. B1ackwell Scientific Publications, Oxford, 210 pp. Tairu FM, Adu AO & Adegbemile CM (2007) Effects of sowing media and depth on transplant quality of garden egg (Solanum gilo L). NIHORT Ibadan, in- house review meeting. Taiz L & Zeiger E (2010) Plant Physiology. Sinauer Associates Inc., USA. Vleeshouwers LM, Bouwmeester HJ and Karssen CM (1995) Redefining seed dormancy: an attempt to integrate physiology and ecology. Journal of Ecology 83: 1031–1037. Warghat AR, Bajpai PK, Srivastava RB, Chaurasia OP, Chauhan RS & Sood H (2014) In vitro protocorm development and mass multiplication of an endangered orchid, Dactylorhiza hatagirea. Turkish Journal of Botany 38: 737–746 Yamaguchi S & Kamiya Y (2000) Gibberellin biosynthesis: Its regulation by endogenous and environmental signals. Plant Cell Physiology 41: 251–257. Yamaguchi S & Kamiya Y (2002) Gibberellins and light-stimulated germination. Journal Plant Growth Regulation 20: 369–376.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 517–521, 2016 DOI: 10.22271/tpr.2016.v3.i3.068 Research article

Some ethnomedicinal plants used against high blood pressure in Bargarh district in Western Odisha (India) S. K. Sen1* and L. M. Behera2 1

Department of Botany, Panchayat College, Bargarh - 768028, India Ex-Reader in Botany, Modipara (Near Water Tank), Sambalpur - 768002, India *Corresponding Author: [email protected] [Accepted: 20 September 2016] 2

Abstract: The knowledge and usage of herbal medicine for the treatment of various ailments among the rural people still a major part of their life and culture. An ethnomedicinal survey was conducted during 2010–13 in different forest pockets and rural areas to collect ethnobotanical information on plant species from the local inhabitants of Bargarh district. Out of a number of collected plant species some are reported to be very useful against high blood pressure. The outcome of this survey is that the local people of the study area still have a strong faith in the efficacy and success of the herbal medicine. Keywords: Ethnomedicine - High blood pressure - Tribals - Bargarh district. [Cite as: Sen SK & Behera LM (2016) Some ethnomedicinal plants used against high blood pressure in Bargarh district in Western Odisha (India). Tropical Plant Research 3(3): 517–521] INTRODUCTION Medicinal plants have been used since time immemorial for the treatment of human as well as animal diseases and ailments. Traditional medicine practice is an important part of health care delivery in moist of the developing countries (Akerel 1998, Truyen et al. 2105, Ngbolua et al. 2016). According to World Health Organization, approximately 80% population in developing countries depends on traditional medicine for the primary healthcare (Mehra et al. 2014). A major portion of these involves the use of medicinal plants. Use of medicinal plants are known by ethnic tribes who resides in the forest area pay a vital role in using forest vegetation of food, cloth, shelter, for the treatment of common ailments, utilize the plants and manage to conserve it to some extent for future use. Odisha State is situated in the eastern part of India having 30 districts. Bargarh is one among them located in western part of Odisha. Prior to 5th November 2011, Bargarh was a subdivision under the district Sambalpur. The district lies between 20°40’–21°49’ N and 82°45’–83°48’ E. The District is surrounded by Chhattisgarh state on the north, Sambalpur District on the east, Bolangir and Subarnapur on the south and Nuapada District on the west. The Bargarh district experiences extreme type of climate with hot and dry summer followed by humid monsoon and chilling winter. The temperature varies between 10°C to 46°C. The average annual rainfall in the district is 1527 mm. The tribals such as Sahanra (Soara), Binjhal, Gond, Kondh, Munda, Kuli, Kalanga, Oran, Mirdha, Dharua, Kisan, Kharia and Parja are inhabited in the Bargarh district. The tribals mostly dependent on their traditional healing system for healthcare and for treating various diseases (Bajpai et al. 2016). Tradition and beliefs are the only basis of use of the herbal medicine. During the ethnobotanical survey in the district, it has been observed that many plants species are being used by the tribals and other rural people for various purposes including herbal medicine to cure and or preventive diseases. Generally the plant parts such as root, leaf, bark, stem, flower, fruit, gum and resin are used as paste, powder, extract, decoction etc. by the people to cure diseases and ailments (Deepa et al. 2016). Some valuable research works have been contributed in the last four to five decades from this locality (Panigrahi 1963, Brahmam & Saxena 1990, Misra et al. 1994, Misra 2004, Pradhan et al. 1999, Sen & Pradhan 1999, Behera & Sen 2007, 2008, Sen & Behera 2003, 2008). But no work on the present subject so far has been contributed from the study area. Therefore, an attempt has been made in this paper to highlights on some ethnomedicinal plants used for the treatment of high blood pressure in Bargarh district. www.tropicalplantresearch.com

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Received: 28 June 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.068

Sen & Behera (2016) 3(3): 517–521 . MATERIALS AND METHODS Field trips were conducted to different forest and rural area of Bargarh district during 2010 2013. The local traditional healers, herbal medicine practitioners, village headman, experienced old men and women were contacted and interviewed to record the ethnomedicinal uses of the plants and their local names (Bajpai et al. 2016). Mostly collections were made from local forests, scrub jungle, village suburbs and cultivated fields. Repeated quarries were made to confirm the authenticity of the information gathered from the local inhabitants. He collected plant specimens were identified with the help of flora books (Haines 1921 25, Saxena & Brahmam 1994 96). The latest botanical nomenclature has been checked with the world renowned and widely accepted website http://www.the plantlist.org. (The Plant List 2010). The herbarium specimens have been deposited in the herbarium of Botany Department, Panchayat College, Bargarh, Odisha. RESULTS Enumeration of species In the enumeration, the names of 10 plant species by the ethnic people of Bargarh district for the treatment of high blood pressure have been arranged alphabetically along with their correct botanical name, family in parenthesis, local names in inverted comma, locality and collection number, parts used, mode of preparation, details of ethnomedicinal uses. 1. Allium sativum L. (Amaryllidaceae), ‘Lesun’, Udepali-529 (Fig. 1A) 2 3 cloves of bulb are soaked in water overnight and either it is swallowed or crushed to paste is taken with a little warm water once daily in empty stomach in the morning. Fresh leaf paste (5 10 g) is taken once daily in empty stomach in the morning. 2. Catharanthus roseus (L.) G. Don (Apocynaceae), ‘Baramasi’, Beherapali-234 (Fig. 1B) Petals (5 numbers) are chewed once in the morning in empty stomach. 3. Cuscuta reflexa Roxb. (Convolvulaceae), ‘Nirmuli’, Khandijharan-276 (Fig. 1C) Whole plant juice (2 teaspoonful) is taken once daily in stomach in the morning. 4. Hygrophila auriculata (Schumach.) Heine, (Acanthaceae), ‘Kuilekha’, Ramkhol-534 (Fig. 1D) Leaf juice (1 teaspoon) and Piper nigrum fruit (5 7 in number) powder are mixed together and taken once daily in empty stomach for 15 days to get relief from high blood pressure at least for a period of one year. 5. Moringa oleifera Lam. (Moringaceae), ‘Munga’, Ramkhol-256 (Fig. 1E) Leaf juice (one teaspoonful) is taken once daily in empty stomach for one month to get relief from hypertension at least for period of one year. Tender leaves are chewed early in the morning for one month to cure blood pressure for one year. 6. Psydrax dicoccos Gaertn. (Rubiaceae), ‘Benimanj’, Ramkhol-376 (Fig. 1F) Equal amount of leaf and bark are crushed together and the juice (5 ml) is taken 1–2 times daily in empty stomach. 7. Rauvolfia serpentina (L.) Benth. ex Kurz (Apocynaceae), ‘Patalgarud’, Nrusinghnath- 519 (Fig. 1G) Root powder/ paste (1.0 1.5 g) is given once daily regularly 15 days and after three days break once again the medicine is continue for 15 days. 8. Senna occidentalis (L.) Link. (Caesalpiniaceae), ‘Chakunda’, Kharmunda-217 (Fig. 1H) Fresh leaf paste (5–10 g) is taken once daily in empty stomach in the morning. 9. Terminalia arjuna (Roxb. ex DC.) Wt. & Arn. (Combretaceae), ‘Ka’, Khandijharan-316 (Fig. 1I) Bark (10 g) is boiled in a glass (250 ml) of water to obtain ¼ decoction is taken once daily in empty stomach early in the morning for one month. Bark powder (50 gm) and old jaggery (50 gm) are mixed together and made into pills of 1 cm size. One pill is taken once daily in warm water. 10. Trigonella foenum-graecum L. (Fabaceae), Methi, Beherapali-236 (Fig. 1J) Seed powder (5g) is taken with warm water in empty stomach once daily. DISCUSSION The present study focuses on traditional medicines used by the tribals to cure or get relief from high blood pressure. Ten plant species from 9 families have been identified with 13 ethnomedicinal prescriptions used by the tribals of the present study. The observation revealed that the herbal medicines used by the tribals are mostly www.tropicalplantresearch.com 518

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Figure 1. Plants used by the ethnic people for the treatment of high blood pressure: A, Allium sativum; B, Catharanthus roseus; C, Cuscuta reflexa; D, Hygrophila auriculata; E, Moringa oleifera; F, Psydrax dicoccos; G, Rauvolfia serpentina; H, Senna occidentalis; I, Terminalia arjuna; J, Trigonella foenum-graecum.

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Sen & Behera (2016) 3(3): 517–521 . administered in the form of paste, powder, juice, decoction, prepared in a crude method from different plant parts such as of root, bulb, bark, leaves, flower, seed and whole plant. Of the various plant parts used, the leaves were most commonly used. Leaf is used maximum (4 prescriptions), followed by bark (3 prescriptions), root (2 prescriptions)and other plant parts are used once in each case and in one case both leaf and bark are used in one prescription (Fig. 2).

Figure 2. Plant parts used in the treatment high blood pressure.

It has also been highlighted that after observing their mode of treatment by different herbal medicine practitioners, which are compared with some of the scientific literatures (Jain 1991, Kirtikar & Basu 1991, Chopra et al. 1992, Ambasta et al. 1992, Warrier 1996, Pal & Jain 1998, Joshi 2004, Paria 2005, Singh 2013) and it has been recorded that out of 13 prescriptions 7 from 6 plant species (with asterisk mark) are new report. CONCLUSION The tribals depend on the plants around them which made them acquire knowledge of economic medicinal properties of many plants by trial and error. Consequently, they act as knowledge saviour for the utilization of many useful plants accumulated and enriched through generations and passed to one another without any written documents. But careful approaches should be followed before administration these drugs. The lack of proper documentation and carelessness towards the knowledge of ethnomedicine are forcing depletion of the traditional knowledge, which has to be preserved for the future benefit of the human civilization. Proper documentation and digitalization of tribal information is utmost importance. However, the present study may create some awareness among the people which might help to conserve their rich and effective ethnomedicinal knowledge in this region. ACKNOWLEDGEMENTS Authors are thankful to Prof N. B. Pradhan, Retired Reader in Botany and Mr. Pareswar Sahu for their kind help during field collection and identification of plant materials. Authors are also thankful to the local informants for sharing their valuable ethnomedicinal knowledge about the plants. REFERENCES Akerel O (1998) Medicinal plants and primary health care agenda for action. Fitoterapia 59: 355–363. Ambasta SP, Ram Chandran K, Kashyappa K & Chand R (1992) Useful plants of India. Publication and Information Directorate, CSIR, New Delhi, 918 p. Bajpai O, Pandey J & Chaudhary LB (2016) Ethnomedicinal uses of tree species by Tharu tribes in the Himalayan Terai region of India. Research Journal of Medicinal Plant 10(1): 19–41. Behera LM & Sen SK (2007) Traditional use of some plants against gynecological disorders by the tribals Ramkhol village forest of Barapahad hill range. Advances in Plant Sciences 20 (2): 555–557. Behera LM & Sen SK (2008) Ethnobotnay of Western Orissa, India. In: Patil DA (eds) Herbal cures: Traditional Approach. Aavishkar Publishers, Distributors, Jaipur, India, pp. 316–331. www.tropicalplantresearch.com

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Sen & Behera (2016) 3(3): 517–521 . Brahmam M & Saxena HO (1990) Ethnobotany of Gandhamardan Hills – Some noteworthy folk-medicinal uses. Ethnobotany 2: 71–79. Chopra RN, Nayar SL & Chopra IR (1996) Glossary of Indian Medicinal Plants (Reprint edition). National Institute of Science Communication, CSIR, New Delhi, 330 p. Deepa MR, Sharma Dharampal P & Udayan PS (2016) Floristic diversities and medicinal importance of selected sacred groves in Thrissur district, Kerala. Tropical Plant Research 3(1): 230–242. Haines HH (1921–25) The Botany of Bihar and Orissa. Arnold & Son &West Nirman Ltd., London, 1348 p. Jain SK (1991) Dictionary of Indian Folk Medicine and Ethnobotany. Deep Publications, New Delhi, 311 p. Joshi SG (2006) Medicinal Plants (Reprint edition). Oxford and IBH publishing Co. Pvt. Ltd., New Delhi, 419 p. Kirtikar KR & Basu BD (1991) Indian Medicinal Plants (Reprint edition). Lalit Mohan Basu, Allahabad. Mehra A, Bajpai O & Joshi H (2014) Diversity, utilization and sacred values of Ethno- medicinal plants of Kumaun Himalaya. Tropical Plant Research 1(3): 80–86. Mishra RC (1994) Studies on the flora and remote sensing of natural resources of Nrusinghnath-Harishankar Complex, Orissa, Ph.D. thesis. Berhampur University, Orissa, India. Mishra RC, Panda PC & Das P (1994) Lesser known medicinal uses of plants among the tribals of Gandhamardan hill range, Orissa. Higher plants of Indian subcontinent (Additional Series of Indian Journal of Forestry No.VI) 3: 135–142. Ngbolua KN, Mihigo SO, Liyongo CI, Ashande MC, Tshibangu DST, Zoawe BG, Baholy R, Fatiany PR & Mpiana PT (2016) Ethno-botanical survey of plant species used in traditional medicine in Kinshasa city (Democratic Republic of the Congo). Tropical Plant Research 3(2): 413–427. Pal DC & Jain SK (1998) Tribal Medicine. NayaProkash, Calcutta, 317 p. Panigrahi G (1963) Gandhamardan Parbat, Orissa A potential source of important indigenous drugs. Bulletin of Regional Research Laboratory, Jammu 1: 111–116. Paria ND (2005) Medicinal Plant Resources of South West Bengal. Directorate of forest, Govt. of West Bengal, Kolkata, 198 p. Pradhan NB, Pradhan RN, Sen SK &Sahu P (1999) Some threatened noteworthy medicinal plants of Bargarh district (Orissa). Neo Botanica 7: 97–100. Saxena HO & Brahmam M (1994–96) The Flora of Orissa. Regional Research Laboratory, Orissa and Orissa Forest Development Corporation Ltd., Orissa, 2918 p. Sen K Sen & Behera LM (2008) Ethnomedicinal plants used by the tribals of Bargarh district to cure diarrhoea and dysentery. Indian Journal Traditional Knowledge 7(3): 425–428. Sen SK & Behera LM (2003) Ethnomedicinal plants used against skin diseases at Bargarh district in Orissa (India). Ethnobotany 15 (1&2): 90–96. Sen SK & Pradhan NB (1999) Conservation of ethnomedicinal plants of Bargarh district in Orissa. Advances in Plant Sciences 12: 207–213. Singh H (2013) Ethnomedicinal uses of some wild flowers in Sundargarh, Mayurbhanj, Angul and Bolangir districts of Odisha. Ethnobotany 25: 115–119. The Plant List (2010) The Plant List: A working list of all plant species. Available from: http://www.the plantlist.org. (accessed: 03 Apr. 2016). Truyen DM, Mansor M & Ruddin AS (2015) A note on Aroids Ethnobotany in Hau River, Vietnam. Tropical Plant Research 2(1): 58–63. Warrier PK, Nambir VPK & Ramankutty G (1996) Indian Medicinal Plants (5 vols). Orient Logman, New Delhi.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 522–535, 2016 DOI: 10.22271/tpr.2016.v3.i3.069 Research article

Uncultivated fodder grass for cattle R. Prameela1* and M. Venkaiah2 1

M.R.College for women, Vizianagaram, Andhra Pradeah, India Department of Botany, Andhra University, Visakhapatnam, India *Corresponding Author: [email protected] [Accepted: 25 September 2016] 2

Abstract: Vizianagaram district, one of the northern districts of Andhra Pradesh lies on the East Coast of India. The district is located between 17o–15' to 19o–15' N and 83o–00' to 83o–45' E. Agriculture is the key occupation of the people of this district. Geographically vizianagaram district divided in to 3 regions, i.e 1) The hilly region 2) The plains and 3) The coasts. The present study has concentrated to generate the information on uncultivated fodder grass. The grass family Poaceae is of a major economic and ecological importance. It is the single most important family of flowering plants for survival of mankind. The grasses form a natural homogenous group of plants with remarkable diversity playing a significant role in the lives of human beings and animals. Studies on grasslands and wild grasses, especially of fodder value have become very important for development of dairy industry, productions of meat and restoration of degraded ecosystems. The grasses have good potentials in sustainable development of the country as well as conservation of both plant and animal diversity. Study areas in vizianagaram district are undisturbed places, open grounds, strip lands, unused rice fields, orchards and sandy areas. During rainy season grass seeds germinate and grow very fast. All these grasses are annuals. Where as in summer, we can see very little pasture. Many grasses are perennials; they can survive in drought conditions, because they possess thick rhizomatous or stoloniferous root system and tufted growth. Perennial grasses form a valuable pasture. Keywords: Annuals - Herbivores - Pasture - Perennials - Strip lands - Vizianagaram. [Cite as: Prameela R & Venkaiah M (2016) Uncultivated fodder grass for cattle. Tropical Plant Research 3(3): 522–535] INTRODUCTION Hooker’s ‘Flora of British India’ accounted for the known grasses of the country (Hooker 1896). Cooke (1901–1908) provided an account of grasses along with other families in his ‘Flora of the Presidency of Bombay’. Gamble (1896) published ‘The Bambuseae of British India’. Blatter & McCann (1935) published an excellent illustrated account of Bombay grasses while Achariyar & Mudaliyar (1921) published an account of South Indian grasses. Fischer (1934) contributed account for the grasses of Madras Presidency. Bor (1960) made extensive studies on grasses of Assam, Uttar Pradesh and published some 125 papers on Indian grasses and finally published a consolidated concise account of grasses of the whole Indian subcontinent ‘The grasses of Burma, Ceylon, India and Pakistan. Several studies were conducted on the flora of North Coastal Andhra Pradesh. Subba rao (1977) studied on the flora of Visakhapatnam; Sriramulu (1986) studied flora of Srikakulam and Venkaiah (2004) flora of Vizianagaram district. In the present studies an attempt made to study the uncultivated fodder grasses of Vizianagaram district. Everybody knows that all herbivorous animals depend upon the grass for their food. We can differentiate herbivorous animals into two categories namely, wild and domesticated or livestock animals. We need not take care about wild herbivores, because they feed on wild grass or green pastures of the forest areas. But we have to take a special care for livestock. Cultivated grass is compulsory for feeding the livestock at home. Generally cultivated grass is growing at the farms for livestock. It is very expensive and limited. In Vizianagaram there are no grasslands, but farmers are growing some types of grasses like Pennisetum spp., Sorghum spp., and also used straw of Rice, Maize, Sugarcane, Finger millet, Proso millet etc. It is limited and not sufficient for livestock, that’s why they are being taken to open areas, where there are some pastures available. The present study concentrated on these uncultivated grasses growing in open lands and used as fodder. www.tropicalplantresearch.com

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Received: 07 June 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.069

Prameela & Venkaiah (2016) 3(3): 522–535 . MATERIALS AND METHODS Study areas in vizianagaram are undisturbed places, open grounds, Striplands, unused rice fields, orchards and Sandy areas. During rainy season grass seeds germinate and grow very fast. All these grasses are annuals. Examples of annual grasses are Brachiaria spp, Digitaria, Echinochloa, Eriochloa, Ischaemum, Paspalidium, Rottboellia, Setaria, Sorghum, Sporobolus and Urochloa etc. These grasses collectively form in a good pasture for the cattle. Where as in summer we can see very little pasture. Many grasses are perennials, they can survive in drought conditions, because they possess thick rhizomatous or stoloniferous root system and tufted growth. Perennial grasses form a valuable pasture. Examples of perennial grasses are Aristida, Chloris, Chrysopogon, Cenchrus, Cynodon, Dactyloctenium, Dichanthium, Eragrostis, Heteropogon, Paspalum, Paspalidium, Perotis and Themeda etc. Regular field trips have been undertaken to the study areas of the district and collected the uncultivated grasses. The collected grasses are identified with the help of regional floras and Herbarium, BSI, Coimbatore. RESULTS Enumeration of Species 1. Alloteropsis cimicina (L.) Stapf in Prain, Fl. Trop. Afr. 9: 487.1919; GS. 357; Bor 276; chowdhary in Ind. For. 86: 90.1960; Gam vol. 3.1766; MV. 197; V. Fl. 213; HS. 52.1972. Milium cimicinum L., Mant. Pl. 2: 184.1771. Herbs, annuals, tufted, decumbent, hairy with bulbous based hairs; nodes hairy, internodes smooth leafy sheaths, leaf blades hairy, 7 cm long, 1–1.3 cm wide, ovate lanceolate, base cordate; base of the internodes purple colour; spikes 4–5, terminal 6 cm long, 20–24 spikelets in each spike. Spikelets 0.3 cm long, elliptic, green, upper glume ciliate, acuminate, upper lemma aristate, keeled, upper palea membranous contains bisexual floret, lower glume mucronate, smaller than the upper one, lower lemma 3 nerved, shortly mucronate and its palea smaller membranous, contains malefloret, stamens 3. 2. Aristida adscensionis L., Sp. Pl. 82.1753; FBI 7: 221; var. adscensionis Bor 407. A. depressa Retz., Obs. Bot 4. 22.1786; GS. 360; AP. Fl. 1130; Gam vol. 3: 1809. Annual, tufted, branched, grows on rocky places; culms erect or decumbent, slender, rootstock creeping, nodes glabrous, leaf blades very narrow or linear; inflorescence contracted panicle, spikelets single, pedicel scaberulous, callus present, glumes 2 unequal, aristate, keel spinuscent, lemma base fringe of hairy, awns 3, unequal, stamens 3, stigmas plumose. 3. Aristida funiculate Trin. & Rupr., Sp. Gram. Stip. 159.1842; var. funiculate; Bor 410.1960; Gam vol. 3: 1809; FBI 7: 226.1896. An annual, tufted grass, culms upto 50 cm tall, geniculately ascending; nodes glabrous, ligule a small ciliate membrane, blade 6–16 × 0.1–0.2 cm linear convolute, base cordate, apex acuminate; inflorescence panicle, lax, narrow, spikelets single, pedicelled, 1-flowered, glumes linear, hyaline, lemma truncate, awn trifid, subequal, stamens 3, caryopsis spiny. 4. Aristida hystrix L.f., Suppl. Pl. 113.1781; FBI 7: 225.1896; Gam vol. 3.1809; Bor 410; AP. Fl. 1131. (Fig. 1A) Annual, small herbs upto 30 cm high; stems shows pseudo dichotomous branching, branches spreading; leaves small, linear, ligule gringe of hairs, axis angled, axis and nodes hairy; inflorescence panicle, effuse, dichotomously branches, spikelets single, pedicelled; glumes unequal, aristate, awns 3, double the length of the spikelets, stamens 3, base of the spikelet black coloured, dry spikelets straw coloured. 5. Bothriochloa pertusa (L.) A. Camus., Soc. Lyon. 1930, Gam vol. 3.1731; Bor 109.1960. A perennial, stoloniferous grass, culms upto 50 cm tall, ascending, nodes sparsely hairy, leaf sheaths 2–6 cm long, glabrous, ligule membranous, blades 3.0–10 × 0.2–0.4 cm, linear, base truncate, apex acuminate, glabrous; racemes 2–6, 5 cm long, slender, digitate, silky hairy, spikelets 6 mm, 2-nate, pitted, sessile spikelet: 4 mm, oblong-lanceolate, pitted, callus bearded, lower glume elliptic-oblong, bearded below, dorsally pitted, subchartaceous, upper glume ovate-lanceolate, finely pointed at the tip, lower lemma lanceolate, nerveless, hyaline, upper lemma reduced to a slender awn, stamens 3, stigmas plumose, caryopsis oblong, pedicelled spikelet: 4mm, male or barren, similar to sessile, spikelet, but awnless. www.tropicalplantresearch.com 523

Prameela & Venkaiah (2016) 3(3): 522–535 . 6. Brachiaria reptans (L.) C.A. Gardner & C.E. Hubb., Hooker’s Icon. Pl. 34: t. 3363.1938; Fl. Trop. Afr. 9: 601.1920. Annuals, culms prostrate or creeping, slender, branched; leaves ovate-lanceolate, cordate at base, margin scaberulous, mouth ciliate, ligule a fringe of hairs, leaf sheath clasping the stem, shorter than the internode; inflorescence panicle, branches up to 9; spikelets 2-nate, both are pedicelled, bears 4–5 setae. 7. Capillipedium assimile (Steud.) A.Camus., Fl. India 15: 6.1984. (Fig. 1B) Andropogon assimillis Steud. in Zoll. Syst. Verz. 48.1854. et in Syn. Pl. Glum. 1: 397.1854; FBI 7: 179.1896. Capillipedium assimile (Steud.) Camus in Lecomte, Fl. Gen. del’ Indo-chine 7: 314.1922; Bor 110.1960. C.glaucopsis (Steud.) Stapf in Hook., lc. Pl. Sub. tab. 3085.1922; Gam vol. 3. 1730. A perennial, tufted grass, culms upto 50 cm long, decumbent, often branched, nodes glabrous or bearded, leaf sheaths usually glabrous, ciliate at mouth, ligule a short membrane, apex ciliate, blades 8–20 × 3–6 cm, linear-lanceolate, apex acuminate, slightly hairy; panicle lax, branches slender, capillary, with long hairs on the axils, spikelets few, distant, joints sparsely ciliate, sessile spikelet: 3 mm, lower glume oblong, ciliate, not pitted, upper glume lanceolate, lower lemma linear, shorter, obtuse, upper lemma reduced to a scale, flattened based awn, pedicelled spikelet: 4mm, not awned, pedicels sparsely ciliate, lower glume lanceolate, lower lemma obovate-oblong, apex ciliate, lower glume lanceolate, lower lemma obovate-oblong, apex ciliate, hyaline, upper lemma absent, stamens 3. 8. Cenchrus ciliaris L., Mant. Alt. 302.1771; Bor 287. Pennisetum cenchroides Rich. in Pers., Syn. Pl. 1: 72.1805, nom. Superfl. FBI 788.1896; AP Fl. 1146; Gam vol. 3.1793; Maha Fl. 421. Tufted herbs, erect or decumbent; leaves linear with tubercle based hairs; racemes cylindric, dense, pale purple, bristles connate at base only, scabrid; involucral spikelets 2, unequal, 2-flowered, large spikelet contains bisexual and male florets, small spikelet contains 2 male florets; stamens 3, green. 9. Chloris barbata Sw., Fl. Ind. Occ. 1: 200.1797; Bor 465; FBI 7: 292; Gam vol. 3.1836; AP Fl. 1148; HS. 508; MV. 199. (Fig. 1C) Annual tufted herbs, base creeping; leaves linear-lanceolate, tip acuminate, ligule membranous; racemes umbellate, spikes 8–12, rachis scaberulous, spikelets 3-flowered, perfect floret 1, glumes linear, densely ciliate on the margin; fertile lemma obovate, densely stiff ciliate on margins, awned; both sterile lemmas boat-shaped, awned, stamens 3, caryopsis oblong, compressed. 10. Chrysopogon aciculatus (Retz.) Trin., Fund. Agrose. 188.1820; Bot. Bihar & Orissa 1035.1924; Gam vol. 3. 1738.1934; Bor 115.1960; V Fl. 209. (Fig. 1D) Andropogon aciculatus Retz., Obs. Bot. 5: 22.1789; FBI 7: 188.1896; AP. Fl. 1155. Perennials with creeping rhizomes, culms decumbent, glabrous, leaf blades sub basal, upto 7 cm long, 0.5 cm wide, glabrous; inflorescence panicle, branches in whorled, in each whorl 5–6 spikes, each spike has long peduncle, tip of the peduncle has 3 spikelets, middle one sessile, remaining two pedicelled, sessile spikelet base hairy awned, bisexual, lower glume bidentate, 3-nerved, 2-keeled, upper glume awned, ciliate, lower lemma epaleate, empty, upper lemma aristate, paleate, contains bisexual floret. 11. Chrysopogon orientalis (Desv.) A. Camus., Bor 118.1960. (Fig. 1E) Rhaphis orientalis Desv., Opuse. 69.1831. Andropogon wightianus Nees ex Steud., Syn. Pl. Glum. 1: 395.1854. FBI 7: 191.1896; AP. Fl. 1155; Gam vol. 3. 1736. A perennial, tufted grass, culms upto 80 cm tall, slender ascending at base, nodes glabrous, leaf sheaths 5–9 cm, shortly ciliate-glabrous at mouth, ligule short, villous; inflorescence panicle, 15 cm long, whorled branches, branches with reddish-brown hair tip, spikelets 3, middle sessile lateral 2 pedicelled, 3 spikelets awned; peduncle very long, slender, spiral, tip hairy, sessile spikelet, lemma awn, very long, as long as peduncle, glume awn as long as spikelet, sessile spikelet contains bisexual floret, pedicelled spikelet lanceolate, pubescent pedicel truncate, margins shortly ciliate, glumes bright red/purple, lower glume ciliate awned, upper glume lanceolate, acuminate, lower lemma, upper lemma also ciliate, staments 3. 12. Cynodon dactylon (L.) Pers., Syn. Pl. 1: 85.1805; FBI 7: 288.1960; Gam vol. 3.1835; Bor 469. www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . Panicum dactylon L., Sp. Pl. 58.1753. Perennial, stoloniferous, culms creeping or decumbent; leaves small, linear, base rounded, tip acuminate, distichous; spikes 4–5, digitate; spikelets sub sessile, laterally compressed; glumes sub equal, lanceolate, 1nerved, lemma oblong, aristate, keels ciliate, palea linear, stamens 3, caryopsis oblong. 13. Dactyloctenium aegyptium (L.) Willd., Ess. Agrost. Expl. t. 15.1812; Gam vol. 3: 1840 (1273). 1934; Bor 489.1960. (Fig. 1F) Cynosurus aegypticum L., Sp. Pl. 72.1753. Eleusine aegyptia (L) Roxb., Fl. Ind. 1: 345.1820; FBI 7: 295.1896. Dactyloctenium aegyptiacum Willd., Enum. Pl. Hort. Berol. 1029.1809. An annual grass, culms erect or creeping, roots at the nodes, branches geniculately ascending, nodes bearded or glabrous, leaf sheaths 2–6 cm long, hairy near mouth, ligule a ciliate rim, blades 2–10 × 0.3–0.8 cm, linear, flat, base rounded, apex acuminate, glabrous or hairy with bulbous hairs; spikes 2–6, digitately radiating, upto 7 cm long, spikelets 3–5 flowered, laterally compressed, sessile, glumes subequal, folded, in lower keels scabrid, in upper keel smooth or hispid, awned, lemmas aristate, palea hyaline, winged on keels, stamens 3, stigmas plumose; caryopsis obovate. 14. Dichanthium annulatum (Forssk.) Stapf., Fl. Trop. Afr. 9: 178.1917; Gam vol. 3.1740; Bor 133.1960; Deshp in Fasc. Fl. India 15: 5.1984. Andropogon annulatus Forssk., Fl. Aegy-Arab. 173.1775; FBI 7: 196.1896. A perennial, tufted grass, culms upto 70 cm tal, erect or ascending, nodes bearded, leaf sheaths 5–10 cm long, glabrous, ciliate near the mouth, ligule membranous, blades 6–20 × 0.3–0.5 cm, linear, base rounded, apex acuminate; racemes 6 cm long, sub digiatately fascicled, peduncles glabrous, spikelets 4 mm, 2-nate, sessile spikelet: 4mm, bisexual, elliptic-oblong, lower glume elliptic-oblong, truncate, keeled, ciliate, upper glume, nerveless, upper lemma narrow, with a scabrid, slender awn, stamens 3, stigmas plumose, caryopsis oblongslightly compressed, pedicelled spikelet: 4mm, male or barren, lower glume elliptic-oblong, 7–11 nerved keeled, upper glume narrow, 3-nerved, lower lemma ciliate, upper lemma small or obsolete. 15. Dichanthium caricosum (L.) A. Camus., Gam vol. 3. 1741; Bor 134.1960; Deshp. in Fasc. Fl. India 15: 7.1986. Andropogon caricosus L., Sp. Pl. 2: 1480. 1763; FBI 7: 196.1896. A perennial, tufted grass, ascending, nodes bearded or with short hairs, leaf sheaths 3-10 cm long, glabrous, ligule shortly ciliate membrane, blades 4–20× 0.3–0.5cm linear, base rounded, apex acuminate, glabroussparsely pubscent, racemes 1–6, upto 8 cm long, both terminal and axillary; joints and pedicels of racemes with short hairs, spikelets 4mm, 2-nate, sessile spikelets: 2mm, oblong, bisexual, lower glume oblong-obvate, 4nerved, truncate, pilose, upper glume elliptic, 3-nerved, hairy, lower lemma lanceolate, nerveless, hyaline, upper narrow, awned, stamens 3, pedicelled spikelet, lower glume obovate, many nerved, pilose, upper glume and lemmas similar to sessile spikelet. 16. Digitaria ciliaris (Retz.) Koeler., Descr. Gramin. 27.1802. (Fig. 1G) Digitaria marginata Link var. fimbriata (Link) Stapf in Prain, Fl. Trop. Afr. 9: 440.1919; Gam vol. 3: 1764 (1222). 1934. D. adscendens (H.B.K.) Henr. in Blumea 1: 92.1934; Bor 298.1960. Panicum adscendens H.B.K. Nov., Gen. Pl. 1: 102.1821. An annual, tufted grass, culms upto 90 cm tall, usually ascending from geniculate or prostrate base, branching from lower nodes, nodes glabrous, leaf sheaths 4–12 cm long, glabrous, ligule membranous, truncate, blades 4–10 × 0.4–0.6 cm, linear-lanceolate, base cordate, apex acute, glabrous, racemes 4–9, 10 cm long, digitate, rachis serrate, trigonous, spikelets 3mm, elliptic-lanceolate, 2-nate, apperssed to the rachis, glabroussparsely ciliate, homomorphous, lower glume reduced to a triangular scale, upper glume linear-lanceolate, 3–5 nerved, pubescent, lower lemma oblong-lanceolate, 5-nerved, marginal nerves pubescent, upper lemma oblonglanceolate, obscurely 3-nerved, stamens 3, stigmas plumose, caryopsis oblong. 17. Digitaria longiflora (Retz.) Pers., Gam vol. 3: 1765; Bor 302.1960. Paspalum longiflorum Retz., Obs. Bot. 4: 5.1786; FBI 7: 17.1896. www.tropicalplantresearch.com

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Figure 1. A, Aristida hystrix L.f.; B, Capillipedium assimile (Steud.) A. Camus.; C, Chloris barbata Sw.; D, Chrysopogon aciculatus (Retz.) Trin.; E, Chrysopogon orientalis (Desv.) A. Camus.; F, Dactyloctenium aegyptium (L.) Willd.; G, Digitaria ciliaris (Retz.) Koeler.; H, Eragrostiella bifaria (Vahl) Bor.; I, Eriochloa procera (Retz) C.E. Hubb.

An annual, tufted grass, culms upto 60 cm tall, ascending, rooting at the nodes, nodes glabrous, leaf sheaths 2–4 cm long, glabrous, ligule membranous, blades 2–7 × 0.2–0.5 cm, linear-lanceolate, base rounded, apex acuminate, glabrous, racemes 2–5, 7 cm long, digitate, rachis serrate, spikelets 2 mm long, elliptic, homomorphous, sparsely pubscent, lower glume absent, upper glume oblong-ovate, 3–5 nerved, hairy, lower www.tropicalplantresearch.com 526

Prameela & Venkaiah (2016) 3(3): 522–535 . lemma similar to upper glume, upper lemma ovate-oblong, subchartaceous, nerveless, palea similar to upper lemma, stamens 3, stigmas plumose, caryopsis ellipsoid. 18. Digitaria sanguinalis (L.) Scop., Fl. Carn. ed. 2.1. 552.1772; Bor 304.1960; T.A. Cope in Nasir & Ali Fl. Pak. 143. 231.1982. Panicum sanguinalis L., Sp. Pl. 57.1753. Paspalum sanguinale (L.) Lam., III. 1: 176.1791; FBI 7: 13.1896. Digitaria sanguinalis (L.) Scop. ssp. aegyptica var. frumentacea Henr. Monogr. Digitaria 985.1950; Bor 304.1960. Digitarua sabguinalis ssp. vulgaris var. rottleriana Henr., 1.c. 986; Bor 304.1960. An annual, tufted grass, culms up to 40 cm tall, erect or ascending from a creeping, branching base, leaf sheaths glabrous, hairy near the mouth, blades 2.5–18×0.4–0.8 cm, linear; spikes, slender, 3–10, 8 cm long, digitate, spikelets 2-nate on abbreviated peduncles, imbricate, lower glume 3–5 nerved, lateral nerves marginal, lower lemma lanceolate or oblong-lanceolate, smooth; caryopsis oblong, whitish. 19. Dinebra retroflexa (Vahl) Panz., Gam vol. 3: 1841 (1274). 1934; Bor 491.1960. Cynosurus retroflexus Vahl, Symb. Bot. 2. 20.1791. Dinebra arabica Jacq., Fragm. Bot. 77. t. 121. f. 1.1807; FBI 7: 297.1896; AP. Fl. 1182. An annual, tufted grass, culms up to 70 cm tall, geniculately ascending below, nodes glabrous, leaf sheaths 5–9 cm long, loose, glabrous, ligule narrow membrane; blades 5–10 × 0.2–0.4 cm linear, base cordate, apex acuminate, glabrous, spikes racemosely arranged along the axis of an inflorescence, up to 20 cm long, rachis stiff, serrate, winged, spikelets 5mm, alternate, 2-flowered, glumes persistent, lanceolate, 1-nerved, strongly keeled, slightly recurved, scaberulous awn, lemma ovate-oblong, hyaline; palea ovate, hyaline, 2-keeled, minutely sparsely ciliate on the keel, stamens 3, stigmas plumose, caryopsis ellipsoid. 20. Echinochloa colonum (L.) Link., Gam vol. 3: 1776; Bor 308.1960. Panicum colonum L., Syst. Nat. (ed.10) 810.1759; FBI 7: 32.1896. An annual, tufted grass, culms up to 70 cm tall, branching from lower nodes, nodes glabrous, leaf sheaths 5– 15 cm long, glabrous, ligule obsolete, blade 5–25 × 0.3–0.6 cm, linear-lanceolate, base cordate, apex acuminate, glabrous; spike like panicle, racemes 8–20, appressed to the rachis, rachis angular, scaberulous, spikelets 2.5 cm, broadly ovate, acute or sub-cuspidate, crowded in rows, second, glumes unequal, lower glume half the length of the upper glume, broadly ovate-orbicular, upper glume broadly ovate, mucronate, puberulous, lower lemma broadly ovate, cuspidate, upper lemma broadly ovate, obtuse, palea oblong, stamens 3, stigmas plumose; caryopsis broadly ellipsoid. 21. Echinochloa crusgalli (L.) Gam, vol. 3: 1777 (1231). 1934; Bor 310.1960. Panicum crusgalli L., Sp. Pl. 56.1753; FBI 7: 30.1896. An annual, tufted grass, culms 70 cm tall, branching from lower nodes, nodes glabrous, leaf sheaths 5–25 cm, glabrous, ligule obsolete, blades 5–20 ×0.4–0.8 cm, linear-lanceolate, base cordate, apex, acuminate, glabrous; spikelike panicle 10 cm long, racemes spreading, rachis rtiquetrous, scabrid, spikelets broadly ovoid, 5 mm, crowded, acute-cuspidate, awned, lower glume broadly ovoid or orbicular, margin ciliate, half as long as the upper glume, upper glume broadly ovate-oblong, concave, cuspidate, hispid on back, lower lemma ovateoblong, stamens 3, stigmas plumose, caryopsis broadly ellipsoid. 22. Echinochloa frumentacea Link., Bor 311.1960. Panicum crusgalli var. frumentaceum Hook.f. FBI 7: 31.1896. Echinochloa colona Link var. frumentacea Ridl., Fl. Malay Penin 5: 223.1925; Gam vol. 3: 1777. An annual, robust grass, culms up to 1 m, leaf sheaths 12 cm, ligule absent, blades to 20 × 2 cm, racemes 18 cm, several seriate, spikelets 4 mm, ovoid-broadly ellipsoid, hispid, glumes subequal, upper with a short mucronate apex, lower lemma mucronate, stiff-hispid outside, upper lemma crustaceous, short mucronate, palea similar to its lemma, caryopsis broadly ellipsoid. 23. Echinochloa stagnina (Retz.) Beauv., Gam vol. 3: 1777 (1231). 1934; Bor 311.1960. Panicum stagninum Retz., Obs. Bot. 5: 17.1789. P.crusgalli sensu Hook.f., FBI 7: 30.1896 p.p. non L. 1753. www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . An annual stoloniferous grass, culms up to 50 cm tall, rooting at the nodes, nodes glabrous, leaf sheaths 5– 10 cm long, loose, glabrous, ligule a fringe of stiff hairs, blades 4–15 × 0.4–0.8 cm, linear-lanceolate, base cordate, apex acuminate, midrib prominent; spikelike panicle 5–10 cm long, racemes interrupted, rachis angular, scabrid, spikelets ovate, crowded, acutely acuminate, awned, secung, pubescent, lower glume broadly ovate, bidentate, pubescent, upper glume broadly ovate, acuminate, hairy at apex and margins, lower lemma ovate, hairy and scabrid at apex, awned, palea oblong-ovate, scabrid margin, male; upper lemma ovate, scabrid along the margins, acute, chartaceous, palea similar to upper lemma, bisexual, stamens 3, stigmas plumose; caryopsis broadly ellipsoid. 24. Enteropogon dolichostachya (Lag.) Keng., Gen. Sp. Pl. 5.1816; Gam vol. 3: 1838 ; Bor. 466. Chloris incompleta Roth, Nov. Pl. Sp. 60.1821; FBI 7: 290.1896; Bot. Bihar & Orissa 968.1924; Gam vol. 3.1838; Suppl. Bot. Bihar & Orissa 164.1950; GS. 374; AP. Fl. 1149; MV. 199; Maha Fl. 426. Slender perennials, upto 93 cm long, culms erect, sometimes decumbent, nodes glabrous, leaf blades linear acuminate, ligule comprise of long hairs, spikes 2–4 digitate, rhachis angled, scabrid on angles, spikelets unilateral, 2-flowered, 2-awned, 2 seriats, lower glume membranous 1-nerved, upper glume short awned, lower lemma membranous, 2-toothed at apex, awned, callus beareded. 25. Eragrostiella bifaria (Vahl) Bor., FBI 7: 325.1896; Gam vol. 3.1828. (Fig. 1H) A perennial, tufted grass, culms up to 50 cm tall, slender, leaf sheaths glabrous, ligule a pubescent line, blades 5–20 × 1.0–1.5 cm, linear, filiform, base narrow, apex acute, glabrous, spike 5–25 cm long, spikelets 5mm long, oblong, second, alternately arranged on the rachis, laterally compressed, 5–40 flowered, glumes subequal, deciduous, lanceolate, 1-nerved, keeled, lemma broadly ovate, 1-nerved, keeled, palea lanceolate, winged on keels, stamens 3, styles plumose; caryopsis globose. 26. Eragrostis cilianensis (All.) Janch., Gam vol. 3.1827; Bor 503.1960. Poa cilianensis All., Fl. Pedem. 2: 246. t. 91. f. 2, 1785. Eragrostis major Host., Icon. Descr. Gram Austr. 4: 14. t. 24.1809; FBI 7: 320.1896; HS. 516. An annual, tufted grass, culms up to 90 cm high, leafy, branched, erect or geniculately ascending, nodes glabrous, leaf sheaths 2–4 cm long, loose ciliate at mouth, ligule a ciliate ridge, blades 5–12 × 0.4–0.6 cm, lanceolate, flat, prominently nerved, base cordate, glandular along the margins, apex acuminate; panicle up to 20 cm long, spreading or contracted, rhachis stiff, spikelets 1 cm long, oblong, breaking up from down wards, 10– 30 flowered, glumes subequal, linear, ovate-lanceolate, scabrid on the nerves at back sides, lemma broadly ovate, acute, 3-nerved, palea linear, ciliate on the margins near the apex, persistent, stamens 3, stigmas plumose, caryopsis subglobose. 27. Eragrostis ciliaris (L.) R.Br., FBI 7:314.1896; Gam vol. 3.1825; Bor 506.1960. Poa cilliaris L., Syst. Nat. ed. 10. 2. 875.1759; HS. 516. An annual, tufted grass, culms up to 40 cm tall, slender, geniculately ascending below, nodes glabrous, leaf sheaths 2.5 cm long, glabrous, ciliate at the mouth, ligule ciliate, blades 2–5 × 0.2–0.4 cm, linear, convolute, base rounded, apex acuminate, glabrous, panicle up to 8 cm long, interrupted, cylindrical, branches short, spikelets 2.5 mm, oblong, subsessile, glumes subequal, ovate-lanceolate, scabrid on the nerve on the backside, lemma ovate, 3-nerved, lateral nerves close to the margin, ciliate, palea obovate, truncate, with soft ciliate, stamens 3, stigmas plumose, caryopsis ovoid. 28. Eragrostis coarctata Stapf., FBI 7: 313.1896; Gam vol. 3.1825; Bor 507.1960; GS. 396. Annual, tufted, 3–4 culms in a tuft, erect, slender, nodes, internodes glabrous, leaf sheath, leaves hairy; panicle dense 15–20 cm long, grayish-purple, spikes up to 15, short 5–6 cm long, villous at nodes, spikelets up to 50 arranged in distichous manner, pedicel short scaberlous, spikelets single contains a bisexual floret, glumes unequal, scaberulous on margins, tip acute or acuminate, 1-nerved, lemma & palea ovate, tip obtuse, margins ciliate, pink, stamens 3, lodicules 2, grain oblong, reddish brown. 29. Eragrostis japonica (Thunb.) Trin., Gam vol. 3.1826; Bor 509.1960. Poa japonica Thunb., Fl. Jap. 51.1784. Eragrostis interrupta Beauv. var. tenuissima Stapf in. FBI 7: 316.1896; GS. 397. www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . Perennial, tufted grass, culms 1.0–1.5 m high, geneculately ascending at base, plants glabrous, ligule membranous, leaf blade linear-lanceolate, tip acuminate, base cordate or rounded; panicle up to 50 cm long, branches whorld, lax, inturrepted, internodes long, spikelets 2–9 flowered, glumes subequal, narrow, 1-nerved, lemma 3-nerved, palea boat shaped, scaberulous, stamens 3 or 2, caryopsis obovoid. 30. Eragrostis tenella (L.) Beauv. ex Roem. & Schult., FBI 7: 315.1896; Bor 513.1960. var. tenella; HS. 518; GS. 398. Annual, densely tufted grass, culms 50 cm high, leaf sheaths glabrous long ciliate at mouth, panicle 20 cm long, decompound, spreading or contracted, spikelets 3–9 flowered, small, palea keeled ciliate, caryopsis ovate. 31. Eragrostis viscosa (Retz.) Trin. Acad. Sci. Petersb. 6, 1: 397.1830; Gam vol. 3.1826; Bor 515.1960. Poa viscose Retz., Obs. Bot. 4: 20.1786/87. Eragrostis tenella var. viscosa (Retz.) Stapf in FBI 7: 315.1896. Perennial, tufted, viscid, scented grass, culms 50 cm high, glandular patches scattered throughout, nodes glabrous ligule fimbriate membrane, panicle 20 cm long, cylindric, dense glandular, branches numerous, spreading spikelets 5–20 flowered, glumes unequal, linear, 1-nerved, lemmas ellipsoid, palea linear, ciliate on the keels, caryopsis ovoid. 32. Eriochloa procera (Retz) C.E. Hubb., Gam vol. 3.1767; Bor 312; AP. Fl. 1202. (Fig. 1I) Eriochloa polystachya Hook.f., FBI 7: 20.1896; Maha Fl. 494. Perennials, densely tufted, up to 1 m high, root stock short; leaves linear, base cordate, ligule rim of hairs. 33. Eriochloa fatmensis (Hochst. & Steud.) Clayton., Bor 312; Maha Fl. 493. Annual, terrestrial, culms 30–60 cm high, geneculately ascending and rooting at lower nodes, ligule hairy; panicle of simple or branched racemes, 3–10 cm long; spikelets paired or rarely solitary, ellipsoid, hispid. 34. Heteropogon contortus (L.) Beauv. ex Roem. & Schult., Gam vol. 3: 1743; Bor 163.1960. (Fig. 2A) Andropogon contortus L., Sp. Pl. 1045.1753; FBI 7: 199.1896; AP. Fl. 1206. An annual, tufted grass, culms up to 60 cm high, erect or decumbent below; nodes glabrous, leaf sheaths 5– 10 cm, glabrous; ligule membranous, fimbriate; blades 4–10 × 0.2–0.4 cm, lanceolate, flat, base rounded, apex acute, scaberulous; racemes solitary, 6 cm, awns forming a twisted spire; lower spikelets homogamous, unawned, either neuter or male; upper spikelets heterogamous, awned, 2-nate, sessile hermaphrodite, pedicelled male or barren; pedicel glabrous; callus pungent, bearded with brown hairs; sessile spikelets: 7 mm, linear, hispidulous, female; callus bearded with brown hairs, lower glume linear-oblong, hispidulous; upper glume lenear, obtuse, margin hyaline, dark brown, hispidulous; lower lemma oblong, truncate, epaleate; upper lemma linear, stipitoform, awned, epaleate; stamens 3, stigma plumose, caryopsis linear; pedicelled spikelets: oblong, hispidulous, lower glume lanceolate, dorsally hispidulous with long bulbous based hairs, winged, serrulate; upper glume oblong-lanceolate, margin hyaline; lemmas hyaline, nerveless, hairy. 35. Oplismenus burmanni (Retz.) P. Beauv., FBI 7.66; Gam vol. 3: 1777; V. Fl. 216. Panicum burmanii Retz., Obs. 3. 10.1783; MV.205; HS. 521; GS. 412. A small procumbent herb, lower nodes bears roots, leaf sheaths, leaf blades hairy, leaf blade ovate lanceolate, base cordate, ligule fringe of hairs; inflorescence panicle, racemes 4, distant, rhachis trigonous, villous, spikelet 2 nate, unequal, sessile spikelet small, pedicelled spikelet large, pedicel villous, 2 flowered, lower glume membranous, ovate, obtuse, pubescent margins ciliate, 3 nerved, awned, awn scaberulous, upper glume similar to lower glume 7 nerved, awn short, lower lemma awned, empty, membranous, 9 nerved, 2 keeled, epaleate; upper lemma chartaceous, its palea similar, contains bisexual floret, stamens 3, stigmas plumose. 36. Oplismenus compositus (L.) P. Beauv., FBI 7: 66; Bor. 317; S. 317; Gam vol. 3.1777; Bot. Bihar & Orissa 3.1045; MV. 205; HS. 521. Pannicum compositus L., Sp. Pl. 1: 57.1753. Oplismenus lanceolatus Kunth, Rev. Gram. 45.1829.

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Prameela & Venkaiah (2016) 3(3): 522–535 . Perennial, stoloniferous up to 60 cm tall, creeping, rooting at nodes, nodes glabrous, leaf sheath 2–9 cm long, margin density liliate, ligule fimbriate membrane; inflorescence terminal, racemes 20 cm long, spikelets 2 flowered, awned, lower glume awned, upper glume aristate. 37. Panicum maximum Jacq., Gam vol. 3.1783; Bor 327. Perennial tufted grass, culms up to 1 m high, stout, nodes hirsute; ligule membranous, blades linear, flat, base cordate, margin scaberulous, apex acuminate, glabrous, mid rib prominent; panicle lax, spikelets linearoblong, dense, lower glume orbicular, half the length of spikelet, 1-nerved, upper glume broadly oblong, margins incurved, 5-nerved; lower lemma oblong, 5-nerved, palea oblong, 2-nerved, male, upper lemma ovate oblong, 3-nerved, transversely rugose, it’s palea similar, nerve less, bisexual. 38. Panicum notatum(Retz.) Bor, 329; Gam vol. 3.1783. Perennial slender grass, culms up to 1 m high, erect from woody stock, branched; nodes glabrous; leaf sheaths ciliate on margin, ligule of soft long hairs, blades linear-oblong, base amplexicaul, margin with tubercled based hairs, apex acuminate, pubescent on both surfaces; panicle up to 25 cm long, lax, open, branches spreading, spikelets few, pedicels very long, filiform; lower glume ovate, 3–5 nerved, pilose, upper glume broadly ovate, 5-nerved; lower lemma ovate, 5-nerved, hairy on outsite, epaleate, upper lemma ovate, crustaceous, indurated, palea coriaceous with involute margins. 39. Paspalidium flavidum (Retz.) A. Camus., Fl. Indo-Chine 7.419.1922; Bot. Bihar & Orissa 1000.1924; Gam vol. 3.1774; Bor 333.1960. (Fig. 2B) Panicum flavidum Retz., obs. Bot. 4: 15.1786; FBI 7. 28.1896; GS. 419; MV. 206; HS. 523; V. Fl. 214. Perennials, tufted, up to 80 cm high, culms erect base decumbent, striate, glabrous, nodes glabrous, leaf blades linear, lanceolate, acute or obtuse, cordate at base, margins scabrid, ligule membranous, base narrow ciliate, sheaths glabrous, striate; inflorescence racemose, racemes distant, up to 2 cm long, rhachis flattened, minutely ciliate, spikelets biseriate, sub sessile, spikelets 2 flowered, lower floret empty, upper floret bisexual, lower glume hyaline, suborbicular, 3-nerved, lower lemma ovate-oblong, subcoriaceous, 5- nerved, palea sililar to its lemma, bidentate, upper glume membranous, sub orbicular, rounded at apex, 7-nerved, upper lemma ovate, acute, coriaceous, granulose, palea similar to its lemma 2 keeled, lodicules 2, broadly cuneate, stamens 3, yellow, ovary triquetrous, styles 2, laterally exserted. 40. Paspalidium geminatum (Forssk.) Stapf., Fl. Trop. Africa 9.583.1920; Bot. Bihar & Orissa 1001.1924; Gam vol. 3.1774.1934; Susppl. Bot. Bihar & Orissa 176.1950; Bor. 333.1960. Panicum geminatum Forssk., Fl. Heg-Arab. 18.1775. P. paspaloides Pers., Syn. 1. 81.1805; FBI 7: 30.1896; GS. 420. Perennial, culms up to 50 cm high, creeping below, branches from the lowernodes, nodes glabrous, ligule a ciliate rim; panicle, spike like, racemes 10–15, alternate on the rhachis; spikelets solitary, 2- flowered, lower male, upper bisexual. 41. Paspalidium punctatum (Burm.) A. Camus., Gam vol. 3.1774; Bor 333. Panicum punctatum Burm., f. Fl. Ind. 26.1768; FBI 7: 29.1896; AP. Fl. 1233. A perennial, tufted grass, culms up to 1m tall, prostrate, rooting at lower nodes, spongy, leaf sheaths 10 cm long, ligule a ridge of hairs, blades 10–15× 0.5–1.0cm, flat or convolute, apex acuminate, glabrous; spike like panicle 30 cm long, racemes 20 cm long, distant, sessile, compressed, appressed to the rachis, spikelets 3mm long, 18–40 per raceme, ovoid, lower glume truncate; upper glume broadly oblong; lower lemma acuminate, palea empty, upper lemma punctate, apiculate, palea coriaceous, margins incurved, stamens 3, stigmas plumose, caryopsis ovoid. 42. Paspalum canarae (Steud.) Veldkamp., Blumea 21: 72 1973. Panicum canarae Steud., Syn. Pl. Glumac. 1: 58.1853. Paspalum compactum sensu FBI 7: 12.1896, non Roth 1821; Gam. vol. 3.1772; Bor 336.1960; GS. 421. Annuals, slender, tufted, culms decumbent glabrous nodes bearded, leaf blades ovate-lanceolate, obtuse, base rounded, hirsute with bulbous based hairs, margins ciliate ligule a rim of hirsute hairs, inflorescence racemose, rhachis angled, winged, hairy at the base of branches, spikelets 2-nate, plano convex, lower glume www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . absent, lower lemma empty as long as the spikelet, 3-nerved epaleate, upper glume as long as the spikelet, broadly ovate, convex, 5-nerved, margins hyalime, upper lemma bisexual, chartaceous, apiculate, palea similar its lemma, 2-keeled, stamens 3, grain reddish-brown, plano convex, granulate. 43. Paspalum scrobiculatum L., FBI 7: 10.1896; Bot. Bihar & Orissa 1000. 1924; Gam vol. 3. 1772; Suppl. Bot. Bihar & Orissa 176.1950; Bor 340.1960; GS. 422; HS. 524. Perennial tufted, culms erect or decumbent, nodes glabrous, leaf blades linear-lanceolate, acuminate, margins white and scabrid, ligules membranous, short, sheaths compressed, keeled, racemes 2–3, spikelets 2 nate, single obovate or suborbicular, rhachis ribbon like, ciliate, pedicels flattened, ciliate, lower glume wanting, lower lemma nerved, epaleate membranous, upper glume membranous, glabrous concave, orbicular or broadly ovate, 7 nerved, upper lemma broadly ovate, turning brown with age, palea similar to its lemma, margins inflexed with flaps on either side at base, lodicules 2, quadrangular, stamens 3. 44. Paspalum vaginatum Sw., Prodr. Veg. Ind. Occ. 21.1788; Bor 341. (Fig. 2C) Aquatic, perennial, rhizomatous, tufted; leaves linear-lanceolate, glabrous, margin puberulous, tip acuminate, sheath hairy, ligule membranous, young leaves 7-nerved, old leaves 1-nerved; spikes 2, sub digitate; spikelets 2-ranked, pedicelled, single, 2-flowered, lower floret empty and upper floret bisexual; pedicel glabrous; Spikelets planoconvex, glabrous; lower glume absent, lower lemma membranous, 1-nerved, it’s palea 7-nerved, thick, stamens 2, orange, lodicules 2. 45. Pennisetum americanum (L.) Leeke., FBI 7: 82.1986; Gam vol. 3.1792; Bor 350.1960; AP. Fl. 1236. An annual, cultivated, tufted grass, culms up to 2 m tall, nodes bearded, leaf sheaths overlapping, sparsely tubercled based hairs, ligule a ciliate rim, blades 30–90 × 0.5–5.0 cm, linear-lanceolate, base rounded, apex acuminate, pilose below, midrib prominent, panicle up to 25 cm long, spiciform, cylindric, dense; rhachis stout, villous below the inflorescence; involucral bristles 4 mm, often purplish-coloured, spikelet 4 mm, oblong, lower glume absent, upper glume minute, truncate, 3-nerved, lower lemma oblong, obtuse, 5-nerved, epaleate, upper lemma ovate-oblong, membranous, 2-nerved; palea very broad, truncate, ciliate at the tip, stamens 3, apex penicillate, stigmas plumose, caryopsis oblong-obovoid or fusiform; glabrous, top exposed. 46. Pennisetum pedicellatum Trin., Mém. Acad. Imp. Sci. Saint-Pétersbourg, Sér. 6, Sci. Math., Seconde Pt. Sci. Nat. 3(2): 184.1834; FBI 1: 86.1896; Gam vol. 3.1792; Bor 346.1960; AP. Fl. 1237. An annual, tufted grass, culms up to 90 cm tall, branches from the base, leafy, leaf sheaths glabrous; ligule a shortly ciliate membrane; blades 10–20 × 3–9 cm, linear, flat, glabrous-sparsely hairy, racemes 13 cm long, cylindric, densely flowered, involucral bristles outer few, slender, short, 3 mm, inner long, 1 cm, numerous, densely villous below the middle, unequal, free from the base, spikelets 4 mm, solitary in the involucral, sub sessile, lower glume very small, woolly; upper glume oblong-lanceolate, hyaline, apiculate; lower lemma oblong, truncate, 3-lobed, hyaline; upper lemma ovate-oblong, obtuse, apex fimbriate ciliate, coriaceous, shining; palea lanceolate, toothed. 47. Pennisetum polystachyon (L.) Schult., Gam vol. 3: 1792 (1241). 1934; Bor 346.1960. Panicum polystachyon L., Syst. Nat. ed. 10. 870.1759; AP. Fl. 1238. An annual, tufted grass, culms up to 40 cm tall, much branched, leaf blades 7.0–35 × 0.5–1.5 cm, panicle 20 cm long, linear, slender; rachis slender, puberulous; bristles densely plumose, especially the inner, spikelets solitary, rarely 2 in an involucre, pedicelled, lower glume minute or suppressed; upper glume puberulous; lower lemma similar to upper glume, 3-toothed; upper lemma chartaceous, fimbriate; palea similar to its lemma. 48. Pennisetum purpureum Schumach., Bor 348.1960; AP. Fl. 1238. A perennial, stoloniferous grass, culms up to 2 m tall, erect, branching from lower nodes; nodes bearded, leaf sheaths 10–15 cm long, glabrous, ligule a dense fringe of hairs; blades 30–60 × 0.7–1.5 cm, linear-lanceolate, base rounded, margins scaberulous, apex acuminate, scabrid, midrib prominent, spikelike raceme up to 18 cm long, solitary, cylindric, yellowish or purplish; involucral bristles numerous of unequal length, one usually very much longer, 8 mm, scabrid, ciliate, inner most scabrid, 6 mm, sparingly plumose towards the base, spikelets solitary, sessile, if in fascicles 2–4, lateral pedicelled, all lanceolate, 5mm, lower lemma absent or if present ovate-lanceolate, truncate, scabrid; upper glume triangular, margin incurved, 3-nerved, scabrid; lower lemma www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . oblong-lanceolate, scabrid on outside surface, 5-nerved, male or barren; upper lemma lanceolate, rolled, scabrid at apex, 2-nerved, stamens 3, penicillate at apex, style united throughout, stigmas plumose, caryopsis ovoid. 49. Pennisetum setosum(Sw) Rich. FBI 7: 87.1896; Bor 340.1960. Cenchrus strosum Sw., Prodr. Veg. Ind. Occ. 26.1788; AP. Fl.1239. A perennial, tufted grass, culms up to 80 cm high, often fastigiately branched at the nodes, leaf sheaths glabrous; ligule with fringed long, soft hairs, blades 10–20 × 5–10 cm, linear, apex acuminate, slightly hairy; racemes 10 cm long, purplish brown, involucral bristles unequal, the outer not ciliate, short, 3mm, the inner longer, long silky hairy below the middle, 1.3 cm, spikelet 4 mm, solitary within the involucral, lower glume minute or completely absent; upper glume ovate-oblong, cuspidate, hyaline; lower lemma oblong, obtuse, bidentate, palea narrowly oblong, hyaline, male: upper lemma ovate-oblong, truncate, coriaceous, shining; palea oblong, truncate, toothed or ciliate at the apex, stamens 3. 50. Perotis indica (L.) Kuntze., Gam vol. 3.1814; Bor 611. (Fig. 2D) Anthoxanthum indicum L., Sp. Pl. 28. Perotis latifolia Ait., Hort. Kew Bull. 1: 96; AP. Fl. 1239. Perennial, tufted, creeping or geniculately ascending from base; ligule absent, blades ovate-lanceolate, base amplexical, margin spinulose, tip acute; inflorescence spike like raceme, terminal, spikelets small, subsessile; glumes subequal, linear, 1-nerved, margin scabrid, awned, lemma and palea contains a bisexual floret, stamens 3. 51. Rottboellia exaltata (L.) L.f., FBI 7: 156.1896; Bot. Bihar & Orissa 1059.1924; Gam vol. 3.1759; Suppl. Bot. Bihar & Orissa 194.1950; Bor. 206; V. Fl. 211; MV. 207; HS. 525. (Fig. 2E) Annuals, erect, tufted, clinging roots from lower nodes, nodes and inter nodes glabrous purple colour, leaf blades up to 60 cm, much longer than inter nodes, acuminate tip, margins & upper surface scaberlous, lower surface smooth, mid nerve prominent white, ligule small membranous, sheaths scaberlous; racemes cylindric, tapering towards the apex into an appendage, greenish yellow, spikelets 2 nate, sessile and pedicelled, sessile spikelet sunk in the receptacle, awnless, lower glume coriaceous, 2 keeled, up to 11-nerved, lower lemma male, ovate-lanceolate, 3-nerved, palea similar to its lemma, lodicules 2, stamens 3, upper glume boat shaped, chartaceous, upper lemma bisexual, hyaline, 1-nerved, palea hyaline, lanceolate, pedicelled spikelet green, 2 flowered, both are male, compressed. 52.Setaria parviflora (Poir.) M. Kerguelen, Lejeunia 120: 161.1987. S. geniculata P. Beauv., Bor 360. Perennial herb, tufted, rhizome short; culms short, flat, procumbent, up to 25cm high; leaf sheath purple, blade 10cm long, 1cm wide, margin purple, ligule membranous; inflorescence panicle cylindric, dense, 4.0– 4.3cm long; spikelets single, if 2-nate, lower spikelets reduced to bristles (about 10), upper half part purple; spikelets ovate, planoconvex, middle part of the pedicel hirsute, lower glume small 3- nerved, half of the spikelet, lower lemma membranous margins inflexed, epaleate, empty; upper glume membranous, upper lemma boat shaped, crustaceous rugose, it’s palea similar, containing a bisexual floret, stamens 3. 53. Setaria pumila (Poir.) Roem & Schult., Gam vol. 3.1789; Bor 363; AP. Fl. 1254. (Fig. 2F) Annual tufted grass, culms up to 50 cm high, nodes glabrous; leaf sheaths glabrous, ligule a fringe of hairs, blades linear-lanceolate, flat, base cordate, apex acuminate; panicle spiciform, cylindric, densely flowered, spikelets ovoid; lower glume, upper glume and higher lemma ovate, hyaline; upper lemma crustaceous, transversely rugose, stamens 3. 54. Setaria verticellata (L.) P. Beauv., FBI 7: 80; Gam vol. 3.1789; Bor 365. Panicum verticillatum L., Sp. Pl. ed. 2.82; AP. Fl. 1255. Annual, tufted, 2–4 culms in a tuft, ererct, culms up to 2 m high; stem flat, nodes swollen, straite, solid; leaves up to 40 cm long, 3 cm wide, linear, base narrow, upper surface scabrid, 10 side nerves, lower surface white, no nerves, margin scaberulous, ligule small membranous; inflorescence 20–23 cm long, rhachis ends with a setae, lower 3 spikelets 3-nate, remaining all 2-nate, spikelet having 1-long setae, setae downward barbelleate.

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Prameela & Venkaiah (2016) 3(3): 522–535 .

Figure 2. A, Heteropogon contortus (L.) Beauv. ex Roem. & Schult.; B, Paspalidium flavidum (Retz.) A. Camus.; C, Paspalum vaginatum Sw.; D, Perotis indica (L.) Kuntze.; E, Rottboellia exaltata (L.) L.f.; F, Setaria pumila (Poir.) Roem & Schult.; G, Sporobolus indicus (L.) R.Br.; H, Urochloa panicoides P.Beauv.; I, Urochloa setigera (Retz.) Stapf.

55. Sporobolus coromandelianus (Retz.) Kunth., FBI 7: 252.1896; Gam vol. 3: 1817 (1258). 1934; Bor 627.1960. Agrostis coromandeliana Retz., Obs. Bot. 4: 1786. Sporobolus commutatus (Trin.) Kunth, Enun. Pl. 1: 214.1833. www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . Vilfa commutate Trin., Diss. Bot. 156.1824; AP. Fl. 1258. Annual, tufted grass, decumbent, nodes glabrous, ligule membranous, blades lanceolate, flat, base cordate, margins cartilaginous spinulosely tooth, apex acute, sparsely tuberculate based hairs, inflorescence panicle, decompound, branches capillary, horizontal, whorled at base, spikelets minute, pedicelled, lower glume minute, suborbicular, upper glume, lemma similar, palea hyaline contains bisexual floret, stamens 3, caryopsis ellipsoid. 56. Sporobolus indicus (L.) R.Br., FBI 7: 247.1896; Gam vol. 3.1817; Bor 629.1960; AP. Fl. 1259. (Fig. 2G) Perennial, tufted grass, culms 60 cm high, nodes, nodes glabrous, leaf sheaths glabrous, ligule fringe of hairs, leaf blades linear flat, base truncate, apex acuminate, inflorescence contracted panicle, branches filiform, spreading, spikelets small, single flowers, lower glume minute, ovate, hyaline, upper glume broadly elliptic, hyaline, acuminate, lemma lanceolate, its palea small hyaline, stamens 2, stigmas plumose, caryopsis obovoid. 57. Themeda laxa (Anderson) A. Camus., Gam vol. 3.1746; Bor 251. AP. Fl. 1264. Perennial, culms up to 40 cm high, tufted; leaf blades filiform, apex acuminate; panicle up to 8 cm long, leafy, few racemes, shortly peduncled outer spathes 2.5 cm, proper spathes glabrous, longer than the spikes; involucral spikelet: 4, all on the same level, male; lower glume glabrous except few bristles near the apex; pedicelled spikelets: similar to involucral spikelet; sessile spikelet: solitary, bisexual, awned. 58. Themeda quadrivalvis (L.) Kuntze., Bot Bihar & Orissa 1050.1924; Gam. vol. 3: 1746.1934; Bor 252; FBI 7: 213.1896; GS. 442; HS. 528; MV. 2008; V. Fl. 210. Annual, erect, culms and nodes glabrous, leaf blades flat, linear lanceolate, tip acuminate, rounded at base, mouth ciliate, ligule membranous; inflorescence leafy panicle, fascicled, erect, spatheoles boat shaped acuminate, tip 3–5 nerved, involucral spikelets pairs situated at the same leaf, middle sessile spikelet awned, contains bisexual floret remaining four spikelets sessile, contains male florets, stamens 3, glumes of male florets 2 keeled with scarious and ciliate margin on one side, pedicelled spikelets 2, linear lanceolate, contains male floret or empty. 59. Themeda triandra Forssk., Gam. vol. 3: 1746.1934; Bor 254: FBI 7: 211.1896. Themeda imberbis (Retz.) Bomb., Fl. 2: 993; Bot. Bihar & Orissa 1049.1924. Perennial, erect, up to 170 cm high, culms & nodes glabrous, leaf-blades flat, up to 41 cm long, linearlanceolate, acute, ligule membranous, rounded, truncate ciliate; inflorescence up to 40 cm long, panicles leafy fascicled, erect or drooping, spathes long, leaf like, acuminate, margins with long hairs, involucral spikelets 2 pairs situated at the same level, shortly acuminate, each pair contains 5 spikelets, middle one sessile awned and contains bisexual floret, callus bearded, sessile spikelets, surrounded by 4 male spikelets, involucral spikelets; lower glume reddish brown, membranous, 2-toothed at apex, teeth unequal, 2-keeled with scarious margins on one side, 11-nerved, covered with stiff bristles, upper glume margins ciliate in the upper half, 3-nerved, 2keeled, lemma male, hyaline, obtuse, margins ciliate, 1-nerved, epaleate, stamens 3, sessile spikelets, lower glume coriaceous truncate at apex, 9-nerced, upper glume coriaceous, obtuse, 3-nerved, lower lemma hyaline, epaleate, upper lemma reduced to an awn, epaleate, pedicelled spikelets male, pedicels glabrous. 60. Urochloa panicoides P. Beauv., Gam vol. 3: 1775; Bor 372. FBI 7: 35 p.p; HS. 530; V. Fl. 214. (Fig. 2H) Annual, tufted, herbs sub erect, nodes swollen; leaves hairy, ligule fringe of hairs or glabrous, cordate at the base, leaf sheath clasping the stem; inflorescence panicle, racemes 5–6, spikelets 2-nate, spikelets ovate to lanceolate, glumes membranous, unequal, upper lemma cuspidate. 61. Urochloa setigera (Retz.) Stapf., Fl. Trop. Africa 9: 598.1920; Bot. Bihar & Orissa 1003.1924; Gam. vol. 3: 1775. (Fig. 2I) Panicum setigerum Retz., Obs. Bot. 4: 15.1786; FBI 7: 36. Brachiaria setigera (Retz.) Hubbard in Hooker’s Icon. Pl. 34. sub. t. 3363.1938; Suppl. Bot. Bihar & Orissa 177.1950; Bor 286. Annuals up to 75 cm high, culms decumbent, rooting at lower nodes, geniculately ascending, nodes pubescent, leaf sheaths hairy, leaf blade lanceolate, 12–14 cm long, wide 2.6 cm, leaf base cordate, acute tip, ligule a ring of white hairs; inflorescence panicle, 10–15 cm long, racemes distant 10–20, crowded 2–3, 2–6 cm long, rhachis triquentrous, hairy on the angles interposed with 3–5 long hairs, each spikelet bears 3–5 long hairs, www.tropicalplantresearch.com

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Prameela & Venkaiah (2016) 3(3): 522–535 . spikelets 2 nate, sessile and pedicelled, spikelets 0.3 cm pedicels pubescent, glumes dissimilar, lower glume one fourth of the spikelet, pink. CONCLUSION Uncultivated grasses are drought tolerant, fast growing, disease resistant, no need to sow, no need to watering, no need to use fertilizers because these soils enriched with humus (cattle dung), nutrients and moisture. They are helpful in controlling soil erosion. Over grazing leads the pastures to disappear, so retire the land from the use by livestock at least till the green cover is sufficiently restored. REFERENCES Achariyar KR & Mudaliyar CT (1921) A Handbook of South Indian Grasses. Madras. Blatter E & McCann C (1935) The Bombay Grasses. Sci. Monogr. No. 5. Imp. Counc. Agric. Rec. India. Bor NL (1960) The Grasses of Burma, Ceylon, India and Pakistan. Pergamon Press, London. Cooke T (1901–1908) The Flora of the Presidency of Bombay. London. Fischer CEC (1934) Gramineae in J.S. Gamble’s Flora of Madras. London. Gamble JS (1896) The Bambuseae of British India. Annals of Royal Botanical Garden Calcutta 7(1): 1–133. Hooker JD & Stapf O (1896) Flora of British India, Vol. 7. Gramineae International, U.K. Sriramulu HS (1986) Flora of Srikakulam district, Andhra Pradesh, India (Flora of India series). Indian Botanical Society, Meerut. Subba rao GV (1977) Flora of Visakhapatnam district, Andhra Pradesh, India (Flora of India series). Indian Botanical Society, Meerut. Venkaiah M (2004) Studies on the Vegetation and Flora of Vizianagaram District, Andhra Pradesh. Andhra University, Visakhapatnam.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 536–542, 2016 DOI: 10.22271/tpr.2016.v3.i3.070 Research article

Ecological studies of mangroves species in Gulf of Khambhat, Gujarat Vandna Devi and Bhawana Pathak* School of Environment and Sustainable Development, Central University of Gujarat, Gandhinagar, India *Corresponding Author: [email protected] [Accepted: 28 September 2016] Abstract: Mangrove forests are of utmost importance due to their role in preventing extreme weather events like tsunamis and cyclones etc. The present study aimed to observe the mangrove plant diversity and edaphic characteristics from Gulf of Khambhat, Gujarat. Ecological parameters and edaphic characteristics were studied for different sites i.e. Navsari, Surat and Bhavnagar. Avicennia marina was found as dominant species at all study sites. Plant species diversity shows increasing tendency with the decrease in plant density. Important Value Index, Shannon-Weaver diversity index and Simpson index of dominance of the mangrove species across the study area were also determined. The present study provides the baseline data of mangrove species and concludes the need of detail study for mangrove species in Gulf of Khambhat, Gujarat for conservation and management strategies. Keywords: Mangrove plants - Avicennia marina - IVI - Plant density - Conservation. [Cite as: Devi V & Pathak B (2016) Ecological studies of mangroves species in Gulf of Khambhat, Gujarat. Tropical Plant Research 3(3): 536–542] INTRODUCTION Mangrove forest ecosystems are significant for the biodiversity, protection of coastal area from erosion and provision of protected nursery breading areas for marine fauna. Ecological study of any area or habitat helps to understand the inter-relationship of all biotic (plants, microbes, other organisms) and abiotic (temperature, moisture and soil etc.) components of environment. Globally mangrove forest cover around 1,46,500.00 km2 of coastline (Alongi 2008) while total mangrove cover India is about 4,662.56 km2. This represents 0.14% of the total geographical area of country and 3% of the global mangrove area. Mangroves are world’s most productive ecosystems, found at the interface between land and sea in tropical and subtropical latitudes. Mangrove forests are only forest on earth where land, freshwater and sea mix together. These forests are specially adapted to high salinity, extreme tides, strong winds, high temperatures, low oxygen and muddy soil (Kathiresan 2010). Gujarat is situated in the west coast of India which is surrounded by Arabian Sea. In maritime states of India; Gujarat has largest coastal area around 28,000 km2 or longest coast line around 1650 km supports variety of marine flora and fauna. The area under mangrove cover (1058 km²) along the Gujarat coast is the second largest block of tidal forest in India, next only to the Sunderbans (2155 km²) (MoEF 2013–14). This state has two gulfs out of three gulfs in India and the coastal area is spread from south Gujarat (high rainfall area about 2500 mm) to north- west of Kachchh (low rain area about 250 mm only). Different range of tides, waves, cyclones and currents in the sea affect the physical as well as the biological conditions of the marine ecosystem whereas clear cutting, hydrological changes, oil spills and climate change are creating more pressure on mangrove forests sites (Blasco et al. 2001). In Gujarat 1103 km2 area is under mangrove which includes 175 km2 moderately dense mangroves (15.86% of mangrove area of state), 928 km2 open mangrove (84.13%). The present research study deals with the ecological status of mangrove species in Gulf of Khambhat, Gujarat. MATERIALS AND METHODS Study area Gujarat state is situated on the west coast of India between 20º06’ N to 24º42’ N latitude and 68º10’ E to 74º28’ E longitude. It is bounded by the Arabian Sea on the west. Ghogha from Bhavnagar (21˚40’ N, 72˚17’ E), Dumas from Surat (21˚4’N, 72˚42’ E), Dandi from Navsari (20˚55' N, 72˚47' E), Dahej from Bharuch were selected from Gulf of Khambat (Cambey) are selected for the present research work (Fig. 1 & 2). www.tropicalplantresearch.com 536 Received: 13 June 2016

Published online: 31 October 2016 https://doi.org/10.22271/tpr.2016.v3.i3.070

Devi & Pathak (2016) 3(3): 536–542 .

Figure 1. Aerial view of four selected sites (Source: Google Earth)

Figure 2. Habitat of mangrove vegetation: A & B, Bhavnagar; C, Navsari, D, Bharuch; E, Surat.

Field methods The mangrove vegetation study was carried out from selected sites during low tide. Quantification of mangrove vegetation at each site was done by quadrate method. 10 quadrates (3⨯3m) were laid randomly at www.tropicalplantresearch.com

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Devi & Pathak (2016) 3(3): 536–542 . each site and in each quadrate, total numbers of trees were counted; tree height and Girth at Breast Height (GBH) was measured for all trees. Quantity parameters like frequency, relative frequency, density, relative density, abundance, dominance, relative dominance, Important Value Index (IVI) was determined Curtis (1959). The distribution pattern of species was determined by ratio of abundance to frequency if the ratio is below 0.025 then it indicates regular distribution, between 0.025–0.050 indicates random distribution and when exceeds 0.050 indicates contagious distribution (Whitford 1949). Species diversity was determines by using Shannon index (H), Simpson’s index of diversity (1-D) were calculated using standard methods (Shannon & Weaver 1963, Simpson 1949, Kerkhoff 2010). Collection and authentication of plant samples Plant samples (leaves, flowers, stem, seeds and roots) were collected from selected sites for authentication and preparation of herbarium. Authentication and identification of collected plant samples was done with the help of Scientist, GEER foundation, Gujarat. Soil sampling and chemical analysis Soil samples were collected from 0–10 cm depths from each site during October and November of the year 2014. Five sets of samples were collected from each study site and mixed together to form a composite soil sample and from which three replicate samples were brought to the laboratory. Collected soil samples were air dried and sieved through a 2 mm mesh and was subjected to routine chemical analysis. Physicochemical characteristics of soil samples were determined using standard methods (APHA 1998). pH of soil was determined using method described by Black (1973). Total organic carbon was determined using method described by Walkey & Black (1934). RESULTS Mangrove plant status

Mangrove associates

True mangrove

Table 1. Mangrove diversity of selected site of Gujarat. Vernacular Species Name name Avicennia marina (Forsk.) Vierh Tivar, Tavarian var. acutissima Mold. Bruguiera gymnorhiza (L) Lam Tavar

Family Avicenniaceae

Status in various sites Bhavnagar Surat Navsari Bharuch + + + +

Rhizophoraceae

-

-

+

-

Sonneratia apetala Buch.-Ham

Motitavar

Lythraceae

-

+

+

-

Acanthus illicifolius Ipomoea pes-carpae (L.) Sw.

Kantaliyo Maryada-vel

Acanthaceae Convolvulaceae

-

-

+ +

-

Sesuvium portulacastrum (L.) L.

Shore purslane

Aizoaceae

+

+

+

-

Salvadora persica L.

Toothbrush tree

Salvadoraceae

-

+

-

-

Suaeda sp.

Seepweeds

Amaranthaceae

-

+

-

-

Note: +, indicates presence; - indicates absence. The patterns of mangroves and associated species distribution in selected study area depict little variation in the species composition (Table 1). At Bhavnagar a total of 886 plants representing two species were identified within 90 m2 area survey. Avicennia marina (Forsk.) Vierh (Avicenniaceae) showed the highest density (87.5 plants/90m2) with 83.33% relative frequency 99.99% relative dominance and 282.09 important value index. Other species recorded with their ecological parameters is presented in table 2. Only two species (Avicennia marina and Sonneratia apetala Buch.-Ham.) were found at Surat site. The maximum number of species was recorded at Navsari site such as Avicennia marina, Sonneratia apetala, Bruguiera gymnorhiza (L.) Lam., and Acanthus ilicifolius L. (Fig. 3). Only one species (Avicennia marina) was recorded at Bharuch site. Avicennia marina has been found common in all selected sites exhibiting maximum density at Bhavnagar (87.5 plants/90m2). Among all species the mean height was maximum of Sonneratia apetala (230.86 cm) followed by Avicennia marina (127.28 cm) and Bruguiera gymnorhiza (109.82 cm). Avicennia marina is dominating species at all selected sites with higher important value index at all sites. In general the distribution pattern of all species was contagiously distributed at all study sites except Sonneratia apetala, which showed random pattern of distribution at Surat and Navsari site. The Shannon-Weaver diversity index and Simpson www.tropicalplantresearch.com

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Devi & Pathak (2016) 3(3): 536–542 .

Figure 3. Some mangrove plants: A, Bruguiera gymnorhiza (L.) Lam.; B, Sonneratia apetala Buch.-Ham.; C, Avicennia marina (Forsk.) Vierh; D, Ipomoea pes-caprae (L.) R.Br.; E, Acanthus illicifolius L.

index of dominance was analyzed for dominance and species diversity (Shannon & Weaver 1963, Simpson 1949). The Shannon-Weaver index showed highest diversity at Navsari site (1.20) followed by Surat (0.20), Bhavnagar (0.07) whereas Simpson index of diversity (1-D) was higher in Navsari (0.66) followed by Surat (0.09), Bhavnagar (0.03) and the value of this index ranges between 0 and 1, the greater the value, the greater the plant diversity. Result shows that Navsari site have been found with more diversity than other sites (Table 3 & 4).

13.7 1.75

Avicennia marina Bruguiera gymnorhiza Sonneratia apetala Acanthus illicifolius

14.9 3.9 3.1 12.4

43.44 11.37 9.04 36.15

14.9 4.88 3.44 13.77

Avicennia marina

18.5

100

18.5

83.33 16.66

71.68 3.30

471.59 0.006

99.99 0.001

0.88 0.28

282.09 17.91

71.43 28.57

86.87 77.01

61.63 2.02

96.83 3.17

0.13 0.04

263.39 36.60

27.03 21.62 24.32 27.03

127.28 109.82 230.86 63.75

122.39 27.56 119.63 -

44.74 10.07 43.73 -

0.15 0.06 0.04 0.14

115.21 43.07 77.09 -

100

114.6

43.09

100

0.185

300

Important value index (IVI)

95.14 4.86

A/F ratio

13.7 0.7

Relative Dominance (%)

Avicennia marina Sonneratia apetala

94.09 100 5.914 20 Site 2 Surat 88.67 100 11.32 40 Site 3 Navsari 40.27 100 13.18 80 9.31 90 37.24 100 Site 4 Bharuch 100 100

Total Basal area (cm2/90m2)

87.5 5.5

Mean Height (cm)

98.76 1.24

Relative Frequency (%)

Abundance (plants/90m2

87.5 1.1

Frequency (%)

Relative density (%)

Avicennia marina Sesuvium portulacastrum

Relative Abundance (%)

Density (plants/90m2)

Table 2. Vegetation characteristics of different selected sites. Site 1 Bhavnagar

Table 3. Diversity indexes of selected mangrove sites.

Diversity indexes Bhavnagar Surat Navsari Shannon-Weaver Index (H) 0.07 0.20 1.20 Simpson index of Diversity (1-D) 0.03 0.09 0.66 Note: At Bharuch site only single species Avicennia marina was recorded in selected sampling area. www.tropicalplantresearch.com

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Devi & Pathak (2016) 3(3): 536–542 . Table 4. Species diversity of different mangrove sites.

Species Name

Sites

Total species

Bhavnagar Surat Avicennia marina Avicennia marina Sesuvium portulacastrum Sonneratia apetala Salvadora persica Suaeda sp. Sesuvium portulacastrum 2

Navsari Bharuch Avicennia marina Avicennia marina Sonneratia apetala Bruguiera gymnorhiza Acanthus illicifolius Ipomoea pes-carpae Sesuvium portulacastrum

5

6

1

Edaphic characteristics Soil characteristics of different selected sites have been presented in table 5. Soil mean temperature was recorded from 29.9–33.5 ºC. pH ranges from 8.37 to 8.68. Similarly, the values of electrical conductivity were from 4.25 mS.cm-1 to 12.28 mS.cm-1. In this study it was found that more water contents were present in soil at Navsari than Bhavnagar and Surat. Table 5. Physicochemical characteristics of different mangrove sites.

Navsari Surat Mean SD SE Mean SD SE 29.9 0.581 0.259 33.5 0.757 0.338 Temperature (˚C) 8.376 0.034 0.015 8.680 0.147 0.066 pH 4.248 0.190 0.085 6.626 1.755 0.785 EC (mS/cm) 60.1 3.021 1.351 23.7 6.485 2.90 M.C. (%) 0.17 0.02 0.01 1.09 0.07 0.04 O.Carbon (%) 0.29 0.04 0.02 1.88 0.12 0.07 O.M. (%) 2.438 --1.208 --Total Nitrogen (%) 4.73 2.73 0.011 0.58 0.33 Av. Phosphorous (%) 0.019 0.081 0.012 0.007 0.111 0.003 0.002 Sulphate (%) 12.23 0.02 0.013 18.24 0.44 0.253 Cl (ppm) 265.33 2.31 1.33 538.67 4.62 2.67 Total Hardness (mg CaCO3/kg) Note: SD, Standard deviation; SE, standard error; n, 3; Av., Average. Soil parameters

Bhavnagar Mean SD 32.5 1.155 8.396 0.096 12.282 4.228 44.2 0.932 1.52 0.04 2.62 0.07 4.734 -0.022 3.61 0.119 0.001 38.16 1.05 1588 41.57

SE 0.516 0.043 1.891 0.417 0.02 0.04 -2.08 0.001 0.604 24.00

DISCUSSION Gujarat has second largest area of mangroves in India (1058 km2). About 99.4% mangrove forest area is represented by three mangrove areas; Gulf of Kachchh (15.2%), Gulf of Khambhat (10.1%) and Kachchh district including Kori creek (74.1%) and remaining 0.6% in Valsad and Navsari district. Even with fewer mangroves area Gulf of Khambhat was reported to have rare mangrove species. The Gulf of Khambhat includes Bharuch, Surat, Navsari and Bhavnagar districts mainly. Therefore coastal area Ghogha from Bhavnagar, Dumas from Surat, Dandi from Navsari, and Dahej from Bharuch has been selected for ecological study. In present study total eight species (Avicennia marina (Forsk.) Vierh, Bruguiera gymnorhiza (L.) Lam., Sonneratia apetala Buch.-Ham., Acanthus ilicifolius, Ipomoea pes-caprae (L.) R.Br., Sesuvium portulacastrum, Salvadora persica and Suaeda sp.) were recorded from Gulf of Khambhat which includes both true mangrove and associate species. Fourteen species of mangrove have already been reported i.e. Avicennia marina (Forsk.) Vierh, Avicennia officinalis L., Avicennia alba Bl., Aegiceras corniculatum (L.) Blanco, Ceriops tagal (Perr.) Robinson, Ceriops decandra (Griff.) Ding Hou, Excoecaria agallocha L., Sonneratia apetala Buch.-Ham., Acanthus ilicifolius L., Bruguiera cylindrica (L.) Bl., Bruguiera gymnorhiza (L.) Savigny, Lumnitzera racemosa Wild, Rhizophora mucronata Lamk., Kandelia candel (L.) Druce (Bhatt et al. 2011). The abundance to frequency ratio indicated that most of the species were contagiously distributed except Sonneratia apetala at two sites (Surat and Navsari), which showed random distribution pattern. Smith (1957), Kershaw (1973), Kumar & Bhatt (2006) have also reported contagious distribution in natural vegetation. In most of places landward

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Devi & Pathak (2016) 3(3): 536–542 . mangrove are under pressure due to clearing of mangrove plants for fodder and conversion of mangrove area to other forms of land use (Farnsworth & Ellison 1997, Ashton & Macintosh 2002). Present study revealed that Avicenniaceae is most dominant mangrove family in all selected sites Avicennia marina was the most frequent, most abundant and most dominant species in all selected sites. Average GBH of selected mangrove species ranged from 0.28 cm to 22 cm. A similar kind of work has also been reported where Avicenniaceae was the dominant mangrove family with 100% frequency and 72.55% relative frequency. Average GBH of mangroves was 21.69 cm. Studies also showed that Avicennia marina can withstand more harsh environmental conditions such as high salinity, high temperature etc (Lunar & Laguardia 2013). It was found that mean height of Avicennia marina was maximum at Navsari (127.28 cm) followed by Bharuch (114.6 cm), Surat (86.87 cm) and Bhavnagar (71.68 cm). Among all species the mean height was maximum of Sonneratia apetala (230.86 cm) followed by Avicennia marina (127.28 cm) at Navsari site and then by Avicennia marina (114.6 cm) of Bharuch site and Bruguiera gymnorhiza (109.82 cm) of Navsari site. In this study it was observed that where plant density is more there plant mean height is less. This may be due to the reason that higher density of Avicennia marina at Bhavnagar may cause reduction in plant growth due to competition for limited resources (Volin et al. 2005, Li et al. 2014). Soil mean temperature was recorded in the range between 29.9ºC to 33.5ºC. A similar study has been done where mean temperature of soil was recorded 31.87ºC at degrading mangrove habitat and 28.26ºC at luxuriant mangrove habitat (Kathiresan 2002). The pH was recorded 8.37 (Navsari), 8.39 (Bhavnagar) and 8.68 (Surat) which is supported by study of Rao & Rao (2014). Study showed that alkaline pH (8.35–8.79) in similar type of habitat. pH have been reported from 7.11–8.52 in Pondicherry mangroves (Satheeshkumar & Khan 2009). In present study it was found that more water contents were present in Navsari mangrove soil than Bhavnagar and Surat mangrove soil. The moisture contents (mean values) were 23.7% (Surat), 44.4% (Bhavnagar) and 60.1% (Navsari). High moisture content at Navsari can either be due to freshwater input of Purna river or frequent tidal inundation (Ashton & Macintosh DJ 2002). It has been observed that 44.4% moisture content is more suitable for plant density at Bhavnagar site where as on other sites with less and more moisture content plant density was less. Kathiresan (2002) also found moisture content 31.49% at degrading sites and 42.2% at luxuriant mangrove habitat. Total nitrogen was recorded from 1.21% to 4.7%. Similar results were observed by Hossain et al. (2012), where total nitrogen has been reported from 0.057% to 0.158% in Sunderban mangrove soil whereas available nitrogen at same site was reported from 0.504 to 2.016 µg.g-1. Available nitrogen has been reported from 29.4 to 81.2 ppm (Rao & Rao 2014). Available phosphorous was found maximum at Bhavnagar (0.022%) followed by Navsari (0.019%) and Surat (0.011%). Available phosphorous of mangrove soil has been reported 3.32 ppm to 5.89 ppm (Rao & Rao (2014). Phosphates have been reported 0.06 mg L-1 by Dogiparti et al. (2014). The values of organic carbon were observed 0.17% at Navsari, 1.09% at Surat and 1.52% at Bhavnagar site and it has been found that with increase in organic carbon plant density is also increasing. Organic carbon has been reported 4.28% and 3.12% at two mangrove areas of Andhra Pradesh (Dogiparti et al. 2014). The organic matter was recorded from 0.29% to 2.62%, which shows similar results studied by Satheeshkumar & Khan (2009) where organic matter has been reported from 0.94% to 3.94% in mangrove soil. CONCLUSION Present study revealed that Avicenniaceae is only dominant family in the Gulf of Khambhat coastal area, Gujarat and due to anthropogenic disturbance i.e. clear cutting, hydrological changes, oil spills and climate change are creating more pressure on mangrove forests in these sites and the other mangrove species either present in very low number or disappeared from that site. The distribution pattern of mangrove species at Gulf of Khambhat is mostly contagious. Availability of eight species but in less number indicated that in near future the coastal region will be dominated by monotonous species (Avicennia marina). Other species regeneration should be focused while making conservation plans. This study will provide baseline for further study in this area and for development of conservation as well as management strategies for mangrove species. ACKNOWLEDGMENTS We are thankful to UGC for providing non-net fellowship. We extend our thanks to Dean, School of Environment and Sustainable Development, CUG, Gandhinagar, India, for providing all necessary facility. We also thank Dr. Harshad Salvi, Scientist, GEER foundation, Gujarat, India for extending help in identifying the plant species. We would like to thanks my colleagues of the development, Central University of Gujarat. www.tropicalplantresearch.com

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Devi & Pathak (2016) 3(3): 536–542 . REFERENCES Alongi DM (2008) Mangrove forests: Resilience, protection from tsunamis, and responses to global climate change, Estuarine. Coastal and Shelf Science 76: 1–13. APHA (1998) Standard method for examination of water and waste water, 19th edition. American Public Health Association, Washington (Method 2130B). Ashton EC & Macintosh DJ (2002) Preliminary assessment of the plant diversity and community ecology of the Sematan mangrove forest, Sarawak, Malaysia. Forest Ecology and Management 166: 111–129. Bhatt JR, Macintosh DJ, Nayar TS, Pandey CN & Nilaratna BP (2011) Towards Conservation and Management of Mangrove Ecosystems in India. International Union for Conservation of Nature and Natural Resources, pp. 65–72, 141–153. Black AL (1973) Soil property changes associated with crop residue management in a wheat- fallow rotation. Soil science Society of America Journal 37: 943–946. Blasco F, Aizpurum M & Gers C (2001) Depletion of the mangroves of the Continental Asia. Wetlands Ecology and Management 9: 245–256. Curtis JT (1959) The Vegetation of Wisconsin. An Ordination of plant communities. University Wisconsin press, Madison Wisconsin, 657 pp. Dogiparti A, Kurapati RK & Duddu SK (2014) Comparison of Selected Soil Chemical Properties of Two Mangrove Areas of East Coast of Andhra Pradesh South India. International Journal of Innovative Research in Science & Engineering 2(5): 319–328. Farnsworth EJ & Ellison AM (1997) The global conservation status of mangroves. Ambio 26, 328-334. Hossain MZ, Aziz CB & Saha ML (2012) Relationship between soil physico-chemical properties and total viable bacterial counts in Sunderban mangrove forests, Bangladesh. Dhaka University Journal of Biological Sciences 21 (2): 169–175. Kathiresan K (2002) Why are mangroves degrading? Current Science 83(10): 1246–1249. Kathiresan K (2010) Importance of Mangrove Forests of India. Journal of Coastal Environment 1(1): 11–25. Kerkhoff (2010) Measuring biodiversity of ecological communities. Ecology Lab 1–3. Kershaw KA (1973) Quantitative and Dynamic Plant Ecology, 2nd Edition. Elbsd & Edward Arnold, London, 308 p. Kumar M & Bhatt V (2006) Plant Biodiversity and Conservation of Forests in Foot Hills of Garhwal Himalaya. Lyonia 11(2):43–59. Li F, Xie Y, Liu Y, Tang Y, Chen X, Deng Z, Hu J & Liu N (2014) Negative influence of burial stress on plant growth was ameliorated by increased plant density in Polygonum hydropiper, Limnologica. Ecology and Management of Inland Waters 45: 33–37. Lunar BC & Laguardia MA (2013) Comparative Study of Diversity of Mangroves in Two Conservation Sites of Calatagan, Batangas, Philippines, IAMURE-International Journal of Marine Ecology 1: 1–8. Rao VVP & Rao BP (2014) Physico-Chemical Analysis of Mangrove Soil in the Machilipatnam Coastal Region, Krishna District, Andhra Pradesh. International Journal of Engineering Research & Technology 3(6): 10–12. Satheeshkumar P & Khan BA (2009) Seasonal Variations in Physico-Chemical Parameters of Water and Sediment Characteristics of Pondicherry Mangroves. African Journal of Basic & Applied Sciences 1: 36–43. Shannon CE & Wiener W (1963) The Mathematical Theory of Communication. University of Illinois press, Urbana. 117 pp. Simpson EH (1949) The Measurement of Diversity. Nature 163: 688. Smith GP (1957) Quantitative Plant Ecology Butterworth. Academic Press, London, 256 pp. Volin HS, Novoplansky A, Goldberg DE & Turkington R (2005) Density regulation in annual plant communities under variable resource levels. Oikos 108: 241–252. Walkey A & Black IA (1934) An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic soil titration method. Soil Science 37: 30–38. Whitford PB (1949) Distribution of woodland plants in relation to succession and clonal growth. Ecology 30: 199–208.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 543–550, 2016 DOI: 10.22271/tpr.2016.v3.i3.071 Research article

Floristic assessment of different habitats of Parvati Aranga wildlife sanctuary and adjacent Tikri forest area, Gonda, Uttar Pradesh, India Vineet Singh1*, S. K. Srivastava2 and L. M. Tewari3 1*

Botanical Survey of India, Central Regional Centre, Allahabad, Uttar Pradesh, India Botanical Survey of India, Northern Regional Centre, Dehradun, Uttarakhand, India 3 Department of Botany, DSB Campus, Kumaon University, Nainital, Uttarakhand, India *Corresponding Author: [email protected] [Accepted: 20 October 2016] 2

Abstract: The Parvati Aranga wildlife sanctuary and adjoining Tikri reserve forest in northeastern Terai region of Uttar Pradesh with its varied ecological habitats and occurrence of patchy wetlands in form of ‘River’ and ‘Tals’ sustains a variety of plant communities. The area also harbours a rich diversity of economical and medicinal plant species, mainly confined to the peripheral region of the forest. A large component of the forest is occupied by diverse forest stands and a number of special habitats portray remarkable vegetational diversity. The present communication reveals that the plant community with special habitat especially in protected and reserve forest area may plays a vital role in the future sustenance of the forest vegetation. Rarity and regeneration pattern of the flora is also discussed. Keywords: Plant community - Special habitats - Terai region. [Cite as: Singh V, Srivastava SK & Tewari LM (2016) Floristic assessment of different habitats of Parvati Aranga wildlife sanctuary and adjacent Tikri forest area, Gonda, Uttar Pradesh, India. Tropical Plant Research 3(3): 543–550] INTRODUCTION The comprehension of relationship between plants and environmental factors can be used as an indicator of environment, in this context a number of plants species used as ecological indicators. In a plant community some plants are dominant and found in abundance, these are important markers because they bear full impact of surroundings. In general, plant communities are better indicators than individual plants and are used to determine the types of soil and other conditions of the environment in a given area. Sometimes these also indicate past or future conditions of the environment. Community structure and composition with special habitats immensely affects the plant diversity pattern in any forest area in terms of the sustenance of a particular community. Forest composition, community structure and diversity pattern are important ecological attributes significantly correlated with prevailing environmental as well as anthropogenic variables (Gairola et al. 2008). The region free from anthropogenic disturbances continues to provide a platform for the microhabitats for an array of local floral elements Wildlife protected areas in India have had a relatively long history of forest management and exploitation as majority of these areas were originally reserved or other categories of government owned forests where focus on management was timber production, meeting the biomass demands of local communities or soil or water conservation (Rodgers & Sawarkar 1988). The special habitats of any forest plays a key role for the state of natural or reserve forest in the area and to suggest conservation measures for the concerned elements. The Terai expanse of eastern Uttar Pradesh is an assortment of human settlement, cultivation fields, natural and semi-natural vegetations comprising of grasslands and forests. In this area most of the primary forests have been substituted by economically and commercially important plants particularly tree species and agricultural fields (Bajpai et al. 2012a). This landscape is listed among the important ecoregions of the world, well known www.tropicalplantresearch.com

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Received: 29 June 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.071

Singh et al. (2016) 3(3): 543–550 . for its unique biodiversity and productivity (Tripathi & Singh 2009). The region is an ecotone between bhabar tract of foot hills of Himalayas and the Gangetic plains (Bajpai et al. 2015a). Several authors have dealt with the vegetation of Terai region of the state (Panigrahi et al. 1969, Maliya 2007, Bajpai et al. 2015a, Kumar et al. 2015) and recently some authors also explored the ecological parameters of some forests of the region (Pandey & Shukla 2003, Chauhan et al. 2008, Behera et al. 2012, Bajpai et al. 2012b, Bajpai et al. 2015b). However the baseline data related to typical vegetation community of the Parvati Aranga wildlife sanctuary (PAWS) has not been documented yet. Thus, this communication deals with the species composition and indicator taxa with special habitats of PAWS. MATERIALS AND METHODS The Study Area, biota and Climate The study was conducted in Parvati Aranga wildlife Sanctuary (PAWS) and adjacent Tikri forest area at a 26° 48'–27° N longitude and 81° 37'–82° 37' E latitude located in Gonda district of north-east Uttar Pradesh (Fig. 1). It is established in 1990, spread over an area of 10 km2 of total 80 km2 area and remaining 70 km2 is of the reserve forest area characterized by typical terai landscape. The sanctuary harbours a rich floral and faunal diversity and is the home for many rare and migratory avifaunas (Singh 2015). The reserve forest is dominated by Shorea robusta as climax species along with other tree species viz. Haldina cordifolia (Roxb.) Ridsdale, Syzygium cumini (L.) Skeels, S. salicifolium (Wight) J. Graham, Tectona grandis L.f., Acacia catechu (L.f.) Willd., Streblus asper Lour., Aegle marmelos (L.) Correa, Madhuca longifolia (J. Koenig ex L.) J. F. Macbr., Barringtonia acutangula (L.) Gaertn., Ficus racemosa L. etc. Understorey species were represented by Clerodendrum serratum (L.) Moon, C. infortunatum L., Mallotus philippensis (Lam.) Muell. Arg., Glycosmis pentaphylla (Retz.) DC. and Carrisa spinarum L. accompanied with climbers and lianas viz. Ichnocarpus frutescens (L.) W.T.Aiton, Tiliacora racemosa Colebr., Bauhinia vahlii Wight & Arn., Cissampelos pareira L. var. hirsuta (Buch. Ham. ex DC.) Forman, Cocculus hirsutus (L.) W. Theob., Abrus precatorius L., Tinospora cordifolia (Willd.) Hook. f. & Thompson.

Figure 1. Location of the study area i.e. Parvati Aranga wildlife sanctuary and Tikri forest area, Gonda.

Along with affluent flora the reserve forest is also endowed with many mammalian fauna viz. Wild boar (Sus scrofa), spotted deer (Axis axis), blue bull (Boselaphus tragocamelus), Indian porcupine (Hystrix indica), Rhesus macaque (Macaca mulata) and grey langur (Semnopithecus ajax) with many reptilian species viz. www.tropicalplantresearch.com

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Singh et al. (2016) 3(3): 543–550 . Bengal monitor (Varanus benghalensis), Indian Cobra (Naja naja), Krait (Bungarus caeruleus), Rat snake (Ptyas mucosa) and Indianpython (Python molurus). The wildlife sanctuary consists of a large wetland in form of lake, rich in avifaunal diversity and different species of fishes. The area also harbours manyrare, threatened and common native and migratory bird viz. Black drongo (Dicrurus macrocercus), Green bee eater (Merops orientalis), Red- wattled Lapwing (Vanellus indicus), Purple swamphen (Porphyrio porphyrio), Sarus crane (Grus antigone), Pied kingfisher (Ceryle rudis), Grey headed fish eagle (Ichthyophaga ichthyaetus) and Red vented Bulbul (Pycnonotus cafer) along with many other species. The people residing near by the sanctuary and the reserve forest are mainly depend upon the natural resources such as fuel wood, fodder, thatched grass and Non- timber forest products for their livelihood and for sacred rituals (Singh & Srivastava 2014). The area is also rich in many medicinal and economically valuable angiosperms and pteridophytes (Singh & Srivastava 2015). The climate is typical monsoon type with three different season’s viz. summer (March–June), Rainy (July– September) and winter (October–February). Mean annual rainfall is about 1240 mm. The driest month is November with 2 mm of rain. The greatest amount of precipitation occurs in July with an average of 356 mm. May is the warmest month of the year; the average temperature is about 34°C during this month. The lowest temperature in the year occurs during January it measures around 15.5°C. The forest of the area have been classified as Eastern Heavy Alluvium plains Sal forest with some part located along the river in swampy areas fall under 4D/SS2- Barringtonia swamp forests and 4D/SS2- Syzygium cumini swamp low forests (Champion & Seth 1968). During the course of exploration (2014–2016), the various ecological habitats were visited in different seasons of the year and the dominant species growing in different communities, which act as a keystone species with special importance as indicator, have been collected randomly along with their field data, dried, preserved and mounted by following the standard herbarium techniques (Jain & Rao 1977–78). These plant specimens were finally identified with the help of floras (Hooker 1872–1897, Duthie 1903–1929) assisted by matching with herbarium specimens for authentication and deposited in BSA and the correct nomenclature of the plants has been provided after consulting recent floras and website like IPNI and The PLANTLIST. The vegetation of the area was observed under different categories viz. top canopy tree species, under-storey, ground flora, lianas and climbers. The relevant information regarding habit, habitat, relative abundance, association, flowering, period, GPS data etc. were collected in the field. RESULTS AND DISCUSSION There are certain plant species which portrays the nature and disposition of habitats commonly referred to as plant indicator. It is found that certain species have one or more specific requirements which may limit their distribution and the occurrence, character and behavior of a plant are thus indicator of the combined effect of all factors prevailing in a habitat. These plant species establish themselves according to their environmental requirement where conditions are favourable. The knowledge of relationship between plants and ecological factors can be used as an indicator of environment. The characteristic species are collectively the best indicators of ecological conditions of the community (Braun-Blanquet 1932). The plants are admittedly a measure of the environment and although the community indicates the nature of the surroundings, only a few key species which are restricted to their habitats are of special importance (Santapau 1958a). The indicator implication of one group of plants must be inferred and applied to an entirely different group of plants. Generally, forest indicators are herbs or shrubs as compared to trees. In the broad sense, forest indicators are site indicators, but rarely do they suggest more than a portion of the several factors that contribute to site. Some plants indicate the characteristic types of forest and they grow in an area which is not disturbed. Narenga porphyrocoma is a grass which binds the soil in which sal (Shorea robusta) can be cultivated. Viola species in eastern Himalayas is a suitable indicator for plantation of Cedrus deodara and Pinus wallichiana. If we know that a particular forest grows better in certain area of specific soil the productivity can be increased. Physical or chemical characteristics of soil moisture relationships, aeration or erosion may be indicated by some species. The nature and composition of flora is manifestation of their cumulative effects of all aspects functioning in a particular habitat. It is usually accepted that a set of species or a whole community is steadier as an indicator than a solitary species and that dominants, particularly of the climax species are more useful indicators than lesser species. Species which are less tolerant to many varying conditions are usually indicators since their www.tropicalplantresearch.com 545

Singh et al. (2016) 3(3): 543–550 . growth requirements are exacting hence, the dominant species from different communities of selected habitats, are of special importance as indicators of the nature of habitat. The vegetation of the area can be broadly classified in to two grops consisting of various types of plant associations, A. Common vegetation and B. Vegetation of special habitats. A. Common Vegetation The common vegetation of the study area is of moist deciduous type with some evergeen and semi-evergreen tree species. The flora under this category dominates the physiognomy of the forest area by forming different phytoassociations which ultimately leads to a healthy forest in this terai region. Some of the important plant associations (Fig. 2) are discussed below.

Figure 2. Number of important plant associates in general vegetation community.

1. Shorea robusta - Mallotus philippensis community: Under this category the other associates are Bridelia retusa (L.) A.Juss., Carrisa spinarum L., Rotheca serrata (L.) Steane & Mabb., Clerodendrum infortunatum L., Curculigo orchioides Gaertn., Ceriscoides turgida (Roxb.) Tirveng., Glycosmis pentaphylla (Retz.) DC. and Haldina cordifolia (Roxb.) Ridsdale. 2. Shorea robusta - Terminalia chebula Community: The other important associates are Aegle marmelos (L.) Correa, Desmodium gangeticum (L.) DC., Phyllodium pulchellum (L.) Desv., Diospyros montana Roxb., Bauhinia vahlii Wight & Arn., Tiliacora racemosa Colebr. and Oplismenus compositus (L.) P. Beauv. 3. Tectona grandis - Steblus asper community: The other important co-existing species are Abrus precatorius L., Alangium salvifolium (L.f.) Wangerin, Cissampelos pareira L. var. hirsuta (Buch.-Ham. ex DC.) Forman, Dioscorea bulbifera L., Cocculus hirsutus (L.) W. Theob., Clerodendrum infortunatum L. and Elephantopus scaber L. 4. Shorea robusta - Terminalia alata community: The other phytoassociates are Abutilon indicum (L.) Sweet, Oroxylum indicum (L.) Kurz., Ailanthus excelsa Roxb., Sida cordata (Burm.f.) Borss. Waalk., Madhuca longifolia (J. Koenig. ex. L.) J.F.Macbr. and Chrysopogon zizanoides (L.) Roberty. 5. Dalbergia sissoo - Acacia catechu community: The other phytoassociates are Ailanthus excelsa Roxb., Albizia procera (Roxb.) Benth., Ampelocissus latifolia (Roxb.) Planch., Kydia calycina Roxb., Abrus precatorius L. and Cardiospermum halicacabum L. 6. Barringtonia acutangula - Syzygium spp. association: In this type of association there may be individual stands of these species or mixed stands at some places. Syzygium cumini (L.) Skeels and S. salicifolium (Wight) J. Graham. are two important species of syzygium in the study area. The other associates are Calamus tenuis Roxb., Saccharum spontaneum L., Oxystelma secamone K. Schum., Tiliacora racemosa Colebr., Smilax zeylanica L., Helminthostachys zeylanica (L.) Hook., and Lygodium flexuosum (L.) Sw. 7. Syzygium spp. - Ficus spp. association: Mainly consists of Syzygium cumini (L.) Skeels and S. salicifolium (Wight) J. Graham. of Syzygium and Ficus racemosa L., F. heterophylla L.f. and F. virens Aiton. Other phytoassociates are Pongamia pinnata (L.) Pierre, Terminalia arjuna (Roxb. ex DC.) Wight & Arn. and Vitex negundo L. www.tropicalplantresearch.com 546

Singh et al. (2016) 3(3): 543–550 . Aquatic flora The common habitations of the hydrophyte are ‘tals’, ‘nalas’ and other water reservoir with low-lying areas. Most of the ‘tals’ and the Parvatiaranga lakes in the area hold water throughout the year, only a few smaller and less deeper ones may dry up during summer season. During rainy season these get filled with water as a part of Saryu river flood area. As the flood water recedes, these water bodies get roofed with a number of hydrophytes viz. Eichhornia crassipes (Mart.) Solms, Hygroryza aristata (Retz.) Nees ex Wight & Arn., Pistia stratiotes L. & Spirodela polyrrhiza (L.) Schleid., Ceratophyllum demersum L., Hydrilla verticillata (L.f.) Royle, Najas graminea Delile, Nechamandra alternifolia (Roxb. ex Wight) Thwaites, Ottelia alismoides (L.) Pers., Potamogeton crispus L., Nelumbo mucifera Gaertn., Nymphaea nouchali Burm.f., Nymphoides indica (L.) Kuntze., Ipomoea aquatica Forssk., Ludwigia adscendens (L.) Hara, Bacopa monnieri (L.) Wettst., Hygrophila auriculata (Schumach.) Heine, Phragmitis karka (Retz.) Trin. ex Steud., Ranunculus sceleratus L., Rumex dentatus L., Typha angustifolia L. and Veronica anagallis-aquatica L. B. Vegetation of Special Habitats The study area is also harbours a rich population of flora with special habitats. There are almost 5 categories (Fig. 3) of special habitats have been observed along with aquatic flora under which the species from the unique and characteristic phytoassociation forms the habitat conditions.

Figure 3. Frequency and percentage of vegetation of special habitats.

1. On marshy shady conditions: Under this situation the following phytoassociates are growing together viz. Bacopa monnieri (L.) Wettst., Centella asiatica (L.) Urb., Oldenlandia corymbosa L., Laphangium luteoalbum (L.) Tzvelev,. Ceratopteris thalictroides (L.) Brongn.,Pycreus pumilus (L.) Nees, Peperomia pellucida (L.) Kunth, Ranunculus muricatus L. and R. scleratoides Perfil. ex Ovczinn. 2. On dry shady situations: In these conditions scattered poulation of Abutilon indicum (L.) Sweet, Desmodium gangeticum (L.) DC., Phyllodium pulchellum (L.) Desv., Leea indica (Burm.f.) Merr., Scoparia dulcis L., Aerva sanguinolenta (L.) Blume and Ageratum conyzoides (L.) L. associations. 3. On drying up beds: In such areas plant species forming large clumps and patches under these associates viz. Coldenia procumbens L., Glinus lotoides L., Grangea maderaspatana (L.) Poir., Heliotropium supinum L., Polycarpon prostratum (Forssk.) Aschers & Schweinf., Polygonum plebeium R. Br., Rumex dentatus L. and Sphaeranthus indicus L. 4. On the bank of water bodies: The following important plants and their associates are observed under this situation viz. Ammannia baccifera L., Lippia javanica (Burm.f.) Spreng., Chrysopogon zizaniodes (L.) Roberty, Arundo donax L., Typha domingensis Pers., Persicaria lapathifolia (L.) Delarbre species. There are also some woody species found along the water bodies in the forest area viz. Barringtonia acutangula (L.) Gaertn., Syzygium cumini (L.) Skeels, S. salicifolium (Wight) J. Graham and Pongamia pinnata(L.) Pierre along with other associates like Oxystelma secamone K. Schum., Tiliacora racemosa Colebr. and Vitex negundo L. 5. On open situations: These conditions support a rich wealth of grasslend flora with some woody species viz. Alangium salvifolium (L.f.) Wang subsp. decapetalum (Lam.) Wang, Alysicarpus monilifer (L.)DC., Apluda mutica L., Biophytum sensitivum (L.) DC., Boerhavia diffusa L., Bothriochloa pertusa (L.) A. Camus, Carissa spinarum L., Chrozophora rotleri A. Juss.Dalz., Clerodendrum infortunatum L., Cynodon dactylon (L.) Pers., www.tropicalplantresearch.com

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Singh et al. (2016) 3(3): 543–550 . Imperata cylindrica (L.) Raeusch., Indigofera linifolia (L.f.) Retz., Solanum virginianum L., Urena lobata L., Chrysopogon zizanioides (L.) Nash, Woodfordia fruticosa (L.) Kurz and Ziziphus nummularia (Burm.f.) Wight & Arn. Parasitic and Epiphytic associations Along with general floral association some unique association in form of parasitic and epiphytic are also found in the area: 1. Cuscuta reflexa Roxb. found on a variety of host plants viz. Streblus asper Lour., Glycosmis pentaphylla Retz. DC., Ipomoea fistulosa Mart. ex Choisy, Vitex negundo L. and on Ziziphus spp. 2. Dendrophthoe falcata (L.f.) Ettingsh. on Bombax ceiba L., Butea monosperma (Lam.) Taub., Mangifera indica L., Dalbergia sissoo DC., Syzygium cumini (L.) Skeels at some places on Tectona grandis L.f. . 3. Vanda tessellata (Roxb.) Hook. ex G. Don mainly growing as an epiphytes on Madhuca longifolia (J. Koenig ex L.) J.F. Macbr., Tectona grandis L.f. and on Shorea robusta Gaertn. (Fig. 4).

Figure 4. Epiphytic association of Vanda tessellata (Roxb.) Hook. ex G. Don on Madhuca longifolia (J. Koenig ex L.) J.F. Macbr.

Pattern of rareness and regeneration potential in the study area Rarity and regeneration of plant species in any forest area plays a significant role in maintenance of a healthy forest. Plumbago zeylanica L., Oroxylum indicum (L.) Kurz., Hymenodictyon orixense (Roxb.) Mabb., Clerodendrum indicum (L.) Kuntze, Gloriosa superba L., Terminalia chebula Retz., Helicteres isoraL., Bauhinia vahlii Wight & Arn., Bacopa monnieri (L.) Wettst., Leea alata Edgew., Habenaria plantaginea Lindl., Tylophora indica (Burm.f.) Merr., Cheilocostus speciosus (J. Koenig) C. D. Specht, Holarrhena pubescens Wall. ex G.Don, Heminthostachys zeylanica were considered most rare plant species found during collection and survey of the area (Table. 1). There are also some common specious occurs frequently in the entire area due to their capacity to produce seedling rapidly viz. Bridelia retusa (L.) A.Juss., Mallotus philippensis (Lam.) Mull. Arg., Mitragyna parvifolia (Roxb.) Korth., Terminalia alata Wall., Rotheca serrata (L.) Steane & Mabb., Clerodendrum infortunatum L., Cayratia trifolia (L.) Domin, Cissampelos pareira L., Ichnocarpus frutescens (L.) W. T. Aiton, Elephantopus scaber L.and Lygodium flexuosum (L.) Sw (Table. 2). Certain rhizomatous species like Curculigo orchioides Gaertn., Typhonium trilobatum (L.) Schott, Helminthostachys zeylanica (L.) Hook. and Gloriosa superba L. growing even in highly stochastic environment. This type of rarity and regeneration among various species are indicative of their ability to reproduce and establish effiently in frequently distributed environment (Shukla 2009). www.tropicalplantresearch.com 548

Singh et al. (2016) 3(3): 543–550 . Table 1. Rare species occurring in the study area.

Name of the species Bacopa monnieri (L.) Wettst Bauhinia vahlii Wight & Arn. Clerodendrum indicum (L.) Kuntze Cheilocostus speciosus (J. Koenig) C.D. Specht Gloriosa superba L. Habenaria plantaginea Lindl. Helminthostachys zeylanica (L.) Hook. Holarrhena pubescens Wall. ex G.Don Hymenodictyon orixense (Roxb.) Mabb. Leea alata Edgew. Oroxylum indicum (L.) Kurz. Passiflora foetida L. Plumbago zeylanica L. Schleichera oleosa (Lour.) Merr. Strychnos nux-vomica L. Terminalia chebula Retz. Tylophora indica (Burm.f.) Merr.

Family Plantaginaceae Leguminosae Verbenaceae Costaceae Liliaceae Orchidaceae Ophioglossaceae Apocynaceae Rubiaceae Vitaceae Bignoniaceae Passifloraceae Plumbaginaceae Sapindaceae Loganiaceae Combretaceae Apocynaceae

Habit Herbs Lianas Shrubs Shrubs Climbers Herbs Herbs Large Shrubs Trees Shrubs Trees Climbers Shrubs Trees Trees Trees Climbers

Phenology July–March Sept.–Jan. April–Dec. Aug.–Jan. July–Nov. Aug.–Nov. Oct.–Jan. May–Feb. July–Feb. June–Sept. June–March Nov.–Jan. Aug.–Oct. April–Aug. March–Feb. March–Oct. May–Sept.

Table 2. Most common species occurring in the study area.

Name of the species Aerva sanguinolenta (L.) Blume Bridelia retusa (L.) A.Juss. Cayratia trifolia (L.) Domin Cissampelos pareira L. Rotheca serrata (L.) Steane Clerodendrum infortunatum L. Phyllodium pulchellum (L.) Desv. Dioscorea bulbifera L. Elephantopus scaber L. Glycosmis pentaphylla (Retz.) DC. Hemidesmus indicus (L.) R.Br. ex Schult. Holoptelea integrifolia Planch. Ichnocarpus frutescens (L.) W.T. Aiton Mallotus philippensis (Lam.) Mull. Arg. Mitragyna parviflora (Roxb.) Korth. Streblus asper Lour. Terminalia alata Wall.

Family Amaranthaceae Phyllanthaceae Vitaceae Menispermaceae Lamiaceae Lamiaceae Leguminosae Dioscoreaceae Asteraceae Rutaceae Apocynaceae Ulmaceae Apocynaceae Euphorbiaceae Rubiaceae Moraceae Combretaceae

Habit Herbs Trees Climbers Climbers Shrubs Shrubs Shrubs Climbers Herbs Shrubs Climbers Trees Climbers Small trees Trees Trees Trees

Phenology July–April July–March Aug.–Nov. June–Dec. Aug.–Oct. March–June Aug. –April June–Nov. Jan.–March Dec.–March Aug.–Jan. Dec.–April July–Feb. Oct.–May Sept.–Jan. May–Sept. April–Nov.

An extensive ecological and floristic study has been conducted in the north-eastern terai region of the Uttar Pradesh with respect to the floral diversity and documentation of vegetational phytosociology. The present communication reveals that the plant community with special habitat specially in protected and reserve forest area may plays a vital role in the future sustenance of the forest vegetation. The area also harbours a rich diversity of economical and medicinal plant species, mainly confined to the peripheral region of the forests. There is need of continued monitoring of various ecological parameters with the help of more accurate and sophasticated ecological tools for the betterment of the plant community of the study area. ACKNOWLEDGEMENTS The authors are thankful to Director, Botanical Survey of India, Kolkata and Scientist- ‘E’ and Head of Office, BSI, CRC, Allahabad for facilities and encouragement. The authors are also grateful to Range Officers and field staffs of Parvati Aranga Wildlife Sanctuary and Tikri reserve forest, Gonda for providing necessary help during field exploration. The authors are thankful to the Head, Department of Botany, D.S.B. campus, www.tropicalplantresearch.com

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Singh et al. (2016) 3(3): 543–550 . Kumaon University, Nainital for valuable suggestions. REFERENCES Bajpai O, Kumar A, Srivastava AK, Kushwaha AK, Pandey J & Chaudhary LB (2015a) Trees species of the Himalayan Terai region of Uttar Pradesh, India: a Checklist. Checklist 11(4): 1–15. Bajpai O, Kushwaha AK, Srivastava AK, Pandey J & Chaudhary LB (2015b) Phytosociological status of a monotypic genus Indopiptadenia: A Near Threatened Tree from the Terai-Bhabar Region of Central Himalaya. Research Journal of Forestry 9(2): 35–47. Bajpai O, Kumar A, Mishra AK, Sahu N, Pandey J, Behera SK & Chaudhary LB (2012b) Recongregation of tree species of Katerniaghat Wildlife Sanctuary, Uttar Pradesh, India. Journal of Biodiversity and Environmental Sciences 2: 24–40. Bajpai O, Kumar A, Mishra AK, Sahu N, Behera SK & Chaudhary LB (2012a) Phenological study of two dominant tree species in tropical moist deciduous forest from the Northern India. International Journal of Botany 8: 66–72. Behera SK, Mishra AK, Sahu N, Kumar A, Singh N, Kumar A, Bajpai O, Chaudhary LB, Khare PB & Tuli R (2012) The study of microclimate in response to different plant community association in tropical moist deciduous forest from northern India. Biodiversity and Conservation 21(5): 1159–1176. Braun-Blanquet J (1932) Plant Sociology- The Study of plant communities, London. Champion HG & Seth SK (1968) A revised survey of the forest Types of India. Manager of Publication, Govt. of India, New Delhi. Chauhan DS, Dhanai CS, Singh B, Chauhan S, Todaria NP & Khalid MA (2008) Regeneration and tree diversity in natural and planted forests in a Terai- Bhabhar forest in Katarniaghat wildlife sanctuary, India. Tropical Ecology 49: 53–67. Duthie JF (1903–1929) Flora of Upper Gangetic Plain and Adjacent Siwalik and Sub-Himalayan Tracts. Vols 1-3. Rep. 1994.Bishen Singh Mahendra Pal Singh, Dehradun. Gairola S, Rawal RS & Todaria NP (2008) Forest vegetation pattern along an altitudinal gradient in sub-alpine zone of West Himalaya, India. African of Plant Science 2(6): 42–48. Hooker JD (1872–1897) The Flora of British India. Bishensingh Mahendra Pal Singh, Dehradun India. Jain SK & Rao RR (1978) A Handbook of Field and Herbarium Methods. Today and Tomorrow ´s Pub. New Delhi. Kumar A, Bajpai O, Mishra AK, Sahu N, Behera SK, Bargali SS & Chaudhary LB (2015) A checklist of the flowering plants of Katerniaghat Wildlife Sanctuary, Uttar Pradesh, India. Journal of Threatened Taxa 7(7): 7309–3408. Maliya SD (2007) Rare species of Katarniyaghat Wildlife sanctuary District Bahraich, Uttar Pradesh, India. Indian Forester 133(8): 1052–1056. Pandey SK & Shukla RP (2003) Plant diversity in managed sal (Shorea robusta Gaertn.) forests of Gorakhpur, India: species composition, regeneration and conservation. Biodiversity and Conservation 12: 2295–2319. Panigrahi G Singh AN Mishra OP (1969) Contribution to the botany of the terai forest of the Bahraich District of Uttar Pradesh.Bulletin of the Botanical Survey of India 11(1&2): 89–114 Rodgers WA & Sawarkar VB (1988) Vegetation management in Wildlife Protected Areas in India. Aspects of Applied Biology 16: 407–422. Santapau H (1958a) Floriistic study in India.Mem. Indian Botanical Society 1: 117–121 Shukla RP (2009) Patterns of plant species diversity across Terai landscape in north-eastern Uttar Pradesh, India. Tropical Ecology 50(1): 111–123. Singh V & Srivastava SK (2014) Utilization of Wild Plants during Tinchhath festival in eastern Uttar Pradesh. Ethnobotany 26(1&2): 101–103. Singh V & Srivastava SK (2015) A note on occurrence and copious growth of Helminthostachys zeylanica (L.) Hook. (Ophioglossaceae) in Tikri forest in Terai region, Uttar Pradesh. Phytotaxonomy 15: 15–16. Singh V (2015) Rare Sighting of Nearly Threatened Grey-Headed Fish-Eagle Icthyophaga Ichthyaetus (Horsfield, 1821) From Tikri Reserve Forest of Eastern Uttar Pradesh, India. Indian Forester 141(10): 1104– 1105.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 551–557, 2016 DOI: 10.22271/tpr.2016.v3.i3.072 Research article

Intra-specific variation in response of Neem (Azadirachta indica A. Juss) to elevated CO2 levels and biochemical characterization of differently responding plants C. Buvaneswaran*, K. Arivoli, T. Sivaranjani, E. Menason, K. Vinothkumar, S. Padmini and S. Senthilkumar Institute of Forest Genetics and Tree Breeding, (ICFRE), R.S.Puram, Coimbatore, Tamil Nadu, India *Corresponding Author: [email protected] [Accepted: 23 October 2016] Abstract: Global climate change the looming environmental threat is mainly due to the increase in atmospheric CO2 levels, which was increasing earlier by about 1.55 ppm per year and currently by 2.76 ppm per year. Thus, CO2 concentration has reached 400.16 ppm in 2015. To understand the response of various tropical tree species to such an elevated CO2, experiments were conducted in Automated Open Top Chambers (AOTC) facility at Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore (India). The results of initial studies indicated the scope for exploring intraspecific variation in response of tropical trees to elevated CO2. Subsequently, experiments were carried out to assess intra-specific variation in Neem (Azadirachta indica). The selected phenotypes of Neem (varieties or clones) were exposed to four treatments viz., i) CO2 of 600 ppm, ii) CO2 of 900 ppm, iii) chamber control- without any CO2 enrichment and iv) ambient conditions. The parameters studied were shoot length, root length, dry matter accumulation in shoot and root. The results of the study showed that there existed significant variation among different treatments of CO2 as well as among various phenotypes or clones in terms of growth characteristics. This intra-specific variation in biomass accumulation under elevated CO2 levels could be exploited for future breeding programmes in developing climate ready genotypes having greater potential to sequester more carbon and produce greater biomass under forecasted elevated levels of atmospheric CO2. Another objective in this study was to analyze intra-specific variation in selected biometrical and biochemical characteristics of leaf samples of neem trees in relation to their differential response to elevated CO2. Among parameters of leaf, Fumaric acid, Malic acid and Oxalic acid, leaf Nitrogen and Specific Leaf Weight may be considered as a biochemical and biometrical marker to categorize the plants adapted to elevated CO2 environments. Keywords: Response to elevated CO2 - Neem (Azadirachta indica) - Open Top Chambers Within species variation in growth. [Cite as: Buvaneswaran C, Arivoli K, Sivaranjani T, Menason E, Vinothkumar K, Padmini S & Senthilkumar S (2016) Intra-specific variation in response of Neem (Azadirachta indica A. Juss) to elevated CO2 levels and biochemical characterization of differently responding plants. Tropical Plant Research 3(3): 551–557] INTRODUCTION Even the most environment friendly emission scenarios lead to an increase in atmospheric CO2 concentration over the next 100 years, to about double the pre-industrial levels up to 550 ppm (IPCC 2007). Currently, CO2 concentration has reached 400.16 ppm in 2015 Mike McGee (2015). This increasing concentration of carbon dioxide (CO2) in the atmosphere may have a direct effect on the physiology of plants: higher CO2 tends to suppress plant transpiration through reduced stomatal conductance (Field et al. 1995). In tree species, short term experiments with Pinus ponderosa, Quercus coccinea, Pinus radiata and Populus deltoides have shown a definite increase in photosynthesis rate up to 40–80% under 600 ppm levels of CO2 (Couteaux et al. 1992). Studies conducted at Institute of Forest Genetics and Tree Breeding (IFGTB) showed the existence of greater inter- and intra-specific variation in tropical tree species in response to elevated CO2 and temperature (Buvaneswaran et al. 2010). With reference to intra-specific clonal variation in tree species, Johanna et al. (2003) reported that two clones of silver birch (Betula pendula) responded differently to elevated CO2 levels. In one clone, total biomass increased by 40% under elevated CO2, but in another clone no response was found. www.tropicalplantresearch.com

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Received: 15 June 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.072

Buvaneswaran et al. (2016) 3(3): 551–557 Similar such intra-specific clonal variations have been reported in Sitka spruce (Murray et al. 1994), Poplar (Ceulemans et al. 1995), Hevea brasiliensis (Devakumar et al. 1998), Populus tremuloides (Isebrands et al. 2003). However, not much study has been conducted on intra-specific variation in response of tree species to elevated CO2 levels, particularly the plantation forestry species of India. Hence, the present study was conducted to generate knowledge on intra-specific variation in important tropical plantation species besides helping to screen assembled productive clones of Neem (Azadirachta indica A. Juss) for higher carbon sequestration potential under elevated CO2. This selection of clones adapted to elevated CO2 levels may also be used for developing ‘climate ready genotypes’ in future through biotechnological interventions. The information generated could also help in identification of biometrical and biochemical markers, if any, for higher carbon sequestration potential of the selected species under elevated CO2. MATERIALS AND METHODS The present research experiment was conducted using Automated Open Top Chambers (AOTC) facility at Institute of Forest Genetics and Tree Breeding (IFGTB), Coimbatore, India. This study site is located on 11°59'0.69" N, 76°57'2.32" E and 437 m MSL. The facility of AOTCs has chambers which are cubical type structures of 3 × 3 × 3 m dimension, fabricated with galvanized iron (GI) pipe frames and covered with polyvinyl chloride (PVC) sheet of 120 micron gauge to have more than 90% transmittance of light. The upper portion of the chamber is kept open to maintain near-natural conditions. A software facility called Supervisory Control and Data Acquisition (SCADA) was used to continuously control record and display the actual and desired CO2 level, relative humidity and temperature in each OTC by feedback control loop passing through Programmable Logical Controllers (PLC). The species selected for the present study was Neem (Azadirachta indica A. Juss) in which 12 clones (varieties) were selected and subjected to various treatments. The initial shoot length was recorded for all the plants. Then, these plants were exposed to four treatments with the different levels of CO2 viz., i) chamber with 600 ppm of CO2, ii) chamber with 900 ppm concentration of CO2, iii) chamber without any elevation of CO2 and iv) ambient control- in open area. CO2 enrichment was done by using CO2 cylinders. The design of the experiment adopted is Completely Randomized Block Design (CRBD). At end of exposure period under various treatments, plants were sampled and growth parameters were measured like shoot length, root length, and fresh weight of leaf, shoot and root. Then, the plant samples were kept in hot air oven at 60ºC till the constant dry weight is obtained. Then dry weight was recorded for in root, shoot and leaves. The data on growth and dry matter production were subjected to analysis of variance for completely randomized design with replications. To analyze intra-specific variation in few biometrical and biochemical characteristics of Neem trees in relation to their differential response to elevated CO2, Four different clones (varieties) of Neem viz. IFGTB-AI9, IFGTB-AI-11, IFGTB-AI-3 and IFGTB-AI-5 were used to assess intra-specific variation in biochemical characteristics in neem trees in relation to their differential response to elevated CO2. Among these four clones selected for the present study, two clones IFGTB-AI-9 and IFGTB-AI-11 were responding positively under elevated CO2 environments in the earlier experiments conducted under Open Top Chambers at Institute of Forest Genetics and Tree Breeding, Coimbatore. On the other hand, clones IFGTB-AI-3 and IFGTB-AI-5 were responding poorly to the elevated CO2 conditions by recording lesser dry matter accumulation in biomass of various plant parts. The mother plants of these four selected clones of neem, belonging to two categories with respect to their response to elevated CO2 environments - were studied for variation in chlorophyll content (chlorophyll - a, chlorophyll - b and total chlorophyll), Organic acids (Fumaric acid, Malic acid, Citric acid and Oxalic acid), Nutrient elements (Nitrogen, Phosphorus, Potassium, Calcium and Magnesium) and also for leaf weight, leaf area and Specific Leaf Weight. All these parameters were assessed using leaf samples collected from the respective clones available in the germplasm assemblage of neem in IFGTB campus, Coimbatore. RESULTS AND DISCUSSIONS A) Response of Azadirachta indica to elevated CO2 in Open Top Chambers A set of 12 phenotypes (clones) of Neem (Azadirachta indica A. Juss) were used in the experiments conducted in Automated Open Top Chambers (AOTC) facility at IFGTB, Coimbatore. The results of the study showed that there existed significant variation among different treatments of CO2 as well as among various www.tropicalplantresearch.com

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Buvaneswaran et al. (2016) 3(3): 551–557 clones in terms of growth characteristics. Overall mean shoot length was 59.73 cm for plants exposed to 600 ppm of CO2 and it was only 28.25 cm under ambient conditions, irrespective of clones (Table 1). Root length was greater (39.33 cm) under 900 ppm CO2 level than that recorded in ambient conditions (30.07 cm). Dry matter accumulation in shoots was 78% more under 600 ppm CO2 level than that of in ambient conditions. Similarly, 58% more dry matter accumulation was recorded in roots of plants grown under 600 ppm of CO 2 levels when compared to the plants grown in ambient conditions (Table 1). Among 12 clones studied, clone IFGTB-AI-12 recorded 86% more total dry matter accumulation in biomass of plants grown under 600 ppm of CO2 as compared to that observed for the same clone under ambient conditions. Clone IFGTB-AI-9 accumulated greater amount of total dry matter in biomass under 900 ppm CO 2 levels that is 89% over and above the total dry matter accumulated in the same clone under ambient environments (Fig 1). In Neem, only one clone that is IFGTB-AI-5 showed negative response in term of total dry matter accumulation under 900 ppm CO2 level by registering 3% less dry matter accumulation in biomass when compared to that of ambient grown plants of the same clone. Followed by clone IFGTB-AI-3 which recorded lesser accumulation of total dry weight when compared to all other positively responding clones. Table 1. Shoot and root characteristics of Azadirachta indica A. Juss plants exposed to various levels of CO2 in open top chamber experiments conducted in IFGTB, Coimbatore, India .

600 ppm Mean shoot length (cm) 59.73 ± 3.48 Mean root length (cm) 36.10 ± 3.11 Leaf dry weight (g per plant) 6.22 ± 0.26 Shoot dry weight (g per plant) 12.24 ± 0.80 Root dry weight (g per plant) 9.12 ± 0.90 Note: Values shown are Mean ± Standard Error

900 ppm Chamber control 54.69 ± 2.12 62.05 ± 2.56 39.33 ± 2.15 33.20 ± 2.79 5.52 ± 0.48 6.85 ± 0.40 10.72 ± 0.61 11.50 ± 0.55 8.29 ± 0.61 7.54 ± 0.57

Ambient 28.25 ± 1.59 30.07 ± 1.01 4.53 ± 0.29 6.86 ± 0.54 5.78 ± 0.49

Figure 1. Increase or decrease in total dry weight (%) under different levels of CO2 as percentage over that of ambient grown respective clones of Azadirachta indica A. Juss.

In the present study, shoot length was increased under elevated CO2 conditions when compared to ambient conditions. As observed in the present study, Pokorny et al. (2012) observed that the shoot length was increased frequently under elevated CO2 conditions. With regard to increased root length under elevated CO2 environment, it is reported that the responses of roots to CO2 are dependent on experimental conditions (Ceulemans & Mousseau 1994). When the plants exposed to increased CO2, root have observed to become more numerous, longer, thicker and faster growing in crops (Chandhuri et al. 1990) with increased root length in many plant species (Norby 1994, Pritchard & Rogers 2000, Bernecchi et al. 2000). Lengths and volumes of tap root and fine roots were higher for CO2 enhanced cotton plants (Rogers et al. 1993). 110% increase in root length of soybean was observed as CO2 concentration increased (Rogers et al. 1992). Increased root length and number under elevated CO2 environments has also been reported in sweet potato (Bhattacharya et al. 1990) and Phaseolus acutifolius and P. vulgaris (Salsman et al. 1999). In the present study, dry matter accumulation both in shoot and root biomass was greater in plants grown under elevated CO2 environments as compared to that in ambient grown plants. Similar findings were reported www.tropicalplantresearch.com

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Buvaneswaran et al. (2016) 3(3): 551–557 by Cao et al. (2008) in Gossypium hirsutum and the above ground biomass was increased under 600 ppm of elevated CO2 level. Lukac et al. (2003) revealed that the CO2 enrichment increases the belowground biomass allocation in three populus species were investigated and the standing root biomass enhanced by 47–76%. Moreover the growth of Minjiang fir showed significantly positive responses of elevated CO2 with greater increases of total biomass than the control conditions (Hou et al. 2011). Ghasemzadeh & Jaafar (2011) reported in two varieties of Zingiher officinale were exposed to different CO2 concentration (400 and 800 ppm) resulted in increasing total plant biomass over the ambient conditions. Mohamed (2013) reported that there is no significant intra specific difference in dry matter production in the tropical dry land forest species of Neem. But when the plants were exposed on increased atmospheric CO2 the final plant biomass, above ground biomass and below ground biomass was significantly increased in tree species (Madhu & Hatfield 2013). Similarly, the shoot biomass was approximately 35% greater for creeping bentgrass plants grown under elevated CO2 compared to plants maintained under ambient CO2, while the root biomass increased by 37% due to elevated CO2 (Burgess & Huang 2014). Wang et al. (2015) studied the responses of rice production under elevated CO2, there was significant stimulation in above ground biomass 28 per cent and below ground biomass of rice was 42 per cent increases. Similar findings of root biomass was higher under elevated CO2 levels has been reported by several authors and it was increased of 55 per cent in Pinus sylvestris (Jach et al., 2000), 32 per cent in Pinus taeda by Jackson et al. (2009). With reference to intra-specific clonal variation in tree species, it is observed in the present study that huge variation existed among the clones in terms of total dry matter accumulation in Neem. Similarly, Johanna et al. (2003) also reported that two clones of silver birch (Betula pendula) responded differently to elevated CO2 levels. In one clone, total biomass increased by 40% under elevated CO2, but in another clone no response was found. Similar such intra-specific clonal variations have been reported in Sitka spruce (Murray et al.1994), Poplar (Ceulemans et al. 1995), Hevea brasiliensis (Devakumar et al. 1998), Populus tremuloides (Isebrands et al. 2003). B) Foliar analysis of differently responding clones for biometrical and biochemical traits In the present study, the existing variation in chlorophyll contents among the clones studied were not corresponding to the growth response observed for the respective clones under the elevated CO2 environments (Table 2). Similarly, Nicolas et al. (2007) also reported that there is no link of chlorophyll with tree growth irrespectively of site and family in hybrids of poplar. However, Kumar & Paramathama (2005) reported that all the traits studied, via., plant height, collar diameter. Number of branches, survival percent, chlorophyll content and suitability index were strongly associated with volume index in 44 clones of Casuarina equisetifolia assembled from Tamilnadu, Andhra Pradesh, Orissa and Pondicherry. Reddy et al. (2003) also reported that chlorophyll a, b and total chlorophyll showed significant positive correlation with leaf area and yield in different genotype of Mulberry. Table 2. Intra-specific Variation in Chlorophyll contents (mg chlorophyll per g leaf tissue) in selected Neem clones.

Clones Chlorophyll-a IFGTB-AI-9 0.0135 ± 0.0016 IFGTB-AI-11 0.0074 ± 0.0025 IFGTB-AI-3 0.0049 ± 0.0023 IFGTB-AI-5 0.0140 ± 0.0014 Note: Values shown are Mean ± Standard Error.

Chlorophyll-b 0.0115 ± 0.0037 0.0155 ± 0.0046 0.0048 ± 0.0025 0.0196 ± 0.0028

Total Chlorophyll 0.0359 ± 0.0072 0.0298 ± 0.0049 0.0245 ± 0.0079 0.0490 ± 0.002

Among various organic acids, Fumaric and Malic acid was in greater quantity in clones positive to elevated CO2 when compared to other two clones which are responding poorly to elevated CO2. On the contrary, Oxalic acid content was observed to be lesser in quantity in clones adaptive to elevated CO2 than in clones less adaptive to elevated CO2. However, there is not much difference among clones in respect of Citric acid content (Table 3). Similar variation in organic acid contents under exposure to stress environments has been reported by Silva et al. (2004) who reported that Gas chromatography/mass spectrometry and ion chromatography analyses indicated that root exposure to AI led to a greater than 200% increase in Malic acid concentration in the root tips of all eucalypt species. The increase in Malate concentration in response to AI treatment is correlated with the tree species. A small increase in citric acid `concentration was also observed in all species, but there were no consistent changes in the concentration of other organic acids in response to AI treatment. In all eucalypt www.tropicalplantresearch.com

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Buvaneswaran et al. (2016) 3(3): 551–557 species, AI treatment induced the secretion of citric acid and Malic acid in root exudates. But no trend with respect to AI tolerance was observed. Thus, although malate and citrate exudation by roots may partially account for the overall high AI tolerance of these eucalypt species, it appears that tolerance is mainly derived from the internal detoxtification of AI by complexation with malic acid. Table 3. Intra-specific Variation in organic acids (mg per g leaf tissue) in selected Neem clones.

Clones Fumaric Acid Malic acid IFGTB-AI-9 11.56 ± 0.022 11.70 ± 0.322 IFGTB-AI-11 10.36 ± 0.093 11.34 ± 0.305 IFGTB-AI-3 9.40 ± 0.066 10.40 ± 0.907 IFGTB-AI-5 8.32 ± 0.057 10.54 ± 0.636 Note: Values shown are Mean ± Standard Error

Citric acid 1.80 ± 0.027 2.04 ± 0.002 1.79 ± 0.032 2.42 ± 0.099

Oxalic acid 0.049 ± 0.001 0.2697 ± 0.012 0.4767 ± 0.065 0.3967 ± 0.085

Significant observation in the present study was that among various nutrient elements studied, Nitrogen content was greater in amount in clones which are categorized as adaptive to elevated CO2 (0.112 to 0.14%) when compared to that in clones which are not adaptive to elevated CO2 environments (0.028 to 0.056%) (Table 4). It is reported that the largest single pool of nitrogen in leaves is RuBisCO and hence it can be inferred that the greater amount of Nitrogen in the leaves of adaptive clones may indicate greater amount of RuBisCO availability. This higher RuBisCO availability is related to higher biomass production, as photosynthesis is catalysed by this RuBisCO, which fixes carbon to form carbohydrates. Moore et al. (1998) also reported that short term exposure of elevated CO2 for plants generally leads to increased rates of leaf-level photosynthesis due to enhanced activity of RuBisCO. On the other hand, photosynthesis down regulation is characterized at the biochemical and leaf levels by reduced chlorophyll content, reduced RuBisCO content and activity and decreased leaf nitrogen concentration on a leaf mass basis (Sage 1994, Tissue et al. 1995). It warrants further research to understand the reason for higher leaf N in some species and lower N levels in leaf in other species when plants are exposed to elevated CO2. Table 4. Intra-specific Variation in nutrient elements (% in leaf tissue) in selected Neem clones.

Clones IFGTB-AI-9 N% P% IFGTB-AI-11 K% Ca IFGTB-AI-3 % Mg % IFGTB-AI-5

N% 0.140 0.112 0.056 0.028

P% 0.196 0.136 0.143 0.135

K% 2.00 2.87 1.97 2.22

Ca 0.56 % 0.28 1.24 1.16

Mg % 0.08 0.21 1.87 1.74

Another important observation made in the present study was that the actual dry weight and leaf area of clones were not differentiated in terms of clonal variation in respect of their response to elevated CO2. However, when Specific Leaf Weight (SLW) was worked out, this SLW parameter clearly got differentiated in clones. Greater SLW was recorded in clones which were responding positively to the elevated CO2 enforcing. Hence, clones IFGTB-AI-9 and IFGTB-AI-11 recorded greater SLW values of 13.06 and 10.75 mg per cm2 respectively (Table 5). On the other hand, clones IFGTB-AI-3 and IFGTB-AI-5 recorded lesser SLW, which were the clones responded poorly to the elevated CO2 environments in the earlier experiments. Sicher et al. (1994) who studied the photosynthetic acclimation to elevated CO2 occurs in transformed Tobacco and reported that the dry weight gain was due to increased specific leaf weight. Specific leaf weight was shown to be a valuable index for comparing photosynthesis various parts of a tree canopy over a season or throughout an entire year. Mean annual photosynthetic rate in five separate portions of a spruce canopy was directly proportional to observed differences in specific leaf weight (Ram 1984). In brief, among various parameters of leaf, organic acids particularly Fumaric acid, Malic acid and Oxalic acid, leaf Nitrogen content and Specific Leaf Weight may be considered to act as a biochemical and biometrical marker to categorize the plants adapted to elevated CO2 environments, specifically to neem trees. Table 5. Intra-specific variation in leaf dry weight, leaf area and Specific Leaf Weight in clones of Neem.

Dry weight Clones (mg leaf) IFGTB-AI-9 389.6per ± 0.129 IFGTB-AI-11 451.7 ± 0.075 IFGTB-AI-3 519.7 ± 0.087 IFGTB-AI-5 221.2 ± 0.037 Note: Values shown are Mean ± Standard Error www.tropicalplantresearch.com

Leaf area (cm2) Specific Leaf Weight (mg per cm2) 13.06 ± 2.16 31.67 ± 5.89 43.00 ± 7.00 10.75 ± 2.31 56.67 ± 6.64 9.36 ± 0.84 28.00 ± 3.05 7.99 ± 0.68

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Buvaneswaran et al. (2016) 3(3): 551–557 CONCLUSION The present study evidently proves that there exists significant intra-specific variation in Azadirachta indica in response to elevated CO2. Greater variation existed in dry matter accumulation in biomass among different clones under elevated CO2 conditions. This variation could be explored in all other commercially important tropical tree species and superior tree varities can be identified for higher productivity and carbon sequestration potential under forecasted elvated CO2 levels of the future envirnment. When four different clones (varieties) of Neem (Azadirachta indica) viz. IFGTB-AI-9, IFGTB-AI-11, IFGTB-AI-3 and IFGTB-AI-5 were used to assess intra-specific variation in biochemical characteristics in neem trees in relation to their differential response to elevated CO2, it is observed that among various parameters of leaf, organic acids particularly Fumaric acid, Malic acid and Oxalic acid, leaf Nitrogen content and Specific Leaf Weight may be considered to act as a biochemical and biometrical marker to categorize the plants adapted to elevated CO2 environments, specifically to neem trees. Further research is suggested to confirm these observations in other tropical tree species which will aid in large scale screening of various tree species for their adaptation potential to ever increasing atmospheric CO2. ACKNOWLEDGMENTS The authors very much thankful to the Director, Institute of Forest Genetics and Tree Breeding, Coimbatore and Director General, Indian Council of Forestry Research and Education, Dehra Dun for providing the opportunity to conduct the experiments in Automated Open Top Chambers. REFERENCES Bernacchi CJ, Coleman JS, Bazzaz FA & McConnaughay KDM (2000) Biomass allocation in old-field annual species grown in elevated CO2 environments: No evidence for optimal partitioning. Global Change Biology 6: 855–863. Bhattacharya NC, Hileman DR, Ghosh PP, Musser RL, Bhattacharya S & Biswas PK (1990) Interaction of enriched CO2 and water stress on the physiology and production in sweet potato grown in open-top chambers. Plant, Cell & Environment 13: 933–940. Burgess P & Huang B (2014) Growth and physiological responses of creeping bentgrass (Agrostis stolonifera) to elevated carbon dioxide concentrations. Horticulture Research 1: 1–8. Buvaneswaran C, Edwin Raj E, Warrier RR & Jayaraj RSC (2010) Scope and opportunities of research on elevated CO2 and plant response in tropical tree species. ENVIS Forestry Bulletin 10(2): 10–16. Cao L, Eby M, Ridgwell A, Caldeira K, Archer D, Ishida A, Joos F, Matsumoto K, Mikolajewicz U, Mouchet A, Orr JC, Plattner GK, Schlitzer R, Tokos K, Totterdell I, Tschumi T, Yamanaka Y & Yoo A (2008) The importance of ocean transport in the fate of anthropogenic CO2. Biogeosciences Discussions 5: 4521–4557. Ceulemans R & Mousseau M (1994) Effects of elevated atmospheric CO2 on woody plants. New Phytologist 127: 425–446. Ceulemans R, Jiang XN & Shao BY (1995) Growth and physiology of one-year old poplar (Populus) under elevated atmospheric CO2 levels. Annals of Botany 75(6): 609–617. Chaudhuri UN, Kirkham MB & Kanemasu ET (1990) Root growth of winter wheat under elevated carbon dioxide and drought. Crop Science 30: 853–857. Couteaux MM, Bottner P, Rouhier H & Billes G (1992) Atmospheric CO2 Increase and Plant Material Quality: Production, Nitrogen Allocation and Litter Decomposition of Sweet Chestnut. In: Teller A, Mathy P & Jeffers JNR (eds) Responses of Forest Ecosystems to Environmental Changes. Springer, pp. 429–436. Devakumar AS, Shesha Shayee MS, Udayakumar M & Prasad TG (1998) Effect of elevated CO2 concentration on seedling growth rate and photosynthesis in Hevea brasillensis. Journal of Bioscience 23(1): 33–36. Field C, Jackson R & Mooney H (1995) Stomatal responses to increased CO2: implications from the plant to the global scale. Plant, Cell & Environment 18: 1214–1255. Ghasemzadeh A & Jaafar HZE (2011) Effect of CO2 Enrichment on Synthesis of some primary and Secondary Metabolites in Ginger (Zingiber officinale Roscoe). International Journal of Molecular Science 12: 1101–1114.

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Buvaneswaran et al. (2016) 3(3): 551–557 Isebrands JG, McDonald EP, Kruger E, Hendrey GR, Percy KE, Pregitzer KS, Sober J & Karnosky DF (2003) Growth responses of aspen clones to elevated carbon dioxide and ozone. Developments in Environmental sciences 3: 411–435. Jach ME, Laureysens I & Ceulemans R (2000) Above and below ground production of young Scots pine (Pinus sylvestris L.) trees after three years of growth in the field under elevated CO2. Annals of Botany 85: 789–798.

Jackson RB, Cook CW, Pippen JS & Palmer SM (2009) Increased belowground biomass and soil CO2 fluxes after a decade of carbon dioxide enrichment in a warm-temperate forest. Ecology 90: 3352–3366. Johanna R, Lindsberg MM, Holopainen T, Oksanen E, Lappi J, Peltonen P & Vapaavuori E (2003) Silver birch and climate change: variable growth and carbon allocation responses to elevated concentrations of carbon dioxide and ozone. Tree Physiology 24(11): 1227–1237. Kumar A & Paramathama M (2005) Correlation and path coefficient studies in Casuarina equisetifolia L. Johnson. Indian Forester 131(1): 47–55. Lukac M, Calfapietra C & Godbold D (2003) Production, turnover and mycorrhizal colonisation of root systems of three Populus species grown under elevated [CO2] (POPFACE). Global Change Biology 9: 838–848. Madhu M & Hatfield JL (2013) Dynamics of Plant Root Growth under Increased Atmospheric Carbon Dioxide. Agronomy Journal 105(3): 657–669. Mike McGee (2015) Available from: https://www.co2.earth (accessed: 10 Mar. 2016) Mohamed EA (2013) Growth Performance and Physiological Characteristics of Seedlings of Six Tropical Dry land Forest Tree Species in the Sudan. Journal of natural resources and environmental studies 1(2): 25–33. Moore BD, Cheng SH & Seeman JR (1998) Effects of short and long term ellevated CO2 on the expression of Ribulose 1-5-bisphosphate carboxylase/oxygenase genes and carbohydrate accumulation in leaves of Arabidopsis thaliana L. Heynh. Plant Physiology 116: 715–723. Murray MB, Smith RI, Leith ID, Fowler D, Lee HSJ, Friend AD & Jarvis PG (1994) Effects of elevated CO2, nutrition and climatic warming on bud phenology in Sitka spruce (Picea sitchensis) and their impact on the risk of frost damage. Tree Physiology 14: 69l–706. Nicolas M, Sophie YD & Reinhart C (2007) Evaluation of leaf traits for indirect selection of high yielding poplar hybrids. Environmental and Experimental Botany 61: 103–116. Norby RJ (1994) Issues and perspectives for investigating responses to elevated atmospheric carbon dioxide. Plant Soil 165: 9–20. Pokorný R, Tomášková I, Drápelová I, Kulhavý J & Marek MV (2012) Long-term effects of CO2 enrichment on bud phenology and shoot growth patterns of Norway spruce juvenile trees. Journal of Forestry Science 56: 251–257.

Pritchard SG & Rogers HH (2000) Spatial and temporal deployment of crop roots in CO2-enriched environments. New Phytology 147: 55–71. Ram O (1984) Leaf area index and specific leaf weight: keys to interpreting canopy photosynthesis and stand growth. Thesis of College of Forestry, Oregon State University, USA. Reddy BK, Rao DMR, Reddy MP, Jayaram RH, Suryanarayana N (2003) Variation of cholorophyll content and its relationship with leaf area and leaf yield in Mulberry genotypes (Morus spp.). Advances in plant sciences 16(1): 277–280. Rogers HH, Peterson CM, McCrimmon JM & Cure JD (1992) Response of soybean roots to elevated atmospheric carbon dioxide. Plant, Cell & Environment 15: 749–752. Rogers HH, Prior SA & O’Neill EG (1993) Cotton root and rhizosphere responses to free-air CO2 enrichment. In: Free-air CO2 enrichment for plant research in the field. CRC Press, Boca Raton, FL. Sage RF (1994) Acclimation of photosyntheiss to increasing atmospheric CO2: the gas exchange perspective. Photosynthesis Research 39: 590–596. Salsman KJ, Jordan DN, Smith SD & Neuman DS (1999) Effect of atmospheric enrichment on root growth and carbohydrate allocation of Phaseolus spp. International Journal of Plant Sciences 160: 1075–1081. Silva IR, Novais RF, Jham GN, Barros NF, Gebrim FO, Nunes FN, Neves JC & Leite FP (2004) Responses of eucalypt species to aluminum: the possible involvement of low molecular weight organic acids in the Al tolerance mechanism. Tree Physiology 24(11): 1267–1277. Tissue DT, Griffin KL, Thomas RB & Strain BR (1995) Effects of low and elevated CO2 on C3 and C4 annuals. II. Photosynthesis and leaf biochemistry. Oecologia 101: 21–28. Wang J, Wnag C, Chen N, Xiong Z, Wolfe D & Zou J (2015) Response of rice production to elevated [CO2] and its interaction with rising temperature or nitrogen supply: a meta-analysis. Climatic Change 130: 529–543. www.tropicalplantresearch.com 557

ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 558–563, 2016 DOI: 10.22271/tpr.2016.v3.i3.073 Research article

Morphological characters of Chaetoceros lorenzianus (Bacillariophyceae) isolated from North Arabian Sea after Tasman Spirit oil spill Asma Tabassum1*, Hina Baig2 and Aliya Rehman1 1

Department of Botany, University of Karachi, Karachi, Pakistan 2 National Institute of Oceanography, Karachi, Pakistan

*Corresponding Author: [email protected]

[Accepted: 26 October 2016]

Abstract: The present study investigated the morphology and taxonomy of marine centric diatom Chaetoceros lorenzianus from Karachi Harbor, Pakistan for the first time for the first time during the incident of Tasman Spirit Oil Spill (2003) in the area. Phytoplankton samples were collected from 5 different locations from the study area. Chaetoceros lorenzianus was found only at one station collected after Tasman Spirit Oil Spill. Moreover morphometric measurements of present record showed narrow range as compared to the records investigated by other workers. Keywords: Chaetoceros lorenzianus - Diatom - Phytoplankton - Tasman Spirit oil spill. [Cite as: Tabassum A, Baig H & Rehman A (2016) Morphological characters of Chaetoceros lorenzianus (Bacillariophyceae) isolated from North Arabian Sea after Tasman Spirit oil spill. Tropical Plant Research 3(3): 558–563] INTRODUCTION Phytoplankton are the major primary producers of marine and fresh water environment (Baliarsingh et al. 2012). Among phytoplankton, the Bacillariophyta (diatoms) contributes at least 40% of the global annual primary productivity (Field et al. 1998). These diatoms are ubiquitous occurrence in marine environment (Sunesen et al. 2008). Genus Chaetoceros Ehrenberg is considered as most diverse and wide spread centric diatom (Cupp 1943, Rines 1999, Hasle & Syvertsen 1997). It comprises of about 400 marine species with few fresh water records (Round et al. 1990). Morphological and taxonomical studies of this genus contributed new records time to time from various parts of the world oceans (Hernandez-Becerril 1993, Hernandez-Becerril 1999, Rines 1999, Trigueros et al. 2002, Murthy et al. 2012, Ozgur et al. 2013). A number of studies have been conducted on distribution and composition of Chaetoceros (Hargraves 1972, Fanuko & Valic, 2009, Tabassum & Saifullah 2010). It was observed to be one of the most frequently occurring genus among centric diatoms (Nwankwo & Onyema 2003, Tabassum & Saifullah 2010). Records of Chaetoceros have also been well observed in sediments with special reference to their resting spores (Stockwell 1991, Witak et al. 2011, Ferrario et al. 1998, Moazzam & Baig 1994). Variation in physiological behavior and their responses to hydrological parameters have also been studied (Johansen et al. 1990). Chaetoceros lorenzianus, is considered to be a harmful bloom forming species (Sunesen et al. 2008). This species was studied by a number of scientists (Cupp 1943, Subrahmanyan 1946, Hendey 1964, Moazzam 1973, Hasle & Syvertsen 1997, Shevchenko et al. 2006, Sunesen et al. 2008, Tabassum & Saifullah 2010). Moreover the lysis of this species by a single stranded DNA virus has also been investigated (Tomaru et al. 2011). The occurrence of Chaetoceros lorenzianus has been discussed from various parts of the world ocean (Cupp 1943, Sunesen et al. 2008, Shevchenko et al. 2006, Wood 1963, Subrahmanyan 1946, Rajasekar et al. 2010, Hendey 1964) It is known from North Arabian Sea bordering Pakistan (Moazzam 1973, Saifullah & Chaghtai 2005, Tabassum & Saifullah 2010). This is the first attempt to study the impact of oil spill in North Arabian Sea on morphological characters of this species.

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Received: 01 July 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.073

Tabassum et al. (2016) 3(3): 558–563 . MATERIALS AND METHODS Phytoplankton samples were collected by net (50 µm) hauls of 5 minute duration at speed of 2 km at 5 sampling stations which were selected in the area affected by oil spill (Fig. 1 and Table 1). Samples were fixed with 10 % buffered formalin immediately after collection. Observations on oceanographic parameters like temperature, salinity and pH were also measured at each station.

Figure 1. Map showing sampling stations of Tasman Spirit Oil Spill effected area. Table 1. Sampling stations in off Karachi Harbour.

Station No. 1 2 3 4 5

Sampling date 19-11-03 19-11-03 19-11-03 19-11-03 20-11-03

Latitude N 24°80‟248N 24°80‟816N 24°80‟771N 24°79‟753N 24°77‟204N

Longitude E 66°89‟938E 66°99‟215E 67°01‟087E 67°02‟718E 67°05‟435E

Samples were observed in light microscope LABX N-400M. Prior to scanning electron microscopy (SEM) the samples were cleaned by cold H2O2 method (Karthick et al. 2010). Cleaned material were coated up to 300 ° A with auto coater using JEOL # JFC 1500 having gold targets. The coated samples were then scanned with JEOL # JSM 6380A microscope. Present paper manifests the light and electron microscopic structures probably for the very first time in this study area. RESULTS Enumeration of species Chaetoceros lorenzianus Grunow, 1863, 157, pl. 5: fig. 13; Cupp 1943, p. 118, Fig. 71; Subrahmanyan 1946, p. 131, Figs. 198–199, 202–204, 206–209 (p. 132); Hendey 1964, p. 124, Plate 16, Fig. 1; Hasle & Syvertsen 1997, p. 204, Plate 42; Shevchenko et al. 2006, p. 249, Figs. 84–89; Sunesen et al. 2008, p. 317 & 318, Fig. 11A–F; Tabassum & Saifullah 2010, p. 1144 & 1146, Fig. 13 (1145). (Fig. 2) Chains straight, cells rectangular, apical axis 11–15 µm. Apertures wide, elliptical to lanceolate, foramina hexagonal, ranges from 10–12 µm. Setae thick, long, spiny, polygonal in cross section, fuse just near the margin, divergent with slight curve forming an angle of 35°–45° to the chain axis. Distribution: During the present study this species was collected only from Station 2. This species is reported by various parts of the world ocean. West Coast of North America (Cupp 1943); Madras, India (Subrahmanyan 1946); Chaleurs Bay Canada (Brunel 1962); Indian Ocean (Wood 1963); British Coastal Waters (Hendey 1964); Manora Channel Karachi (Moazzam 1973); Indian Ocean (Simonsen 1974); Peter the Great Bay, Sea of Japan (Shevchenko et al. 2006); Buenos Aires Argentina (Sunesen et al. 2008). www.tropicalplantresearch.com 559

Tabassum et al. (2016) 3(3): 558–563 . Observation from the study area: The valve profile of Chaetoceros lorenzianus is nearly close to the findings of numerous workers (Cupp 1943, Subrahmanyan 1946, Hendey 1964, Moazzam 1973, Hasle & Syvertsen 1997, Shevchenko et al. 2006, Sunesen et al. 2008, Tabassum & Saifullah, 2010) except the size of apical axis which is within a narrow range (Table 2). This may be attributed to their presence in the environment which was polluted with the crude oil because of Tasman Spirit oil spill which might have affected the cell metabolism. Parab et al. (2008) observed morphological changes in another centric diatom Thalassiosira because of oil exposure.

Figure 2. Chaetoceros lorenzianus (SEM, pair of sibling cells in girdle view): A, Scale bar: 10 µm; B, Sibling cells with long setae. Scale bar: 20 µm; C, Sibling valves with wide aperture. Scale bar: 5 µm. Table 2. Comparison of morphometric data among Chaetoceros lorenzianus of present study with the previous records.

Apical axis Pervalvar axis

Cupp, 1943 (Pacific Ocean)

Subrahman yan, 1946 (Indian Ocean)

Hendey, 1964 (Atlantic Ocean)

7 µm – 48 µm -

16 µm – 58 µm -

26 µm – 60 µm -

Moazzam , 1973 (North Arabian Sea) 5 µm – 40 µm -

Hasle and Syvertsen , 1997 7 µm – 80 µm -

Sunesen et al., 2008 (Atlantic Ocean) 16 µm – 36 µm -

Tabassum and Saifullah, 2010 (North Arabian Sea) 15 µm – 35 µm 10 µm – 20 µm

Present study (North Arabian Sea) 10 µm – 15 µm 7 µm – 11 µm

DISCUSSION Any change in the ecosystem of an aquatic environment can be determined by analysis of phytoplankton composition of that area (Guilloux et al. 2013). An extensive amount of studies have been conducted on effects of oil pollution on ecosystem of water bodies which showed deleterious effects on growth of phytoplankton community structure (Parab et al. 2008, Jiang et al. 2010). www.tropicalplantresearch.com 560

Tabassum et al. (2016) 3(3): 558–563 . Genus Chaetoceros is termed as fast growing diatom and its domination among other members of phytoplankton was also observed during the studies conducted in the other parts of the world during stress condition of oil spill (Hallare et al. 2011). Chaetoceros lorenzianus collected from North Arabian Sea bordering Pakistan belongs to sub-genus Hyalochaete (Hasle & Syvertsen 1997). In the present study morphometric data including apical axis and pervalver axis of Ch. lorenzianus was found in narrow range but at the same time within the range of the results recorded earlier by other workers (Table 2). It is recorded that the traces of oil (raw or refine) are lethal to autotrophic life forms as they can cease metabolic activities by limiting their enzymatic activities (Lewis & Pryor 2013) and decrease chlorophyll „a‟ concentration (Lee et al. 2009). As it is evident that the toxicity of crude oil is concentration dependent (Sheekh et al. 2000) and the species were recorded during the initial days of spill so recorded narrow range of morphometric measurements of the cells may accounted due to the effect of crude oil. Moreover previous findings showed that Kuzmenko (1975), Tabassum & Saifullah (2010) observed 16 species of Chaetoceros in the month of February from Arabian Sea whereas seven species of this genus including C. lorenzianus were reported in the month of October from Kuwait Bay, Arabian Sea (Heil et al. 2001). Present study manifests sporadic occurrence of the species in the month of November immediate after Tasman Spirit Oil Spill which may have attributed to the effect of oil spill in this area. ACKNOWLEDGEMENTS Authors are deeply indebted to National Institute of Oceanography, Pakistan for providing the samples of project entitled “Tasman Spirit Oil Spill” for this study. And we are also thankful to Dr. Ines Sunesen from Departamento Cientifico Ficologia, Universidad Nacional de La Plata, Argentina for constant support in this study. REFERENCES Baliarsingh, SK, Biraja KU, Srichanda S & Sahu KC (2012) Seasonal variation of phytoplankton community in Navigable Channel of Gopalpur Port, East coast of India: A Taxonomic study. International Journal of Modern Botany 2(3): 40–46. Cupp EE (1943) Marine plankton diatoms of the West Coast of North America, Bulletins of the Scripps Institution of Oceanography 5: 1–238. Fanuko N & Valic M, 2009 Phytoplankton composition and biomass of the northern Adriatic lagoonof Stella Maris, Croatia Acta Botanica Croatica 68(1): 29–44. Ferrario ME, Sar EA, & Vernet M (1998) Chaetoceros resting spores in the Gerlache Strait, Antarctic Peninsula. Polar Biology 19: 286–288. Field CB, Behrenfeld MJ, Randerson JT & Falkowski P (1998) Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281(5374): 237–240. Guilloux L, Riguat-Jalbert F, Jouenne F, Ristori S, Viprey M, Not F, Vaulot S & Simon N (2013) An annotated checklist of Marine Phytoplankton taxa at the SOMLIT-Astan time series off Roscoff (Western English Channel, France): data collected from 2000 to 2010. Cahiers De Biologie Marine 54: 247–256. Hallare AV, Lasafin KJA, & Magallanes JR (2011) Shift in Phytoplankton community structure in a tropical marine reserve before and after a major oil spill event. International Journal of Environmental Research 5(3): 651–660. Hargraves PE (1972) Studies on marine plankton diatom.1. Chaetoceros diadema (Her.) Gram: life cycle, structural morphology and regional distribution. Phycologia 11(3&4): 247–257. Hasle GR, Syvertsens EE (1997) Marine Diatoms. In: Tomas CR (ed) Identifying Marine Phytoplankton. Academic Press, San Diego, California, pp. 1–385. Heil CA, Patricia M, Glibert MA, Al-Sarawi MF, Manaf B & Muna H (2001) First record of a fish killing Gymnodinium sp. bloom in Kuwait Bay, Arabian Sea: Chronology and potential causes. Marine Ecology Progress Series 214: 15–23. Hendey NI (1964) An introductory account of the smaller algae of British coastal waters. Part 5: Bacillariophyceae (Diatoms). Her Majesty‟s Stationery Office, London. Hernandez-becerril DU (1999) Chaetoceros sumatranus, a member of Chaetoceros section Coarctati, sect. nov. (Bacillariophyceae). Cryptogamie Algologie 20(2): 95–104. www.tropicalplantresearch.com

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Tabassum et al. (2016) 3(3): 558–563 . Jiang Z, Huang Y, Xu X, Liao Y, Shou L, Liu, J, Chen Q & Zeng J (2010) Advance in the toxic effects of petroleum water accommodated fraction on marine plankton. Acta Ecologia Sinica 30(1): 8–15. Johansen JR, Barclay WR & Nagle N (1990) Chaetoceros muelleri (Bacillariophyceae): Physiological variability within ten strains. Journal of Phycology 26: 271–278. Karthick B, Jonathan CT, Mahesh MK & Ramachandra TV (2010) Protocols for collections, preservation and enumeration of diatoms from aquatic habitats for water quality monitoring in India. The IUP Journal of Soil & Water Sciences III(1): 25–60. Kuzmenko V (1975) Systematic composition of phytoplankton of Arabian Sea. Biology of the Sea, 34: 15–261. Lee CII, Kim MC & Kim HC (2009) Temporal variation of chlorophyll a concentration in the coastal waters affected by the Hebei Spirit oil spill in the west sea of Korea. Marine Pollution Bulletin 58: 496–502. Lewis M & Pryor R (2013) Toxicities of oils, dispersants and dispersed oils to algae and aquatic plants: Review and database value to resource sustainability. Environmental Pollution 180: 345–367. Martha EF, Eugenia AS & Maria V (1998) Chaetoceros resting spores in the Gerlache Strait, Antarctic Peninsula. Polar Biology 19: 286–288. Moazzam M & Baig HS (1994) Species composition of phytoplankton in Antarctic waters observed during Pakistan‟s Antarctic Expedition. Marine Research 3(2): 1–43. Moazzam M (1973) Taxonomic and seasonal studies of planktonic centric diatoms from Manora channel (Lower Harbour) Karachi, M.Sc. Thesis. Department of Marine Biology. University of Karachi. Murthy KN, Babu MN, Annapurna C & Sarma NS (2012) First record of Chaetoceros minims (Bacillariophyceae) from the Indian waters. Marine Biodiversity Records 5: 1–4. Nwanko DI & Onyema IC (2003) A check list of planktonic algae off lagos coast. Journal of Marine Science Research & Development 9: 75–82. Ozgur B, Ojvind M, Nina L & Arif G (2013) Contributions to the Diatom flora of the Black Sea from ultrastructural and molecular studies: new records of Skeletonema marinoi, Pseudo-nitzschia pugens var. aveirensis and Chaetoceros tenuissimus for the marine flora of Turkey. Nova Hedwigia Band 96(3-4): 427– 444. Parab SR, Pandit RA, Kadam AN & Indap MM (2008) Effect of Bombay high crude oil and its water-soluble fraction on growth and metabolism of diatom Thalassiosira sp. Indian Journal of Marine Sciences 37(3): 251–255. Rajasekar T, Rajkumar M, SUN J, Ashok PV & Perumal P (2010) Seasonal variation of phytoplankton diversity in the Coleroon coastal waters, southeast coast of India. Acta Oceanologica Sinica 29. Rines JEB (1999) Morphology and taxonomy of Chaetoceros contortus Schutt 1895, with preliminary observations on Chaetoceros compressus Lauder 1864 (Subgenus Hyalochaete, Section Compressa). Botanica Marina 42: 539–551. Round FE, Crawford RM & Mann DG (1990) The Diatoms, Biology & Morphology of the Genera. Cambridge University Press, Cambridge, pp. 1–747. Saifullah SM & Chaghtai F (2005) Effect of “Tasman Spirit” oil spill on marine plants in the coastal area of Karachi. International Journal of Biology Biotechnology 2(2): 299–306. Sheekh MM, Nagger AE, Osman MEH & Haieder A (2000) Comparative studies on the green algae Chlorella homosphaera and Chlorella vulgaris with respect to oil pollution in the river Nile. Water, Air, & Soil Pollution 121: 187–201. Shevchenko OG, Orlava TY & Hernandez-Becerril DU (2006) The Chaetoceros (Bacillariophyta) from Peter the Great Bay, Sea of Japan. Botanica Marina 49: 236–258. Simonsen R (1974) The diatom plankton of the Indian Ocean Expedition of R/V “Meteor” 1964-1965. “Meteor” Forchungsergebnisse, 41pl. Gebruder Borntrager, Berlin, pp. 66. Stockwells DA (1991) Distribution of Chaetoceros resting spores in the quaternary sediments from Leg 119. In: Barron J, Larsen B et al. (eds) Proceedings of Ocean Drill. Program, Scientific Results 119: 599–610. Subrahmanyan R (1946) A systematic account of the marine plankton diatoms of the Madras coast. Proceedings of the Indian Academy of Science 24B: 85–197. Sunesen I, Hernandez-Becerril DU & Sar EA (2008) Marine diatoms from Buenos Aires coastal waters (Argentina). V. Species of the genus Chaetoceros. Revista de Biologia Marina Oceanografia 43(2): 303– 326. www.tropicalplantresearch.com

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Tabassum et al. (2016) 3(3): 558–563 . Tabassum A & Saifullah SM (2010) The planktonic diatom of the genus Chaetoceros Ehrenberg from northwestern Arabian Sea bordering Pakistan. Pakistan Journal of Botany 42(2): 1137–1151. Tomaru Y, Yoshitake T, Hidekazu S, Tamostsu N, Kanae K & Keizo N (2011) Isolation and characterization of a single-stranded DNA virus infecting Chaetoceros lorenzianus Grunow. Applied and Environmental Microbiology 77(15): 5285–5293. Trigueros JM, Orive E & Arriluza J (2002) Observations on Ch. Salsugineus (Chaetocerotales, Bacillariophyceae): first record of this bloom-forming diatom in a European estuary. European Journal of Phycology 37: 571–578. Witak M, Dunder J & Leniewska M (2011) Chaetoceros resting spores as indicators of Holocene paleoenvironmental changes in the Gulf of Gdansk, Southern Baltic Sea. Oceanological and Hydrobiological Studies 40: 21–29. Wood EJF (1963) Checklist of diatoms recorded from the Indian Ocean. Rep Div Fish Oceanogr CSIRO 36: 1– 304.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 564–568, 2016 DOI: 10.22271/tpr.2016.v3.i3.074 Research article

Diversity and distribution of Litsea in Chikkamagaluru, Karnataka S. G. Srinivas and Y. L. Krishnamurthy* Dept. of P.G Studies and Research in Applied Botany, Kuvempu University, Jnanasahyadri, Shankaraghatta-577451, Shivamogga, Karnataka, India *Corresponding Author: [email protected] [Accepted: 29 October 2016] Abstract: The study gives a report on the diversity of Litsea (Lauraceae) occur in Chikkamagaluru district of Karnataka, India. Study was conducted in two habitats of Kemmannugundi and Mullayyanagiri regions. Extensive field surveys were conducted for survey of the species by laying six belt transects of 250×4 m size. The data indicated that four species of Litsea occurred in the study sites; namely, Litsea floribunda, Litsea stocksii, Litsea glabrata and Litsea mysorensis. L. floribunda showed higher density when compared with other species, all the four species distributed frequently in Kemmannugundi whereas; in Mullayyanagiri only L. floribunda species is present. These trees commonly associated with other tree species are Cinnamomum verum, Neolitsea cassia, Maesa indica, Memecylon malabaricum and Syzizium cumini. Keywords: Associated species - Kemmannugundi - Lauraceae - Mullayyanagiri - Western Ghats. [Cite as: Srinivas SG & Krishnamurthy YL (2016) Diversity and distribution of Litsea in Chikkamagaluru, Karnataka. Tropical Plant Research 3(3): 564–568] INTRODUCTION The genus Litsea consists of about 400 species which is largest genus in the family Lauraceae distributed in tropical and subtropical Asia, Australia, New Zealand, North America and subtropical South America (Chaing et al. 2012). In India about 45 species are distributed in evergreen and semi evergreen forests of the Western Ghats (Bhuniya et al. 2010), 12 species are also found in Meghalaya, Manipur, Assam and Sikkim. Among 45 species 40 of which are endemic to peninsular India, 11 species are found in Karnataka (Saldanha 1996). The Litsea trees are evergreen dioecious with alternate or whorled leaves, inflorescence is pedunculate axillary umbellate or corymbose racemes. Bracts are present, 4–6 in numbers perianth tube companulate, anthers four celled. Ovary free or coverved by perianth, style curved, stigma dilated, fruit ovoid or globose (Gamble & Fischer 1998). Leaves and barks of Litsea stocksii and L. glutinosa are used as medicines. Essential oils like citral, lauric acid and oleic acid extracted are used commercially for the preparation of insecticides, perfumes, flavours and colognes. Oil extracted from Litsea cubeba is a good competitor of Chinese lemon oil due to its low cost of production and easy method of cultivation of the species. Decoction of different parts of the plant used to cure burns, sprains, cough, bronchitis and paralysis (Bhuniya et al. 2009). The taxonomy of the family Lauraceae is still not settled compare to other families. It is poorly understood due to its great diversity, inadequate morphological characters and lack of investment in taxonomic work. Litsea is a very interesting tree species in Western Ghats of India occur in evergreen and semi evergreen forests, information on its diversity, distribution and genetic relatedness within populations are not fully explored. Hence in this present study we focussed to study the diversity and distribution of Litsea species in Chikkamagaluru district, Karnataka. MATERIALS AND METHODS Study area The study area covers Kemmannugundi, Mullayyanagiri in Chikkamagaluru district situated between 12°54' to 13°53' N and 75°04' to 76°21' E in the Western Ghats regions of Karnataka (Fig. 1). The sampling sites have www.tropicalplantresearch.com

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Received: 13 July 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.074

Srinivas & Krishnamurthy (2016) 3(3): 564–568 . rich forest vegetation such as evergreen and semi evergreen forests, the wide range of ecological conditions and altitudinal variation resulted in diverse vegetation in study area. Mullayyanagiri is the highest elevated region in Karnataka. In Kemmannugundi, Mullayyanagiri region the temperature varies between 10° to 32° C across the different months of the year.

Figure 1. Map showing sampling sites in Chikkamagaluru District, Karnataka.

Tree sampling and Data analysis Extensive field surveys carried out throughout the year to know the diversity, distribution and phenology of the Litsea species. Stratified random sampling method is used to collect the tree data, three belt transects of 250×4 m was laid in each study sites and girth was measured at breast height using a girth tape. Species density, frequency, abundance, importance value index and basal area of plant were calculated by following Mishra (1968), Mueller-Dambois & Ellenberg (1974). The importance value index was calculated by summing of relative density, frequency and relative dominance. Species diversity index was calculated by Shannon Wiener index (1963); the species dominance index was calculated by using Simpson (1949). RESULTS AND DISCUSSION The four species of Litsea occurred in the two study sites; namely, Litsea floribunda, Litsea stocksii, Litsea glabrata and Litsea mysorensis (Fig. 2). These four species collected from the study sites, identified through some morphological characters using standard floras and herbarium samples were prepared. L. floribunda is present in Kemmannugundi and Mullayyanagiri, but the L. stocksii, L. glabrata, L. mysorensis only present in Kemmannugundi region absent in Mullayyanagiri (Table 1). The results showed that L. floribunda frequently present in all transects, the frequency of L. glabrata is 0.67, L. mysorensis 0.33, L. stocksii 1.0. The L. floribunda showed highest density 46.67 and 33.67 it covers a basal area of 1904.79 m2.ha-1 and 885.27 m2.ha-1 (Table 1) in Mullayyanagiri and Kemmannugundi respectively, L. mysorensis showed lesser density and basal area compare to all the species. Abundance and frequency (A/F) ratio of all the Litsea species in the study sites is >0.05, it showed a clumped or contagious pattern of distribution this is because it is a dioecious tree, clumping of individuals of the same species is often clearly related to gap formation and dispersal, pollination mechanism of the species. Upadhaya et al. (2003) investigated on the same family members Cinnamomum and Neolitsea it also showed clumped pattern of distribution. www.tropicalplantresearch.com

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Srinivas & Krishnamurthy (2016) 3(3): 564–568 .

Figure 2. Litsea species in Chikkamagaluru district, Karnataka: A–B, Litsea floribunda; C–D, Litsea stocksii; E–F, Litsea mysorensis; G–H, Litsea glabrata.

A total of 15 associated species belongs to 10 families were recorded in both Kemmannugundi and Mullayyanagiri study sites (Table 2). Five species belongs to family Lauraceae, this is because of preference of same environmental factors from the genera. Callicarpa, Cinnamomum, Cryptocarya, Neolitsea and Syzygium species are frequently distributed in all the transects of Kemmannugundi. Cinnamomum showed high density (21.33) per transect in Mullayyanagiri whereas Memecylon, Ochlandra, Psychotria showed low density (0.33) www.tropicalplantresearch.com 566

Srinivas & Krishnamurthy (2016) 3(3): 564–568 . in both the study sites (Table 2). Actinodaphne, Cryptocarya, Macaranga and Neolitsea cassia only present in Kemmannugundi absent in Mullayyanagiri region. Table 1. Frequency (Fre), density per transect (Den), abundance (Abun), IVI, A/F ratio of Litsea spp.

Species Kemmannugundi Litsea floribunda Litsea glabrata Litsea mysorensis Litsea stocksii Mullayyanagiri Litsea floribunda

Fre

Den

Abun

RF

RD

RA

IVI

A/F Basal area m2/ha

1 0.67 0.33 1

33.67 2 1 7

33.67 3 3 7

7.32 4.88 2.44 7.32

37.14 2.21 1.1 7.72

29.93 2.67 2.67 6.22

74.39 9.76 6.21 21.26

4.6 0.61 1.23 0.96

885.27 1.77 0.48 39.48

1

47.67

47.67

10

50

44.9

104.9

4.77

1904.79

Table 2. Frequency (Fre), density per transect (Den), abundance (Abun), IVI, A/F ratio of the major associated species.

Species

Fre Actinodaphne sp. 0.33 Callicarpa tomentosa 1 Cinnamomum verum 1 Cryptocarya sp. 1 Glochidion sp. 0.67 Macaranga peltata 0.67 Maesa indica 0.67 Memecylon malabaricum 0.33 Neolitsea cassia 1 Neolitsea zeylanica 0.67 Nothapodytes foetida 0.33 Ochlandra travancorica 0.33 Psychotria nigra 0.33 Syzygium cumini 1 Vernonia arborea 0.33

Kemmannugundi Den Abun RF RD 1.67 5 2.44 1.84 2.67 2.67 7.32 2.94 11 11 7.32 12.13 4.67 4.67 7.32 5.15 3.67 5.5 4.88 4.04 1.67 2.5 4.88 1.84 4.33 6.5 4.88 4.78 0.33 1 2.44 0.37 5 5 7.32 5.52 2.33 3.5 4.88 2.57 1.33 4 2.44 1.47 0.33 1 2.44 0.37 0.33 1 2.44 0.37 4 4 7.32 4.41 1 3 2.44 1.1

RA 4.44 2.37 9.78 4.15 4.89 2.22 5.78 0.89 4.44 3.11 3.56 0.89 0.89 3.56 2.67

IVI 8.72 12.63 29.23 16.62 13.81 8.94 15.44 3.7 17.28 10.56 7.47 3.7 3.7 15.29 6.21

A/F 2.05 0.36 1.5 0.64 1.13 0.51 1.33 0.41 0.68 0.72 1.64 0.41 0.41 0.55 1.23

Fre 1 1 0.67 1 0.33 1 0.67 0.33 0.33 0.67 0.33

Mullayyanagiri Den Abun RF RD 1.67 1.67 10 1.75 21.33 21.33 10 22.3 3 4.5 6.67 3.15 3.67 3.67 10 3.85 0.33 1 3.33 0.35 4.33 4.33 10 4.55 2.33 3.5 6.67 2.45 0.33 1 3.33 0.35 0.33 1 3.33 0.35 4.67 7 6.67 4.9 0.33 1 3.33 0.35

RA

IVI

1.57 20 4.24 3.45 0.94 4.08 3.3 0.94 0.94 6.59 0.94

13.32 52.3 14.06 17.3 4.62 18.63 12.42 4.62 4.62 18.16 4.62

Shanon index is a diversity index taking into account of number of individuals as well as number of taxa. The Shanon and Simpson index of Kemmannugudi is 2.32, 0.82 respectively and 1.65, 0.69 in Mullayyanagiri respectively (Fig. 3). According to Shanon and Simpson indices Kemmannugundi has highest species richness area compare to the Mullayyanagiri region. The Shanon index of the Kemmannugundi region is lower (2.32) compare to Sulimudi forests of Western Ghats, Kerala (2.64) (Magesh & Menon 2011) and Simpson value higher (0.82) compare to Vagamon region (0.36) (Brilliant et al. 2012).

Figure 3. Shanon and Simpson diversity index in Kemmannugundi and Mullayyanagiri.

CONCLUSION www.tropicalplantresearch.com

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A/F 0.17 2.13 0.68 0.37 0.3 0.43 0.53 0.3 0.3 1.05 0.3

Srinivas & Krishnamurthy (2016) 3(3): 564–568 . This study revealed that the two study sites harboured four Litsea species and Litsea floribunda showed good species richness. Litsea stocksii, Litsea glabrata and Litsea mysorensis showed low species richness in Kemmannugundi whereas these species absent in Mullayyanagiri. A total of 15 associated species belongs to 10 families were recorded; Laural members are the dominant associated species in both the study sites. ACKNOWLEDGEMENTS The authors are thankful to the Department of Science and Technology (DST) New Delhi, for providing financial assistance as an Inspire Fellowship (IF140097) to Srinivas SG and authors also acknowledge thanks to Kuvempu University to providing research facilities. The author also acknowledges special thanks to Shravan Kumar S., Avinash K.S., Ashwini H.S., for their help in field collections in the studies. REFERENCES Bhuniya T, Singh P & Mukherjee SK (2009) Distribution of the genus Litsea Lam. (Lauraceae) in India with special reference to rare and endemic species. Phytotaxonomy 9: 116–121. Bhuniya T, Singh P & Mukherjee SK (2010) An account of the species of Litsea Lam. (Lauraceae) endemic to India. Bangladesh Journal of Plant Taxon 17: 183–191. Brilliant R, Varghese VM, Paul J & Pradeepkumar AP (2012) Vegetation analysis of montane forest of Western Ghats with special emphasis on RET species. International journal of Biodiversity and Conservation 4: 652– 664. Chaing YC, Huei CS, Min CH, Li PJ & Hsiang HK (2012) Characterization of microsatellite loci from Litsea hypophaea, a tree endemic to Taiwan. American Journal of Botany 99(6): e251–e254. Gamble JS & Fischer CEC (1998) Flora of Presidency of Madras, Vol. 1–3. Adlard and Son Limited, 21, Hart street, WC. Magesh G & Menon ARR (2011) Vegetation status, species diversity and endemism of Sulimudi forests in southern Western Ghats of Kerala, India. The Indian Forester 2: 304–311. Mishra R (1968) Ecology work book. Oxford and IBH publishing company Calcutta India. Mueller DD & Ellenberg H (1974) Aims and methods of vegetation ecology. John Wiley and Sons New York USA. Saldanha CJ (1996) Flora of Karnataka, Vol. 1–4. Oxford and IBH publishing Ltd, New Delhi. Shanon CE & Weiner W (1963) The Mathematical theory of communication. University of Illinois press Urbana. Simpson EH (1949) Measurement of diversity. Nature 163: 688. Upadhaya K, Pandey HN, Law PS & Tripathi RS (2003) Tree diversity in sacred grooves of the Jaintia hills in Meghalaya, Northeast India. Biodiversity and Conservations 12: 583–597.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 569–572, 2016 DOI: 10.22271/tpr.2016.v3.i3.075 Research article

Alternaria polypodiicola, a new foliicolous fungus discovered on Microsorum punctatum from Uttar Pradesh, India Shambhu Kumar1* and Raghvendra Singh2 1

2

Department of Forest Pathology, Kerala Forest Research Institute, Thrissur, Kerala, India Centre of Advanced Study in Botany, Institute of Sciences, Banaras Hindu University, Varanasi. Uttar Pradesh, India

*Corresponding Author: [email protected]

[Accepted: 31 October 2016]

Abstract: Alternaria polypodiicola sp. nov., is described, illustrated and discussed, causing foliar disease on a pteridophytic plant Microsorum punctatum (Polypodiaceae) from Uttar Pradesh, India. The present species was compared with closely similar species based on morphological characters. This species is characterized by having well developed stromata, unbranched and shorter conidiophores with shorter smooth conidia. A key is provided to all species of Alternaria reported on Polypodiaceae. The description and nomenclatural novelty details were deposited in Mycobank. Keywords: Taxonomy - Foliicolous - Hyphomycetes - Microsorum - Alternaria - New species. [Cite as: Kumar S & Singh R (2016) Alternaria polypodiicola, a new foliicolous fungus discovered on Microsorum punctatum from Uttar Pradesh, India. Tropical Plant Research 3(3): 569–572] INTRODUCTION Microsorum punctatum (L.) Copel. is a small evergreen ornamental pteridophytic plant belongs to family Polypodiaceae of Plant kingdom. It is a common fern species in Africa and Asia and occurs naturally in various forest types of tropics and subtropics from sea level up to 2800 m elevation (Nooteboom 1997, Bosman 1991). The plant shows good medicinal properties. The leaf and juice are used as purgative, diuretic and for healing wounds (May 1978, Sharma & Pegu 2011). During the regular observation of plants of the BSIP garden, Lucknow, the living leaves of Microsorum punctatum exhibiting foliar blights was encountered. However, it differs morphologically from previously described Alternaria species and therefore is proposed here as new based on critical microscopic examination and comparison of morphological features with those of the closely similar forms. The details description and illustration of Alternaria polypodiicola is presented here. MATERIALS AND METHODS The diseased plant leaves samples were collected from BSIP Campus, Lucknow during September 2012. The photographs of the infection spots were taken by using a Sony DSC-5730 camera during the time of collection. The collected samples were carried to the laboratory and processed by following the standard techniques (Castañeda-Ruiz 2005, Hawskworth 1974, Savile 1962). The sun dried and pressed leaf specimens were placed in air tight polyethylene bags and then kept in ziplock polythene bag along with collection details. The surface scrapping and free hand cut sections of infected leaf samples were taken through infection spots and mounted in cotton-blue lactophenol mount mixture for microscopic examination. Detailed observations of morphological characters were carried out by means of an Olympus CX31 light microscope (400×) and measurement was done by micrometry. Morphotaxonomic determination was made with the help of current literature pertaining to Alternaria. The holotype specimen has been deposited in Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute, Pune, India future reference. Description and nomenclatural detail were deposited in MycoBank (www.MycoBank.org). The systematics position of the taxa is given in accordance with Cannon & Kirk (2007), Kirk et al. (2008), Seifert et al. (2011), Farr & Rossman (2015) and the Index Fungorum (www.indexfungorum.org; accessed 30 April 2015). www.tropicalplantresearch.com

569

Received: 14 July 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.075

Kumar S & Singh (2016) 3(3): 569–572 . RESULTS Taxonomic descriptions Alternaria polypodiicola Sham. Kumar & Raghv. Singh sp. nov. (Fig. 1, 2) MycoBank MB 817343 Anamorphic fungus, hyphomycetes, Foliicolous, Infection spots amphigenous, initially circular to irregular (5-25 mm diam.), brown, but later on severe infection it spreading on entire surface of the leaves. Colonies amphiphyllous, effuse, brown. Mycelium internal. Stromata present (15µm in diam), pseudoparenchymatous. Conidiophores macronematous, fasciculatous (5–7 in a fascicle), straight to curved, simple, cylindrical, unbranched, thick walled, smooth, 1–3 septate, brown, 10–55 × 3–5 μm. Conidiogenous cells integrated, terminal, monotretic, scars thickened. Conidia simple, acropleurogenous, solitary to catenate, dry, smooth obclavate to ellipsoidal to ovoid (muriform), rostrum present, 2–4 transversely septate and 2–3 obliquely septate, brown, base obtuse, 20–50 × 10–18 µm, hilum thickened (1.5–2.0 µm), germinating conidium present. Material examined: India, Uttar Pradesh, Lucknow, BSIP Campus, on living leaves of Microsorum punctatum (L.) Copel. (Polypodiaceae), 2nd September, 2012, coll. Shambhu Kumar, AMH-9515 (holotype). Etymology: The specific epithet polypodiicola in reference to host family. Teleomorph: Undetermined.

Figure 1. Microsorum punctatum: A, Host Plant; B, Infection spots on upper surface of leaf (Scale bars: B = 20 mm); C, Infection spots on lower surface of leaf.

Identification key to Alternaria spp. reported on Polypodiaceae 1 Stromata absent………………………………………………………………………………………...………..2 1* Stromata present….……….………………………………………………..….……………………………….3 2 Conidiophores up to 50 × 3–6 µm, branched.…………...……………….……..……………………………….4 2*Conidiophores up to 115 × 4–6 μm, simple or unbranched…...………………………….….…………....……5 3 Conidiophores 10–55 µm × 3–5 μm, unbranched….……………………………….………………..…….....…6 4 Conidia 20–63 × 9–18 µm, 8 transversely septate and several obliquely septate, verruculose......... A. alternata 5 Conidia 22–95 × 8–19 µm, 4–7 transversely septate and several obliquely septate,verruculose… A. tenuissima 6 Conidia 20–50 × 10–18 µm, 2–4 transversely septate and 2–3 obliquely septate, smooth……. A. polypodiicola DISCUSSION Perusal of literatures indicated that there was no record of Alternaria on this host (Farr & Rossman, 2015, Bilgrami et al. 1991, Jamaluddin et al. 2004). Alternaria polypodii (invalidly published) on Polypodium sp. and Alternaria sp. on Platycerium bifurcatum and Platycerium sp. from Florida (Farr & Rossman 2015) have been reported on Polypodiaceae which were very similar to A. alternata. From India, A. alternata was previously reported on Polypodium vulgare L. (Narang et al. 1978) from Allahabad and A. tenuissima (Kunze ex Pers) www.tropicalplantresearch.com

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Kumar S & Singh (2016) 3(3): 569–572 . Wilt. on Polypodium multilineatum L. (Kanaujia et al. 1978) from Faizabad respectively on the family Polypodiaceae. Therefore, the present fungus was compared with these two earlier reported species. The stromata is present in A. polypodiicola while absent in earlier described species. The conidiophores are unbranched and very much shorter (10–55 × 3–5 μm) in A. polypodiicola while branched and longer (up to 50 × 3–6 µm) in A. alternata and A. tenuissima (simple or unbranched and up to 115 × 4–6 μm). The Conidia of A. polypodiicola are shorter (20–50 × 10–18 µm) than both previously described A. alternata (20–63 × 9–18 µm) and A. tenuissima (22–95 × 8–19 µm). The conidia of novel species are smooth while verruculose in both the earlier described species. Thus, A. polypodiicola is treated as a new species.

Figure 2. Alternaria polypodiicola (AMH-9515, holotype): A–D, Conidiophores; E–H, Conidia. (Scale bars A–H = 5 μm)

ACKNOWLEDGEMENTS Authors’ are thankful to the Director Kerala Forest Research Institute, Peechi for encouragement and necessary facilities. Thankfulness is also due to the Curator, Ajrekar Mycological Herbarium (AMH), Agharkar Research Institute, Pune (MS), India for depositing specimen and providing accession number thereof. We are also grateful to Dr. Ajit Pratap Singh, Senior Scientist, CSIR-NBRI, Lucknow, for the host identification. Shambhu Kumar is grateful to Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, New Delhi for financial support (SB/YS/LS-288/2013). REFERENCES Bilgrami KS, Jamaluddin & Rizwi MA (1991) Fungi of India: List and references. Today’s and Tomorrow’s Printers and Publishers, New Delhi. pp. 798. Bosman MTM (1991) A Monograph of the Fern Genus Microsorum (Polypodiaceae). Rijksherbarium/Hortus Botanicus, Leiden, the Netherlands. Cannon PF & Kirk PF (2007) Fungal Families of the World. Wallingford, UK: CAB International. pp. 456. Castañeda-Ruiz RF (2005) Metodologíaen el estudio de loshongosanamorfos. Anais do V Congresso Latino Americano de Micología. Brasilia, pp. 182–183. www.tropicalplantresearch.com

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Kumar S & Singh (2016) 3(3): 569–572 . Farr DF & Rossman AY (2015) Fungal Databases, Systematic Mycology and Microbiology. ARS, USDA. Available from: http://nt.ars-grin.gov/fungaldatabases/ (accessed: 30 Apr. 2015). Hawskworth DL (1974) Mycologist’s Handbook. CMI, Kew. pp. 231. Index Fungorum (2015) Index Fungorum. Available from: http://www.indexfungorum.org/ (accessed 30 Apr. 2015). Jamaluddin, Goswami MG & Ojha BM (2004) Fungi of India 1989–2001. Scientific Publishers, Jodhpur, Rajasthan, India. pp. 326. Kanaujia RS & Raj Kishore (1978) Annoted list of Fungi from Faizabad (U.P.), India. Indian Journal of Mycology and Plant Pathology 8: 188–194. Kirk PF, Cannon PF, Minter DW & Stalpers JA (2008) Dictionary of the Fungi, 10th ed. Wallingford, UK: CAB International. pp. 402. May LW (1978) The economic uses and associated folklore of ferns and fern allies. Botanical Review 44(4): 491–528. MycoBank (2015) Fungal databases nomenclature and species banks. Available from: http://www.mycobank.org/ (accessed: 30 Apr. 2015). Narang M & Chandra S (1978) Some new leaf spot diseases of ferns from India. Acta Biologica Indica 6: 108– 114. Nooteboom HP (1997) The microsoroid ferns (Polypodiaceae). Blumea 42: 261–395 Savile DBO (1962) Collection and care of Botanical specimens. Department of Agriculture, Canada, pp. 1113. Seifert K, Morgan-Jones G, Gams W & Kendrick B. (2011) The Genera of Hyphomycetes. CBS Biodiversity Series 9: 1–997. Sharma UK & Pegu S (2011) Ethnobotany of religious and supernatural beliefs of the Missing tribes of Assam with special reference to the 'Dobur Uie'. Journal of Ethnobiology and Ethnomedicine 7(1): 16.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 573–585, 2016 DOI: 10.22271/tpr.2016.v3.i3.076 Research article

Floristic composition and biological spectrum of weeds in agroclimatic zone of Nalbari district, Assam, India D. K. Bhattacharjya1* and S. K. Sarma2 1

Corresponding author, Dept. of Botany, M.C. College, Barpeta-781301, Assam, India 2 Department of Botany, Gauhati University, Guwahati-781014, Assam, India

*Corresponding Author: [email protected]

[Accepted: 02 November 2016]

Abstract: Present paper deals with the study of floristic composition and biological spectrum of the agro-climatic zone of Nalbari district of Assam. Field observation encompasses a total of 217 weed species from both angiosperms and pteridophytes belonging to 150 genera under 60 families. Asteraceae, being the largest family possesses 19 genera and 22 species. Out of the total species, 183 species (84.33 %) showed the annual life span as against 34 species (15.67 %) showing the perennial life span. One hundred thirty eight species (63.59 %) were recorded to be propagated by seeds/spores; 66 species (30.41 %) were found to opt for both seeds and vegetative propagules and only 13 species (5.99 %) were listed to be propagated exclusively by vegetative propagules. The phytoclimate of the agro-climatic zone can be regarded as thero-cryptophytic since major percentage of the species falls under the life forms Therophytes and Cryptophytes which has been assessed after comparison with the normal world spectrum as proposed by Raunkiaer. Keywords: Agro-climatic zone - Weed flora - Life span - Phytodiversity - Propagation. [Cite as: Bhattacharjya DK & Sarma SK (2016) Floristic composition and biological spectrum of weeds in agroclimatic zone of Nalbari district, Assam, India. Tropical Plant Research 3(3): 573–585] INTRODUCTION Agro-climatic condition includes such basic factors of plant growth like soil types, temperature, rainfall and water availability which directly influence the vegetation of an area. Agro-climatic zone is a unit of land use in terms of major climates and the zone is favourable for certain range of crops and cultivars. Such climatic division is meant for effective and efficient management of local phytoresources, i.e. crops to meet the growing demand of food, fodder, fibre, fuel wood, timber etc. without adversely affecting the environment and causing any sort of loss to the nature (http://vikaspedia.in). India comprises a total of 15 agro-climatic zones and Assam belongs to the Eastern Himalayan zone of 15 such zones (Singh 2012). Nalbari district of Assam is basically an agriculture dependent district. The richness of the flora of this district with a healthy combination of both primitive and advanced families is supported by the geographical position of the district and also the favourable climatic conditions. Varieties of weed species are found to infest the crop-lands, waste lands, aquatic bodies etc. in the district throughout the year. Particularly in the crop-lands, the obnoxious weeds create severe problems interfering the yield of the crops in the area. Varieties of crop cultivations are practiced throughout the year in the agro-climatic zones in Nalbari district of Assam. Along with the crops, the crop fields in the agro-climatic zones are found to be infested by a variety of weed species throughout the year. Weeds are the plants grown in the places where they are not desired. Weeds may include all types of undesirable plants like grasses, sedges, forbs, aquatic plants, parasitic angiosperms, pteridophytic plants etc. Such plants may be the constant associates of the cultivated plants also, comprising the crop-field weeds, i.e. the vegetation infesting the human maintained crop-fields. Still the weeds have been occupying an integral part of the phytodiversity of a region. The vegetation infesting the crop-fields of the agro-climatic zones is very often ignored and not included in the study in terms of ecology and taxonomy in comparison to forest, grassland, wetland and others. Weeds, www.tropicalplantresearch.com

573

Received: 19 July 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.076

Bhattacharjya & Sarma (2016) 3(3): 573–585 . although are very frequently termed as obnoxious, harmful to both human and animals and reducer of crop yield, yet a lot of weed species are useful in many ways (Bhattacharjya & Borah 2006, Bhattacharjya et al. 2006). Apart from these, weeds have an important contribution towards the phytodiversity of a region. Survival of mankind is directly related with the survival of phytodiversity. Being rich in phytodiversity the entire country in general and the state of Assam and entire North-East in particular possess a large number of plant species growing in a variety of climatic and edaphic conditions resulting in the formation of a wide range of habitats (Reddy et al. 2008). Floristic richness of a region enables one to have the idea about the design and functioning of the communities along with the pattern and process of the community structure (Thakur 2015). Study of floristic composition is considered as fundamental and regarded as prerequisite for all kinds of ecological research. All other works revolve around the floristic study (Naveed et al. 2012). Climatic, edaphic and biotic factors prevailing in an area influence the formation of vegetation of that area (Shahid & Joshi 2015). Existing vegetation is also an indicator of the climate, soil and anthropogenic influences occurring in a region (Sharma et al. 2014). The major community description and its appearance depend upon the occurrence of life forms which are based on the position and degree of protection of regenerating parts with respect to the ground surface (Cain 1950). The physical appearance of vegetation chiefly depends on the life form of dominant plant species (Hanson & Churchill 1961). Life form pattern of the species and the proportion of life forms in an area reflect a complete ecological picture of the community as well as provide a good indication of the climatic zone of the community (Cain 1950, Kershaw 1973). The plant species of any community can be classified in one or the other life forms. The ratio of the life forms of different species in terms of numbers or percentages in any floristic community is the biological or phytoclimatic spectrum. The biological spectrum is also regarded as the indicative of the prevailing environment as the life forms are related to the environment around the plants (Sudhakar Reddy et al. 2011). Plant’s life form is one among its most striking characteristics. In classifying vegetation, Raunkiaer’s life form system is very widely used as such or with a few modifications (Braun-Blanquet 1951, Dansereau 1957a, Mueller-Dombois & Ellenberg 1974). Analyzing the life forms of various regions of the world and comparing them with a normal spectrum based on 1,000 species selected at random, Raunkiaer reported a predominance of Phanerophytes in tropical moist regions. Following the same principle, a high preponderance of Therophytes and not insignificant proportions of Chamaephytes and Hemicryptophytes may be found from a desert area (Raunkiaer 1934). There has been an increasing effort on the study of vegetation in connection with floristic composition, life forms and biological spectrum in different times (Gillespie 2004, Batalha & Martins 2004, Reddy et al. 2011, Theilade et al. 2011, Saikia et al. 2012, Naveed et al. 2012, Burja et al. 2013, Aye et al. 2014, Thakur 2015, Alemu et al. 2015). However, no such study has been conducted hitherto in the Nalbari district of Assam. Accordingly, no work is reported from the agro-climatic zone of the district in this regard. The present work, therefore, aims at to study the floristic composition of the agro-climatic zone of the district along with the preparation of biological spectrum of the agro-zone. MATERIALS AND METHODS The study area Nalbari districtof Assam lies between 26º 10' N to 26º 47' N latitude and 90º 15' E to 91º 10' E longitude which occupies an area of about 1009.57 km2. The area is mainly plain. The northern side of the district is bounded by the Baksa district. The southern side by the mighty Brahmaputra. The Kamrup District falls in the east and the Barpeta District in the west. The entire area of the District is situated at the plains of the Brahmaputra Valley. The tributaries of the Brahmaputra, Nona, Buradia, Pagaldia, Borolia and Tihu which are originated from the foothills of the Himalayan Range are wild in nature and have enormous contribution towards the agrarian economy of the district. The Soil condition of the district is heterogeneous one. The Soil of the northern part is clayey and loamy, whereas middle part is loamy and sandy. The southern part is characterized by sandy soil (Industrial Profile of Nalbari district, ministry of MSME, Govt. of India). The soil pH varies from 5.05 to 7.22. The District has a sub-tropical climate with semi dry hot summer and cold winter. During summer, generally during the months from May to August, heavy rainfall occurs for which the district experiences flood. The District experiences annual (average) rainfall of 1500 mm and its humidity hovers around 80%. The average temperature during summer and winter are 27.00º C and 16.21º C. respectively (Office of the Deputy Commissioner, Nalbari, Assam, India). Several crop cultivations belonging to both www.tropicalplantresearch.com 574

Bhattacharjya & Sarma (2016) 3(3): 573–585 . summer and winter seasons are practiced throughout the year in the district. The major crops include rice, wheat, lentil, pea, mustard, jute, sugarcane, chillis, onion, turmeric, vegetable yielding species etc. Data collection Frequent field visits were made for a period of three years from 2013 to 2015 to collect the weed species infesting the crop-fields under the agro-climatic zone of Nalbari district of Assam. Cultivated crop species were not taken into consideration as the species are fully human maintained and not part of natural vegetation. Method(s) of propagation and the position of the perennating buds of each weed species were recorded on the spot by thorough observation. Life spans of the species were recorded after close observation of the life cycle right from seedling stage till death. For determination of the life-forms and analysis of the biological spectrum, Raunkiaer’s system as modified by Braun-Blanquet (1951) has been followed. The percentage of each life form was calculated by using the following formula: Number of species in any life form % Life form =  100 Total number of species of all life forms The data recorded have been presented in tabular form by arranging the families according to Bentham & Hooker’s system of classification. RESULTS A total of 217 weed species belonging to 150 genera and 60 families were encountered during the field study in the agro-climatic zone of Nalbari district of Assam. Normally highest number of angiospermic species (210) has been recorded from the study area in comparison to the pteridophytic species (7) (Table 1, 2; Fig. 1, 2). Table 1. Enumeration of Angiospermic weed species and their basic ecological features. (Abbreviations: A: annual, Bl: bulbil, Ch: chamephyte, Cr: cryptophyte, Hm: hemicryptophyte, P: perennial, Of: offset, Ph: phanerophyte, R: runner, Rs: root stock, Rz: rhizome, S: seed, Sc: sucker, Sm: stem, St: stolon, Tb: tuber, Th: therophyte)

Family Nymphaeaceae

Species

Euryale ferox Salisb. Nelumbo nucifera Geartn. Nymphaea alba L. N. nouchali Burm.f. N. rubra Roxb. ex Salisb. Nymphoides cristata (Roxb.) Kuntze Papaveraceae Argemone mexicana L. Brassicaceae Capsella bursa-pastoris (L.) Medikus. Rorippa benghalensis (DC.) H. Hara. Capparaceae Cleome viscosa L. Caryophyllaceae Drymaria diandra Blume. Polycarpon prostratum (Forsk.) Asch. & Schweinf. Stellaria media (L.) Vill. S. wallichiana Haines. Portulacaceae Portulaca oleracea L. Hypericaceae Hypericum japonicum Thunb. ex Murray Malvaceae Abutilon indicum (L.) Sweet. Malvastrum coromandalianum (L.) Garcke Sida cordifolia L. Sida rhombifolia L. Tiliaceae Corchorus aestuans L. Grewia sapida Roxb. Triumfetta rhomboidea Jacq. Linaceae Linum ustitatissimum L. Balsaminaceae Impatiens glandulifera Royle Oxalidaceae Oxalis corniculata L. O. debilis H.B.K. var. corymbosa Sapindaceae Cardiospermum halicacabum L. Fabaceae: Mimosoideae Mimosa pudica L. Caesalpinioideae Cassia sophera L. C. tora L. www.tropicalplantresearch.com

Life span P P P P P P A A A A A A A A A A A P P P A P P A A A A A A A A

Method of Life form Propagation S, Rz Cr S, Rz Cr Rz, St Cr Rz, St Cr Rz, St Cr Rz Cr S Th S Ph S Th S Th S Ch S Th S Th S Th S Th S Th S Ph S Ph S Th S Th S Ph S Ph S Ph S Th S Th S, R Hm S, Bl Hm S Ph S S S

Th Th Ph 575

Bhattacharjya & Sarma (2016) 3(3): 573–585 . Papilionatae Aeschynomene aspera L. A S Cr Aeschynomene indica L. A S Ph Crotalaria juncea L. P S Ph Desmodium gangeticum (L.) DC. A S Th D. laxiflorum DC. A S Th D. triflorum (L.) DC. A S Th D. triquetrum (L.) DC. ssp. pseudotriquetrum A S Ph Lathyrus aphaca L. A S Th Tephrosia purpurea (L) Pers. P S Ph Rosaceae Duchesnea indica (Andrews) Focks. A S, R Hm Haloragaceae Callitriche stagnalis Scop. A S Th Lythraceae Ammannia baccifera L. A S Th A. multiflora Roxb. A S Th Cuphea carthagenensis (Jacq.) J.F.Macbr. A S Th Rotala indica (Willd.) Cochne. A S Th Onagraceae Ludwigia adscendens (L.) Hara. A S, Of Cr L. octavalvis (Jacq.) Raven. A S Th L. perennis L. P S Th Trapaceae Trapa bispinosa (Roxb.) Makino P S, Rz Cr T. natans L. P S, Rz Cr Molluginaceae Glinus lotoides L. A S Th Mollugo pentaphylla L. A S Th Apiaceae Centella asiatica (L.) Urban. A S, R Hm Hydrocotyle javanica thunb. A S, R Hm H. sibthorpioides Lam. A S, R Hm Oenanthe javanica (Blume) Dc. A S Th Rubiaceae Dentella repens (L.) J.R. & G. Forst. A S Th Oldenlandia diffusa (Willd.) Roxb. A S, R Ch Richardia scabra L. A S, R Th Asteraceae Blumea densiflora DC. A S Th B. lacera (Burm. f.) DC. A S Ph Cosmos sulfureus Cav. A S Th Cotula hemisphaerica Wall. A S Th Dichrocephala integrifolia (L.f.) O.Ktze. A S Th Eclipta prostrata L. A S Th Elephantopus scaber L. A S Th Enhydra fluctuans Lour. A S Cr Chromolaena odorata (L.) R.M.King & H.Rob. A S Ph Gnaphalium luteo-album L. A S Th G. pensylvanicum Willd. A S Th G. polycaulon Pers. A S Th Grangea maderaspatana (L.) Poiret. A S Ch Mikania micrantha Kunth. A S, R Ph Parthenium hysterophorus L. P S Th Sonchus wightianus DC. A S Th Sphaeranthus indicus L. A S Th Spilanthes paniculata Wallich ex DC. A S, Sc Ch Taraxacum officinale Wigg. A S Ph Vernonia cinerea (L.) Less. A S Th Xanthium indicum Koenig. in Roxb. A S Th Youngia japonica (L.) DC. A S Th Campanulaceae Lobelia zeylanica L. A S Th Wehlandbergia marginata (Thunb.) DC. A S Th Hydrophyllaceae Hydrolea zeylanica (L.) Vahl. A S Cr Boraginaceae Cynoglossum zeylanicum (Vahl.) Brand A S Ph Heliotropium indicum L. A S Th Convolvulaceae Evolvulus mummularis L. A S, R Hm Ipomea aquatica Forst. A S Cr I. carnea (Mart. ex Choisy) Austin. P S, Sm. Ch www.tropicalplantresearch.com

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Solanaceae

Scrophulariaceae

Lentibulariaceae Acanthaceae

Verbenaceae Lamiaceae

Amaranthaceae

Chenopodiaceae Polygonaceae

Euphorbiaceae

Urticaceae Cannabinaceae Ceratophyllaceae Hydrocharitaceae

Nicotiana plumbaginifolia Viv. Solanum nigrum L. S. torvum Swartz. Limnophila hirsuta Benth. Limnophila indica (L.) Druce L. heterophylla (Roxb.) Ben Lindernia anagallis (Burm. f.) Pennell. L. antipoda (L.) Alston. L. ciliata (Colsm.) Pennell. L. cordifolia (Colsm.) Merr. L. crustacea (L.) Muell. L. parviflora (Roxb.) Haines. L. ruelloides (Colsm.) Pennell. L. tenuifolia (Colsm.) Alston. L. viscosa (Hornem) Boldingh. Mazas pumilus (Burm.f.) Steen. Mecardonia procumbens (Mill.) Small Scoparia dulcis L. Torenia diffusa D. Don. Utricularia aurea Lour. Hygrophila polysperma (Roxb.) T. Anders. Lepidagthis incurva D. Don. Rostellularia japonica (Thunb.) Ellis. Rungia pectinata (L.) Nees. Clerodendrum viscosum Vent. Phyla nodiflora (L.) Greene. Leonurus japonicus Houtt. Leucas Plukenetii (Roth.) Spreng. Ocimum Basilicum L. Pogostemon Fraternus Mig. P. Strigosus Benth. Achyranthes aspera L. Alternanthera philoxeroides (Mart.) Griseb. A. sessilis (L.) R.Br. ex DC. Amaranthus hybridus L. A. spinosus L. A. viridis L. Celosia argentea (L.) Schinz. Chenopodium album L. Polygonum barbatum L. P. glabrum Willd. P. chinense L. P. hydropiper L. P. orientale L. P. plebeium R.Br. P. strigosum Br. Prodr. Rumex dentatus L. Rumex maritimus L. Rumex nepalensis Spreng. Acalypha indica L. Croton bonplandianum Baill. Euphorbia hirta L. E. thymifolia L. Phyllanthus fraternus Webster. Pouzolzia zeylanica (L.) Bennett. Cannabis sativa L. Ceratophyllum demersum L. Hydrilla verticillata (L.f.) Royle. Ottelia alismoides (L.) Pers. Valisnaria spiralis L.

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Bhattacharjya & Sarma (2016) 3(3): 573–585 . A S Ph A S Th A S Ph A S Th A S Cr A S Cr A S Th A S Th A S Th A S Th A S Th A S Th A S Th A S Cr A S Th A S Th A S Th A S Th A S Th A S, Of Cr A S Cr A S Ph A S Th A S Th P S Ph A S Th A S Ph A S Th P S Ph A S Th A S Th A S Th A S Ch A S, R Ch A S Th A S Th A S Th A S Th A S Th A S Th A s Th A S Th A S Th A S Th A S Th A S Th A S Th A S Th A S Th A S Th A S Th A S, R Ch A S Th A S Th A S Th A S Th A S, Rs Cr A Rz Cr A S Cr P S, Rz Cr 577

Zingiberaceae Pontederiaceae

Alpinia allughas (Retz.) Rosc. Eichhornia crassipes (Mart.) Solms. Monochoria hastata (L.) Solms. M. vaginalis C.Presl. Commelinaceae Commelina benghalensis L. C. diffusa Burm. Cyanotis axillaris (L.) Don. Floscopa scandens Lour. Murdania nudiflora (L.) Brenan. Typhaceae Typha elephantina Roxb. Araceae Amorphophallus campanulatus (Roxb.) Bl. Colacasia esculenta (L.) Schott. Lasia spinosa Thw. Pistia stratiotes L. Lemnaceae Lemna purpusilla Torrey Spirodela polyrrhiza (L.) Schl. Najadaceae Najas indica (Willd.) Cham. Najas minor All. Aponogetonaceae Aponogeton appendiculatus H. Brug Potamogetonaceae Potomogeton crispus L. Alismataceae Alisma plantago L. Sagittaria guayanensis H.B. & K. Sagittaria sagittifolia L. Eriocaulaceae Eriocaulon viride Koern. Cyperaceae Cyperus bulbosus Vahl. C. corymbosus Rottb. C difformis L. C. halpan L. C. iria L. C. pilosus Vahl. C. pumilus L. C. rotundus L. C. sanguinolentus Vahl. C. tenuispica Steud. Elaeocharis dulcis (Burm.f.) Henschel. Fimbristylis aestivalis (Retz.) Vahl. F. dichotoma (L.) Vahl. F. littoralis Gaud. F. miliacea (L.) Vahl. F. tomentosa Vahl. Kyllinga monocephela Roxb. Schoenoplectus articulatus (L.) S. grossuss (L.f.) Scirpus articulatus L. S. juncoides Roxb Poaceae Andropogon ascinoidis C.B.Clarke. Axonopus compressus (Sw.) Beauv. Brachiaria distachya (L.) Stapf. Cynodon dactylon (L.) Pers. Dactyloctenium aegyptium (L.) P.Beauv. Digitaria ciliaris (Retg.) Koel. D. sanguinalis Scop. Echinochloa colonum Link. Eleusine indica (L.) Gaertn. Eragrostis coarctata Stapf. E. tenella (L.) P. Beauv. E. unioloides (Retz.) Nees ex Steud. E. viscosa Trin. Eriochloa procera (Retz.) C.E.Hubb Phragmites karka Trin. ex Steud. www.tropicalplantresearch.com

Bhattacharjya & Sarma (2016) 3(3): 573–585 . P Rz Cr A Of Cr A Rz Cr A Rz Cr A S,R Ch A S,R Ch A S,R Ch A S,R Ch A S Ch P S, Rz Cr P Tb Ch P Sc, Rz Hm P S, St Ch A S, St Cr A S Cr A S, B Cr A S Cr A S Cr A S, Rs Cr A S,B Cr P S Cr A Tb Cr P Tb Cr A S. Tb Cr A S, Tb Cr P S, Tb Cr A S, Tb Ch A S, Tb Ch A S, Tb Ch A S, Tb Ch A S, Tb Ch A S, Tb Ch A S, Tb Ch A S, Tb Ch A S Cr A S, Tb Hm A S, Tb Ch A S, Tb Hm A S, Tb Hm P S, Tb Ch A S, Rs Cr P S, Tb Cr P S, Tb Cr A S, Tb Hm A S, Tb Hm A S Ch A S,R Hm A S Hm A S,R Hm A S, Tb Hm A S Ch A S Ch A S Ch A S, Rs Hm A S Hm A S, Tb Ch A S, Tb Hm A S, Tb Ch A S, Rz Ch A S Th 578

Bhattacharjya & Sarma (2016) 3(3): 573–585 . Pteridophytes, 7 Angiosperms: Monocot, 63

Angiosperms: Dicot, 147

Figure 1. Plant groups and constituent number of species. Table 2. Enumeration of Pteridophytic weed species and their basic ecological features. (Abbreviations: A: annual, Ch: chamephyte, Cr: cryptophyte, Hm: hemicryptophyte, P: perennial, R: runner, Rs: root stock, Sp: spore)

Family

Species

Selaginellaceae Equisetaceae Dryopteridaceae Thelypteridaceae Marsiliaceae

Selaginella descipiens Warb. Equisetum ramosissimum Desf. ssp. debile Hauke. Diplazium esculentum (Retz.) Swartz. Christella parasitica (L.) Holttum Marsilea minuta L. M. quadrifolia L. Azolla pinnata R.Br.

Azollaceae

Life span A A P P A A A

Method of Propagation Sp Sp, Rs Sp, Rs Sp, Rs Sp, R Sp, R Sp

Life form Ch Ch Hm Hm Cr Cr Cr

Among the angiosperms, 147 species belonging to 104 genera and 40 families fall under dicotyledons, whereas 63 species belonging to 40 genera and 14 families undergo monocotyledons comprising a dicot-monocot ratio of 2:1, 3:1 and 3:1 for species, genera and families respectively. Among the pteridophytes, 7 species could be collected from the study area belonging to 6 genera under 6 families (Table 2). Among all the families (Angiospermic and Pteridophytic), Asteraceae (Dicot) was found to be the largest one comprising 19 genera (12.67 %) followed by Poaceae (Monocot) comprising 11 genera (7.33 %), Fabaceae (Dicot) comprising 7 genera (4.67 %), Scrophulariaceae (Dicot) comprising 6 genera (4.00 %) etc. (Table 3). Regarding species content, the family Asteraceae (Dicot) was found to be the largest comprising 22 species (10.14 %) followed by Cyperaceae (Monocot) comprising 21 species (9.68%), Scrophulariaceae (Dicot) comprising 16 species (7.37 %), Poaceae (Monocot) comprising 15 species (6.91), Fabaceae (Dicot) comprising 12 species (5.53 %) etc. (Table 3). Thus the family Asteraceae has been found to possess highest number of both genera and species and can be regarded richest of all observed families in the agro-climatic zone of the district. Table 3. Different taxa and their constituent numbers in the study area.

Sl. No. Family Angiosperms (Dicotyledons): 1 Nymphaeaceae 2 Papaveraceae 3 Brassicaceae 4 Capparaceae 5 Caryophyllaceae 6 Portulacaceae 7 Hypericaceae 8 Malvaceae 9 Tiliaceae 10 Linaceae 11 Balsaminaceae 12 Oxalidaceae 13 Sapindaceae www.tropicalplantresearch.com

No. of genera

%

No. of species

%

4 1 2 1 3 1 1 3 3 1 1 1 1

2.67 0.67 1.33 0.67 2.00 0.67 0.67 2.00 2.00 0.67 0.67 0.67 0.67

6 1 2 1 4 1 1 4 3 1 1 2 1

2.76 0.46 0.92 0.46 1.84 0.46 0.46 1.84 1.38 0.46 0.46 0.92 0.46 579

Bhattacharjya & Sarma (2016) 3(3): 573–585 . 14

Fabaceae: Mimosoideae Caesalpinioideae Papilionatae 15 Rosaceae 16 Haloragaceae 17 Lythraceae 18 Onagraceae 19 Trapaceae 20 Molluginaceae 21 Apiaceae 22 Rubiaceae 23 Asteraceae 24 Campanulaceae 25 Hydrophyllaceae 26 Boraginaceae 27 Convolvulaceae 28 Solanaceae 29 Scrophulariaceae 30 Lentibulariaceae 31 Acanthaceae 32 Verbenaceae 33 Lamiaceae 34 Amaranthaceae 35 Chenopodiaceae 36 Polygonaceae 37 Euphorbiaceae 38 Urticaceae 39 Cannabinaceae 40 Ceratophyllaceae Angiosperms (Monocotyledons): 41 Hydrocharitaceae 42 Zingiberaceae 43 Pontederiaceae 44 Commelinaceae 45 Typhaceae 46 Araceae 47 Lemnaceae 48 Najadaceae 49 Aponogetonaceae 50 Potamogetonaceae 51 Alismataceae 52 Eriocaulaceae 53 Cyperaceae 54 Poaceae Pteridophytes: 55 Selaginellaceae 56 Equisetaceae 57 Dryopteridaceae 58 Thelypteridaceae 59 Marsiliaceae 60 Azollaceae Total

1 1 5 1 1 3 1 1 2 3 3 19 2 1 2 2 2 6 1 4 2 4 4 1 2 4 1 1 1

4.67 0.67 0.67 2.00 0.67 0.67 1.33 2.00 2.00 12.67 1.33 0.67 1.33 1.33 1.33 4.00 0.67 2.62 2.62 2.67 2.67 0.67 1.33 2.67 0.67 0.67 0.67

1 2 9 1 1 4 3 2 2 4 3 22 2 1 2 3 3 16 1 4 2 5 7 1 10 5 1 1 1

5.53 0.46 0.46 1.84 1.38 0.92 0.92 1.84 1.38 10.14 0.92 0.46 0.92 1.38 1.38 7.37 0.46 1.84 0.92 2.30 3.23 0.46 4.61 2.30 0.46 0.46 0.46

3 1 2 4 1 4 2 1 1 1 2 1 6 11

2.00 0.67 1.33 2.67 0.67 2.67 1.33 0.67 0.67 0.67 1.33 0.67 4.00 7.33

3 1 3 5 1 4 2 2 1 1 3 1 21 15

1.38 0.46 1.38 2.30 0.46 1.84 0.92 0.92 0.46 0.46 1.38 0.46 9.68 6.91

1 1 1 1 1 1 150

0.67 0.67 0.67 0.67 0.67 0.67 100.00

1 1 1 1 2 1 217

0.46 0.46 0.46 0.46 0.92 0.46 100.00

Regarding life span of the species, 183 species (84.33 %) were found to be annual as against 34 (15.67 %) perennial species (Table 1; Fig. 3). Mode of propagation reveals the predominance of seed producing species. One hundred thirty eight (63.59 %) species were found to propagate only by seeds and spores (in case of pteridophytes), 66 species (30.41 %) by both seed/spore and vegetative propagules. Only 13 species (5.99 %) were found to propagate exclusively by vegetative means (Fig. 4). The vegetative propagules were recorded to be runner, sucker, corm, tuber, offset, bulbil, rhizome, stolon etc. (Table 1). www.tropicalplantresearch.com 580

Bhattacharjya & Sarma (2016) 3(3): 573–585 .

Figure 2. A, Acalypha indica; B, Ageratum conyzoides; C, Alternanthera sessilis; D, Amaranthus spinosus; E, Amaranthus viridis; F, Andropogon ascinoidis; G, Cassia tora; H, Commelina benghalensis; I, Cyanotis axillaris; J, Cyperus bulbosus; K, Cyperus iria; L, Echinochloa colonum; M, Eclipta alba; N, Eichhornia crassipes; O, Eleusine indica; P, Eragrostis viscosa; Q, Euphorbia hirta; R, Evolvulus nummularis; S, Ipomea aquatica; T, Leucas plukenetii; U, Ludwigia octavalvis; V, Mikania micrantha; W, Mimosa pudica; X, Oldenlandia diffusa; Y, Persicaria hydropiper; Z, Ricinus communis; Aa, Scoperia dulcis; Ab, Urena lobata.

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Bhattacharjya & Sarma (2016) 3(3): 573–585 . Perennial, 15.67%

Annual, 84.33%

Figure 3. Life span of the species.

Only by vegetative propagules, 5.99%

Both by seeds/spores and vegetative propagules, 30.41% Only by seeds/spores, 63.59%

Figure 4. Propagation method of the species.

The current work was based on extensive explorations of the agro-climatic zone of the district. Out of the 217 species collected from different locations of the district, 23 species (10.60 %) belong to the life-form class Phanerophyte, 34 species (15.67 %) to Chamaephyte, 22 species (10.14 %) to Hemicryptophyte, 47 species (21.65 %) to Cryptophyte and 91 species (41.94 %) to Therophyte (Table 1, 4). The analysis clearly indicates the total deviation of the biological spectrum from the normal spectrum as proposed by Raunkiaer (Table 4). Study reveals the Therophytes to have the highest percentage followed by Cryptophytes and Chamephytes. On the other hand, Hemicryptophytes showed the lowest percentage. Thus, Therophytes are more abundant in the study area of the district and in contrary; the hemicryptophytes are the rare life form in the study area (Fig. 5). Table 4. Comparative Biological spectrum.

No. of species in Percentage distribution of the species among Raunkiaer’s normal each life form different life forms (Observed spectrum) spectrum Phanerophytes (Ph) 23 10.60 46 Chamaephytes (Ch) 34 15.67 9 Hemicryptophytes (Hm) 22 10.14 26 Cryptophytes (Cr) 47 21.65 6 Therophytes (Th) 91 41.94 13 Total 217 100.00 100.00 Life forms

DISCUSSION AND CONCLUSION Agro-climatic zones are rich in weed diversity. Apart from the cultivated crops, the zones are good habitat for a variety of weed species grown throughout the year (Sarma & Bhattacharjya 2006, Bhattacharjya & Sarma 2007, Bhattacharjya & Sarma 2008, Padal et al. 2013, Rana & Masoodi 2013, Dhole et al. 2013, Talukdar 2013). Present study reveals the predominance of annual weed species over the perennial ones. This is due to the anthropogenic activities including various cropping practices, weeding, collecting food and fodder species and overall control on the engineered ecosystem to gain maximum output which may reduce the growth of perennial species. Annual weeds produce very high amount of seeds to ensure propagation and survival. Sufficient amount of small seeds also ensures high probability of dispersal and re-infestation (Shivakumar et al. 2014). Thus in the www.tropicalplantresearch.com

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% of Species

Bhattacharjya & Sarma (2016) 3(3): 573–585 . study area, predominance of seed producing annuals is well marked. The dual method of propagation including seeds and vegetative propagules offer few weed species extra advantages to survive even in the extremes of environmental conditions. 50 45 40 35 30 25 20 15 10 5 0

Observed spectrum

Raunkiaer’s normal spectrum

Life forms Figure 5. Comparison of observed & normal spectrum.

The dominant life forms in biological spectrum of a region indicate the phytoclimate of that region (Yadava & Singh 1977, Dagar & Balakrishnan 1984, Al-Yemeni & Sher 2010, Reddy et al. 2011, Sharma et al. 2014, Thakur 2015, Shahid & Joshi 2015). Since the present observation indicates a higher percentage of the Therophytes, hence, the weed flora of the study area is a therophytic one which, in turn, indicates a therophytic phytoclimate prevailing in the agro-climatic zone of the district. The rich therophytic flora is due to the grazing, weeding or other human interference in the area during the process of cultivation which reduces the number of other life forms. However the human interference has not affected the dominance of the therophytes as they produce a large number of viable seeds which, in turn, are able to establish themselves to continue their generations. Although the Hemicryptophytes are able to withstand various adverse climatic conditions along with the biotic pressure, their lower percentage value (10.14 %) kept them less significant in the area. This is due to the constant human interference in the area through ploughing, hoeing, slashing, burrowing etc. associated with the agricultural practices. It can be concluded that the vegetation of the agro-climatic zone of Nalbari district is mostly seasonal and annual weeds predominate, majority of which continue to survive in subsequent periods by their seeds or vegetative propagules and therefore their presence remains almost unchanged. Agricultural practices, grazing, scrapping by animals, collection of plants for different purposes etc. are the disturbing factors operating in the area which contribute to higher the number of therophytes and overall deviation of the biological spectrum from the normal one. ACKNOWLEDGEMENT Authors are thankful to Mrs. Kaberi Saikia Das, Head, Dept. of Botany, M.C. College, Barpeta (Assam) for her encouragement to conduct the work. REFERENCES Alemu B, Hundera K & Abera B (2015) Floristic composition and structural analysis of Gelesha forest, Gambella regional State, Southwest Ethiopia. Journal of Ecology and the Natural Environment 7(7): 218– 227. Al-Yemeni M & Sher H (2010) Biological spectrum with some other ecological attributes of the flora and vegetation of the Asir Mountain of South West, Saudi Arabia. African Journal of Biotechnology 9(34): 5550–5559. Aye YY, Savent P, Chanin U, Kanita T & Nophea S (2014) Floristic Composition, Diversity and Stand Structure of Tropical Forests in Popa Mountain Park. Journal of Environmental Protection 5: 1588–1602. www.tropicalplantresearch.com

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Bhattacharjya & Sarma (2016) 3(3): 573–585 . Batlaha MA & Martins FR (2004) Floristic, frequency and vegetation life form spectra of a cerrado cite. Brazilian Journal of Biology 64 (2): 203–209. Bhattacharjya DK & Borah PC (2006) Importance of medicinal weeds and role of women in rural health and hygiene: a case study in Nalbari district of Assam. Indian Journal of Traditional Knowledge 7(3): 501–504. Bhattacharjya DK & Sarma SK (2007) Systematic Enumeration of Weeds occurring in Summer Crop-fields: a case study in Nalbari district, Assam. In: Proceeding, National Seminar on “Biodiversity Conservation- the Post-Rio Scenario in India”, Assam University, Silchar. Bhattacharjya DK & Sarma SK (2008) Crop Field Weeds in Nalbari District of Assam, India. Pleione 2(2): 182–189. Bhattacharjya DK, Devi B, Sarma SK & Das S (2006) Edible weeds in crop-fields of Nalbari district, Assam. Journal of Non-timber Forest Products 13(4): 281–286. Braun-Blanquet J (1951) Pflanzensoziologie. Springer Verlag, Vienna. Burju T, Hundera K & Kelbessa E (2013) Floristic Composition and Structural Analysis of Jibat Humid Afromontane Forest, West Shewa Zone, Oromia National Regional State, Ethiopia. Ethiopian Journal of Education and Sciences 8(2): 11–33. Cain SA (1950) Life-forms and phytoclimate. The Botanical Review 16(1): 1–32. Dagar JC & Balakrishnan NP (1984) Life form and biological spectrum of Andaman and Nicobar Islands. Bulletin of Botanical Survey of India 26: 154–159. Dansereau Pierre (1957a) Biogeography: an ecological perspective. The Ronald Press Co., New York, xiii + 394 pp. Dhole1 JA, Lone KD, Dhole, NA & Bodke SS (2013) Studies on weed diversity of Wheat (Triticum aestivum L.) crop fields of Marathwada Region. International Journal of Current Microbiology and Applied Sciences 2(6): 293–298. District portal of Nalbari district, Assam: Office of the Deputy Commissioner, Nalbari, Assam, India. Available from: www.nalbari.nic.in. (accessed: 16 June 2016). Gillespie TW, Brock J & Wright CW (2004) Prospects for quantifying structure, floristic composition and species richness of tropical forests. International Journal of Remote Sensing 25(4): 707–715. Hanson HC & Churchill ED (1961) The plant community. Reinhold Publishing Corp, New York. Available from: http://vikaspedia.in/agriculture/crop-production/weather-information/agro-climatic-zones-in-india (accessed: 22 May 2016). Kershaw KA (1973) Quantitative and dynamic plant ecology (2nd edn.). ELBS and Edward Arnold (Publ) Ltd., London. Mueller- Dombois, D & Ellenberg H (1974) Aims and methods of vegetation ecology. John Wiley & Sons, Inc. New York. Naveed S, Hussain F, Khattak, I & Badsha L (2012) Floristic Composition and Ecological Characteristics of Olea Acacia Forest of Shamshokii District Karak. Global Journal of Science Frontier Research Biological Science 12 (8): 30–36. Padal SB, Sandhya Sri B, Raju Buchi & Rama Krishna B (2013) Floristic Diversity and Indigenous Uses of Dominated Weeds in Maize Crop of Chinthapalli mandal, Visakhapatnam district, Andhra Pradesh, India. IOSR Journal of Agriculture and Veterinary Science 2(6): 56–63. Rana D & Masoodi H (2013) Studies on floristic diversity of an organic farm of Himachal Pradesh, India: Transformation of a barren land to a productive niche. International Journal of Biodiversity and Conservation 5(12): 810–816. Raunkiaer C (1934) The life forms of plants and statistical plant geography. Oxford University Press, Oxford. Reddy CS, Rao KT, Krishna ISR & Javed SMM (2008) Vegetation and floristic studies in Nallamalais, Andhra Pradesh, India. Journal of Plant Sciences 3 (1): 85–91. Saikia P, Choudhury BI & Khan ML (2012) Floristic composition and plant utilization pattern in homegardens of Upper Assam, India. Tropical Ecology 53(1): 105–118. Sarma SK & Bhattacharjya DK (2006) Systematic study of weeds occurring in different winter crop-fields of Nalbari district of Assam. Journal of Assam Science Society 46: 27–31. Sasaki N (2013) Floristic Composition, Diversity and Stand Structure of Tropical Forests in Popa Mountain Park. Journal of Environmental Protection 5: 1588–1602. www.tropicalplantresearch.com

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 586–591, 2016 DOI: 10.22271/tpr.2016.v3.i3.077 Research article

Differential responses of pea seedlings to salicylic acid under UV-B stress Chanda Bano, N. B. Singh* and Sunaina Plant Physiology Laboratory, Department of Botany, University of Allahabad, Uttar Pradesh, India *Corresponding Author: [email protected]

[Accepted: 05 November 2016]

Abstract: In nature, plants are continuously exposed to solar light. They cannot avoid exposure to UV-B radiation. The purpose of this investigation was to examine how UV-B radiation affects seed germination, seedling growth, protein and sugar contents and activities of antioxidant enzymes in pea (Pisum sativum) seedlings. Salicylic acid mediated physiological responses in UVB stressed pea seedlings. UV-B exposure adversely affected germination and physiology of Pisum sativum L. Salicylic acid mitigated the impacts of UV-B stress. Seed germination decreased with increased duration of UV-B exposure. Enhanced activities of antioxidant enzymes in response to UV-B radiation played a protective role against UV-B radiation. Keywords: Antioxidants - Oxidative stress - Pisum sativum L. - UV-B radiation - Salicylic acid. [Cite as: Bano C, Singh NB & Sunaina (2016) Differential responses of pea seedlings to salicylic acid under UV-B stress. Tropical Plant Research 3(3): 586–591] INTRODUCTION Plants are exposed to various abiotic and biotic stresses including solar ultraviolet-B (UV-B) radiation in the natural environment. UV–B radiation is the biggest challenge to life forms on the earth. Over the last few decades degradation of ozone layer leads to enhanced solar UV-B radiation on the earth (McKenzie et al. 2003, Liu et al. 2013). The damage of the ozone layer caused by human activities may result in increased level of ultraviolet radiation reaching the earth surface which is harmful for all living beings including crop plants (Shaukat et al. 2011). UV-B exposure alters biochemical processes in crop plants and affects the morphological parameters (Casati & Walbot 2003, Zu et al. 2010). The exposure of plant to UV-B damages macromolecules viz., nucleic acid, lipid and proteins (Ries et al. 2000, Frohnmeyer & Staiger 2003) decreases photosynthetic rate (Feng et al. 2003) and alters activities of several antioxidant enzymes (Agrawal & Mishra 2009). The UV-B radiation has adverse impact on growth and decreases productivity of crop plants (Shaukat et al. 2011). Plant growth regulators mitigate adverse effects of various environmental stresses. Salicylic acid (SA), a secondary phenolic metabolite, is considered as plant hormone. It is naturally found in plants and acts as a signaling molecule (Davies 2004, Amin et al. 2013). SA plays an important role in regulating the metabolic activities of plants (Davies 2004, Amin et al. 2013). SA application enhances the biomass production and yield in a variety of plants like maize (Amin et al. 2013), wheat (Arfan et al. 2007) under adverse conditions. SA also activates the defense system of plants to protect them from deleterious impact of abiotic stress. Pisum sativum L. (pea) is one of the most economically important pulse crops in India because of its high protein content. Cultivation of pea is common in India due to its ability to fix atmospheric nitrogen. It is used as soil primer for other crops. The aim of the present study was to examine the impact of UV-B exposure on seed germination and growth of pea and the stress responses of SA in UV-B stressed pea seedlings. This study may help regulate the tolerance of abiotic stress like UV-B by plant growth hormone. MATERIALS AND METHODS Seeds and chemical Seeds of Pisum sativum L. cv. Rachana were procured from the Seed Agency at Allahabad, Uttar Pradesh, www.tropicalplantresearch.com

586

Received: 04 August 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.077

Bano et al. (2016) 3(3): 586–591 . India. Salicylic acid (molecular weight 138.121 g.mol-1) was purchased from Loba Chemie Pvt. Ltd., Mumbai, India. Salicylic acid and ultraviolet-B treatments Salicylic acid was prepared by dissolving a requisite amount of SA in 1.0 mL of ethanol and the volume was made 100 mL with double distilled water and used for treatment. Seeds were exposed to UV-B radiation for 15, 30, 60 and 90 min. Fluorescent UV-B tube (TL 40 W/12 Philips, Holland) was used for UV-B irradiation. The UV-B tube was wrapped with cellulose acetate filter (Johnston Industrial Plastics, Toronto, Canada) to avoid all incidents of UV-C (< 280 nm). The UV-B irradiation intensity was measured with the help of power meter (Spectra Physics, USA model 407, A-2). Petri-plate assay Seeds were soaked in two groups each in 100 mL of SA (0.5 mM). The seeds soaked in double distilled water (DDW) for 4 hours, separately. After completion of course of time the seeds of the two groups were further divided into five sets. Out of five, four sets of each group was exposed to UV-B radiation for 15, 30, 60 and 90 min. The complete experimental setup has ten different combinations such as: control (without treatment), SA (0.5 mM), UV-B1 (15 min), UV-B2 (30 min), UV-B3 (60 min), UV-B4 (90 min), UV-B1+SA, UV-B2+SA, UV-B3+SA, UV-B4+SA. Ten seeds of each treatment were placed at equal distance in sterilized petriplates (dia 9 cm, depth 1.5 cm ) lined with double layer of Whatman No. 1 filter papers. Filter papers were moistened with 5 mL of SA for respective treatments and DDW in control and incubated at 28°±2° C for germination in growth chamber. The experiment was conducted in replicate of three. Germination was initiated at 48 hrs after sowing. Germination and seedling growth were recorded till 7 days after sowing at the interval of 24 hours. Radicle and plumule length was recorded with the help of metric scale. Protein content Protein content was determined according to the method of Lowry et al. (1951). The amount of protein was calculated with reference to the standard curve obtained from bovine serum albumin. Sugar content The determination of total soluble sugars (TSS) was done following Hedge & Hofreiter (1962). About 0.1 g radicle was homogenized in 5 mL 95% (v/v) ethanol. After centrifugation, 1 mL supernatant was mixed with 4 mL anthrone reagent and heated on boiling water bath for 10 min. Absorbance was recorded at 620 nm after cooling. The amount of sugar was determined by the standard curve prepared from glucose. Extraction and assay of antioxidant enzymes Fresh sample of radicle (0.25 g) was homogenized with 0.1 M sodium phosphate buffer containing 1% (w/v) polyvinyl pyrrolidone (pH 7.0) in a pre-cooled mortar and pestle. The extract was centrifuged at 4◦ C at 14,000g for 30 min in cooling centrifuge (Remi instruments C 24). The supernatant was used for the assay of antioxidant enzymes viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) activities. The SOD activity (EC 1.15.1.1) was estimated by the nitroblue tetrazolium (NBT) photochemical assay following Beyer and Fridovich (1987). The reaction mixture (4 mL) consisted of 20 mM methionine, 0.15 mM ethylene diamine-tetra acetic acid (EDTA), 0.12 mM NBT and 0.5 mL supernatant. The test tubes were exposed to fluorescent lamps for 30 min and identical unilluminated assay mixture served as blank. One unit of enzyme was measured as the amount of enzyme which caused 50% inhibition of NBT reduction. Catalase (CAT, EC1.11.1.6) activity was assayed following the method by Cakmak & Marschner (1992). Assay mixture (2 mL) contained 25 mM sodium phosphate buffer (pH 7.0), 10 mM H 2O2, and 0.5 mL enzyme extract. The rate of H2O2 decomposition for 1 min was monitored at 240 nm and calculated using extinction coefficient of 39.4 mM− 1cm− 1 and expressed as enzyme unit mg− 1 protein. One unit of CAT was determined as the amount of enzyme required to oxidize 1 mM H2O2 min− 1. Ascorbate peroxidase (APX, EC1.11.1.11) activities were assayed following Nakano & Asada (1981). Assay mixture (2 mL) contained 25 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM ascorbate, 1.0 mM H2O2, and 0.2 mL enzyme extract. H2O2 was the last component to be added. The absorbance was recorded for 1 min at 290 nm (extinction coefficient of 2.8 mM− 1cm− 1). Enzyme specific activity was measured as enzyme unit per one milligram protein as the amount of enzyme required to oxidize 1 mM H2O2 min− 1. www.tropicalplantresearch.com

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Bano et al. (2016) 3(3): 586–591 . Guaiacol peroxidase (GPX, EC 1.11.1.7) activities were assayed following Hemeda and Klein (1990). The reaction mixture (2 mL) contained 25 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.05% (v/v) guaiacol, 1.0 mM H2O2, and 0.2 mL of enzyme extract. The increase in absorbance due to oxidation of guaiacol was monitored at 470 nm. The enzyme activities were measured using extinction coefficient of 26.6 mM− 1cm− 1 and expressed as enzyme unit per mg protein. RESULTS UV-B decreased seed germination in dose dependent manner. However, in combination with SA gradual increase in germination was recorded (Table 1). Maximum 53.84% reduction in seed germination was observed in the pea seedlings exposed in UV-B for 90 min. Growth was measured in terms of radicle and plumule length. UV-B radiation induced impact on growth of pea seedlings was measured and results are presented in tables 1. UV-B significantly declined the radicle and plumule length which was concentration dependent. Maximum 59.11% and 90.66% decrease in radicle and plumule length in the pea seedlings over control was recorded respectively in 90 min single UV-B treatment. Application of SA with UV-B exposure exhibited positive effects on radicle and plumule length of the seedlings. Table 1. Mitigating effects of salicylic acid on UV-B stressed Pisum sativum L.

Plumule length Protein Sugar (cm) (mg.g-1 FW) (mg.g-1 FW) C 97.5±0.21a 9.05±0.31b 3.75±0.02b 21.47±0.05b 29.66±0.26b S 100.0±0.23a 10.3±0.57a 4.4±1.0a 25.80±1.48a 32.80±0.29a UV1 82.5±0.33b 7.85±0.20c 2.05±0.60bc 16.34±0.36c 28.20±0.53bcd UV2 72.5±1.43d 7.1±0.17c 1.65±0.83c 9.33±1.48d 26.33±0.26de UV3 57.5±1.33f 5.2±0.05de 1.25±0.54de 6.03±0.014e 24.10±0.27fg UV4 45.0±0.76g 3.7±0.34f 0.35±0.14e 4.23±0.014f 20.67±0.26h UV1+S 85.0±0.32b 8.1±0.41c 3.4±0.28abc 19.34±1.45b 29.16±0.02bc UV2+S 77.5±1.43c 7.85±0.02c 2.6±0.80cd 10.66±1.47d 27.64±0.08cde UV3+S 62.50±1.3e 6.1±0.23d 2.7±0.11cd 7.95±0.34e 25.72±0.20ef UV4+S 55.0±0.67f 4.35±0.54ef 0.55±0.25e 5.75±0.08f 22.59±0.32g Note: Mean±SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n =3. C= control, S= 0.5mM concentration of salicylic acid, UV 1= 15, UV2 = 30, UV3 = 60 and UV4 = 90 min treatment of ultraviolet-B radiation, UV1+S, UV2+S, UV3+S and UV4+S are combined treatments of UV-B and salicylic acid.

Treatments

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The results pertaining to protein and sugar in seedlings are depicted in table 1. The increase in duration of UV-B radiation from 15 min to 90 min caused progressive decrease in protein and sugar contents. A steep decline of 80.29% protein and 30.33% sugar in pea was recorded following 90 min of UV-B exposure. The seedlings showed significant increase in activities of antioxidant enzymes viz., SOD, CAT, APX and GPX following UV-B exposure from 15 to 90 min (Fig. 1). The seedlings exposed to UV-B alone exhibited an increase in the activities of SOD, CAT APX and GPX by 59.78%, 88.76%, 81.56% and 62.80% respectively over the control. Simultaneous treatment with SA and UV-B caused significant improvement in the activities of SOD, CAT, APX and GPX. SA manifested positive impact on UV-B stressed seedlings to avoid oxidative damage caused by UV-B exposure. DISCUSSION UV-B radiation is one of the important abiotic stress factors manifesting significant impact on growth and physiological processes in plants (Mishra et al. 2009, Dwivedi et al. 2015). This problem is aggravated due to further increase in UV-B radiation in troposphere. Previously it has been demonstrated that SA priming reduced susceptibility towards different kinds of abiotic stresses by modulating defense system (Horva´th et al. 2007) Decline in seed germination under UV-B exposure was reported (Shaukat et al. 2011).Our results are in agreement with the previous findings of Shaukat et al. (2011) on Helianthus annuus. SA in low doses significantly increased seed germination in Arabidopsis under different abiotic stresses (Rajjou et al. 2006). The positive effect of SA on seed germination under abiotic stress is due to decreased level of oxidative damage (Peykarestan et al. 2012) and it also initiates translation and elongation factors, proteases and two subunits of the 20S proteasome which consequently revamp seed germination by encouraging protein synthesis that are indispensable for seed germination and the movement or protein breakdown gather during the course of seed www.tropicalplantresearch.com

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Figure 1. Mitigating effects of salicylic acid on antioxidant enzyme activities of UV-B stressed Pisum sativum L. Mean±SE values followed by same letters within each column are not significantly different at 0.05 (ANOVA and Duncan’s multiple range test), n =3. C= control, S= 0.5mM concentration of salicylic acid, UV1= 15, UV2 = 30, UV3 = 60 and UV4 = 90 min treatment of ultraviolet-B radiation, UV1+S, UV2+S, UV3+S and UV4+S are combined treatments of UV-B and salicylic acid.

maturation and also in the synthesis of various enzymes used in several metabolic pathways like gycolysis, glyoxylate cycle, pentose phosphate pathway, and gluconeogenesis (Rajjou et al. 2006). The results of the present study showed that UV-B alone decreased growth of pea seedlings in dose-dependent manner. The declined growth in Helianthus annuus exposed to UV-B is reported (Shaukat et al. 2011). In contrast, SA priming of seedlings /plants ameliorated the toxic effect of UV-B and promoted growth as compared to the pea seedlings exposed to UV-B treatments alone. SA pretreatment may mask impact of the UV-B response of the seedlings by modulating the antioxidant system. UV-B induced reduction in protein content of pea at higher duration of UV-B exposure may be linked with UV-B induced disturbance in protein synthesis. Like protein content we recorded reduced sugar content under UV-B stress. Seed germination and early development of seedlings exploit their storage food material (Rosa et al. 2009) and when plants are exposed to stress, some protective mechanisms can also form in plants and it requires energy and resources and the resource of energy in plants is mostly sugar (Ho 1988). Similar to our results decreased sugar content observed in maize (Correia et al. 2005) Different metabolic pathways in plants continuously produced reactive oxygen species as byproduct. Plants exposed to increased UV-B radiation induced ROS accumulation which includes not only free radicals such as superoxides but also hydrogen peroxide and singlet oxygen which caused oxidative damage to nucleic acid and proteins (Foyer et al. 1994) and metabolic system. The plants have efficient antioxidative defense system to mitigate the harmful impact of oxidative stress induced by UV-B stress. The plant defense system provides protection against free radicals and reactive oxygen species.SA pre-treatment alleviates the adverse effect of UV-B by modulating activity of antioxidant (Horva´th et al. 2007). The activities of antioxidant enzymes induced by UV-B exposure varied with plant species. Generally, activities of antioxidants like SOD, CAT, APX and GPX were found to be increased by UV-B radiation (Mishra et al. 2009, Dwivedi et al. 2015). SA treatment inhibits oxidative damage by modulating antioxidant enzymes activity and detoxifies superoxide www.tropicalplantresearch.com

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Bano et al. (2016) 3(3): 586–591 . radicals (Farooq et al. 2008). SOD present in different part of cell acts as first line of defense in plant. It acts first on free radicals and converts to hydrogen peroxide, CAT in peroxisomes and APX in the cell as whole have potential to convert hydrogen peroxide into water and oxygen (Noctor & Foyer 1998). Kondo and Kawashima have also demonstrated the enhanced antioxidant activity to UV-B exposure (Kondo & Kawashima 2000). The growth of plant is the result of equilibrium between ROS produced and antioxidant enzymes induced. Enhanced activities of antioxidant evinced the plant tolerance against UV-B radiation. Several studies reported the effect of UV-B and salicylic acid on plants but here we studied the effect of UV-B and salicylic acid on seed germination and metabolic activity of pea seedlings. CONCLUSION The present study showed that UV-B exposure exhibited adverse effects on seed germination, seedlings growth of Pisum sativum and their defence system. The seed priming with salicylic acid, as plant growth regulator, in appropriate concentration alleviate the adverse effects of UV-B radiation in pea seedlings. ACKNOWLEDGMENTS The authors are thankful to the UGC, New Delhi and University of Allahabad, Allahabad for providing financial assistance to Chanda Bano. REFERENCES Agrawal SB & Mishra S (2009) Effects of supplemental ultraviolet-B and cadmium on growth, antioxidant and yield of Pisum sativum L. Ecotoxicology and Environmental Safety 72: 610–618. Amin AA, El-Kader AAA, Shalaby MAF, Gharib FAE, Rashad ESM & da Silva JAT (2013) Physiological effects of salicylic acid and thiourea on growth and productivity of maize plants in sandy soil. Communication in Soil Science and Plant Analysis 44: 1141–1155. Arfan M, Athar HR & Ashraf M (2007) Does exogenous application of salicylic acid through the rooting medium modulate growth and photosynthetic capacity in differently adapted spring wheat cultivated under salt stress? Journal of Plant Physiology 6: 685–694 Beyer WF & Fridovich I (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Analytical Biochemistry 161: 559–566. Cakmak I & Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiology 98: 1222–1227. Casati P & Walbot V (2003) Gene expression profiling in response to ultraviolet radiation in maize genotypes with varying flavonoid content. Plant Physiology 132: 1739–1754. Correia CM, Pereira M, Coutinho JF, Björn Lars O, José MG & Torres-Pereira (2005) Ultraviolet-B radiation and nitrogen affect the photosynthesis of maize: a Mediterranean field study. European Journal of Agronomy 22: 337–347. Davies PJ (2004) The plant hormones: their nature, occurrence and function. In: Davies PJ (ed) Plant Hormones: Biosynthesis, Signal Transduction, Action, 3rd ed. Kluwer Academic Publishers, Dordrecht, pp. 1–15. Dwivedi R, Singh VP, Kumar J & Prasad SM (2015) Differential physiological and biochemical responses of two Vigna species under enhanced UV-B radiation, Journal of Radiation Research and Applied Science 8: 173–181. Farooq M, Aziz T, Basra SMA, Cheema MA & Rehman H (2008b) Chilling tolerance in hybrid maize induced by seed priming with salicylic acid. Journal of Agronomy and Crop Science 194: 161–168. Feng H, An L, Chen T, Qiang W, Xu S, Zhang M, Wang X & Cheng G (2003) The effects of enhanced ultraviolet-B radiation on growth, photosynthesis and stable carbon isotope composition (13C) of two soybean cultivars (Glycine max) under field conditions. Environmental and Experimental Botany 49: 1–8. Foyer CR, Lelandais M & Kunert KJ (1994) Photooxidative stress in plants. Physiologia Plantarum 92: 696– 717. Frohnmeyer H & Staiger D (2003) Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection. Plant Physiology 133: 1420–1428. www.tropicalplantresearch.com

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Bano et al. (2016) 3(3): 586–591 . Hedge JE & Hofreiter BT (1962) Estimation of carbohydrate. In: Whistler RL & BeMiller JN (eds) Methods in Carbohydrate Chemistry. Academic Press New York, pp. 17–22. Hemeda HM & Klein BP (1990) Effects of naturally occurring antioxidants on peroxidase activity of vegetable extracts. Journal of Food Science 55: 184–185. Ho LC (1988) Metabolism and compartmentation of imported sugars in sink organs in relation to sink strength. Annual Review in Plant Physiology and Plant Molecular Biology 39: 355–79. Horva´th E, Szalai G & Janda T (2007) Induction of abiotic stress tolerance by salicylic acid signaling. Journal of Plant Growth Regulation 26: 290–300. Kondo N & Kawashima M (2000) Enhancement of the tolerance to oxidative stress in cucumber (Cucumis sativus L.) seedlings by UV-B irradiation: possible involvement of phenolic compounds and antioxidative enzymes. Journal of Plant Research 113: 311–31. Liu B, Liu XB, Li YS & Herbert SJ (2013) Effects of enhanced UV-B radiation on seed growth characteristics and yield components in soybean. Field Crops Research 154: 158–163. Lowry OH, Rosenbrough RJ, Farr AL & Randall RJ (1951) Protein measurement with Folin phenol reagent. Journal of Biological Chemistry 193: 265–275. McKenzie RL, Björn LO, Bais A & Ilyas M (2003) Changes in biologically active ultraviolet radiation reaching the earth's surface. Photochemistry and Photobiological Science 2: 5–1. Mishra V, Srivastava G & Prasad SM (2009) Antioxidant response of bitter gourd (Momordica charantia L.) seedlings to interactive effect of dimethoate and UV-B irradiation. Scientia Horticulturae 120: 373–378. Nakano Y & Asada K (1981) Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiology 22: 867–880. Noctor G & Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Annual Review in Plant Physiology and Molecular Biology 49: 249–279. Peykarestan B, Seify M, Fadaei MS & Hatim M (2012) UV Irradiation Effects on Seed Germination and Growth, Protein Content, Peroxidase and Protease Activity in Portulaca grandiflora and Portulaca oleracea, World Applied Science Journal 17 (7): 802–808. Rajjou L, Belghazi M, Huguet R, Robin C, Moreau A, Job C & Job D (2006) Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. Plant Physiology 141: 910–923. Ries G, Heller W, Putchta H, Sandermann H, Seidlitz HK & Hohn B (2000) Elevated UV-B radiation reduced genome stability in plants.Nature 406: 98–101. Rosa M, Prado C, Podazza G, Interdonato R, González JA, Hilal M & Prado FE (2009) Soluble sugarsMetabolism, sensing and abiotic stress A complex network in the life of plants. Plant Signaling and Behaviour 4(5): 388–393. Shaukat SS, Zaidi S & Khan MA (2011) Effect of supplemental UV-B radiation on germination, seedling growth, and biochemical responses of sunflower (Helianthus annuus L.). Fuuast Journal of Biology 1(1): 27–33. Zu YG, Pang HH, Yu JH, Li DW, Wei XX, Gao YX & Tong L (2010) Responses in the morphology, physiology and biochemistry of Taxus chinensis var. mairei grown under supplementary UV-B radiation. Journal of Photochemistry and Photobiology 98: 152–158.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 592–599, 2016 DOI: 10.22271/tpr.2016.v3.i3.078 Research article

Exotic species invasion threats to forests: A case study from the Betla national park, Palamu, Jharkhand, India Preeti Kumari and A. K. Choudhary* Department of Botany, Ranchi University, Ranchi-834002, Jharkhand, India *Corresponding Author: [email protected]

[Accepted: 08 November 2016]

Abstract: Exotic Species Invasion (ESI) cause a little recognized, but very substantial impact to forest ecosystems worldwide. Climatic variability, physiographic range, increasing trade, travel and tourism have accelerated the spread of unwanted non-native species to conservation areas, making vulnerable to the establishment of ESI. Exotic Invasive Plants (EIPs) are known to displace native plants, alter ecosystems processes, hydrology, primary productivity, nutrient cycling and soil structure and most importantly reduce native biodiversity. There is evidence to suggest that the threats due to ESI may increase with climate change and associated changes in habitats. In this paper, we assess the threat of EIPs to natural forests in Betla National Park (BNP), Palamu in Jharkhand State, India. Based on intensive field surveys and using quadret method we identified 142 EIPs in the BNP forest. 21 plots of 20 × 20 m for trees, 5 × 5 m for shrubs and 1 × 1 m for herbs were laid randomly adjoining the forest at 10 to 100 m distance from the road and settlement area. Total 14 EIPs were recorded among which Lantana camara and Parthenium hysterophorus were found to be the most dominant species. . The survey revealed that apart from the ecological harm, invasive plants adversely affect the livelihood of all those who are dependent on forests. The paper identifies impact, early detection and rapid control, prevention of spread and habitat restoration as urgent measures for combating the threats. Keywords: Exotic invasive plants - Risk assessment - BNP forest - Climatic change impact. [Cite as: Kumari P & Choudhary AK (2016) Exotic species invasion threats to forests: A case study from the Betla national park, Palamu, Jharkhand, India. Tropical Plant Research 3(3): 592–599] INTRODUCTION Exotic Species that become invasive are considered to be main direct drivers of biodiversity loss across the globe. Management of Exotic Species Invasion (ESI) is seen as major challenge in the field of biodiversity conservation. ESI, the non-native species threaten ecosystems, destroy habitats and create problems to other native species through invasion. It is considered as the second greatest agent of species endangerment and extinction. The ecological cost is often the irretrievable loss of native species and ecosystems. It also causes heavy economic loss, in terms of reduced crop and livestock production, reduced native biodiversity, increased production costs and so forth. Biodiversity has become one of the most popular topics for discussion at local, national and global level. Biodiversity entails all forms of biological entities inhabiting the earth including prokaryotes–wild plants and animals, microorganisms, domesticated animals and cultivated plants and even genetic material like seeds and germ-plasma (Kothari 1993). Exotic Invasive Species (EIS) are species, native to one area or region, that have been introduced into an area outside their normal distribution, either by accident or on purpose and which have colonized or invaded their new home, threatening biological diversity, ecosystems and habitats, and human wellbeing (CBD 2000). Biological invasion worldwide threatens biodiversity, ecosystem dynamics, resource availability, national economy and human health studied by (Ricciardi et al. 2000). The spread of EIS is now recognized as one of the greatest threat to the ecosystem. Exotic invasive species (animal pests, viruses, pathogens and plants) have become one of the most serious threats to the ecological and economic well-being of every habitat and region on the Earth (Boy et al. 2013). The introduction of alien species to a new location can either be accidental or intentional studied by (Enserink 1999). Accidental www.tropicalplantresearch.com

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Received: 06 August 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.078

Kumari & Choudhary (2016) 3(3): 592–599 . introductions are helped by travel across countries and continents and import of various items such as timber, food grains, fodder etc. (Shimono et al. 2008). Intentional introductions are for a variety of purposes such as agriculture, horticulture, forestry and ornamental (Cremer 2003). All invasive species possess certain biological attributes which contribute to their success as invaders in a new habitat. For invasive alien plants (IAPs), these attributes include production of a large number of easily dispersible, light weight seeds, fast growth rate and better competitive resource capture and utilization abilities compared to native plants (Burns 2006). The economic damage due to ESI is estimated to be to the tune of 1.4 trillion dollars globally. In many countries, the overall loss due to invasions is over 1% of the GDP. In the United States alone, for example, the annual costs of containing the spread of IAS are reported to be more than US$ 135 billion (Boy et al. 2013). The impacts of IAPs include displacement of native plant species, change of soil chemical profile, rewarding pollinators better than the native species thereby reducing the reproductive success of local species, changing hydrological regimes, making the new habitat fire prone and limiting the photosynthetic efficiency of the local species by reducing light availability (Nilsson & Grelsson1995). Subsequent impacts would happen by reduced availability of forest resources like medicinal plants from natural forests and timber from forest plantations. As in the classical case of Kaziranga National Park (Assam, India) where in the movement of the endangered one horned rhinoceros was limited by thickets of Mimosa diploticha var. diplotricha, the impact on fauna would also be critical studied by (Vattakkavan et al. 2002). Indirect impacts occur by way of complete elimination of food plants of the fauna and by making the habitat fire prone. It was believed that the threat of IES would be much low in natural habitats as compared to disturbed habitats. Forests were considered to be immune to large scale plant invasions. Climate change and the emergence of invasive alien plant species (IAPS), which are commonly referred as weeds, are two of the greatest threats to biodiversity and ecosystem services (Burgiel & Muir 2010, IUCN 2000) defines IAPS as plants that have become established in natural or semi-natural ecosystems or habitats, an agent of change, and threatens native biological diversity. A study of (IPCC 2007) identified that climate change is one of the factors for emergence of invasive species. Increase in atmospheric temperatures and CO2 concentrations are likely to increase opportunities for the introduction of invasive species because of their adaptability and ability to disturb a broader range of bio geographic conditions and environments (Mooney 2000). A study by (Lodge et al. 2006) showed that IAPS endanger the environment, the economy and human welfare. It also reduces biodiversity, replaces important native species and increases investment in agriculture and silviculture operations (Ricciardi et al. 2000) and disrupts prevailing vegetation dynamics and nutrient cycling (Richardson & Higgins 1998). The estimated damage from IAPS worldwide totals more than US $1.4 trillionna year (5 percent of the global economy). Impacts affect a wide range of sectors including agriculture, forestry, aquaculture, transportation, trade, energy and recreation (Stern 2006). The prime objective of the present paper is to report the Exotic Species Invasion (ESI) Threats to Forests in Betla National Park (BNP) Palamu, Jharkhand, India. We have also reported effect of ESI on different plant species, environment and different ecosystem etc. In addition to the above the preventive measures of ESI of National Park have been also studied. MATERIALS AND METHODOLOGY Study Area Betla National Park (BNP) situated between latitude 23°25' N to 23°55' N and longitude 83°50' to 84°36' E, was notified in 1973 as one of India’s first nine tiger reserves established under Project Tiger . It is located in the western part of the Chhotanagpur Plateau and spans an area of 1129.93 km2 comprising the Palamau Wildlife Sanctuary and Betla National Park is spread over Latehar, Palamau and Garhwa District in Jharkhand (Fig. 1). It is also part of the Central India Landscape and extends into the Sanjay-Dubri Tiger reserve and Achanakmar-Kanha tiger landscape through the Jashpur and Mahan forest of Chhattisgarh. The vegetation types mainly categorized as dry moist forest, dry Sal forest, moist Sal forest, high level plateau Sal forest and moist forest. BNP is also becoming home to many unwanted non-native plants. Very limited research has been carried out about ESI in BNP. Data collection Questionnaire survey was carried out from October 2015 to June 2016. Total 140 individuals who have long been inhabited in the study area and utilizing the local resources for their livelihood were interviewed to explore their perception regarding ESI. Total 21 sampling plots of various sized quadrates 20 × 20 m for trees, 5 × 5 m www.tropicalplantresearch.com

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Kumari & Choudhary (2016) 3(3): 592–599 . for shrubs and 1 × 1 m for herbs were laid at 10 to 100 m distance in adjoining forest from road and human settlement area. Nested plots 5 × 5 m and 1 × 1 m quadrates were allocated randomly in two corners of 20 × 20 m plot (Tiwari et al. 2005, Bajpai et al. 2015). Community consultations, individual interviews, field observations, literature review, group discussions were conducted to collect data.

Figure 1. Study Area of Betla National Park, Palamu, Jharkhand, India.

Data analysis Both quantitative and qualitative techniques were used for data analysis. By using following method density and frequency were calculated. The analysis was interpreted in a simple and understandable chart form. Density is an expression of numerical strength of a species in a community studied by using formula (Mishra 1968). ( ) Frequency denotes the number of sampling unite in which, a particular species occurs and so it expresses the distribution of dispersion of various species in community. ( ) RESULTS AND DISCUSSION ESI in study area All together 14 EIPs were encountered in the sampled areas (Table 1). Among the ESI observed, Lantana camara & Parthenium hysterophorus was most common in the study area. It has the highest cover in the adjoining forest, near settlement where human disturbances were high. Based on household survey Lantana camara & Parthenium hysterophorus was found to be the most problematic ESI. An area where tree canopy is dense and the undergrowth do not find sufficient sunlight, invasion of species is low compared to open and degraded land. Therefore, with increasing tree canopy there is decreasing invasion of unwanted species. After Lantana camara & Parthenium hysterophorus, Agertum houstonianum and Ipomoea purpurea, Ipomoea hederfolia were other to ESI found with high density in the study area. The spread of EIPs especially Perthanium hysterophorus complex and is threatening both the natural biological richness and livelihood of inhabitants. Many locals have stopped grazing their livestocks in forest as the palatable grasses in the forest like Imperata cylindrical, Cynodon dactylon are rapidly being replaced by the ESI especially by Perthanium hysterophorus. www.tropicalplantresearch.com

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Kumari & Choudhary (2016) 3(3): 592–599 . Table 1. Exotic Species Invasion (ESI) Observed in Betla National Park, Palamu, Jharkhand, India.

Specie Alternanthera brasiliana (L.) Kuntze Alternanthera philoxeroides (Mart.) Griseb. Amaranthus spinosus L. Ipomoea purpurea (L.) Roth Ipomoea hederifolia L. Jatropha gossypifolia L. Lantana camara L. Leucaena leucocephala (Lam.) de Wit Merremia vitifolia (Burm.f.) Hallier f. Mimosa pudica L. Parthenium hysterophorus L. Prosopis juliflora (Sw.) DC. Sphagneticola trilobata (L.) Pruski Synedrella nodiflora (L.) Gaertn.

Introduction Intentional Unknown Accidental Intentiona International Intentional Intentional Intentional Accidental Intentional Accidental Intentional Intentional Accidental

Purpose Ornamental Unknown NA Ornamental Ornamental Hedge plant Ornamental Social forestry NA Ornamental NA Fire wood Ornamental NA

Habitat Subshrub Herb Herb Climber Climber Shrub Shrub Tree Climber Herb Herb Tree Herb Herb

Origin Central & South America South America South & Central America Central America Cenral America Tropical America Central & South America Mexico & Central America South Asia Tropical America North & South America Central & South America South America & West Indies Tropical America

Effect of Exotic Species Invasion Effects on ecosystem After studies we found that invasive exotic plant species (IEPS) threaten the environment, reduce biodiversity, replace economically important plant species and increase the investment in agriculture and silviculture practices, prevail vegetation dynamics and alter nutrient cycling (Richardson & Higgins 1998). They can promote hazards like forest fire. Plant invasions dramatically affect the distribution, abundance and reproduction of many native species (Sala et al. 1999). In the study area too, impacts of Invasive Exotic Plant species especially Parthenium hysterophorus was well observed and none of the ecosystems were free from their impact. Edges of forests, agricultural lands and wetlands have been severe IEPS intrusion. Although all ecosystems are susceptible to invasion, ecosystems entwined with higher level of human interventions (e.g. forestry, agriculture, wetland and rangelands) are likely to pose greater susceptibility (Yelenik et al. 2007). In the study site, rangeland, agriculture land as well as fallow lands and roadsides were highly susceptible to invasion of Ipomoea purpurea (L.) & Ipomoea hederifolia L. IEPS (Fig. 2). Both IEPs Lantana Perthanium

20 18

Number of site observed

S.N. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

16 14 12 10 8 6 4 Forest

Agriculture Grazing land Wetland Road side Different types of ecosystem Figure 2. showing impact of invasive exotic plant species on important ecosystem.

Effects on Forest As per surveys revealed that the forest, roadsides and fall low lands previously dominated by Alternanthera, Ipomoea, Jatropha, Lantana etc. were invaded by IEPS. Some plants failed to maintain their biomass in the changing climate and their dominance was fairly slacked off by IEPS. As a result, dense and diverse forests are www.tropicalplantresearch.com

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Kumari & Choudhary (2016) 3(3): 592–599 . more resistant to ecological invasion (Pimm 1984). However, forests in studied area has invaded about 20% area particularly forest edges and plantation forests of the studied ecosystem. These species suppress the growth of native trees, shrubs and grasses growing beneath or close to them. Subsequently foliage for wildlife animals is reduced leading to starvation and death of the animals. Effects on human ecosystem The full costs of invasions also include the social and health impacts of exotic invasive species on humans, in particular to the rural communities depending on forests. As a result of the impacts of exotic invasive species on native forest biodiversity, a loss of food sources and traditional medicines may be experienced thereby compromising not only the health of local people but also the livelihoods of those dependent on the collection and sale of such items for income. For small-scale landowners, exotic invasive species can also decrease the value of their land. Forest workers, as part of their jobs, and people living in and around forests are more exposed to exotic invasive species such as the reservoirs and hosts of many emerging infectious diseases. Examples of such diseases include Lyme disease, hay fever also known rhinitis, some immune disorders, eczema, Ebola and Marburg hemorrhagic fevers, malaria, yellow fever, leishmaniasis, trypanosomiasis and Kyasanur forest disease. People living in and around invaded forest areas may also suffer allergic or other negative reactions to the exotic invasive species themselves or to the measures used to control them such as chemical and biological pesticides. A commonly planted tree for land restoration and as a source of forest products, mesquite (Prosopis juliflora) is a major cause of allergies in India. Sensitivity to mesquite pollen has been shown to result in asthma, rhinitis and conjunctivitis. In some places, children living close to areas infested with Dendrolimus sibiricus have experienced significant allergic reactions to the hairy caterpillars that have entered their homes. The hairs on larvae and egg masses of gypsy moth (Lymantria dispar) also cause allergies in some people. All these invasive plants and trees have had serious socio-economic impacts and ultimately increased poverty in the local communities. Some area of the study site is densely populated by subsistence farmers and livestock rearing is an integral part of their livelihood. 30

PNS

PIS

25 20 15 10 5 Achyranthus aspera Anagallis arvensis Ageratum conyzoides Allium cepa Alternanthera paronichyoides Argemone mexicana Blumea sp. Borreria stricta Cajanus cajan Chrysophogon aciculatus Commelina benghalensis Cynodon dactylon Desmodium triflorum Dichanthium sp. Dichanthium sp2 Digitaria ciliaris Dolichos lablab Heteropogon contortus Leucas aspera Lindernia crustata Parthenium hysterophorus Raphanus sativus Solanum surratense Tridax procumbans Triticum astevum Vernonia cinneria Xanthium strumarium Zea mays

0

Figure 3. Reperesenting the comparative account of parthenium (exotic species invasion) non invaded and invaded study sites.

Impacts on Wetland and Rangeland Ecosystems P. hysterophorus and I. purpurea is distributed throughout the study site; it can be just in any type of soil and environment. Nearby wetlands and throughout the rangeland is the most favourable environment for the www.tropicalplantresearch.com

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Kumari & Choudhary (2016) 3(3): 592–599 . invasion of invasive species. Apart from those species, water bodies of the study area were invaded by Jalkumbhi (Pistia stratiotes L.) and unwanted water favoring plants. I. purpurea mostly prefer damps areas such as wetland margins, drainage lines and gullies. Impact on Agricultural Ecosystem IEPS are also considered biological polluters and are capable of hybridizing with native plant relatives that result in genetically modified to a plant’s genetic make-up results into great peril of biodiversity, which has also same impact in the study site. Parthenium hysterophorus has adverse impacts on most of the agricultural crops as nutrients and fertilizers supplied to the main crop are being exploited by this species (Fig. 3). Agricultural crops, particularly ginger, millet, rice and grasses, were outcompeted by others and their productivity declined. Reduction in production of cereals and grasses in studied area as a result of invasion by Lantana, Perthanium. According to (Oerke et al. 1995) there was a loss of 13 percent of agricultural outputs due to weeds. Many grasses species, such as Artemisia spp, Solanum xanthocarpum and Urtica sp. of fallow lands and Scrophularia species, Hypoxis aurea etc. of agricultural lands were threatened by invasion of Perthaniums, Mimosa, and Sphagneticola invasive exotic plant species in the study site. Impact of P. hysterophorus on livestock is more severe in the study site. There are a number of livestock mortalities, particularly of buffaloes. The cases generally happened in the spring when the plant flowers are in full bloom. Similarly, IEPS affect the dynamics and composition of soil and have impacts on ecosystem functions, such as soil nutrient cycling and soil chemistry. In the study site, IEPS were growing in a wide range of soils but not flourishing in shade. Soil texture in agriculture land of the study area is silt clay. Methods to control of Exotic Species Invasion The following steps are proposed to manage existing EIPs and prevent any new incursions. Preventive measures This study revealed the presence of invasion exotic plants in all the forest areas surveyed. It also showed direct and indirect impacts due to these invasions. It is recommended that a more comprehensive forest surveillance covering all the forest divisions in the State needs to be carried out before evolving proper control strategies. To prevent new incursions of ESI into forests, the following steps are to be adopted: i. All plants, plant prop gules and soil intended for transportation into forest areas (soil for civil works, seedlings of forest tree species) should be thoroughly monitored for the presence of seeds and other prop gules of ESI. ii. Import of seeds, seedlings and other prop gules of all plant species should be done only after risk assessment and observing proper quarantine procedures. iii. Forest areas, especially those which are tourist destinations, need to have water filled dips at the entry point so as to wash agricultural implements and tires of vehicles free of ESI propagules before entering into forest areas. Early detection and rapid control The most economical way to contain ESI is to establish an efficient surveillance system so as to detect ESI soon after their arrival and eradicate them when their population is small and the spread is limited. To achieve this, sea ports, airports and tourist and pilgrimage routes into forest areas are to be monitored regularly for new invasive species using proper tools and methods. The staff of quarantine/customs and forest department need be trained to identify ESI which are potential threats so as to adopt measures to stop incursions and contain the population. Prevention of spread For ESI which have already established in some areas and immediate eradication is difficult, efforts should be focused on preventing their spread by: 1) Restricting the movement of soil and plant parts from infested areas to un-infested areas and 2) Removing the weeds manually or mechanically (cutting or pulling) before flowering and fruiting and burning them at the site. Habitat restoration Manual/mechanical control may be difficult, costly and unsustainable for exotic weeds which have established in large areas. In such cases, systematic restoration strategies should be taken up. To achieve this, www.tropicalplantresearch.com

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Kumari & Choudhary (2016) 3(3): 592–599 . remove the weeds manually or mechanically (pulling along with roots/tubers) in small areas at a time and subsequently plant the area with fast growing native species. Assisted regeneration may also be attempted in such areas. CONCLUSION Exotic Species Invasion (ESI) is a serious threat to forests of Jharkhand since they impact heavily on native biodiversity, productivity and result in landscape level changes. In this observation we have found that total 14 IEPs in covered area and some species like Lantana, Perthanium and Ipomoea purpurea are highly dominated over native resources. The invasion is more serious in agriculture land followed by disturbed sites, such as newly constructed earthen roads. The invasion is seriously affecting agriculture productivity. A few households are using these species for making compost manure and producing bio-briquettes for energy. Promoting biobriquette production and market linkage would help to improve the health condition of different ecosystems and provide additional income to local community. Furthermore, it supports controlling forest degradation and loss of biodiversity. The problem demands urgent attention. Prevention of new incursions can be achieved by adopting risk assessments before import of plants and planting material. Forest surveillance, early detection and rapid control, manual/mechanical removal of weeds followed by planting of native species and assisted regeneration are suggested as immediate steps to control invasion and reduce impacts. Herbicidal application in forest areas need to be avoided as far as possible. REFERENCES Bajpai O, Kushwaha AK, Srivastava AK, Pandey J & Chaudhary LB (2015) Phytosociological status of a monotypic genus Indopiptadenia:A Near Threatened Tree from the Terai-Bhabar Region of Central Himalaya. Research Journal of Forestry 9(2): 35–47. Boy G & Witt A (2013) Invasive alien plants and their management in Africa. In: Synthesis Report of the UNEP/GEF Project ‘Removing barriers to Invasive Plant Management in Africa (RBIPMA)’, CABI, Africa. Burgiel SW & Muir A (2010) Invasive Species, Climate Change and Ecosystem-based Adaptation: Addressing Multiple Drivers of Global Change, GISP, Washington DC, USA and Nairobi, Kenya. Burns JH (2006) Relatedness and environment affect traits associated with invasive and non-invasive introduced Commelinaceae. Ecological Applications 16: 1367–1376. CBD (Convention on Biological Diversity) (2002) D ecision VI/23: Alien species that threaten ecosystems, habitats or species (Endnote I). Secretariat of the Convention on Biological Diversity, Montreal. Cremer K (2003) Introduced willows can become invasive pests in Australia. Biodiversity 4: 17–24. Enserink M (1999) Biological invaders sweep. Science 285(5435): 1834–1836. IPCC (2007) Climate Change 2007: Impacts, Adaptation, and Vulnerability. Working Group II Contribution to the Intergovernmental Panel on Climate Change. Fourth Assessment Report. Summary for Policy Makers, IPCC, Geneva, Switzerland IUCN (2000) IUCN Guidelines for the Prevention of Biodiversity Loss due to Biological Invasion. IUCN, Gland, Switzerland. Kothari A (1993) Biodiversity conservation: for those vanishing species. Survey of environment, The Hindu, Madras. Lodge DM, Williams S, MacIsaac HJ, Hayes KR, Leung B, Reichard S, Mack RN, Moyle PB, Smith M, Andow DA, Carlton JT & McMichael A (2006) Biological invasions: recommendations for policy and management. Ecological Applications 16(6): 2034–2054. Mishra R (1968) Ecology Work Book. Oxford and IBH Company, New Delhi, India pp. 244 Mooney HA (2000) Invasive Species in a Changing World. Island Press, Washington DC. Nilsson C and Grelsson G (1995), The fragility of ecosystems: A review. Journal of Applied Ecology 32: pp. 677–692. Oerke EC, Dehne H-W, Schönbeck F & Weber A (1995) Crop Production and Crop Protection: Estimated Losses in major Food and Cash Crops. Elsevier, Amsterdam and New York. Pimm SL (1984) the complexity and stability of ecosystems. Nature 307(5949): 321–326. Ricciardi A, Steiner WWM, Mack RN & Simerloff D (2000) Towards a global information system for invasive species. Bioscience 50(3): 239–244. www.tropicalplantresearch.com

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Kumari & Choudhary (2016) 3(3): 592–599 . Ricciardi A, Steiner WWM, Mack RN & Simeroff D (2000) Towards a global information system for invasive species. Bioscience 50(3): 239–244. Richardson DM & Higgins SI (1998) Pines as invasion in the Southern hemisphere. In: Richardson DM (ed) Ecology and biogeography of Pinus. Cambridge: Cambridge University Press. Sala OE et. al. (1999) Global change, biodiversity and ecological complexity. In: Walker B, Steffen WL, Canadell J & Ingram JSI (eds) The Terrestrial Biosphere and Global Change: Implications for Natural and Managed Ecosystems. Cambridge University Press, Cambridge, UK. Shimono Y & Konuma A (2008) Effects of human-mediated processes on weed species composition in internationally traded grain commodities. Weed Research 48(1): 10–18 Stern N (2006) The Economics of Climate Change. HM Treasury, London, UK. Tiwari S, Adhikari B, Siwakoti M & Subedi K (2005) An inventory and assessment of invasive alien plant species of Nepal. IUCN, The World Conservation Union, Nepal. Vattakkavan J, Vasu NK, Varma S, Gureja N & Aiyadurai A (2002) Silent stranglers: Eradication of Mimosa in Kaziranga National Park, Assam. Wildlife Trust of India, New Delhi. Yelenik SG, Stock WD & Richardson DM (2007), Functional group identity does not predict invader impacts, Differential effects of nitrogen-fixing exotic plants on ecosystem function. Biological Invasions 9(2): 117– 125.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 600–605, 2016 DOI: 10.22271/tpr.2016.v3.i3.079 Research article

Disease progression in potato germplasm from different reaction groups against potato virus Y in relation to environmental factors Ata-ul-Haq1, Yasir Iftikhar1, Muhammad Irfan Ullah2, Mustansar Mubeen3*, Qaiser Shakeel3, Faheema Bakhtawar1 and Iram Bilqees1 1

Department of Plant Pathology, University College of Agriculture, University of Sargodha, Pakistan Department of Entomology, University College of Agriculture, University of Sargodha, Sargodha, Pakistan 3 State Key Laboratory of Agricultural Microbiology and Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China 2

*Corresponding Author: [email protected]

[Accepted: 20 November 2016]

Abstract: Germplasm of 10 potato varieties/lines were screened out against PVY. Out of 10, five (Tota-704, FD 71-1, FSD WHITE, FD 8-1, FD 76 24) were found susceptible, three (FD 74-41, FD 74-50 and N-96-25) moderately susceptible and two (Kuruda and SH 216 A) moderately resistant varieties. The mean incidence of PVY was 61%. ELISA confirmed virus in the samples from all varieties showing moderately yellow to yellow color in the ELISA plate. There was significant correlation between PVY and maximum, minimum temperature and relative humidity. Disease severity was maximum in the range of 20–29°C for maximum temperature, 5–6°C for minimum temperature in all three groups of Varieties. 82–83% relative humidity favors the disease severity. Keywords: Correlation - Disease severity - Potyvirus - Relative humidity - Temperature. [Cite as: Ata-ul-Haq, Iftikhar Y, Ullah MI, Mubeen M, Shakeel Q, Bakhtawar F & Bilqees I (2016) Disease progression in potato germplasm from different reaction groups against potato virus Y in relation to environmental factors. Tropical Plant Research 3(3): 600–605] INTRODUCTION Potato (Solanum tuberosum L.) is an important vegetable crop all over the world. It is a staple food in different part of the world because of its high nutrition value. The production of potato is threatened by different biotic and abiotic factors not only in the world but also in Pakistan Qamar et al. (2015). Potato has very low production in Pakistan as compared to world production due to biotic and abiotic factors. Among those biotic factors viruses are the most devastating pathogens. Potato crop is affected by 40 viruses Valkonen (2007). In Pakistan, Eight potato viruses viz., Potato virus Y (PVY), Potato virus X (PVX), Potato leaf roll virus (PLRV), Potato mop top virus (PMTV), Potato virus S (PVS), Potato virus A (PVA), Alfalfa mosaic virus (AMV) and Potato virus M (PVM) have been reported Mughal et al. (1988). Among these PVY, PVX and PLRV are widely distributed in Pakistan. PVY alone or along with PVX cause a huge loss to potato crop. Potato virus Y belongs to family Potyviridae, which contains economically most important and largest group of plant viruses Buchen & Osmend (1987). Ahmad et al. (2003) during a survey declared the PVY, PVX and PVS as major diseases in fields of potato. Losses due to PVY are up to 83% in Pakistan Mughal & Khalid (1985) and Khalid et al. (2000). Potato virus Y is ssRNA Potyvirus having different strains; PVYN, PVYO and PVYC de Box & Huttinga (1981). It is transmitted mechanically and through insect vector in non-persistent manners. Green peach Aphid (Myzus persicae Sulzer) plays vital role in disease transmission. Environmental factors play an important role in disease development and their information will not only be helpful in understanding importance of epidemiological factors but also in formulating management strategies. Ahmad et al. (2011) studied the relation of environmental factors (rainfall, temperature and humidity) with incidence of PVY and PVX. They concluded that rainfall and www.tropicalplantresearch.com

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Received: 16 August 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.079

Ata-ul-Haq et al. (2016) 3(3): 600–605 . high humidity help to spreading of incidence. Environmental factors have positive relation with PVY and PVX incidence. PVY have significant and non-significant result at high and low temperature but it is negatively correlated Qamar et al. (2003). The current research work was carried out to monitor the disease incidence of PVY and influence of environmental factors on disease severity. MATERIALS & METHODS Screening of Potato germplasm The research trial comprising of 10 potato varieties was planned to screen the potato germplasm against potato virus Y (PVY). Symptoms were observed carefully for screening using the disease rating scale Qamar et al. (2003) with slight modification (Table 1). Disease incidence was calculated as follows; Percent disease incidence (%) = Table 1. Rating scale for the screening of potato germplasm.

Rating Scale 0 1 2 3 4

Response No visible symptom Mosaic pattern starts on leaves( 25% leaves showing symptoms) Mosaic and Mottling (50% leaves have symptoms) Dwarfing, rugosity and mottling of leaves (75% leaves affected) Leaf drooping severe mosaic and mottling (100)

Reaction Immune Resistance Moderately resistance Moderately susceptible Susceptible

Enzyme Linked Immunosorbent Assay (ELISA) Samples from 10 varieties on the basis of symptoms were collected and confirmed through DAS-ELISA by Iftikhar et al. (2009). Polyclonal antibodies diluted in coating buffer were coated in ELISA plate and incubation at 4°C for 24 hours was followed by washing for three times at five minutes interval. After washing wells of ELISA plate were charged with antigens. Incubation and washing were repeated prior to add conjugate antibody. After incubation and washing pNP (para nitro phenyl) was added as substrate. Reaction was observed visually after the incubation of 30–60 minutes at room temperature. Correlation of Environmental factors Environmental factors (Maximum and minimum temperature, rainfall and relative humidity) were recorded for crop duration (October–January). Statistical Analysis Correlation between environmental factors and disease severity in different varieties was analyzed by using “R for Windows”. RESULTS Screening and Incidence

Incidence (%)

80 70

70 63

63

60

FSD WHITE

FD 8-1

63

60

60

63

60

57

57

50 40 30 20 10 0 Tota-704 FD 71-1

FD 76 24 FD 74-41 FD 74-50 N-96-25

Kuruda SH 216 A

Varieties

Figure 1. Incidence of PVY in different potato varieties.

Ten Varieties/lines were screened out against PVY. The disease incidence was in range from 70% (Tota704) to 57% in two varieties Kuruda and SH 216A (Fig. 1). Varieties were grouped into three categories; www.tropicalplantresearch.com

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Ata-ul-Haq et al. (2016) 3(3): 600–605 . Susceptible (Tota-704, FD 71-1, FSD White, FD 8-1 and FD 76-24) Moderate susceptible (FD 74-41, FD 74-50 and N-96-25) and Moderate resistant (Kuruda and SH 216 A) on the basis of disease rating scale (Fig. 2). 4.5

4

4

4

4

4

Rating Scale

4 3.5

3

3

3

3 2.5

2

2

2 1.5 1 0.5 0 Tota-704 FD 71-1

FSD WHITE

FD 8-1 FD 76 24 FD 74-41 FD 74-50 N-96-25 Kuruda SH 216 A Varieties

Figure 2. Disease severity in different potato varieties.

Enzyme Linked Immunosorbant Assay (ELISA) ELISA confirmed the PVY in the samples collected from ten varieties on the basis of symptoms resembling to the virus. Light yellow to moderate yellow colour was observed in virus infected samples. The samples were tested against PVY and PVX antibodies separately on a same microplate and found the positive results (Fig. 3).

Figure 3. ELISA plate (wells) showing the reaction against positive samples of PVY.

Correlation of environmental factors with disease severity All the environmental factors such as maximum, minimum temperature and relative humidity had highly significantly correlation with disease progression in susceptible varieties while maximum and minimum in moderately susceptible and only minimum temperature in moderately resistant (Table 2). Disease was progressed in susceptible varieties maximum severity was observed at temperature of 19–22ºC and started decrease gradually as the temperature increases. Similarly high disease severity was observed minimum temperature of 5–6ºC and started decrease with the increase in temperature. 82–83% relative humidity was found favorable for disease severity (Fig. 4). www.tropicalplantresearch.com

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Ata-ul-Haq et al. (2016) 3(3): 600–605 . Table 2. Correlation between varieties and environmental factors.

Varieties Tota-704 FD 71-1 FSD WHITE FD 8-1 FD 76-24 FD 74-41 FD 74-50 N-96-25 Kuruda SH 216 A

Group

Max. temperature

Min. temperature

Humidity

Susceptible

0.00063 ***

2.31e-09 ***

0.01192 *

Moderate Susceptible

0.00361 **

3.41e-06 ***

0.28519

Moderate Resistance

0.10435

0.00182 **

0.13293

Figure 4. Disease progression in susceptible varieties against PVY in Sargodha.

Moderately susceptible varieties showed that highest disease severity at 22ºC in case of maximum temperature and increase in temperature reduced the disease severity. In minimum temperature disease severity was maximum at 6ºC and minimum disease severity was recorded at 10ºC. Intensity of disease decreased with the increase in relative humidity (Fig. 5). Moderately resistant varieties showed minimum temperature was highly significant correlation with disease intensity. Disease severity was at peak at 19ºC in case of maximum temperature while at 5ºC in minimum temperature. Maximum disease intensity was recorded at the 83% relative humidity (Fig. 6). DISCUSSION Potato virus potyvirus (PVY) is one of the most economically declared threatening viruses in potato not only in Pakistan also all over the world. Variety of symptoms exhibited by PVY depends upon the viral strains. PVYO is the most prevailing strain. Potato varieties/lines were screened out to record the disease incidence and disease severity according to disease rating scale Qamar et al. (2003) with slight modification. Ten varieties were divided to three groups i.e. moderately resistant, moderately susceptible and Susceptible. Out of ten varieties no variety showed immune or resistance response with the mean disease incidence of 62% was recorded. Out of ten varieties five were susceptible, three moderately susceptible and two moderately resistant. Our results were in accordance with the results of Abbas et al. (2012). They reported disease incidence of 55% www.tropicalplantresearch.com

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Ata-ul-Haq et al. (2016) 3(3): 600–605 . during a survey in the different localities of Punjab. Similarly Ahmed et al. (2013) recorded the incidence of PVY up to 67% in Egypt. They reported that PVYO and PVYN were most prevalent strains.

Figure 5. Disease progression in moderately susceptible varieties against PVY in Sargodha.

Figure 6. Disease progression in moderately resistant varieties against PVY in Sargodha.

Mughal et al. (1988) detected the presence of PVY through ELISA. Similarly Ahmed et al. (1995) confirmed the PVY, PLRV and PVX in potato samples through ELISA. Susceptible Verities significantly correlated with maximum, minimum temperature and relative humidity. While maximum and minimum temperature showed significant correlation with moderately susceptible varieties. Whereas moderately resistant www.tropicalplantresearch.com 604

Ata-ul-Haq et al. (2016) 3(3): 600–605 . varieties had significantly correlated only with minimum temperature. Our results confirmed the work of Qamar et al. (2003) who studied the correlation of environmental variables (maximum and minimum air temperature, pan evaporation, wind velocity, wind speed, clouds and relative humidity) with PVY disease on six Potato varieties viz; 394017-45, TPS-8901, 394029-129, TPS- 9620, 3912202-103 and TPS 9804. Maximum disease severity was recorded at 24–28ºC as maximum temperature and 9–12ºC as minimum temperature. He also indicated the increasing trend of PVY disease development at minimum temperature of 5–13ºC and 15–31ºC maximum temperature. He also indicated the increasing trend of PVY at minimum temperature of 5–13ºC. Therefore it is concluded that environmental factors have significant impact on disease severity and will be helpful not only in formulating management strategies but also finding a resistant source against PVY. ACKNOWLEDGEMENTS Authors are highly thankful to Dr. S.M. Mughal for providing polyclonal antibodies against PVY and Director, Potato Research Institute, Sahiwal, Pakistan, for providing us potato germplasm. REFERENCES Abbas MF, Hammed S, Rauf A, Nosheen Q, Ghani A, Qadir A & Zakia S (2012) Incidence of six viruses in potato growing areas of Pakistan. Pakistan Journal of Phytopathology 24: 44–47. Ahmad N, Khan MA, Ali S, Khan NA, Binyamin R & Sandhu AF (2011) Epidemiological studies and management of potato germplasm against PVX and PVY. Pakistan Journal of Phytopathology 23(2): 159– 165. Ahmad W, Iman D & Jan HU (2003) Recent trend of potato virus prevailing in potato growing areas of Punjab. Pakistan Journal of Phytopathology 15: 21–24. Ahmed M, Ahmed W & Hussain A (1995) Detection of major potato viruses from different potato growing localities of Punjab. In: Research and development of potato production in Pakistan. Proceedings of the National Seminar held (23–25 Apr) at NARC Islamabad, Pakistan pp. 175–179. Ahmed RZ, Ibrahim IAM, Hassan HMS & El-Wakil DA (2013) Incidence of virus Y Strains and Effect of Infection on the Productivity of Potato Tubers. Journal of Agriculture and Vegetable Science 2: 58–64. Buchen & Osmend C (1987) Plant viruses online descriptions and list from VIDE database. Potato virus Y, Potyvirus. de Box JA & Huttinga H (1981) CMI/AAB description of plant viruses. Potato virus Y 242. Available from: www.dpvweb.ent/dprv/showdpv.php?dpvNo=242. (accessed: 10 Sep. 2016). Iftikhar Y, Khan MA, Rashid A, Mughal SM, Iqbal Z, Batool A, Abbas M, Khan MM, Muhammad S & Jaskani MJ (2009) Occurrence and distribution of citrus tristeza closterovirus in the Punjab and NWFP, Pakistan. Pakistan Journal of Botany 41: 373–380. Khalid S, Iftikhar S, Munir A & Ahmad I (2000) Potato diseases in Pakistan. PARC Islamabad, Pakistan pp. 165. Mughal SM & Khalid S (1985) Virus diseases in relation to potato production in Pakistan. In: National Seminar on Potato in Pakistan (2–4 Apr 1985). PARC Islamabad, Pakistan. Mughal SM, Khalid S, Gillani TS & Devaux A (1988) Detection of potato viruses in Pakistan. In: Proceeding of 2nd Triennial Conference. PARC Islamabad, Pakistan pp. 12–26. Qamar MI, Iftikhar Y, Iqbal Z, Mubeen M & Haq A (2015) Screening of Potato Germplasm through ELISA against Potato Virus X (PVX). Universal Journal of Plant Science 3(2): 21–24. Qamar N, Khan M & Rashid A (2003) Correlation of Environmental Conditions with Potato Virus X (PVX) and Y (PVY) Disease Severities Recorded on 21 Advance Lines/Varieties of Potato (Solanum tuberosum L.). International Journal of Agriculture and Biology 5: 181–184. Valkonen JPT (2007) Viruses, Economical losses and Biotechnological potential. In: Vreugdenhil J (ed) Potato Biology and Biotechnology. Elsevier, New York, pp. 619–641.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 606–610, 2016 DOI: 10.22271/tpr.2016.v3.i3.080 Research article

A note on precocious pollen germination in Woodfordia fruticosa (L.) Kurz. Kanak Sahai*, Krishna Kumar Rawat and Dayanidhi Gupta CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India *Corresponding Author: [email protected]

[Accepted: 22 November 2016]

Abstract: The unusual phenomenon of pollen germination prior to their release from anthers was studied in detail in different samples of flowers collected from different geographical regions of India viz. Eastern Himalayan region (Teesta valley near Darjeeling, West Bengal), Western Himalayan region (Yamuna bridge near Mussoorie and Pauri Garhwal district), Gangetic plains (Katerniaghat Wildlife Sanctuary, Uttar Pradesh) and arid region of Rajasthan (Mount Abu). Though pollen germination prior to their release was regularly observed during the study, it varied at different level of anther and stigma and in different geographical regions. Highest i.e. 7.31% of such type of pollen germination was recorded in the samples of Pauri Garhwal of Western Himalayan region followed by 3.45% from Mussoorie of the same region when flower was at its initial stage of opening and pistil was short and below the level of anther. This phenomenon of pollen germination was regularly absent in the samples of Eastern Himalayan region. Keywords: Precocious pollen germination - Himalayan - Gangetic plain - Arid region. [Cite as: Sahai K, Rawat KK & Gupta D (2016) A note on precocious pollen germination in Woodfordia fruticosa (L.) Kurz. Tropical Plant Research 3(3): 606–610] INTRODUCTION Precocious pollen germination inside the anther loculi are commonly reported to occur in cleistogamous flowers (Maheshwari 1962, Frankel & Galun 1977, Lord 1979). However, It has also been observed in some chasmogamous flowers by many workers like Trigonella foenum-graecum (Joshi & Raghuvanshi 1967), Lathyrus sativus (Verma & Grewal 1971), Bergenia crassifolia (L.) Fritsch, Citrus limon (L.) Burm. Fil, Cucumis melo L. cv. Giant stide (Tezier), Prunus avium L. cv. Mora di Cazzano, (Pacini & Franchi 1982); Malus pumila, Prunus amygdalus (Koul et al. 1985), Catharanthus roseus (Mishra & Kumar 2001), Arabidopsis thaliana (Johnson & McCormick 2001, Xie et al. 2010), Biophytum sensitivum (Sreedevi & Rekha 2003), Glycine max (Kaur et al. 2005) etc. While working on various samples of Woodfordia fruticosa (L.) Kurz. collected from different parts of the country, the interesting phenomenon of pollen germination inside indehisced anther lobe was observed and investigated further in detail for the present study. Since Woodfordia fruticosa is an important dye yielding (Dymock et al. 1890, Uphof 1959, Das et al. 2007) and medicinally important species (Kuramochi-Motegi et al. 1992, Liu et al. 2004, Chandan et al. 2008), its overexploitation reported it threatened species (Kokkirala et al. 2012) and even now listed as Low risk/Least concerned species (IUCN 1998). A perusal of literature reveals though, multifarious properties of Woodfordia fruticosa were highlighted from time to time, studies on reproductive behavior are almost lacking so far. Woodfordia fruticosa with chasmogamous flowers regularly showed pollen germination inside anther lobes. Hence, this phenomenon not reported so far in this plant species was considered interesting for the present study. MATERIAL AND METHODS Study species Woodfordia fruticosa (Lythraceae) commonly known as ‘Fire flam bush’ due to brilliant red flowers is a shrub/small tree. It is endemic to India and also distributed in Bhutan, China, Indonesia, Laos, Madagascar, Myanmar, Nepal, Pakistan, Sri Lanka, Thailand and Tropical Africa (Abbasi et al. 2012, Meena & Kumar 2015). The plant is known for various medicinal properties including anti-tumor, antioxidant, www.tropicalplantresearch.com

606

Received: 17 August 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.080

Sahai et al. (2016) 3(3): 606–610 . immunomodulatory, antiproliferative, antihyperglycemic, antimicrobial, anti-inflammatory and analgesic, hepatoprotective, cytotoxic, preventive & curative and anti-ulcer activities (Rani et al. 2015). The flowers have wider use in Ayurvedic formulations like ‘arishta’ and ‘asava’ (Kores et al. 1993). Of the 18 aristas, 17 have been found to contain Woodfordia fruticosa (Meena & Kumar 2015). Study sites Flowers and buds of various stages of Woodfordia fruticosa were collected from four different geographical regions of the country viz. Eastern Himalayan (Teesta valley near Darjeeling, West Bengal), Western Himalayan (Yamuna bridge near Mussorie and Pauri Garhwal district), Gangetic plain (Uttar Pradesh: Katerniaghat wildlife sanctuary) and Arid region of Rajasthan (Mount Abu). Sample preparation and observation The randomly collected flowering material from different geographical regions of India was fixed in FAA and later preserved in 70% alcohol. Flowers and buds of different stages were observed under dissecting microscope for morphological studies. Indehiscent anthers and stigmas of different developmental stages were selected, dissected and examined under microscope. Since stigma was present at the different level of anthers, the pistil was categorized as short (stigma below the anther level), medium (stigma equal to anther level) and long (stigma above the anther level) and the stigma receptivity was tested for each category with Benzedine test (Galen et al. 1985). Pollen grains were extracted from anther lobes corresponding to the entire three pistil category and stained with Alexander’s stain (Alexander 1980). Observations were made under stereoscopic zoom microscope to check the pollen viability, sterility as well as their germination inside the anther lobe and photographed with the help of Nikon Eclipse 80i microscope. Percentage of fertile pollen grains and their germination inside anther was assessed by counting the pollen grains in randomly selected microscopic fields. Average score was calculated from 25 such ramdom counts for each sample. For histological observation, samples were dehydrated in tertiary-butyle-alcohol series and infiltrated and embedded in paraffin wax. Thin sections of 10µm were cut through rotary microtome, double stained with Safranine and Fast green and finally mounted onto Canada balsam. Microphotographs were taken with the help of microscopic camera attachment. RESULTS AND DISCUSSION Study species The plant has simple, opposite, sessile or shortly petiolate, ovate-lanceolate or lanceolate leaves. Flowers are irregular, born in axillary cymes, bright red and bisexual; calyx tubular; petal small; stamen 12; ovary superior with numerous ovule. Fruit membranous capsule; seeds numerous, wedge-shaped, brown and smooth (Jayaweera 1981, Napagoda & Yakandawala 2008). Table 1. Pollen status and its perecocious germination along with different developmental stages of stigma in Woodfordia fruticosa of different geographical region. (S = Short pistil, M = Medium pistil, L = Long pistil)

Geographical regions

Pollen/ anther

Mount Abu, Rajasthan (Arid region)

59375-146875, avg. 93075±30824

Katerniaghat Wildlife Sanctuary (Gangetic plains)

33125-111250, avg. 66875±21308

Pauri Garhwal (W. Himalaya)

38000-91667, avg. 64200±14051

Teesta valley, near Darjeeling (E. Himalaya)

46667-112667 avg. 73667±18522

Near Yamuna bridge, Moosoorie (W. Himalaya)

37666-85666, avg. 58760±14180

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Fertile pollens (%) 91.28 (S) 87.33 (M) 50.00 (L) 98.87 (S) 97.00 (M) 40.90 (L) 83.94 (S) 88.82 (M) 94.93 (L) 99.27 (S) 98.09 (M) 91.67 (L) 99.37 (S) 89.35 (M) 93.53 (L)

Precocious pollen germination (%) 0.89 (S) 0.66 (M) 0.68 (L) 0.00 (S) 1.19 (M) 0.00 (L) 7.31 (S) 0.43 (M) 0.53 (L) 0.00 (S) 0.00 (M) 0.00 (L) 3.45 (S) 0.00 (M) 0.00 (L) 607

Sahai et al. (2016) 3(3): 606–610 .

Figure 1. Precocious pollen germination in Woodfordia fruticosa. A, section of anther lobe showing germinated pollen grains; B–D, germinating pollen grains with pollen tubes of varying length; E, germinated pollen grains forming cluster.

Stigma became receptive along the flower opening and the receptivity reached at peak at the level of anthers (medium sized pistil), which gradually decreased when stigma passes the anthers and reached above the anther level (long pistil). However, pollen viability has no concern with the in-situ pollen germination (Table 1). Pollen germination inside anther (Fig. 1A–E) was observed in all the samples of three different eco-geographic regions, viz. western Himalayan and Arid region and Gangetic plain except in the samples of eastern Himalayan region where this phenomenon was absent in spite of repeated visits and random collections (Table 1). Pollen germination prior to their release from anther (precocious pollen germination) greatly varied provenance to provenance. It was highest i.e. 7.31% in the samples collected from Pauri Garhwal of western Himalayan region followed by 3.45% from Mussoorie of the same region (Table 1). Interestingly in both the samples, the maximum in-situ germination was recorded at the initial stage of flower opening when pistil was short and below the level of anthers. However, in open flower stage, when pistil reached either at the level of the stamen (medium sized pistil) or above them (long pistil), the pollen germination was poor or even absent in some samples (Table 1). Such phenomenon of pollen germination did not occur at the same rate in all the samples; it varied at different levels of anthers in respect to stigma and in different geographical regions (Table 1). In some www.tropicalplantresearch.com 608

Sahai et al. (2016) 3(3): 606–610 . cases pollen germination inside the anther was observed in clusters and therefore, eventually released together (Fig. 1E). These pollen clusters may also create obstruction for other pollen to discharge freely at once from anther and thus act as a hindrance to selfing (Verma & Grewal 1971). On the other hand, as a result of in-situ germination and subsequent obstruction, the pollen will come out slowly and therefore, pollen discharge may lost longer, which may attribute to long lasting chances of selfing as suggested by Kaur et al. (2005) who described in-situ pollen germination in Glycine max as a strategy to facilitate a high degree of selfing. However, Koul et al. (1985) suggested that in-situ pollen germination is probably controlled by genetic factors as they observed the phenomenon in specific varieties of apples growing along with many other varieties in the same environment. According to them such type of pollen germination does not occur year after year which indicates that certain environmental condition is necessary for the expression of particular gene or genes responsible for precocious pollen germination in particular plant species. They speculated the reason of limited number of precocious pollen germination either they do not carry the same potential of germination or not exposed strictly to similar conditions inside the anthers. Similar observations were made by Mishra & Kumar (2001) in Catharanthus roseus and found this mechanism as genetically controlled. Dhar et al. (2002) co-related precocious pollen germination with high relative humidity and high soil moisture. This fact is evident from our observation as the maximum pollen germination inside the anther lobe was observed in Western Himalayan samples where high humid conditions were usual. Interestingly, contrary to them, the samples collected from dry arid area (Rajasthan) regularly revealed precocious pollen germination at all the stages of pistil, though the percentage was not very high (Table 1). During the normal case of pollen germination the moisture content of pollen grains as well as of the microsporangium is reduced as the anther ages (Koul et al. 1985). The pollen grains get partially dehydrated and the locular fluid disappears before anthesis to promote anther opening and pollen grain dispersal. Reaching onto the stigma, the pollen grains rehydrate before germination by absorbing the water provided by the receptive stigma (Heslop-Harrison 1979a, 1979b). The extent of dehydration varies plants to plants according to pollination strategies and meterological conditions at anthesis (Baranas & Rajki 1976). Partially dehydrated pollen grains could easily germinate in-situ, where probably the locules remain wet. Precocious pollen germination with partly dehydrated anther has been observed in seagrass Thalassodendron ciliatum (Ducker et al. 1978). The wet loculus and absence or delay of pollen dehydration, was speculated to trigger pollen germination inside anthers by Pacini & Franci (1982). However, in our opinion pre dispersal pollen maturity is more responsible for precocious pollen germination. Since all the pollen grains inside the anther lobe did not take part in germination (Fig. 1A), only mature pollen grains that got moisture might be able to germinate. Though, it is very difficult to speculate whether the pre- germinated pollen grains takes part in self or cross pollination/fertilization, once the pre-germinated pollen or pollen cluster reaches to the receptive stigma of same flower, it can enhance the possibility of self-pollination to some extent due to already grown pollen tubes. Cross pollination due to precocious pollen germination cannot be successful as the dispersal of germinated pollen grains and their landing on to the stigma surface in a healthy condition cannot be expected because they can be damaged mechanically by exposure to dry air. However, this mechanism of precocious pollen germination needs further analysis to find out its real causes and significance. ACKNOWLEDGEMENTS The authors are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow for kindly providing the necessary facilities. The study was conducted with the financial support from Council of Scientific and Industrial Research (CSIR), New Delhi under project head OLP-0083. REFERENCES Abbasi AM, Khan MA, Ahmad M & Zafar M (2012) Medicinal plant biodiversity of lesser Himalaya-Pakistan. Springer, New York, London, 220 p. Alexander MP (1980) A versatile stain for pollen, fungi, yeast and bacteria. Stain Technology 55: 13–18. Baranas B & Rajki E (1976) Storage of maize (Zea mais L.) pollen at -196ºC in liquid nitrogen. Euphytica 25: 747–752. Chandan BK, Saxena AK, Shukla S, Sharma N, Gupta DK, Singh K, Suri J, Bhadauria M & Qazi GN (2008) Hepatoprotactive activity of Woodfordia fruticosa Kurz. flowers against carbon tetrachloride induced hepatotoxicity. Journal of Ethnopharmacology 119(2): 218–224. Das PK, Goswami S, Chinnaiah A, Panda N, Banerjee S, Sahu NPB & Achari B (2007) Woodfordia fruticosa: Traditional uses and recent findings. Journal of Ethnopharmacology 110: 189–199.

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Sahai et al. (2016) 3(3): 606–610 . Dhar R, Sharma N, & Koul AK (2002) Intra population variation in site of pollen germination and pollination in Trifolium fragiferum L. Phytomorphology 52: 323–330. Ducker SC, Pettitt SM & Knox RB (1978) Biology of Australian seagrasses: pollen development and submarine pollination in Amphibolis antarctica and Thalassodendron ciliatum (Cymodoceaceae). Australian Journal of Botany 26: 265–285. Dymock W, Warden CJH & Hooper D (1890) Pharmacographia Indica. Education Society’s Press, Byculla, Mumbai, 206 p. Frankel R & Galun E (1977) Pollination mechanisms, reproduction and plant breeding. Springer Verlag, Berlin, 284 p. Gale C, Plowright RC & Thompson JD (1985) Floral biology and regulation of seed set and size in the lily, Clintonia borealis. American Journal of Botany 72(10): 1544–1552. Heslop-Harrison J (1979a) An interpretation of the hydrodynamics of pollen. American Journal of Botany 66: 737–743. Heslop-Harrison J (1979b) Aspects of the structure, cytochemistry and germination of the pollen of rye (Secale cereale L.). Annals of Botany 44 (suppl.1): 1–47. IUCN (1998) CAMP workshop on Medicinal plants, India (January, 1997) 1998. Woodfordia fruticosa. The IUCN Red List of Threatened Species 1998: e.T39058A10160263. Available from: http://dx.doi.org/10.2305/IUCN.UK.1998.RLTS. T39058A10160263.en. (accessed on 02 August, 2016). Jayaweera DMA (1981) Medicinal plants (indigenous and exotic) used in Ceylon. The National Science Council of Sri Lanka, Colombo. Johnson SA & McCormick S (2001) Pollen germinated precociously in the anthers of raring-to-go, an Arabidopsis gametophytic mutant. Plant Physiology 126: 685–695. Joshi S & Raghuvanshi SS (1967) B-chromosomes, pollen germination in situ and connected grains in Trigonella foenumgraecum. Phyton 12: 278–282. Kaur S, Nayyar H, Bhanwra RK & Kumar S (2005) Precocious germination of pollen grains in anthers of soybean (Glycine max (L.) Merr.). Soybean Genetics Newsletter 32: 1–10. Kokkirala VR, Kota S, Yarra R, Bulle M, Aileni M, Gadidasu KK, Teixeira da Silva JA & Abbagani S (2012) Micropropagation via nodal explants of Woodfordia fruticosa (L.) Kurz. Medicinal and Aromatic Plant Science and Biotechnology 6(1): 50–53. Kores BH, Vanden Berg AJJ, Abeysekera AM, DeSilva KTD & Labadie RP (1993) Fermentation in traditional medicine. The impact of Woodfordia fruticosa flowers on the immunomodulatory activity and the alcohol and sugar content of Nimba arishta. Journal of Ethnopharmacolgy 40: 117–125. Koul AK, Singh A, Singh R & Wafai BA (1985) Pollen grain germination inside the anthers of two chasmogamous angiosperms: Almond (Prunus amygdalus L. Batsch) and Apple (Malus pumila Mill.). Euphytica 34: 125–128. Kuramochi-Motegi A, Kuramochi H, Kobayashi F, Ekimota H, Takahashi K, Kadota S, Takamori Y & Kikuchi T (1992) Woodfruticosin (Woodfordin C), a new inhibitor of DNA topoisomerase II. Experimental antitumor activity. Biochemical Pharmacology 44: 1961–1965. Liu MJ, Wang Z, Li HX, Wu RC, Liu YZ & Wu QY (2004) Mitochondrial dysfunction as an early event in the process of apoptosis induced by Woodfordin I in human Leukemia K562 cells. Toxicology and Applied Pharmacology 194: 141–145. Lord E (1979) The development of cleistogamous and chasmogamous flowers in Lamium aplexicaule (Labiatae); an example of heteroblastic inflorescence development. Botanical Gazette 140: 39–50. Maheshwari JK (1962) Cleistogamy in angiosperms. In: Maheshwari P, Johri BM & Vasil IK (eds) Proceedings of the Summer School of Botany. Ministry of Scientific Research and Cultural Affairs, New Delhi, India, pp. 145–155. Meena V & Kumar S (2015) Woodfordia fruticosa (L.) Kurz.: A high demand threatened plant with potential medicinal values. Indian Journal of Plant Sciences 4(3): 100–106. Mishra P & Kumar S (2001) A monogenic recessive mutant with precocious in situ pollen germination in Catharanthus roseus L. Journal of Medicinal and Aromatic Plant Sciences 23: 277–279. Napagoda NADR & Yakandawala K (2008) Woodfordia fruticosa (Lythraceae): A medicinal plant for landscaping. In: Proceedings of 6th Agricultural Research Symposium, 13-14 August 2008. Makandura, Wayamba University of Sri Lanka, pp. 294–298. Pacini E & Franchi GG (1982) Germination of pollen inside anthers in some non-cleistogamous species. Caryologia 35: 205–215. Rani S, Rahman K, Younis M & Basar SN (2015) Dhawa (Woodfordia fruticosa (L.) Kurz.): A versatile medicinal plant. International Journal of Pharmaceutical Sciences and Drug Research 7(4): 315–320. Sreedevi P & Rekha B (2003) Pollen features and germination of Biophytum sensitivum var. sensitivum DC. Phytomorphology 53(2): 157–164. Uphof JC Th (1959) Dictionary of Economic Plants. H.R. Engelmann (J. Crammer), Weinheim, Germany, 400 p. Verma SC & Grewal SS (1971) Precocious germination of pollen in Lathyrus sativus and its significance. Phytomorphology 21: 362–367. Xie B, Wang X & Hong Z (2010) Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis. Planta 231: 809–823.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 611–615, 2016 DOI: 10.22271/tpr.2016.v3.i3.081 Research article

New species and new records of Graphis (Ostropales: Graphidaceae) from Eastern Ghats, India Satish Mohabe1, Anjali Devi B.1, Sanjeeva Nayaka2 and A. Madhusudhana Reddy1* 1

Department of Botany, Yogi Vemana University, Vemanapuram, Kadapa-526003, Andhra Pradesh, India 2 Lichenology Laboratory, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, Uttar Pradesh, India

*Corresponding Author: [email protected]

[Accepted: 25 November 2016]

Abstract: A new species Graphis neeladriensis, and two new records, G. plumierae and G. subalbostriata are described from the Eastern Ghats of India. The newly described species is characterized by crustose, UV+ yellow thallus, sub-immersed to erumpent, short to elongate and simple to sparingly branched lirellae, 2–4 striate labia, laterally carbonized exciple, clear hymenium and terminally muriform ascospores. Keywords: Rayalaseema - Seshachalam Biosphere Reserve - Lichens - Taxonomy. [Cite as: Mohabe S, Anjali DB, Nayaka S & Reddy AM (2016) New species and new records of Graphis (Ostropales: Graphidaceae) from Eastern Ghats, India. Tropical Plant Research 3(3): 611–615] INTRODUCTION Recent studies on the global diversity within the lichen family Graphidaceae indicates that there are large numbers of undiscovered species in the family and at least 175 species have been discovered since 2002. Further analysis predicts that geographically Graphidaceae have a concentrated diversity in a few regions of the world including Southern India (Lücking et al. 2014). Graphis Staiger (2002) is a major genus under the lichen family Graphidaceae, comprising of around 370 species worldwide (Kirk et. al. 2008, Lucking 2009, Joshi et al. 2010, Bárcenas-Peñta et al. 2014, Singh et al. 2014, Joshi et al. 2014). 111 species of Graphis are known from tropical, subtropical and temperate regions of India (Singh & Sinha 2010) and recent studies added 17 more species to the genus (Chitale et al. 2011, Singh & Swanlatha 2011a,b, Gupta & Sinha 2012, Singh et al. 2014). Within the genus Graphis species having lichexanthone are rare and so far only five species such as G. stipitata A. W. Archer, G. sauroidea Leight., G. haleana R. C. Harris, G. lucifica R. C. Harris and G. flavopalmicola Y. Joshi, Lücking & Hur are reported (Lücking 2009, Joshi et al. 2010). During the exploration on lichen in Rayalaseema forest of Andhra Pradesh a total of 126 species have been reported (Reddy et al. 2011, Nayaka et al. 2013, Anjali et al. 2013, Mohabe et al. 2014a,b & 2016) out of which only a single species Diorygma junghuhnii (Mont. & Bosch) Kalb, Staiger & Elix belonged to Graphidaceae. The explorations resulted in collection of several interesting specimens belonging to Graphidaceous taxa of which recently Mohabe et al. (2015a) described a new species Diorygma kurnoolensis Mohabe, Nayaka & Reddy. In the present communication a new species Graphis neeladriensis and new records for India, G. plumierae Vain. and G. subalbostriata Lücking are reported. The new species is unique in having lichexanthone in chemistry and terminally muriform ascospores. MATERIALS & METHODS The present study is based on recently collected specimens from Neeladri range of Tirumala hills (Fig. 1A) and Mallaiah Konda hills of Thambalapalli from Chittoor district which comes under Seshachalam Biosphere Reserve in Andhra Pradesh of India. The external morphology of the specimens were observed under a Magnüs MS 24/13 stereo-zoom microscope while anatomical characters of the thallus and apothecia were observed under a ZEISS Axiostar plus compound microscope. Thin hand cut sections of the thallus and apothecia were initially mounted in water to record the colour and measurements of various structures. The apothecial sections were then observed after applying aqueous 10% KOH solution while Lugol’s solution (I) was used for iodine www.tropicalplantresearch.com

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Received: 27 August 2016

Published online: 30 November 2016 https://doi.org/10.22271/tpr.2016.v3.i3.081

Mohabe et al. (2016) 3(3): 611–615 . reactions. The colour tests were performed by using routine reagents; aqueous solution of KOH (K), calcium hypochlorite (C), and paraphenylenediamine (P). The lichen substances were identified by thin-layer chromatography following literature (White & James 1985, Orange et. al. 2001) and specimens were identified through world key to the genus Graphis (Lücking et al. 2009) and by comparison with the protologues. The nomenclature of Lücking et al. (2009) was followed for lirellae morphology. The specimens of the new species have been deposited in the Herbarium of CSIR-National Botanical Research Institute Lucknow (LWG), Uttar Pradesh, India and specimens of new records have been deposited in the Lichen Herbarium, Department of Botany, Yogi Vemana University (YVUH) Kadapa, Andhra Pradesh, India. RESULTS & DISCUSSION NEW SPECIES Graphis neeladriensis Mohabe S, Anjali DB & Nayaka S sp. nov.

(Fig. 1A–F) MycoBank No.: MB 819499 This species is characterized by sub-immersed to erumpent, short to elongate and simple to branched lirellae, 2–4 striate labia, laterally carbonized exciple, clear hymenium, 8–spored ascus, transversely 4–12 and terminally 1–2(–3) vertically septate ascospores and presence of lichexanthone in thallus. Type: INDIA, Andhra Pradesh, Chittoor district, Tirumala hills, Neeladri range, on bark of Artocarpus heterophyllus, alt. ca. 650 m, 06.07.2014, Satish Mohabe & Anjali Devi B. 4097 (holotype-LWG). Thallus greenish grey to grey, smooth to cracked, shiny, 80–160 µm thick, corticated; cortex hyaline, 20–35 µm thick; algal layer continuous 75–120 µm thick, medulla with oxalate crystals, 10–25 µm thick; prothallus indistinct or white. Ascomata lirellate, variable, numerous, sub-immersed to erumpent, simple, short to elongate (towards centre) and sparingly branched (towards periphery), 0.2–3.5 mm long, 0.1–0.4 mm wide, end acute to obtuse, laterally covered by thalline margin in younger parts; labia epruinose, 2–4 striate; disc slit like closed, rarely open, epruinose; exciple dark brown to black, 50–90 µm thick, convergent, laterally carbonized; hymenium hyaline, clear, without oil globules, 175–225 µm wide, 85–200 µm high; hypothecium hyaline; ascus cylindrical, 40–150 × 14–20 µm, 8–spored, I− ; ascospores hyaline, transversely 4–12 and vertical cells 1–3 in end locules (terminally muriform), 24–77 × 7–12 µm, I+ blue. Table 1. Comparison of Graphis species containing lichexanthone. (New species is in bold)

Species name 1. Graphis flavopalmicola

Lirellae morph handelii-morph

Labia Exciple Hymenium Septation entire completely clear transversely carbonized septate

No. of septa 5–9-septate

Spore length Substance Unknown 19–27 μm

2. Graphis haleana striatula-morph

striate completely clear carbonized

transversely septate

9–19-septate

50–85 μm

Nil

3. Graphis lucifica striatula-morph

striate completely clear carbonized

transversely septate

5–9-septate

20–40 μm

Nil

4. Graphis neelladriensis sp. nov.

tenella-morph

striate laterally clear carbonized

terminally muriform

4–12 transverse 24–77 μm to 1–3 vertical septate

Nil

5. Graphis sauroidea

hossei-morph

entire completely clear carbonized

transversely septate

4–5-septate

45–60 μm

Nil

6. Graphis stipitata

hossei-morph

entire laterally clear carbonized

transversely septate

7–15-septate

15–20 μm

Norstictic, connorstictic acid

Chemistry: Thallus K− , P− , C− , KC− , UV+ yellow; TLC: lichexanthone present. Distribution and Ecology: Graphis neeladriensis is found growing on bark of Arctocarpous heterophylla trees growing in the In–situ conservation garden of medicinal plants in Neeladri range of Tirumala hills, Chittoor district of Andhra Pradesh at an altitude of around 650 m. It was found growing in association with other crustose lichens such as Bacidia sp., Graphis sp., Lecanora achroa Nyl. and a foliose species Hyperphyscia adglutinata (Flörke) H. Mayrhofer & Poelt. Etymology: The specific epithet is named after type locality Neeladri range which is the highest peak of Tirumala hills. www.tropicalplantresearch.com 612

Mohabe et al. (2016) 3(3): 611–615 .

Figure 1. Graphis neeladriensis (holotype): A–C, Habit showing variations in lirellae; D, transverse section of lirellae; E, ascus with ascospores; F, terminally muriform ascospores in iodine.

Remark: Graphis neeladriensis resembles Graphis neoelongata Lücking and Graphis dichotoma (Müll. Arg.) Lücking in having striate labia, laterally carbonized exciple, clear hymenium, terminally muriform or submuriform ascospores but both the species differs chemically by lacking lichexanthone in thallus. The lichexanthone containing species, G. haleana, and G. lucifica differs from new species with completely carbonized exciple and transversely septate ascospores, while G. stipitata differs in having entire labia, smaller ascospores and presence of norstictic and connorstictic acid. Further G. flavopalmicola and G. sauroidea have entire labia, completely carbonized exciple, transversely septate and smaller to medium ascospores. The comparative status of the new species among other lichexanthone containing species are given in table 1. Additional specimen examined: INDIA, Andhra Pradesh, Chittoor district, Tirumala hills, Neeladri range, on bark of Artocarpus heterophyllus, alt. ca. 650 m, 06.07.2014, Satish Mohabe & Anjali Devi B. 4098 (isotypeLWG). www.tropicalplantresearch.com

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Mohabe et al. (2016) 3(3): 611–615 . NEW RECORDS 1. Graphis plumierae Vain. Ann. Acad. Sci. fenn., Ser. A 6(7): 161 (1915). (Fig. 2A) Thallus whitish to greenish grey, smooth to cracked, corticated, 190–297 µm thick; cortex hyaline, 23–39 µm thick; medulla white with many crystals, 10–28 µm wide; algal layer continuous 75–95 µm thick; prothallus indistinct to brownish. Apothecia lirellate, numerous, immersed to sub-immersed, simple to branched, 1.0–2.5 mm long, 0.3–0.7 mm wide, with rounded to acute ends; labia pruinose, laterally covered by thalline margin; disc concealed, epruinose; epihymenium 14–19 µm, exciple dark brown to black, laterally carbonized, 25–62 µm thick; hymenium inspersed, with oil globules, 122–149 µm wide; hypothecium hyaline to yellowish brown 20–36 µm high; ascus cylindrical, 64–100 × 9–16 µm, 8–spored; ascospores colourless, transversely 5–11 septate, 18–58 × 6–10 µm. Chemistry: K+ yellow turning red, P+ yellow, KC–, C–; TLC: Norstictic acid, stictic and salazinic acid present. Distribution: It is a Neotropical species earlier known from Guadeloupe, Mexico and in the present study it is found on tree trunks in tropical forests of Eastern Ghats in India. Remark: G. plumierae Vain. has resemblance to G. brevicarpa M. Nakan., Kashiw. & K.H. Moon and G. crebra but differs by its concealed disc, white pruinose labia, immersed lirellae with lateral thalline margin and presence of norstictic acid with stictic and salazinic acid. Further G. brevicarpa differs by its apically thick complete thalline margin, epruinose labia and smaller ascospores while G. crebra has erumpent lirellae with lateral thalline margin, exposed disc with white pruina (scripta-morph). Specimens examined: INDIA, Andhra Pradesh, Chittoor district, Thambalapalli, Mallaiah Konda Hills, alt. ca. 956 m, on bark, 05.01.2013, A. Madhusudhana Reddy & Satish Mohabe 2812 (LWG); Tirumala hills, Dharmagiri, alt. ca. 937 m, on bark, 07.02.2013, Anjali Devi B. & Satish Mohabe 3414 (YVUH).

Figure 2. Habit of Graphis species A, Graphis plumierae Vain.; B, Graphis subalbostriata Lücking.

2. Graphis subalbostriata Lücking, Lichenologist 41(4): 363–452 (2009). (Fig. 2B) Thallus crustose, corticolous, whitish grey, thin, shiny, smooth to rough, continuous to discontinuous ecorticated; prothallus indistinct or absent. Apothecia lirellate, lirellae simple, small, rarely branched, 0.5–1.5 mm long, 0.1–0.5 mm wide, erumpent to prominent, laterally covered by thallus; disc concealed, epruinose; labia partially black, with 7–8 striation, distinct white lines in-between striation, formed by clusters of calcium-oxalate crystals; exciple apically to peripherally carbonized, 21–27 µm thick; hymenium clear, without oil globules; ascus cylindrical, 125–235 × 20–30 µm, 8–spored; ascospores colourless, fusiform, transversely 5–11–septate, 45–95 × 8–12 µm. Chemistry: Thallus K− , KC− , C− , P− ; TLC: No chemicals. Distribution: It is a Neotropical species earlier recorded from Guadeloupe and in the present study it was found on tree trunks in tropical forest of Eastern Ghats in India. Remark: G. subalbostriata is close to G. patwardhanii Kulk. but latter species has isidiate thallus. G. subalbostriata is also close to G. olivacea but it has dark olive-grey thallus, erumpent lirellae with apically thin and complete thalline margin, elongate and irregularly branched lirellae. www.tropicalplantresearch.com 614

Mohabe et al. (2016) 3(3): 611–615 . Specimen examined: INDIA, Andhra Pradesh, Chittoor district, Tirumala hills, Dharmagiri, on bark, alt. ca. 937 m, 07.02.2013, Anjali Devi B. & Satish Mohabe 3396, 3449, 3402, 3420 (YVUH); Shilathoranam, on bark, alt. ca. 958 m, 05.07.2013, Satish Mohabe & Anjali Devi B. 4034 (YVUH). ACKNOWLEDGMENTS The authors are very grateful to Council of Scientific and Industrial Research, New Delhi and Department of Science and Technology (INSPIRE), New Delhi for financial support; to the Director and Dr. D.K. Upreti, Chief Scientist, CSIR-National Botanical Research Institute, Lucknow for providing laboratory facilities; to Forest Officials of Andhra Pradesh for their cooperation during the exploration. REFERENCES Bárcenas-Peñta A, Lücking R, Miranda-González R & Herrera-Campos MA (2014) Three new species of Graphis (Ascomycota: Ostropales: Graphidaceae) from Mexico, with updates to taxonomic key entries for 41 species described between 2009 and 2013. Lichenologist 46: 69–82. Chitale G, Makhija U & Sharma B (2011) Additional species of Graphis from Maharashtra, India. Mycotaxon 115: 469–480. Gupta P & Sinha GP (2012) A new record of lichen in the genus Graphis for India from Assam. Indian Journal of Forestry 35(1): 133–134. Joshi S, Jayalal U, Oh SO, Nguyen TT, Dzung NA & Hur JS (2014) A New Species of Graphis and New Lichen Records from Vietnam, Including a Second Worldwide Report of Sarcographina cyclospora. Mycobiology 42(1): 17–21. Joshi Y, Lücking R, Yamamoto Y, Wang XY, Jin KY & Hur JS (2010) A new species of Graphis (lichenized Ascomycetes) from South Korea. Mycotaxon 113: 305–309. Lücking R (2009) The taxonomy of the genus Graphis sensu Staiger: (Ostrapales: Graphidaceae). Lichenologist 41: 319–362. Lücking R, Archer AW & Aptroot A (2009) A worldwide key to the genus Graphis (Ostropales: Graphidaceae). Lichenologist 41: 363–452. Lücking R, Mark K, Johnston, Aptroot A et al. (2014) One hundred and seventy-five new species of Graphidaceae: closing the gap or a drop in the bucket? Phytotaxa 189 (1): 007–038. Mohabe S, Nayaka S, Reddy MA & Anjali DB (2015a) Diorygma kurnoolensis (Graphidaceae), a new species of saxicolous lichen from Southern India. Geophytology 45(1): 47–50. Mohabe S, Reddy MA, Anjali DB, Nayaka S & Chandramati PS (2014a) Further new addition to the lichen mycota of Andhra Pradesh, India. Journal of Threatened Taxa 6(8): 6122–6126. Mohabe S, Reddy MA, Anjali DB Nayaka S & Chandramati PS (2014b) Assessment of lichens diversity in Rayalaseema Forest of Andhra Pradesh, India. In: National Conference on Plant Biology-2014. held at Department of Botany, Yogi Vemana University, Vemanapuram, Kadapa, Andhra Pradesh. pp. 50–51. Mohabe S, Anjali DB, Reddy AM, Nayaka S & Chandramati PS (2016) An appraisal of lichen biota in Chittoor district of Andhra Pradesh, India. In: Pulaiah T, Sandhya R (eds) Biodiversity in India. pp. 247–296. Nayaka S, Reddy MA, Ponmurugan P, Devi BA, Ayyappadasan G & Upreti DK (2013) Eastern Ghats, biodiversity reserves with unexplored lichen wealth. Current Science 104(7): 821–825. Orange A, James PW & White FJ (2001) Microchemical methods for the identification of lichens. British Lichen Society, U.K. Reddy MA, Nayaka S, Shankar PC, Reddy SR & Rao BRP (2011) New distributional records and checklist of lichens for Andhra Pradesh, India. The Indian Forester 137: 1371–1376. Singh KP & Swarnlatha G (2011a) New records of Graphis (Lichenized fungi) from India. Indian Journal of Forestry 34: 243–244. Singh KP & Swarnlatha G (2011b) A note on Graphidaceous lichens from Arunachal Pradesh, India. Indian Journal of Forestry 34: 353–360. Singh P & Singh KP (2014) Two new species of Graphis (Ascomycota: Ostropales: Graphidaceae), from Indo-Burma biodiversity hotspot. Mycosphere 5(4): 504–509. Staiger B (2002) Die Flechtenfamilie Graphidaceae: Studien in Richtung einer natürlicheren Gliederung. Bibliotheca Lichenologica 85: 1–526. White FJ & James PW (1985) A new guide to microchemical techniques for the identification of lichen substances. Bulletin British Lichen Society 57(Suppl.): 1–41.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 616–633, 2016 DOI: 10.22271/tpr.2016.v3.i3.082 Research article

Seed priming with spermine ameliorates salinity stress in the germinated seedlings of two rice cultivars differing in their level of salt tolerance Saikat Paul and Aryadeep Roychoudhury* Post Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata-700016, West Bengal, India *Corresponding Author: [email protected]

[Accepted: 02 December 2016]

Abstract: The present study was aimed to assess the efficacy of the tetramine, spermine (Spm) as a seed priming agent in attenuating oxidative damages and improving salt tolerance in salt-stressed seedlings of IR-64 (salt-sensitive) and Nonabokra (salt-tolerant) rice cultivars. The extent of damages was lesser in Nonabokra due to higher cysteine and ascorbic acid (AA), reducing power ability, concomitant with unaltered ascorbic acid oxidase (AAO) activity, and elevated ascorbate peroxidase (APX) and α-amylase activity. Spm priming alleviated salt stress injury by lowering the malondialdehyde and H2O2 content and avoiding chlorophyll degeneration in both the cultivars, the effect being more pronounced in IR-64 in terms of H2O2 reduction. The intrinsic property of Spm in stress amelioration was highly evident with respect to the reduction in the levels of anthocyanin, total phenolics and cysteine, and activity of AAO and superoxide dismutase (SOD) in IR-64, whereas lowered guaiacol peroxidase (GPX), catalase (CAT) and SOD activity in Nonabokra, as compared to Spm non-primed stressed-seedlings. However, Spm priming enhanced the reducing power ability, GPX, α-amylase and polyphenol oxidase (PPO) activities in IR-64, and anthocyanin, AA and CAT activity in Nonabokra, as means of mitigating cellular NaCl toxicity. A clear-cut variation in GPX, CAT, SOD and esterase isozyme profile was discernible between the two cultivars during salinity stress, with specific isoform(s) being up regulated or down regulated with Spm pre-treatment. In terms of osmolyte regulation, Spm priming appeared to be more promising in Nonabokra, because of the enhanced levels of reducing sugar, amino acids and proline. All these results indicated that seed priming with Spm at the pre-sowing stage can promote salinity tolerance with varying degrees in the two rice cultivars by attenuating oxidative damages, triggering the antioxidants and osmolytes, and activating the antioxidative enzymes at the protein level. Keywords: Antioxidants - Osmolytes - Salt stress - Seed priming - Spermine. [Cite as: Paul S & Roychoudhury A (2016) Seed priming with spermine ameliorates salinity stress in the germinated seedlings of two rice cultivars differing in their level of salt tolerance. Tropical Plant Research 3(3): 616–633] INTRODUCTION Soil salinity is one of the brutal abiotic stress factors affecting crop productivity worldwide. Rice sensitivity to salt varies according to growth stage and also among the cultivars. The high yielding rice cultivars like IR-29, IR-64, IR-72, M-1-48 are salt-sensitive, whereas cultivars like Pokkali, Nonabokra, Oormundakon, etc. are low yielding, but salt-tolerant (Soda et al. 2013). Salt stress involves a combination of dehydration or osmotic stress damages due to excess accumulation of Na+ ions and loss of K+ ions, which adversely affects plant growth and development. Oxidative damages, invoked under salinity stress, as an early rapid response is due to the formation of reactive oxygen species (ROS), like superoxide anion (O2-), singlet oxygen (1O2), hydroxyl radicals (OH-) and hydrogen peroxide (H2O2). The ROS also trigger peroxidative reactions and cause serious damages to phospholipids, nucleic acids and proteins. To ward off such damages, plants have evolved a complex www.tropicalplantresearch.com

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Received: 07 September 2016

Published online: 31 December 2016 https://doi.org/10.22271/tpr.2016.v3.i3.082

Paul & Roychoudhury (2016) 3(3): 616–633 . antioxidative system, participated by the non-enzymatic and enzymatic antioxidants (Gill & Tuteja 2010). The enzymatic antioxidants typically include guaiacol peroxidase (GPX, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) and enzymes belonging to the ascorbate-glutathione cycle (AsAGSH cycle) such as ascorbate peroxidase (APX, EC 1.11.1.11). In perennial rye grass (Lolium perenne L.), NaCl treatment enhanced the expression of SOD, CAT and peroxidase (POD) (Hu et al. 2011). Four forms of CAT isozymes, three APX isozymes and seven GPX isozymes were identified in plants under salinity stress (Lee & An 2005). Differential changes in the level of isozyme forms might be important signals in salt stress response (Parida et al. 2004). Under osmotic stress, the compatible solutes or osmolytes accumulate intracellularly in order to lower the osmotic potential, so as to drag more water within the cell. The common osmolytes include sucrose, proline (Pro) and glycine betaine, in addition to other molecules accumulating to high concentrations in certain species (Munns & Tester 2008). Accumulation of Pro and mannitol has been reported under drought stress (Sickler et al. 2007). Polyamines (PAs) constitute another class of low molecular weight, nitrogeneous, aliphatic osmoprotectants. The common PAs in plants are spermidine (Spd3+), spermine (Spm4+) and their precursor, putrescine (Put2+). Being polycationic in nature at physiological pH, PAs can readily bind to the negatively charged phospholipid head group or other anionic sites on the membrane, regulating the integrity of the membrane. They also play vital role during multiple abiotic stresses including salinity, drought, low or high temperature and heavy metal toxicity (Roychoudhury et al. 2008). Being a tetramine, Spm is presumed to be a protective agent against various oxidative damages. Yamaguchi et al. (2006) found that a Spm-deficient Arabidopsis mutant exhibited hypersensitivity to NaCl stress. The NaCl-hypersensitivity of the mutant could be cured by Spm but not by Put and Spd, suggesting a close link between NaCl hypersensitivity and Spm deficiency. Spm was found to have a protective role in detached rice leaves during water stress induced by the application of polyethylene glycol 6000 (Cheng & Kao 2010). Numerous attempts have been made to improve the salinity tolerance of various crops by traditional breeding program, but the progress has been quite slow with limited commercial success. Exogenous PA application increased endogenous PA levels and alleviated salt stress damages in vegetative tissues of several plants including rice. Such protective roles have been attributed to the reduction of salinity stress-induced damages (Liu et al. 2006) by inducing the activity of antioxidative enzymes and increasing the synthesis of nonenzymatic antioxidants and compatible osmolytes (Roychoudhury et al. 2011, Saleethong et al. 2011). Transgenic plants overproducing PAs, via modulating PA-metabolic pathways, rendered protection against abiotic stress conditions, while reduced in vivo PA levels resulted in decreased stress tolerance (Alet et al. 2012). Exogenously applied PAs have also been shown to effectively alleviate stress injury caused by acid rain, ozone, heavy metals, chilling and water stress (Alcázar et al. 2011). Shi et al. (2010) found that Spm pretreatment confers dehydration tolerance of Citrus plants through modulation of antioxidative potential and stomatal regulation. Since the distinct functional regulation of PAs largely depend on plant species as well varieties of the same species, deciphering the precise significance of PAs in stress response is quite complicated. The use of some simple, cost-effective methods, such as seed priming is regarded as a pragmatic approach to overcome plant growth retardation under saline condition. Seed quality, seed germination rate and seedling vigor are altogether vital factors for sustainable crop production, particularly under adverse environmental conditions (Sun et al. 2007). Priming is a process by which seeds are exposed to restricted water availability under controlled conditions, allowing some of the pre-germination metabolic activities (physiological and chemical) to proceed, before completion of germination. This is followed by short-term storage through redrying before ultimate sowing (Farooq et al. 2010a, b). There are adequate reports showing that under diverse environmental stresses such as salinity, water deficit and extreme temperatures, osmopriming leads to cellular, subcellular and molecular changes in seeds, subsequently promoting seed vigor during germination and seedling emergence in different plant species (Beckers & Conrath 2007). However, the efficacy of different priming agents varies under different stresses as well as in different crop species. Although the effects of seed osmopriming with different compatible solutes and growth regulators on different plant species have been reported from time to time, little evidence exists on the effects of seed priming with Spm on germinated rice seedlings, subsequently exposed to salt stress. Using two rice cultivars with varying degrees of salt tolerance, viz., IR-64 (salt-sensitive) and Nonabokra (salt-tolerant) as experimental models, the focus of this study was to investigate if pre-soaking the seeds with Spm has the potentiality to www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . ameliorate the oxidative damages in the germinated seedlings exposed to salinity stress. The efficacy of Spm as a seed priming agent in improving salt tolerance was investigated on the basis of measuring certain biochemical stress-markers (lipid peroxidation, hydrogen peroxide formation and chlorophyll degradation), analyzing the level or activity of antioxidants (non-enzymatic and enzymatic), osmolyte regulation, isozyme expression patterns and activities of the enzymes like α-amylase and polyphenol oxidase. MATERIALS AND METHODS Plant materials, growth conditions and stress treatment The seeds of Oryza sativa L. cv. IR-64 were obtained from Chinsurah Rice Research Station (Hooghly, West Bengal, India) and Nonabokra seeds from Central Soil Salinity Research Institute (Canning, West Bengal, India). The initial seed moisture content ranged between 8.5-9.5% (on a dry weight basis). The seeds were surface sterilized with 0.1% (w/v) HgCl2 for 20 min, and washed extensively with sterile water. The seeds were pre-treated with 2.5 mM of Spm, the concentration of Spm was chosen on the basis of earlier work by Iqbal et al. (2006). Healthy rice seeds (500 seeds for each treatment) were primed separately in 50 ml solution of Spm or in distilled water for 8 h at room temperature (25oC) in plastic cups. After pre-soaking, the seeds were surface dried on filter paper and allowed to dry for 12 h at room temperature (25oC). The air-dried seeds of both the cultivars were placed on two layers of filter paper and supplemented with 75 mM NaCl for stress treatment, while distilled water was used as control (untreated). Solutions were renewed every two days. Four sets of samples were maintained: Set 1. Water-primed seed without salt stress Set 2. Water-primed seed with salt (75 mM NaCl) stress Set 3. Spm-primed seed without salt stress Set 4. Spm-primed seed with salt (75 mM NaCl) stress The 10 day-old seedlings from each of the above sets were germinated at 32oC, under 16 h light and 8 h dark photoperiodic cycles with 50% relative humidity and 700 µmol photons m-2 s-1. The seedlings were harvested, frozen in liquid nitrogen, and 0.5 g of each sample was used for the following estimation. Estimation of oxidative damages The malondialdehyde (MDA) content was estimated from 0.5 g of each of the samples, using MDA extinction coefficient 155 mM-1cm-1 (Roychoudhury et al. 2012). Hydrogen peroxide levels from 0.5 g of samples were determined spectrophotometrically at 390 nm according to Velikova et al. (2000). Total chlorophyll content from 0.5 g of leaf samples was estimated according to Roychoudhury et al. (2007). Estimation of non-enzymatic antioxidant parameters [anthocyanin, cysteine (Cys), total phenolic content (TPC) ascorbic acid (AA), reducing power] and ascorbic acid oxidase (AAO, EC 1.10.3.3) activity For anthocyanin determination, 0.5 g of samples was extracted with acidified [1% (v/v) HCl] methanol (25 mg ml-1) for 24 h at 4oC with occasional shaking. The absorbance of the extract was recorded at 525 nm and the amount of anthocyanin was calculated using a millimolar extinction coefficient of 31.6 (Roychoudhury et al. 2007). For Cys estimation, 0.5 g of samples was homogenized in 5% (v/v) chilled perchloric acid (PCA) and centrifuged at 10,000 × g for 10 min at 4oC. The absorbance of the supernatant was measured using acidninhydrin reagent at 560 nm (Roychoudhury et al. 2007). The TPC in the extract was determined according to Jayaprakasha et al. (2001) with some modifications. About 0.5 ml of the aqueous extract of each sample was mixed with 2.5 ml of 10-fold-diluted Folin–Ciocalteu reagent and 2 ml of 7.5% (w/v) sodium carbonate. The mixture was allowed to stand for 30 min at room temperature (25oC) and the absorbance was measured at 760 nm. The final results were expressed as tannic acid equivalents. The assay of AA was performed by macerating the leaf tissues with metaphosphoric acid and titrating with 0.1% 2, 6-dichlorophenolindophenol (DCPIP) (Roychoudhury et al. 2007). The AAO activity was assayed following the method of Oberbacher & Vines (1963). The reducing power of the rice extract was determined following Kumaran & Karunakaran (2006), with some modifications and the absorbance was measured at 700 nm. Estimation of activity of antioxidant enzymes: guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) Total protein was extracted from the samples by the method of Anderson et al. (1995). The GPX activity was determined using the method of Srinivas et al. (1999) following the formation of tetraguaiacol by www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . measuring the absorbance at 470 nm and using an extinction coefficient 26.6 mM-1cm-1. The activity of APX was measured according to the method of Nakano & Asada (1981) by estimating the rate of ascorbate oxidation (extinction coefficient 2.8 mM-1 cm-1). The CAT activity was assayed by measuring the initial rate of H2O2 disappearance at 240 nm using the extinction coefficient 40 mM-1cm-1 for H2O2 (Velikova et al. 2000). The SOD activity, the basis of which is its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) (Beauchamp & Fridovich 1971), was assayed following Alonso et al. (2001) with certain modifications. The unit of SOD activity was defined as that amount of enzyme which caused 50% inhibition of the initial rate of reaction in the absence of enzyme. In gel analysis of isozymes of GPX, CAT, SOD and esterase (EST, EC 3.1.1.43) For in gel analysis of the isozymes of GPX, 50 μg of protein was run in a non-denaturing 10% polyacrylamide gel in dark under cold condition. The specific bands were detected by submerging the gel in a staining solution containing 50 mM potassium phosphate buffer pH 7.0, 0.46% (v/v) guaiacol and 13 mM H2O2 until red bands appeared. For in gel studies of CAT, 80 μg of protein was loaded in non-denaturing 10% polyacrylamide gel under cold condition. The electrophoresed samples in the gel was incubated in 0.05% H2O2 (v/v) for 10 min and subsequently developed in 2% (w/v) FeCl3 and 2% (w/v) K3FeCN6 solution for 10 min. For in gel staining of isozymes for SOD, 80 μg of protein was run through 10% native polyacrylamide gel electrophoresis in dark under cold conditions, followed by completely submerging the gel in freshly prepared staining buffer, containing 50 mM phosphate buffer pH 7.0, 0.1 ml EDTA, 28 mM TEMED, 0.003 mM riboflavin and 0.25 mM nitroblue tetrazolium for 30 min in dark condition. Thereafter, the gel was placed on an illuminated glass plate until the bands become visible. For detection of isozymes for EST, about 50 μg of protein was run in a non-denaturing 10% polyacrylamide gel in dark under cold condition. For staining of the bands, α-naphthyl acetate was used as a substrate and Fast Blue RR salt as a dye coupler. The staining solution consisted of 100 ml of 0.1 M sodium phosphate buffer pH 6.0, 100 mg Fast Blue RR salt and 0.1 g of αnaphthyl acetate (acetone : water, 1: 1). The gel was incubated in the filtered solution for 30 min at 37oC in dark, and then fixed in 50% (v/v) ethanol. Estimation of reducing sugars, total amino acids and proline The reducing sugar content from 0.5 g of samples was determined spectrophotometrically at 630 nm with freshly prepared anthrone reagent (Irigoyen et al. 1992). The total amino acids were quantified by the ninhydrin method according to Moore (1968). Free proline (Pro) content from the leaf samples was determined at 520 nm according to the procedure of Bates et al. (1973). Estimation of α-amylase (EC 3.2.1.1) and polyphenol oxidase (PPO, EC 1.14.18.1) activity The assay of α-amylase activity was performed from 1 g of tissues (Tarrago & Nicolas 1976) after inactivating β-amylase by heating at 70ºC for 5 min with 9 mM CaCl2 and performing the assay following the standard method (Chrispeels & Varner 1967). The PPO activity was assayed spectrophotometrically at 480 nm using 1 g of samples (Mayer & Harel 1979). Protein estimation For all the enzyme assays, protein contents were estimated using bovine serum albumin (BSA) as standard (Lowry et al. 1951). Equal amount of total protein from all the test samples were used in our assay. Statistical analysis The experiments were carried out in a completely randomized design (CRD) with three replicates; each replication comprised an average of 50 seeds, and the results presented as means ± standard error (SE). The data and significant differences among mean values were compared by descriptive statistics (± SE) followed by Student’s‘t’-test. The statistical significance was calculated at P ≤ 0.05. RESULTS MDA, H2O2 and chlorophyll content During salt stress, the MDA content increased in both the cultivars raised from water pre-treated seeds, viz., 2.2 times and 1.2 times respectively in IR-64 and Nonabokra, when compared to the control. When Spm pretreated seeds were subjected to stress imposition, the MDA content in the seedlings was reduced 1.7 folds and 1.4 folds respectively in IR-64 and Nonabokra, as compared with seedlings raised from Spm non pre-treated seeds under salinity stress conditions (Fig. 1A). Salinity stress increased H2O2 content 1.7 times and 1.1 times www.tropicalplantresearch.com 619

Paul & Roychoudhury (2016) 3(3): 616–633 . respectively in IR-64 and Nonabokra seedlings germinated from water pre-treated seeds, as compared with control. The induction of H2O2 by salt was reduced in the seedlings raised from Spm pre-treated seeds, viz., 1.9 times and 1.2 times in IR-64 and Nonabokra respectively, as compared to the stressed seedlings without Spm pre-treatment (Fig. 1B). The chlorophyll content in salt–stressed seedlings decreased 58% and 49% respectively in IR-64 and Nonabokra, as compared with unstressed seedlings. However, seedlings raised from Spm pretreated seeds could overcome the salinity-induced chlorophyll loss by 32% and 33% respectively in IR-64 and Nonabokra, as compared to the seedlings raised from Spm non-pretreated seeds (Fig. 1C).

Figure 1. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on oxidative damage indices, viz., MDA content (A), H2O2 content (B) and chlorophyll degeneration (C), in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. Data are the mean value (n = 3) ± SE. The SE in each case is represented by the vertical bar in each graph. Statistical differences at P ≤ 0.05 have been calculated for each t-test.

Non-enzymatic antioxidant levels and AAO activity Salinity stress triggered the anthocyanin level almost 1.6 times in IR-64 and 1.2 times in Nonabokra, as compared with control seedlings. The stressed seedlings from Spm-treated seeds showed 2.1 times reduction in anthocyanin in IR-64, whereas 1.3 times increment in Nonabokra, relative to the Spm non-treated stressed seedlings. The anthocyanin level was considerably higher in Nonabokra (1.5 times) than IR-64 in Spm pretreated stressed seedlings (Fig. 2A). For Cys, a decrease in the level was recorded for both the cultivars during salinity stress, viz., 1.4 times and 1.1 times in IR-64 and Nonabokra respectively. Following Spm priming, the stressed seedlings showed further reduction in Cys level in both the cultivars, viz., 1.6 times and 1.8 times in IRwww.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . 64 and Nonabokra respectively, as compared with stressed seedlings without Spm pre-treatment (Fig. 2B). Salt stress increased the AAO activity by 1.2 times in IR-64, whereas the activity remained unchanged in Nonabokra, relative to the control seedlings. Spm pre-treatment lowered the AAO activity 1.3 times in IR-64, while remaining unchanged in Nonabokra seedlings, when subjected to salt stress (Fig. 2C). The TPC in saltstressed seedlings showed 1.3 times and 1.1 times increase in IR-64 and Nonabokra respectively, as compared with control seedlings. However, exposure of seedlings from Spm pre-treated seeds to salt stress lowered the TPC, viz., 1.8 times and 1.4 times respectively, relative to the stressed seedlings in absence of Spm (Fig. 2D). With salinity stress, the AA content remained unaltered in IR-64, whereas it was induced 1.2 times in Nonabokra, as compared with non-stressed seedlings. When Spm-primed seeds were grown under salt stress, the AA content again remained almost unchanged in IR-64, whereas Nonabokra showed 1.2 times enhancement, with respect to Spm-non primed salt-treated seedlings (Fig. 2E). The reducing power in salt-stressed seedlings of IR-64 decreased drastically by 11.4 times, while 1.8 times decrease was recorded in Nonakora seedlings, with respect to the unstressed seedlings. The reducing power was enhanced particularly in IR-64 seedlings by 1.5 times, when Spm-pre-treated seeds were used, as compared with Spm non-primed samples (Fig. 2F).

Figure 2. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on antioxidant parameters, viz., anthocyanin content (A), Cys content (B), TPC (C), AA content (D), AAO activity (E) and reducing power (F), in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The data represented are means of three observations (n = 3) ± SE. Statistical differences at P ≤ 0.05 have been calculated for each t-test.

Antioxidant enzyme activity Salinity stress increased the GPX activity 1.8 times in both IR-64 and Nonabokra, relative to the control. Spm pre-treatment drastically induced further the GPX activity, viz., 5.8 times in IR-64, while in the tolerant cultivar Nonabokra, the GPX activity was lowered 1.5 times, as compared with stressed seedlings in absence of Spm pre-treatment (Fig. 3A). The APX activity, on the contrary, showed differential response in the two cultivars. While the activity decreased 1.8 times in IR-64, it increased 1.3 times in Nonabokra after salinity stress. Seed priming with Spm induced the activity slightly in IR-64, while the activity was lowered by 1.7 times in Nonabokra during salinity stress, as compared to stressed seedlings in absence of Spm (Fig. 3B). Salinity stress increased the CAT activity 2.1 times and 1.6 times in IR-64 and Nonabokra respectively, when compared to the control. Spm pre-treatment of seeds increased the CAT activity particularly in Nonabokra (1.3 times), while in IR-64, it was almost unaltered, as compared with Spm non-treated plants under salinity stress condition www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . (Fig. 3C). Salinity stress raised the SOD activity 3.7 times in IR-64 and 4.4 times in Nonabokra seedlings relative to the control (unstressed seedlings). Spm pre-treatment of seeds, however, lowered the SOD activity in the salt-stressed seedlings of both the cultivars, viz., 3.9 times and 1.6 times respectively in IR-64 and Nonabokra, when compared to the stressed seedlings raised from Spm non-primed seeds (Fig. 3D).

Figure 3. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on the activity of antioxidative enzymes, viz., GPX (A), APX (B), CAT (C) and SOD (D) in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The data represented are means of three observations (n = 3) ± SE. Statistical differences at P ≤ 0.05 have been calculated for each t-test.

Isozyme profile for the antioxidative enzymes While five GPX isozymes (GPX1, GPX2, GPX3, GPX4 and GPX5) were noted in IR-64, GPX5 was uninduced in Nonabokra, so that four isozymes (GPX1, GPX2, GPX3 and GPX4) were prominent. The GPX1 expression was more prominent in IR-64, while lesser induced only in the stressed seedlings of Nonabokra, whether in absence or presence of Spm pre-treatment. However, GPX2 expression was higher in Nonabokra, especially in the stressed seedlings, while weakly induced in IR-64 (Fig. 4A, 4B). Three CAT isozymes (CAT1, CAT2 and CAT3) were found in Nonabokra, while CAT3 was undetected in IR-64. The most abundant isozyme was CAT1, whose expression was almost constitutive in Nonabokra, while in IR-64, it was better induced after salinity stress and with Spm pre-treatment. Both salinity stress and Spm pre-treatment increased the intensity of CAT2 in IR-64, but undetected under control condition. The CAT2 and CAT3 inductions were higher in Nonabokra during salinity stress, while the expression was lower after Spm pre-treatment (Fig. 4C, 4D). Four SOD isozymes (SOD1, SOD2, SOD3 and SOD4) were observed in Nonabokra, of which SOD4 was undetected in IR-64. The abundance of SOD1, SOD2 and SOD3 was enhanced in IR-64 stressed seedlings raised from Spm pre-treated seeds (Fig. 4E, 4F). In case of EST, three isozymes, namely EST1, EST2 and EST3 were noted in both the cultivars. The most abundant isozyme was EST2, which was constitutively expressed in both IR-64 and Nonabokra under all the experimental conditions. The EST3 isozyme induction was considerably higher in Nonabokra than IR-64, where EST3 induction was noted upon salinity stress, with or without Spm pretreatment. Of all the isozymes, the EST1 was the most feebly induced in both the cultivars; the expression was raised after salinity stress in Nonabokra, whereas Spm pre-treatment induced the isozyme appreciably in IR-64 www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . (Fig. 4G, 4H).

Figure 4. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on isozyme profile of GPX, CAT, SOD and EST in IR-64 (A, C, E and G respectively) and Nonabokra (B, D, F and H respectively) seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The bands were resolved in non-denaturing polyacrylamide gel.

Reducing sugar, total amino acid and proline levels Salinity stress lowered the reducing sugar content in both the cultivars, viz., 1.1 times and 1.3 times respectively in IR-64 and Nonabokra, as compared with unstressed seedlings. When Spm pre-treated seeds were germinated in presence of salt, the reducing sugar level in IR-64 seedlings remained unaffected, while that in Nonabokra seedlings increased 1.2 times, compared with the stressed seedlings without Spm pre-treatment (Fig. 5A). Upon salt stress exposure, the total amino acid level in Nonabokra decreased 1.3 times, while it remained unaltered in IR-64, with respect to the control conditions. Seedlings raised from Spm pre-treated seeds showed differential response during salinity stress. While in IR-64, the amino acid level was lowered 1.1 times, the level enhanced almost 1.8 times in Nonabokra, as compared with stressed seedlings in absence of Spm (Fig. 5B). The Pro level in both the cultivars increased with salinity stress, 1.6 times in IR-64 and 1.9 times in Nonabokra, as compared with non-stressed seedlings. Nonabokra seedlings raised from Spm primed seeds showed further enhancement in Pro level, viz., 1.2 times, while IR-64 seedlings registered 1.2 times lowered Pro level, with respect to stressed seedlings without Spm pre-treatment (Fig. 5C). www.tropicalplantresearch.com

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Figure 5. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on osmolyte regulation, viz., reducing sugar content (A), amino acid content (B) and Pro content (C), in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The data represented are means of three observations (n = 3) ± SE. Statistical differences at P ≤ 0.05 have been calculated for each ttest.

α-amylase and PPO activity The α-amylase activity showed a differential pattern of activity in the two cultivars during salt stress. While a decreased activity was noted in IR-64, Nonabokra seedlings registered 1.4 times enhanced activity, as compared with unstressed seedlings. When Spm-primed seeds were exposed to salinity stress, the enzyme activity increased 1.1 times in IR-64, whereas it remained unaltered in Nonabokra, with respect to the salttreated seedlings without Spm pre-treatment (Fig. 6A). Salinity stress lowered the PPO activity 1.8 times in Nonabokra, while the activity was slightly triggered in IR-64, with respect to control seedlings. When Spmprimed seeds were grown under salt treatment, the PPO activity was lowered to a small extent (1.1 times) in Nonabokra, while showing a small increment (1.2 times) in IR-64, relative to the stressed seedlings without Spm application (Fig. 6B). DISCUSSION Priming, a prior encounter with a particular type of chemical or stress condition, is known to endow plants with greater tolerance to subsequent stress exposure of the same or different kind. Seed priming is a pre-sowing strategy that influences the seedling development at a later stage, via modulation of pre-germination metabolic activity, preceding the protrusion of the radicle (Patane et al. 2009). During priming, the seeds may be partially treated with water or various chemical solutions, so that the pre-germination metabolic activity starts, but the radicle emergence is prevented, followed by drying the seed. Improved growth and stress tolerance of the www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . primed seedlings have been reported for wheat, maize, cucumber, sugarcane, lentil and capsicum (Yadav et al. 2011). Although the phenomenon is known for decades, the underlying mechanism responsible for the better performance of the primed plants under stress regimes is not well explained or understood.

Figure 6. Effect of Spm (2.5 mM) pre-treatment of seeds (8 h) on α-amylase (A) and PPO (B) activity in IR-64 and Nonabokra seedlings under 75 mM NaCl; the stress was imposed for 10 days. The untreated seedlings (with or without Spm pre-treatment of seeds) served as experimental control. The data represented are means of three observations (n = 3) ± SE. Statistical differences at P ≤ 0.05 have been calculated for each t-test.

The PA accumulation in many plants is the immediate response observed in different crop species after exposure to saline conditions (Roychoudhury & Das 2014). Most significant changes in PA levels upon salinization appear to be those of Spm, according to the data reported in rice (Maiale et al. 2004), maize (Jimenez-Bremont et al. 2007) and wheat (El-Shintinawy 2000). In most cases, Spm is more potent than Spd and considerably more efficient than Put. The more pronounced protective effect of Spm in comparison with other PAs could be accounted for by its longer chain and greater number of positive charges which allows greater neutralizing and membrane stabilizing ability. The Spm-deficient mutant Arabidopsis was found to be hypersensitive to high salt stress and this phenotype was abrogated by exogenously applied Spm (Yamaguchi et al. 2006). The ameliorative role of exogenous Spm in reproductive phase of soybean during polyethylene glycol (PEG)-induced osmotic stress has been shown, thereby improving the plant reproductive health (Radhakrishnan & Lee 2013). Since chemical priming is one of the prominent pre-germination strategies to overcome the detrimental effects associated with osmotic stresses, the effect of seed priming with Spm was assessed in the present study on the performance of seedlings of two rice cultivars, IR-64 and Nonabokra, during subsequent exposure to salinity stress. Salinity stress led to a significant decline in the seedling quality of both the varieties by causing chlorophyll degeneration, increased MDA content and higher production of H2O2, though the salt-tolerant cultivar suffered lesser damages. Both chlorophyll degradation and increment of MDA content, which is a product of lipid peroxidation, are associated with the accumulation of ROS, pointing towards the damages incurred by the cell www.tropicalplantresearch.com 625

Paul & Roychoudhury (2016) 3(3): 616–633 . membrane and chloroplast membrane in plants (Halliwell 2006). Gill & Tuteja (2010) reported that higher accumulation of MDA declines the membrane fluidity, inactivates the receptors and degrade the membrane proteins, enzymes and ion channels. However, seed pre-treatment with Spm considerably lowered the MDA and H2O2 content, along with partial recovery of chlorophyll during salinity stress. The effect of Spm was more prominent in IR-64 in terms of considerable lowering of the H2O2 production under saline conditions. All these results are consistent with the previous studies in Arabidopsis, sunflower and soybean (Kusano et al. 2007, Radhakrishnan & Lee 2013) where exogenous Spm could effectively overcome the deleterious effects of salinity stress. Among the non-enzymatic antioxidants, the levels of anthocyanin and TPC showed increment with salinity stress in both the cultivars, with somewhat greater enhancement in the susceptible cultivar IR-64. Such increase is well supported by our earlier observations in rice during salinity (Roychoudhury et al. 2008) and drought (Basu et al. 2010) stress. The concerted action of low molecular weight antioxidants like anthocyanins (ChalkerScott 1999) and polyphenols (Sgherri et al. 2004) can effectively scavenge harmful radicals and stabilize the membranes against lipid oxidation under stressed conditions. Genisel et al. (2015) have reported the mitigating effect of Cys on growth inhibition in salt-stressed barley seedlings related to its own antioxidant properties. The lowered Cys content in both the cultivars, especially in IR-64, with salinity stress could be explained by the fact that more and more endogenous Cys pool is being utilized probably for the synthesis of thiol-related compounds, like glutathione or γ-glutamylcysteine-containing homologues, which maintain cellular redox homeostasis by quenching of ROS (Das & Roychoudhury 2014). Shalata & Neumann (2001) have shown the role of AA in increasing resistance to salt stress in tomato. Exogenous AA also increased the endogenous AA content, thereby reducing oxidative damages, with more pronounced effect in the tolerant cultivar Pokkali than in Peta, the sensitive cultivar (Wang et al. 2014). These data are in agreement with our observation where salinity stress enhanced the AA content only in the tolerant cultivar Nonabokra, concomitant with unaltered AAO activity. On the other hand, the AA content remained unaltered in IR-64, with a slightly enhanced AAO activity, signifying the better protective role of AA in Nonabokra. The suppressed expression of AAO in Nonabokra also justifies greater tolerance to salt stress than IR-64 (Yamamoto et al. 2005). The higher reducing power capability of Nonabokra during salinity stress depicts more efficient antioxidative mechanism in this cultivar in terms of free radical scavenging. The reduction in anthocyanins, TPC and Cys levels in stressed seedlings raised from Spm-primed seeds (as compared to stressed seedlings in absence of Spm) in IR-64 clearly highlights the role of Spm itself as antioxidant in mitigating oxidative stress in the salt-sensitive cultivar. However, Spm priming further elevated the anthocyanin and AA content in the stressed Nonabokra seedlings as a means to ward off the damaging symptoms. This increase is well supported by the earlier observation where Spm application induced an increment in certain antioxidants like reduced glutathione and polyphenols (Radhakrishnan & Lee 2013). The lowered AAO activity and increased reducing power in stressed IR-64 seedlings raised from Spm-primed seeds also reflect the importance of Spm in maintaining higher AA pool with better defense mechanism in IR-64, the sensitive cultivar. To prevent ROS from damaging various cellular components, plants have developed multiple detoxification mechanism, including the activation of various antioxidative enzymes like SOD, POD, CAT, APX and glutathione reductase. In our case, salinity stress increased the activities of GPX and CAT in both the cultivars. The elevated activities of CAT and POD were also observed in salinity-stressed cucumber (Duan et al. 2008). The activity of APX, that uses AA as the specific electron donor, increased in the salt-tolerant cultivar during stressed conditions, concomitant with the increase in AA. A similar increase in APX activity was observed in Hordeum vulgare, Plantago maritima and Brassica campestris in response to salt stress (Hernández et al. 2010). However, in our study, a decreased APX activity was recorded in the salt-sensitive cultivar IR-64. Such decrease is in accordance with earlier studies in wheat (Heidari & Mesri 2008) and Eugenia (Acosta-Motos et al. 2015). In response to salt stress, the SOD activity increased in both IR-64 and Nonabokra seedlings, the activity being more pronounced in the sensitive cultivar. A similar increase in SOD activity was observed in salt-stressed pea, beet, maize and tomato (Azevedo-Neto et al. 2005, Koca et al. 2006). Seed priming with Spm followed by seedling exposure to salinity stress showed quite a contrasting response of all the antioxidative enzymes examined in the two cultivars. With respect to the GPX, Spm priming during salinity stress was particularly significant in the sensitive cultivar, showing a direct elevation or stimulatory effect of GPX activity, thereby rendering the tolerance mechanism in IR-64. The GPX activity, on the contrary, was down regulated by www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . Spm in Nonabokra. A similar trend in Nonabokra was noteworthy with respect to APX and SOD as well, where the stressed seedlings from Spm-primed seeds showed lowered enzyme activity, compared to the stressed seedlings without Spm. This showed that Spm at the applied concentration has an intrinsic protective role during salinity stress, so that the much heightened activity of such enzymes observed during salt stress is not that prerequisite in presence of Spm. The same explanation could also hold for SOD in Spm-primed, salt-treated IR-64 seedlings, where the down regulation is strikingly noteworthy. Our observation differs from many of the earlier observations, where exogenous PA application in the form of foliar spray or hydroponic culture during salinity or drought stress, mostly increased the activity of CAT, SOD, APX and POD (Roychoudhury et al. 2011, Shi et al. 2013, Zhu et al. 2014). However, Velikova et al. (2000) showed that pre-treatment with PAs led to a reduction of POD activity in acid rain-treated bean plants. Spm priming possibly pre-disposes the seedlings to and mimics a stress-like condition and enables them for better acclimatization when they encounter the actual stress situation, so that the higher activity of the antioxidant enzymes is not obligatory even in presence of NaCl. Yet the seedlings can manage to survive with a lowered activity of the majority of antioxidant enzymes. Thus, the anti-stress effect of PA pre-treatment during salinity stress is actually supported by the reduced defensive response rendered by the antioxidative enzymes in presence of Spm, as reported in the present work. The enhanced CAT activity with Spm in salt-treated Nonabokra is, however, in accordance with the earlier observation by Farooq et al. (2009). Several investigations have reported varietal differences in isozyme profile with salinity stress. While four SOD activity bands were identified in the leaves of salt-tolerant Plantago maritima, only two bands were observed in salt-sensitive P. media. Likewise, five POD activity bands were identified in the leaves of P. maritima, whereas only two bands in P. media (Sekmen et al. 2007). A differential POD and SOD isozyme activities among the four potato cultivars, Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive) were noted under saline conditions (Rahnama & Ebrahimzadeh 2006). Short-term salinity induced the expression of POD and SOD isozymes in cucumber seedlings (Du et al. 2010). In our experiment, five isozymes of GPX (GPX1-5) were noted in the sensitive cultivar, while four (GPX1-4) in the tolerant cultivar. The effect of Spm pre-treatment during salinity stress was evident from the higher induction of GPX1 in IR-64. GPX1 was however feebly induced in the stressed Nonabokra seedlings, even after Spm pretreatment. GPX5 was almost undetected in IR-64 upon Spm priming, as compared to control. Unlike IR-64, the GPX2 expression was sharper in the stressed Nonabokra seedlings, whether Spm pre-treated or not. Thus, the two varieties responded differentially with respect to GPX isozyme induction during Spm pre-treatment, with GPX1 playing more vital role in IR-64 and GPX2 in Nonabokra. In case of SOD, SOD4 was undetected in IR64, and the expression of SOD1, SOD2 and SOD3 was enhanced in the stressed seedlings of IR-64, raised from Spm pre-treated seeds. SOD3 expression was higher in Nonabokra under salinity stress. El-baky et al. (2003) observed that POD and CAT isozymes for different onion cultivars differed in number and relative concentration due to salt stress. In our study, the induction of CAT2 and CAT3 was higher in Nonabokra during salinity stress, while the expression was lower after Spm pre-treatment. The CAT1 expression in IR-64 was saltinducible and dependent on Spm treatment, whereas CAT3 expression was sharper in Nonabokra. Puyang et al. (2015) showed that pre-treatment with Spd increased the intensity of APX and CAT isozymes in both the cultivars, Kenblue and Midnight, and POD isozymes only in Kenblue cultivar of Poa pratensis. Exogenous Spd elevated the intensities of isozymes of APX, POD and SOD in alfalfa (Zhu et al. 2014). Application of exogenous Spd could also overcome oxidative damages in cucumber during salt and heat stress by causing certain changes in the zymogram expression of some antioxidant enzymes (Du et al. 2010, Tian et al. 2012). Our observation on EST showed that EST3 induction was considerably higher in Nonabokra than IR-64, where the induction was noted only after salinity stress, with or without Spm pre-treatment. EST1 was also expressed at a higher level in Nonabokra during stress, either in absence or presence of Spm. Such EST isozyme variation was reported earlier by Swapna (2002) in four rice cultivars, viz., Pokkali (moderately salt-tolerant) and M-1-48, Annapoorna and Jyothi (salt-sensitive), which indicated that this isozyme is also linked with salt tolerance. Mung bean and Sueada maritima plants, cultured under in vitro conditions, exhibited the highest EST activity between 150 and 400 mM NaCl. Overall, a clear-cut variation in GPX, CAT, SOD and EST isozyme profile was discernible between the two cultivars in our work, with specific isoform(s) being up regulated or down regulated with Spm pre-treatment.

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Paul & Roychoudhury (2016) 3(3): 616–633 . One of the strategies to regain turgor and resume growth during ionic stresses is to accumulate osmolytes like reducing sugar, including major carbohydrates, total free amino acids and especially Pro, which synergistically reduce the osmotic potential of the cytosol to facilitate water uptake (Roychoudhury et al. 2015). Zahra et al. (2010) reported that sugar levels in rice leaves increased significantly under salt stress. However, Alamgir & Ali (1999) observed that salinity reduced sugar content in four varieties, but increased sugar content in five other varieties. Pro and free amino acid content in the salt-stressed tissues of Pennisetum glaucum increased with increase in salt concentration as well as with duration of salt stress, thereby protecting the cellular macromolecules, maintaining the osmotic balance and also scavenging the free radicals (Sneha et al. 2013). Chutipaijit et al. (2009) reported that free Pro content of rice varieties was significantly increased with increasing salinity levels. Our observation showed a reduction in the level of reducing sugars in both the cultivars, as well as decrease in total amino acids in Nonabokra during salinity stress. Salinity stress might have led to a significant decrease in the efficiency of photosynthesis, thereby reducing the supply of soluble sugars. The soluble sugars, under such challenging situations, are probably utilized extensively for growth and maintenance of the osmotic homeostasis of cells. The reduced amino acid level could be accounted for by the fact that the amino acids are probably channelized towards the production of novel proteins during stress, rendering better tolerance in Nonabokra. Increase in free Pro level in the salt-stressed cultivars could either be due to enhanced synthesis, decreased degradation or both. The implication of Spm in increasing the level of reducing sugar in Nonabokra during salt stress is clear from our observation. Amino acids, especially Pro detoxifies plants by scavenging ROS or preventing them from damaging cellular structures (Roychoudhury et al. 2015). The constructive role of Spm priming in stress alleviation in terms of enhanced amino acid and Pro accumulation seems to be more promising in Nonabokra. The reverse trend, viz., decrease in amino acid or Pro content during salinity stress with Spm pre-treatment in IR-64 suggests an alternate mechanism by which Spm priming reduce the impact of salinity stress in the sensitive cultivar. The influence of NaCl on α-amylase activity was different in the two rice cultivars. While the activity was lowered in the sensitive cultivar, it increased to an appreciable extent in Nonabokra, signifying that sugar mobilization and hence germination was not compromised in the latter. Ashraf et al. (2002) have suggested that salt stress led to a decrease in α-amylase activity and break down of starch into reducing and non-reducing sugars in cotton. The amylase activity decreased with increasing salinity in Phaseolus vulgaris and increased in maize. In case of rice, salt stress significantly inhibited the activity of α-amylase during germination stage (Hualong et al. 2014), supporting our observation with IR-64. The reduction in α-amylase activity in IR-64 could account for the reduction in concentration and translocation of free sugars into the embryo axes during germination and early growth. Exogenous Put treatment during salinity stress increased the α-amylase activity in the growing seeds of P. vulgaris, thereby increasing the germination percentage of salt-stressed seeds (Zeid 2004). Tipirdamaz et al. (1995) also reported that Put, Spd and Spm significantly increased α-amylase activity in barley seeds. Our data, showing slightly improved α-amylase activity in stressed seedlings of IR-64 raised from Spm-primed seeds, correlates with earlier observations, indicating that adverse effect of salt stress on germination could be partially rectified in the salt-sensitive cultivar. The PPO activities were higher in bean and maize seedlings treated with NaCl (Tuna et al. 2013). A higher PPO activity in the leaf tissues of groundnut var. TAG-24 than in the variety TG-26 during water stress ensured better drought tolerance mechanism in the former (Shinde & Laware 2015). Quite a contrasting result was derived in maize and Phaseolus mungo where PPO activity was decreased with increasing salinity (Dash & Panda 2001). In our case, the PPO activity followed a different trend in the two cultivars during salinity stress, with a significant decrease in the tolerant cultivar, thereby enabling conservation of phenolics as antioxidants, while increasing to a small extent in the sensitive cultivar. Earlier reports have shown that application of Spd increased the PPO activity in the leaves of sugarbeet during salinity stress (Hajiboland & Ebrahimi 2013). Treatment with Spd also caused PPO activation and mitigated the salinity effect in cucumber plants (Radhakrishnan & Lee 2014). In our case, the small increment in PPO activity in salt-stressed IR-64 seedlings raised from Spm-primed seeds is in agreement with the available reports. Although enhanced stress tolerance via exogenous PA application as foliar spray or in hydroponic culture are well documented in the available literatures, the mechanism of the protective effect of Spm pre-soaking of seeds on the performance of NaCl-stressed, germinated seedlings is poorly studied and understood. The present communication showed that oxidative damages encountered in the two examined rice cultivars could be www.tropicalplantresearch.com

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Paul & Roychoudhury (2016) 3(3): 616–633 . effectively attenuated with varying degrees, if the Spm pre-treated seeds are germinated and grown under salt stress. Spm itself exerts a direct antioxidative role, particularly in IR-64, thereby reducing the detrimental effect of salt stress by lowering the MDA and H2O2 content, and retrieving the endogenous chlorophyll content. The protective effect of Spm in Nonabokra was evidenced from the increase in osmolyte levels, rather than regulating the antioxidant machinery. Spm also effectively rendered protection against salt stress in IR-64 by enhancing the α-amylase and PPO activity. In the nutshell, seed priming with Spm at the pre-sowing stage holds a great promise as a traditional method of agriculture in growing major staple food crop like rice under salinity regimes, so as to prevent cumulative damages and widespread crop losses. ACKNOWLEDGEMENTS Financial assistance from Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India through the research grant (SR/FT/LS-65/2010) and from Council of Scientific and Industrial Research (CSIR), Government of India, through the research grant [38(1387)/14/EMRII] to Dr. Aryadeep Roychoudhury is gratefully acknowledged. REFERENCES Acosta-Motos JR, Diaz-Vivancos P, Álvarez S, Fernández-García N, Sanchez-Blanco MJ & Hernández JA (2015) Physiological and biochemical mechanisms of the ornamental Eugenia myrtifolia L. plants for coping with NaCl stress and recovery. Planta 242: 829–846. Alamgir ANM & Ali MY (1999) Effect of salinity on leaf pigments, sugar and protein concentrations and chloroplast ATPase activity of rice (Oryza sativa L.). Bangladesh Journal of Botany 28: 145–149. Alcázar R, Marco F, Cuevas JC, Patrón M, Ferrando A, Carrasco P, Tiburcio AF & Altabella T (2006) Involvement of polyamines in plant response to abiotic stress. Biotechnology Letters 28: 1867–1876. Alet AI, Sánchez DH, Cuevas JC, Marina M, Carrasco P, Altabella T, Tiburcio AF & Ruiz OA (2012) New insights into the role of spermine in Arabidopsis thaliana under long-term salt stress. Plant Science 182: 94– 100. Alonso R, Elvira S, Castillo FJ & Gimeno BS (2001) Interactive effects of ozone and drought stress on pigments and activities of antioxidative enzymes in Pinus halepensis. Plant, Cell & Environment 24: 905–916. Anderson MD, Prasad TK & Stewart CR (1995) Changes in isozyme profiles of catalase, peroxidase and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiology 109: 1247–1257. Ashraf MY, Sarwar G, Ashraf M, Afaf R & Sattar A (2002) Salinity induced changes in α-amylase activity during germination and early cotton seedling growth. Biologia Plantarum 45: 589–591. Azevedo-Neto AD, Prisco JT, Eneas-Filho J, de Abreu CEB & Gomes-Filho E (2005) Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and roots of salt tolerant and salt sensitive maize genotypes. Environmental and Experimental Botany 56: 87–94. Basu S, Roychoudhury A, Saha PP & Sengupta DN (2010) Differential antioxidative responses of indica rice cultivars to drought stress. Plant Growth Regulation 60: 51–59. Bates LS, Waldren RP & Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39: 205–207. Beauchamp C & Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 44: 276–287. Beckers GJM & Conrath U (2007) Priming for stress resistance: From the lab to the field. Current Opinion in Plant Biology 10: 425–431. Chalker-Scott L (1999) Environmental significance of anthocyanins in plant stress response. Photochemistry & Photobiology 70: 1–9. Cheng DG & Kao CH (2010) Effect of exogenous spermine on polyethylene glycol-induced responses in rice leaves. Crop, Environment & Bioinformatics 7: 233–242. Chrispeels MJ & Varner JE (1967) Gibberellic acid-enhanced synthesis and release of α-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiology 42: 398–406. Chutipaijit S, Cha-Um S & Sompornpailin K (2009) Differential accumulations of proline and flavonoids in indica rice varieties against salinity. Pakistan Journal of Botany 41: 2497–2506. www.tropicalplantresearch.com

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 634–641, 2016 DOI: 10.22271/tpr.2016.v3.i3.083 Research article

Isolation and characterization of lectin from the leaves of Euphorbia tithymaloides (L.) Aruna A. Jawade, Shubhangi K. Pingle*, Rajani G. Tumane, Anvita S. Sharma, Archana S. Ramteke and Ruchika K. Jain National Institute of Miners’ Health, JNARDDC Campus, Wadi Nagpur-440023, Maharashtra, India *Corresponding Author: [email protected]

[Accepted: 03 December 2016]

Abstract: Lectins or glycoproteins from the leaves of Euphorbia tithymalo were isolated after screening of different flora from Central India. The crude extract of leaves was dialyzed andammonium sulfate precipitation done and followed by dialysis for the purification of lectins. Protein concentration in the purified extract was 10 mg.ml-1 measured by Biuret Method. The purified lectins were able to agglutinate human erythrocytes of ABO blood group system. Agglutination was also visible with animal erythrocytes. Lectin of ET was Galactose/Lactose specific and shows maximum activity at pH-7 and temperature between 40–60º C. Molecular weight of purified extract of ET was determined by 1D- SDS Polyacrylamide gel electrophoresis which was found to be 70.24, 28.53 and 14.68 kDa. Keywords: Agglutination - Blood group - Electrophoresis - Euphorbia - Glycoprotein. [Cite as: Jawade AA, Pingle SK, Tumane RG, Sharma AS, Ramteke AS & Jain RK (2016) Isolation and characterization of lectin from the leaves of Euphorbia tithymaloides (L.). Tropical Plant Research 3(3): 634– 641] INTRODUCTION Lectins are proteins or glycoproteins of non-immune origin which possess the ability to agglutinate erythrocytes or precipitate glycoconjugates by binding to the recognized and specific carbohydrate residue present on cell surface (Ramteke 2009). Lectins are widely distributed in nature and can be found in many plants, animals and microorganisms. Plant lectin contains at least one catalytic domain and has the ability to recognize complex glycoconjugates (Peumans & Damme 1998). Lectins present in leaves, roots, stems and seeds of plants perform different biological activities and help in secondary metabolism such as defense mechanism. Due to specific binding capabilities, lectins involve in endocytosis, intracellular translocation of glycoproteins, cellular regulation, migration and adhesion, phagocytosis, binding of microorganisms to target tissues, control of morphogenesis, metastasis and many other activities (Sharon & Lis 2004, Abreu & Matthew 2006). ABO blood group system with Rhesus factor comprises of distinct determinant and lectins agglutinate with specific type of antigen present on RBCs (Ajit & Kanjaksha 2016). Euphorbia tithymaloid (ET) is a perennial succulent spurge. It is native to tropical and sub-tropical North America and Central America. These shrubs are 6 to 8 feet long and 18 to 24 inch wide. ET grows in fertilized sandy soil rich in metal concentration like boron, copper, iron, manganese, molybdenum and zinc. Their leaves are alternate, sessile, glabrous and acuminate in shape. It is a carcinogenic plant thus has the ability to grow in toxic soil very easily and rapidly. Sometimes, ET is also used to remediate soil and can be used as border of garden. Lectins present in this plant have many medicinal use and help in curing many diseases also. It shows anti-inflammatory, anti-bacterial, anti-septic, anti-hemorrhagic, anti-viral, anti-tumor and abortive properties. In this study lectins from the leaves of ET were characterized in terms of their physical, chemical and biological properties. MATERIALS AND METHODS Leaves of ET were collected from Nagpur, Maharashtra in Central India and used as source of lectins. www.tropicalplantresearch.com

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Received: 15 September 2016

Published online: 31 December 2016 https://doi.org/10.22271/tpr.2016.v3.i3.083

Jawade et al. (2016) 3(3): 634–641 . Human blood of groups A Rh+, B Rh+ and O Rh+ were collected from healthy persons. Animal bloods were collected from veterinary hospital of Nagpur, Maharashtra. Ammonium sulfate, sodium chloride, dialysis membrane-50, total protein kit (BioSystem-Reagent and chemicals), different sugars, pH buffers (pH 2–12), acrylamide, bis-acrylamide, β-Mercaptoethanol, coomassie brilliant blue, urea, thiourea, DDT, CHAPS, IPG strips, bromophenol blue, glycerol, SDS, TEMED and other chemicals were purchased from Himedia and Serva. Preparation of crude extract Leaves of ET were collected from the road side area of Nagpur, Maharashtra. Leaves were washed 2–3 times with tap water and then with distilled water and soaked in tissue paper. Leaves were homogenized with minimum amount of saline by using mortar and pestle at 4ºC. Homogenized extract was allowed to filter by using muslin cloth. The filtrate was centrifuged at 5000 rpm for 20 minutes. The obtained supernatant was stored at 4ºC and used for further analysis (Patil & Despande 2015). Purification of crude extract The crude extract was dialyzed for the separation of proteins and removal of impurity and small moleculesby dialysis membraneinnormal saline at 4ºC. This membrane contains micro pores through which the small molecules easily escaped. Therefore, protein molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis bag. Ammonium sulfate (AS) was used to precipitate lectins from the dialyzed extract by gradualaddition of ASat 4ºC. The precipitation obtained after 0–100% saturation of AS was centrifuged at 6,000 rpm for 30 minutes. Then, precipitate was dissolved in normal saline and again dialyzed till the solution was free from ammonium sulfate fraction (ASF). Protein Estimation Protein was estimated by using commercially available protein kit. Preparation of 2% erythrocytes The human blood samples werecollected in heparinized tubes and stored at 4ºC. Erythrocytes were washed 3–4 times with normal saline. Washed RBCs were used for preparation of 2% erythrocytes solution (Olsen 1944). Similarly, animal bloodswere also preceded as mentioned above. This 2% erythrocytes solution was used for determination of hemagglutination activity. Agglutination assay Agglutination test of purified extract was done by using 2% suspension of erythrocytes (Deshpande & Patil 2003). Hemagglutination activity was determined in 96 wells plate by using serial dilution. Agglutination was observed visually with carpet and button pattern after 5 hrs. Hemagglutination unit (HAU) which represents the titer strength was calculated with the reciprocal of last dilution of agglutination. Specific activity (SA) which is HAU per mg protein was also calculated. Agglutination inhibition assay Agglutination inhibition assay was done by testing the ability of different carbohydrates like disaccharides, pentoses, hexoses, oligosaccharides etc. to inhibit the agglutination (Kurokawa et al. 1976). 100 µl of 500 mM sugar solutions were incubated with 100 µl lectin for 30 minutes at room temperature. The agglutination in the presence of sugar was examined with 2% erythrocytes by the above described method. Minimum inhibitory concentration was taken which did not agglutinate the erythrocytes. pH stability studies The pH dependence of ET leaves lectin was determined by using buffer ranging from pH 1–13. For pH 1: 0.1N HCl, for pH 2 & 3: 0. 2M glycine - HCl buffer, for pH 4 & 5: 0.2M sodium acetate buffer, for pH 6 & 7: 0.2M sodium phosphate buffer, for pH 8: 0.2M Tris HCl buffer, for pH 9: 0.2M glycine-NaOH buffer and for pH 10 & 13: 0.2M carbonate-bicarbonate buffers were used. 100 µl of lectin was incubated with 100 µl of different buffer solutions for 30 minutes at room temperature and then assayed for agglutination with 2% erythrocytes (Suseelan et al. 1997). Temperature stability study Effect of temperature on the lectins of ET was observed by incubating 100 µl of leaves extract at different temperature ranging from 10–100ºC for 30 minutes. Agglutination test was carried out with 2% erythrocytes solution. www.tropicalplantresearch.com

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Jawade et al. (2016) 3(3): 634–641 . SDS polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE was done to determine the molecular weight of lectins of ET by the method of Weber & Osborn (1969). A 10% stacking gel and 5% running gel was used in SDS-PAGE with a standard marker protein (Aprotinin-6.5kDa, Lyzozyme-14.3 kDa, Soyabean Trypsin Inhibitor-20.1 kDa, Carbonic Anhydrase-29 kDa, Ovalbumin-43 kDa, Bovine Serum Albumin-66 kDa and Phosphorylase b-97.4 kDa). After electrophoresis the gel was stained with 0.2% coomassie brilliant blue (R250) and then destained in 10% acetic acid. Isoelectric focusing In 2-D electrophoresis, IPG (Immobilized pH gradient) strip of pH ranging 3–10 was used to perform isoelectric focusing of the sample. The strip containing sample were rehydrated using rehydrating buffer (6M Urea, 4% CHAPS, ampholyte, 0.1% Bromophenol blue) at room temperaturefor 18 hrs. 1D was performed according to the standard method after which the strip was treated with equilibration buffer (1.5 M TrisHCl, 6M Urea, 30% glycerol, 2% SDS, 0.01% Bromophenol blue and 200 mg of dithiothreitol) and with blocking buffer (1.5 M TrisHCl, 6M Urea, 30% glycerol, 2% SDS, 0.01% Bromophenol blue and 250 mg of iodoacetamide). Then 2D SDS-PAGE was allowed to run with 10% running gel at 15ºC. The spots appeared after 0.2% Coomassie blue stain was destained by using 10% acetic acid. RESULTS A total 120 herbs and shrubs from Nagpur District were randomly selected and screened for identification of agglutination activity. The crude extract of 25 plants showed agglutination activity with erythrocytes of different blood groups of human and animals. On the basis of literature survey and information obtained from civilian about medicinal valuesthree plantswere selected for further study. ET was selected as it showed good agglutination activity against blood group system (ABO). Protein estimation Protein content in the dialyzed extract of ET was found to be 10 mg.ml-1 by using Biuret kit. Agglutination assay Table 1. Agglutination study of lectins of Euphorbia tithymaloides leaves with human and animal erythrocytes.

Erythrocytes Human ‘O’ Human ‘B’ Human ‘A’ Cow Dog Fish Hen

Agglutination +++ ++ + + -

Hemagglutination results in table 1 revealed that human blood group ‘O’ showed strong agglutination as compared to ‘A’ and ‘B’ with lectin from the leaves of ET. In figure 1, blood group ‘A’, ‘B’ and ‘O’ gives carpet pattern till 6, 7 and 8 times dilutions respectively and on further dilution button pattern starts appearing which represents no more further precipitation of lectins. Plant lectin has also showed agglutination against cow erythrocyte but no agglutination was found in case of dogs, fish and hen (table 1). On the basis of result it was observed that minimum concentration of lectin was required for agglutination of group ‘O’ erythrocyte and was followed by group ‘B’ and group ‘A’. Hemagglutination unit (HAU) and specific activity (SA) were also calculated and depicted in the table 2.

Figure 1. Hemagglutinationtiterwith ABO blood group.

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Jawade et al. (2016) 3(3): 634–641 . Table 2. Protein concentration in leaves of Euphorbia tithymaloides.

HAU/ml ‘A’ ‘B’ ‘O’ 10 320 640 1280 Dialyzed extract Note: HAU-Hemagglutination Unit, SA-Specific activity. Protein (mg.ml-1)

‘A’ 32

SA ‘B’ 64

‘O’ 128

Agglutination inhibition assay Agglutination activity of lectin from leaves of ET was inhibited by D-Galactose and Lactose as showed in table 3. Result indicated inhibition of lectin was due to galactose/lactose specific sugars. Table 3. Inhibition of agglutination with different sugars by lectins of Euphorbia tithymaloides leaves.

Sugars D-Glucose Sucrose Lactose Sorbitol D-Fructose D-Maltose D-Arabinose D-Galactose D-Xylose D-Mannose D-Ribose

Minimum concentration required to inhibit the hemagglutination (mM) No inhibition No inhibition 500 No inhibition No inhibition No inhibition No inhibition 500 No inhibition No inhibition No inhibition

pH stability According to the result the optimum pH for maximum agglutination by leaves of ET was found to be neutral (pH 7). The activity varies in pH ranging from 3 to 11. Agglutination activity was lost below pH 4 and above pH 11 as mentioned in figure 2.

Figure 2. Effect of pH on Agglutination activity of lectin of Euphorbia tithymaloides leaves.

Effect of temperature and thermal inactivation Lectin from leaves of ET shows 100% agglutination between temperatures ranging from 40- 60ºC when tested after heated for 1 h at temperature above 20ºC. Below 40ºC and above 60ºC till 70ºC agglutination activity was half of initial and as the temperature increases the activity decreases and finally lost at 100ºC. According to the observed result lectin was stable for long period of time but it thermally inactive after heating at 100 ºC as showed in figure 3.

Figure 3. Effect of temperature on Agglutination activity of lectin of Euphorbia tithymaloides leaves.

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Jawade et al. (2016) 3(3): 634–641 . 1D-SDS polyacrylamide gel electrophoresis SDS-PAGE resulted into the appearance of bands with molecular weight of 70.24, 28.53 and 14.68 kDa in the purified extract from leaves of ET showed in figure 4. Thus, these may be the molecular mass of lectins present in ET.

Figure 4. Band pattern of lectins of ET on 1-D SDS-PAGE.

2D-SDS polyacrylamide gel electrophoresis On the basis of analyses of 12 observed spots, which were ranging in between pIs of 6.5 to 29 as showed in the figure 5.

Figure 5. Isoelectric focusing of lectins of ET on 2-D PAGE.

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Jawade et al. (2016) 3(3): 634–641 . DISCUSSION This study focused on identification and characterization of the lectin activities from ET plant species. Lectins were extracted and purified by using conventionalammonium sulfate precipitation method. The purification procedure consists of 0–100% ammonium sulfate saturation, which was followed by dialysis. The conventional ammonium sulfate precipitation method was very useful technique for initial purification of lectins from crude extract of Tridax procumben leaves (Ramteke & Patil 2005) and Volvariella volvacea (Mothong 2009). The extract agglutinates the erythrocytes of ABO blood group which resembles the characteristics of most of the glycoproteins. Nagata & Burger (1974) had reported that wheat germ lectins (WGL) were non blood group specific lectins. The present study also convinced with the above report as lectins of ET agglutinated RBC of all blood groups with similar competence indicating that there is no specificity towards blood groups. This may be due to absence of lectins specific receptors on the surface of RBSs. Lectins of many plant species also agglutinate with erythrocytes of different animals. Ramteke & Patil (2005) noted that lectin of Tridax procumbans agglutinates dog erythrocyte. Similar results were also observed in case of lectins of ET which can be also supported by the study of Ahmed & Chatterjee (1987). Lectins have the specific ability to bind carbohydrates which was examined by hemagglutination inhibition assay. Goldstein & Hayes (1978) has reported that the lectins of Euphorbiaceae family are galactose specific. In the same line Irazoqui et al. (2005) and Lubaki et al. (1983) noted that E. miliiand, E. heterophylla species are galactose specific lectins. Our findings are also on the same line as lectins of ET were inhibited by D-galactose and lactose which shows galactose specificity. Similar inhibition was observed in Tridax procumbens (Ramteke & Patil 2005), tubers of Dioscorea opposite (Chan & Ng 2013) and Zizyphus oenoplia (Butle & Patil 2015). Optimum pH for maximum activity of lectins varies in different plant species. In present study lectins of ET shows optimum activity at neutral pH-7 and at the same time it is inactive at extreme acidic and basic pH. Similar activity had been observed in many species like Spatholobus parviflorus (Geethanandan 2010), Volvariella volvacea lectins (Mothong 2009), bioactive lectin from Zizyphus oenoplia (Butle & Patil 2015), calyx lectin of Tridax procumbens (Ramteke et al. 2005), Apios tuber lectin (Kenmochi et al. 2015) and Jackfruit (Artocarpus integrifolia) lectin (Ahmed & Chatterjee 1987). Thermal stability of lectins was studied in ET shows maximum activity at temperature range from 40–60ºC respectively and loses its activity at 100ºC. Similar results were observed in case of Jackfruit (Artocarpus integrifolia) lectins (Ahmed & Chatterjee 1987) and Apios tuber lectin (Kenmochi et al. 2015) in which the activity was lost after 85ºC. Geethanandan (2010) and Pereira et al. (2015) mentioned that lectins of Spatholobus parviflorus and Colocasia esculenta respectively, lose their activity after 100ºC. The purified lectins from ET were processed on One Dimensional SDS-PAGE to get molecular mass as shown in figure 4. Lectins with molecular mass 70.24, 28.53 and 14.68 kDa were obtained. MW of lectin present in E. heterophylla (Lubaki et al. 1983) resembles with the result as it also possesses dimeric protein with two identical subunits of 32 kDa. Thus, it may be possible that ET have subunits of 33.91 and 31.33 kDa. Two dimensional SDS-PAGE isoelectric points (pI) of ET were obtained as shown in figure 5. According to Pereira et al. (2015) in Colocasia esculenta16 spots with pIs ranging from 6.5 to 9.5 were reported. Similarly, in our study 12 spots with pIs ranging from 6.5 to 29 were obtained. Similar results were also obtained in Volvariella volvacea (Mothong 2009) and Cyphomandra betacea (Xu 1991). CONCLUSION Partial purified ET plants lectins exhibited strong agglutination with erythrocytes of different species however, the titer against O Rh + was higher than B Rh + and A Rh + and cow. In contrast, no haem agglutination of goat, dog and fish erythrocyte was observed. Result would be explained by differences in glycosylation of the surface protein in different species of erythrocytes. Agglutination activity of ET was completely inhibited by Galactose and lactose sugars it exhibited that lectins of ET are galactose specific. Plant lectins showed optimum activity at different pH and temperature range. Extreme acidic and basic pH inhibits the lectin activity of ET whereas at neutral pH gives optimum activity. ET lectins works between temperatures range 40–60 ºC and gradually loses its activity at higher temperature and completely loss after 100ºC. It indicated that this lectin is not thermostable. Based on one dimensional study, bands with molecular mass 70.24, 28.53 and 14.68 kDa were expressed in purified lectins samples. Galactose specific lectins isolated www.tropicalplantresearch.com

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Jawade et al. (2016) 3(3): 634–641 . from other plants have been reported to be dimeric or tetremeric proteins. Thus, it may be possible that ET have subunits of 33.91 and 31.33 kDa. From the literature surveys and reports it can be said that the importance of lectin is widely spread in cellular and molecular biology. There are several applications of lectins including treatment of various diseases, as a potential therapeutic agent and lectins also act as markers. The role of lectins in research has been steadily increasing these days. There are many unidentified flora lectins. So, those plants should be explored for experimental investigation to make lectins next generation’s medicine and food. ACKNOWLEDGEMENTS Authors are thankful to Director of National Institute of Miners’ Health for their valuable support and encouragement. We are also grateful to local people for their help during the field study. REFERENCES Abreu P & Matthew S (2006) Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthusti thymaloides. Life Sciences 78: 1578–1585. Ahmed H & Chatterjee BP (1987) Further Characterization and Immuno chemical Studies on the Carbohydrate Specificity of Jackfruit (Artocarpus integrifolia) lectin. The Journal of Biological Chemistry 264(16): 9365– 9372. Ajit CG & Kanjaksha G (2016) Use of lectins in immunohematology. Asian Journal of Transfusion Science 10(1): 12–21. Butle AB & Patil MB (2015) Isolation and characterization of a bio reactive lectin from Zizyphus oenoplia. World Journal of Pharmaceutical Sciences 3(7): 1413–1420. Chan YS & Ng TB (2013) A lectin with highly potent inhibitory activity toward breast cancer cells from edible tubers of Dioscorea opposite cv. Nagaimo. Plos one 8(1): e54212: 1–11. Deshpande K & Patil M (2003) Studies of lectins of wild medicinal plant, Ph.D. Thesis. Nagpur University, Nagpur, Maharashtra, India. Geethanandan K (2010) Isolation, purification and crystal structure analysis of a new lectin from Spatholobus parviflorus, Ph.D. Thesis. Kannur University, Kerela, India. Goldstein IJ & Hayes CE (1978) The lectins: carbohydrate-binding proteins of plants and animals. Advances in Carbohydrate Chemistry and Biochemistry 35: 127–340. Irazoqui FJ, Hamp MMV, Lardone RD, Villarreal MA, Sendra VG, Montich GG, Trindade VM, Clausen H & Nores GA (2005) Fine carbohydrate reconition of Euphorbia milii lectins. Biochemical and biophysical research communications 336: 14–21. Kenmochi E, Kabir SR, Ogawa T, Naude R, Tateno H & Hirabayashi J (2015) Isolation and biochemical characterization of Apios tuber lectin. Molecules 20: 987–1002. Kurokawa T, Tsuda M & Sugino Y (1976). Purification and characterization of lectin from Wistaria floribunda seeds. The Journal of Biological Chemistry 251: 5686–5693. Lubaki MN, Peumans WJ & Carlier AR (1983) Isolation and partial characterization of a lectin from Euphorbia heterophylla seeds. Biochemical Journal 215: 141–145. Mothong N (2009) Lectins from straw mushroom cultivated in north- eastern Thailand, Ph.D. Thesis. Suranaree University of technology, Nakhon Ratchasima, Thailand. Nagata Y & Burger MM (1974) Wheat Germ Agglutinin: Molecular characteristics and specificity for sugar binding. The Journal of Biological Chemistry 249: 3116–3122. Olsen ID (1944) The use of lectins (Agglutinins) to study cell surface, Ph.D. Thesis. Columbia University, New York City, USA. Patil MB & Deshpande KV (2015) Isolation and characterization of lectin from leaves of Dregea volubilis. Journal of Global Biosciences 4: 2496–2503. Pereira PR, Winter HC, Vericimo MA, Meagher JL, Stuckey JA, Goldstein IJ, Paschoalin VMF & Silva JT (2015) Structural analysis and binding properties of isoform of tarin, the GNA-related lectin from Colocasia esculenta. Biochimica et Biophysica Acta 1854(1): 20–30. Peumans WJ & Damme EJMV (1998) Plant lectins: Versatile proteins with important perspectives in biotechnology. Biotechnology and Genetic Engineering Reviews 15: 199–228. www.tropicalplantresearch.com

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Jawade et al. (2016) 3(3): 634–641 . Ramteke AP & Patil MB (2005) Purification and characterization of Tridax procumbens calyx lectin. Biosciences Biotechnology Research Asia 3: 103–110. Ramteke AP (2009) Studies on the lectins of some medicinal plants, Ph.D. Thesis. Nagpur University, Nagpur, Maharashtra, India. Sharon N & Lis H (2004) History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14: 53–62. Suseelan KN, Bhatia CR & Mitra R (1997) Purification & characterization of twomajorlectins from Vignamungo. Journal of Biosciences 22 (4): 439–455. Weber K & Osborne M (1969) The reliability of molecular weight determination by sodium dodecyl sulphatepolyacryl amide gel electrophoresis. The Journal of Biochemistry 244: 4406–4412. Xu C (1991) Purification and characterization of a lectin from tamarillo fruits (Cyphomandra betacea), Ph.D. Thesis. Massey University Library, Palmerston North, New Zealand.

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ISSN (E): 2349 – 1183 ISSN (P): 2349 – 9265 3(3): 642–648, 2016 DOI: 10.22271/tpr.2016.v3.i3.084 Research article

Nutritional composition and fungi deterioration of canned tomato products collected from Ibadan, South-western Nigeria S. G. Jonathan1, B. J. Babalola1, O. J. Olawuyi2, J. A. Odebode3* and A. O. Ajayi4 1

Mycology & Biotechnology Unit, Department of Botany, University of Ibadan, Ibadan, Nigeria Genetics and Molecular Biology, Department of Botany, University of Ibadan, Ibadan, Nigeria 3 Mycology Unit, Department of Botany, University of Lagos, Akoka Nigeria 4 Department of Microbiology, Federal University of Oye-Ekiti, Ekiti State, Nigeria

2

*Corresponding Author: [email protected]

[Accepted: 11 December 2016]

Abstract: The present study was conducted in order to evaluate fungi and proximate analysis in three popularly consumed canned tomato products in Ibadan, Nigeria, Neurospora crassa was isolated from Pomo and Terra products while Aspergillus flavus and Macrophomina phaseolina was only isolated from Terra tin tomato products. The presence of Saccharomyces cerevisiae and Cercospora sp. was also observed in Pomo tin tomato products. Penicillium chrysogenum and Fusarium oxysporum was observed in Gino tin tomato products and Terra also shows the presence of F. oxysporum. The proximate analysis shows that the crude protein, ash content, ether extract and dry matter compositions of canned tomato products were significantly influenced by the brands of tomato product analyzed and it was indicted that the tomato products were very rich in nutrient. The Gino tin tomato presented the highest mean ash concentration with significant differences with respect to the Pomo and Terra tin products. There were no significant differences between the ether extract content when compared and significant differences were found within the replicates of the three tomato tin tomato products. The Gino tin products had the least mean value of crude protein which might be as a result of only two fungi isolates present while the high crude protein in Terra tin products is as a result more fungi contaminants that were present during isolation. The variations in aflatoxins levels in all the three mouldy tomato products indicates that they pose a threat to human health since there was invasion by toxigenic fungi after three weeks of storage. However, the opening of tin tomato products allows easy colonization of fungi and this has health implications on human being, Therefore, tin tomato products should be used immediately after opening. Keywords: Proximate analysis - Tomato - Nutrient - Aflatoxins - Toxigenic fungi. [Cite as: Jonathan SG, Babalola BJ, Olawuyi OJ, Odebode JA & Ajayi AO (2016) Nutritional composition and fungi deterioration of canned tomato products collected from Ibadan, South-western Nigeria. Tropical Plant Research 3(3): 642–648] INTRODUCTION Tomato is a herbaceous plant (Solanum lycopersicum L.) and a member of the Solanaceae. It products are widely consumed by humans all over the world as processed products such as canned tomato, sauce, juice ketchup, stews and soup (Lenucci et al. 2006). Tomato products are essential source of vitamin A, vitamin C, potassium, fiber (Herson & Hulland 1980, USDA 2012) and are considered as one of the most important ingredient in many dishes. It is desirable as dietary choices for vulnerable population groups such as the elderly (Banwart 1981, 2001, Buchann 2008) and it is associated with a reduced risk of chronic degenerable diseases (Agarwa & Aai 2000, Rao & Agarwal 1998).Tomato seeds contain high quality plant proteins that can be supplemented into various food products (Sogi et al. 2005). In recent years, tomato has received a considerable increment in its horizontal and vertical total annual production (FAO 1999). In Nigeria, the demand for canned tomato products has increased considerably, because of its prevention of www.tropicalplantresearch.com

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Received: 02 October 2016

Published online: 31 December 2016 https://doi.org/10.22271/tpr.2016.v3.i3.084

Jonathan et al. (2016) 3(3): 642–648 . heart diseases and prostate cancer (Jones 2008) and in the lowering of high blood pressure and because of its fresh taste for salad. Tomatoes are now consumed world over. Tomatoes also contain calystegine alkaloids (polyhydroxylated nortropane alkaloids) (Asano et al. 1997, 2001). Tomato products make a significant contribution to human nutrition due to the concentration and availability of several nutrients in these products and to their widespread consumption (Sahlin et al. 2004). The processing of canned tomato paste seems to increase nutrient bioavailability, which could be due to the fact that the nutrients are detached or extracted from their structures. This is particularly true for lycopene (Rao et al. 1998, Shi & Le 2000). Tomatoes and its byproducts serve as raw materials for several secondary products. A very valuable constituent of tomato is the red pigment carotenoid lycopene, an exceptionally efficient quencher of singlet oxygen and therefore an important anti-oxidant. Lycopene, as well as other valuable substances such as beta-carotene, alphacarotene, alpha-tocopherol, gamma-tocopherol and delta-tocopherol can be effectively extracted from tomato skins, seeds, and other by-products using supercritical fluid extraction technology (Baysal et al. 2000, Rozzi et al. 2002). Canned tomato pastes are packed in tin or steel cans, an air-tight container for distribution, storage or preservation. Fungi may be found in canned tomato paste due to corrosion and leakage of the metals or from tin foils used in packaging. These canned containers have a high potential of harboring toxigenic fungi. In this study, the aim was to determine the nutritional analysis and the common fungi associated with the deterioration of canned tomato pastes MATERIALS AND METHODS Study area and sample collection This study was conducted at the Mycology/pathology unit of the Department of Botany, University of Ibadan, Ibadan, Nigeria. Gino, Pomo and Terra, three popular brands of canned tomato products which are widely consumed among the University of Ibadan students were used in this research study and were purchased at Bodija markets in Ibadan, Nigeria. These samples were collected in sterile nylon and transported to the laboratory immediately. Sterilization of Materials and Media Preparation The canned tomato products were aseptically opened using a sterile tin cutter in a microbial free environment. The media used were sterilized at 121ºC for 15minutes in an autoclave and were prepared according to the manufacturers’ instruction. Culture media generally used for the study is potato dextrose agar (PDA). All glass ware were sterilized in the hot air oven at 160ºC for two hours. The inoculating needle were sterilized by flaming in the spirit lamp until red hot, working surface were sterilized by the application of sodium hypochlorite and absolute ethanol Isolation of pure cultures 5 ml of the canned tomato products was measured into each of the sterilized McCartney bottles labeled accordingly. This was vigorously shaken and 1 ml of sample was pipette into a sterile McCartney bottles containing 9 ml of distilled water. The sample was serially diluted and 1 ml each of aliquots of 106 and 107 were added to molten PDA plates. The plates were allowed to solidify and incubated at 30°C for 3–5 days. The fungal colonies were counted every 24 hours. Successive hyphae tip were transferred until pure cultures of each of fungus was obtained. Pure culture were obtained by picking distinct colonies of fungi from the pour plate using inoculating needle and subculture into freshly prepared plates of PDA. The plates were incubated at room temperature. After which the pure culture was transferred into slant. Morphological and Microscopic Identification With the aid of the sterile inoculating needle, pure fungi isolates were inoculated into the centre of sterile potato dextrose agar plate to allow uniform growth distribution, hyphae formation with the colour and shape. A sterile inoculating needle was used to pick a thin films 48–72 hours old mycelium from a pure culture and was transferred to a drop of Lactophenol cotton blue in a clean, grease free glass slide and was gently teased in the stain to ensure mixing by using an inoculating needle. The slide was covered with a cove slip. Identification was done with the aid of microscope X10 and X40 objective lenses. The shape and arrangement of the fruity body was noted. This was done for the different isolates and the observations were recorded. Analysis of Nutrient Composition of Kilishi The crude protein, ether extract, ash content, and dry matter of the canned tomato products were determined www.tropicalplantresearch.com

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Jonathan et al. (2016) 3(3): 642–648 . according to AOAC (2005). The experimental plates were arranged in triplicates. Screening for aflatoxin B1 was also carried out using the procedure of AOAC Offical methods of analysis. Data analyses The data obtained were subjected to Analysis of Variance (ANOVA) using SPSS version 16.0. Duncan Multiple Range Test (DMRT) was further used to separate treatment means where there was significant difference. Tables, plates and graphs were also used to illustrate results as appropriate. RESULTS Different fungi were isolated from the various canned tomato products obtained from Bodija market, Ibadan. Neurospora crassa Shear & B.O. Dodge was found in Pomo and Terra canned tomato products but was not present in Gino products. Aspergillus flavus Link and Macrophomina phaseolina (Tassi) Goid. was only isolated from terra canned tomato products. Saccharomyces cerevisiae Meyen ex E.C. Hansen and Cercospora sp. was also isolated from Pomo canned tomato products but was not found in Terra and Gino products. Penicillium chrysogenum Thom and Fusarium oxysporum Schlecht. emend. Snyder & Hansen was observed in Gino tomato products (Table 1). Terra also shows the presence of Fusarium oxysporum but did not occur in Table 1. Fungi isolated from canned tomato products purchased from Bodija market in Ibadan.

Fungi Neurospora crassa Shear & B.O. Dodge Aspergillus flavus Link Macrophomina phaseolina (Tassi) Goid. Aspergillus terreus Thom Saccharomyces cerevisiae Meyen ex E.C. Hansen Penicillium chrysogenum Thom Cercospora sp. Fusarium oxysporum Schlecht. emend. Snyder & Hansen Note: +, - indicates present and not present respectively.

Pomo + + + -

Terra + + + + +

Gino + +

Pomo products. The mean square effect of replicate, day after inoculation on the growth area of fungi found in POMO, TERRA and GINO Tomato Canned products in presented in table 2. The effect of replicate is nonsignificant for the growth area of the fungi isolated from Pomo and Terra tomato canned products but highly significant for the growth area of fungi found in Gino tomato canned products. The effect of day after inoculation is also significant for the growth area of the fungi found in Pomo tomato canned products but nonsignificant for the growth area of the fungi isolated from Gino tomato canned products but highly significant for the growth area of the fungi found in Terra Tomato canned products. The effect of replicates on the growth area of fungi found in POMO, TERRA and GINO Tomato Canned products is shown in table 3. Replicate 1 is significantly different from second replicate and third replicate. The least growth is of fungi isolated from Pomo products were found in third replicate. Table 2. Effect of Mean Square of Replicate, Day after inoculation on the growth area of fungi found in POMO, TERRA and GINO Tomato Canned products.

Source of variation Df GAP GAT 2 19.27ns 4.50ns Rep * 4 19.74 8.48** DAI 38 6.72 3.00 Error 45 Total 44 Corrected total Note: GAT= Growth area of fungi found in Terra, GAG= Growth area of fungi found in GINO. *= P< 0.01 highly significant, **= P< 0.05 significant, ns= Non-significant.

GAG 10.20** 6.48ns 5.77

Table 3. Effect of Replicates on the growth area of fungi found in POMO, TERRA and GINO Tomato Canned products.

Replicate 1 2 3

GAP 6.79a 6.08ab 4.57b

GAT 7.35a 8.06a 6.98a

GAG 4.69b 3.28b 7.19a

Note: GAP= Growth area of fungi found in Pomo, GAT= Growth area of fungi found in Terra, GAG= Growth area of fungi found in GINO. Means with the same letter in the same column are not significantly different at P< 0.05 using Duncan’s Multiple Range Test (DMRT).

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Jonathan et al. (2016) 3(3): 642–648 . Table 4. Effect of Day After Inoculation on the growth area of fungi found in POMO, TERRA and GINO Tomato Canned products.

DAI GAP GAT GAG 3.80b 4.89c 3.89a 3 4.75ab 6.74b 4.72a 6 6.30ab 7.94ab 4.97a 9 7.03a 8.87a 5.53a 12 7.18a 8.87a 6.14a 15 Note: DAI= Day ater inoculation, GAP=Growth area of fungi found in Pomo, GAT= Growth area of fungi found in Terra, GAG= Growth area of fungi found in GINO Means with the same letter in the same column are not significantly different at P< 0.05 using Duncan’s Multiple Range Test (DMRT). There are non-significance differences exhibited by the replicate on the growth area of all the fungi isolated from Gino tomato canned products. However, third replicate is significantly different from first replicate and second replicate which are non-significantly different from each other for the growth area of fungi isolated from Gino Tomato Canned products. The Effect of Day after inoculation on the growth area of fungi isolated in POMO, TERRA and GINO Tomato Canned products is shown in table 4. There is non-significance differences between the growth area of the fungi isolated from Pomo tomato canned products at 12 and 15 DAI, but significantly different from 6 and 9 DAI which are non-significantly different from each other. The growth are of fungi found in Pomo products at 3DAI is significantly different with the least mean value of 3.80. Also, the growth area of the fungi isolated from terra at 12 and 15 DAI are non-significantly different from each other but significantly different from 3, 6 and 9 DAI which are significantly different from each other. There are nonTable 5. Mean square table of Tomato product showing the proximate analysis.

Source df CP (%) AS (%) EE (%) DM (%) 2 57.26** 22.71** 0.00ns 0.00ns Tomato ns 2 2.95 6.02** 0.05** 1676.30** Rep 4 4.77 0.18 6.11 1.88 Error 9 Total 8 Corrected total Note: CP= Crude protein, AS= Ash, EE= Ether extract, DM= Dry matter. *= P< 0.01 highly significant, **= P< 0.05 significant, ns= Non-significant. significant differences between all the growth areas of the fungi isolated from Gino for all the days after inoculation. The mean square effect shows that the Crude protein, Ash content were highly significant for the tomato products but Ether extract, and dry moisture were non-significant for the tomato paste products. The mean effect on the replicate also shows that it is highly significant for ash content, ether extract and dry moisture but non-significant for crude protein (Table 5). Table 6. Effect of Tomato Canned products on the proximate analysis.

Tomato products CP (%) AS (%) EE (%) DM (%) 3.11c 9.50a 0.86a 49.99a Gino 5.26b 7.08b 0.87a 50.00a Pomo 11.52a 4.01c 0.88a 50.00a Terra Note: CP= Crude protein, AS= Ash, EE= Ether extract, DM= Dry matter. *= P< 0.01 highly significant, **= P< 0.05 significant, ns= Non-significant. Table 6 shows the effect of tomato products on the proximate analysis. The Gino tomato product shows the least mean value for CP and it is significantly different from the CP of POMO and TERRA. The highest mean value for crude protein was shown in Terra. There is a significant difference among the ash content of Gino, Pomo and Terra Tomato products. The least ash content was obtained from terra. For the ether extract and moisture content, there were non-significant differences between Gino, Pomo and Terra. Table 7 shows the Table 7. Effect of replicate on the proximate analysis of tomato products.

Replicate CP (%) AS (%) EE (%) DM (%) 7.67a 8.33a 1.00a 26.36bc 1 6.54ab 5.51c 0.74c 73.63a 2 5.69b 6.75b 0.87b 50.00b 3 Note: CP= Crude protein, AS= Ash, EE= Ether extract, DM= Dry matter. *= P< 0.01 highly significant, **= P< 0.05 significant, ns= Non-significant. www.tropicalplantresearch.com

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Jonathan et al. (2016) 3(3): 642–648 . effect of replicate on the proximate analysis of tomato products. First replicate has the highest crude protein and its significantly different from second replicate, third replicate has the least protein value of 5.69. For Ash content, Replicate 1 is significantly different from second replicate and third replicate while second replicate shows the least value for the ash content 5.51. The ether extract of first replicate is also significantly different from second replicate and third replicate, second replicate shows the least value of 0.74. The effect of second replicate on dry weight is significantly different from first and third replicates. First replicate has the least mean value of 26.36. Figure 1 shows the presence of aflatoxin B1 in a mouldy kilishi.

Figure 1. Aflatoxin B1(ug/kg) Assay in Mouldy Tomato Canned products.

DISCUSSION AND CONCLUSION The analysis carried out showed that various fungi can be isolated from canned tomato products. This study agrees with the work of Kalyoncu et al. (2005) who reported the presence of Aspergillus flavus, A. terreus Thom, Fusarium oxysporum, Penicillium ochraceus in home-made tomato paste samples from the fields and markets in Manisa Province of Turkey. High rate of fungi found in Pomo and Terra canned tomato products could be as a result of leakage in the packaging tin can. There might be two factors such as packaging and processing method that influence growth of fungi and the proximate composition of tomatoes. The result of this study is in accordance with the report made by Alabi & Esan (2013) who identified Aspergillus flavus, A. fumigatus Fresenius, A. niger van Tieghem and Fusarium sp. associated with the spoilage of the industrial tomato paste. The processing method is a more influential factor than production method. Some differences in the ripening stage could decisively influence the studied proximate parameters. The aflatoxins detected in all the three mouldy tomato products indicates that they pose a threat to human health since there was invasion by toxigenic fungi after three weeks of storage. Microorganisms isolated from the tin tomato products are in accordance with previous report where enzymes of A. flavus and A. fumigatus were found to be responsible for the deterioration of tomato fruit (Adisa 1985). Aspergillus sp. is very common and is involved in spoilage of food items. This work is also in line with the work of Kolawole et al. (2010) who reported the presence of Aspergillus sp., Aspergillus niger, Rhizopus stolonifer (Ehrenb.: Fr.) Vuill. and Penicillium chrysogenum in dried tomato products. The health status of man can be compromised with aspergillosis if after large amounts of spores are inhaled. ANOVA reveals that the tomato pastes were very rich in nutrient and can easily out rank all other vegetables in total contribution to human nutrition (Grubben & Denton 2004). The proximate analysis showed that the crude protein, ash content, ether extract and dry matter compositions of tomato fruits were significantly influenced by the brands of tomato product analysed. The Gino tin tomato presented the highest mean ash concentration with significant differences with respect to the Pomo and Terra tin products. The high ash content obtained in Gino tomato products might be due to the phosphorus fertilizer supplementation that acted on the tomato fruit on the field (Oke et al. 2005). However, there were no significant differences between the ether extract content when compared and significant differences were found within the replicates of the three tomato tin tomato products. The mean content of total crude protein in the analyzed Terra tin tomatoes was 11.52 which were significantly higher than other Gino tomato products. The GINO tin products had the least mean value of crude protein (3.11) possibly as a result of the low level of fungi present www.tropicalplantresearch.com 646

Jonathan et al. (2016) 3(3): 642–648 . (fungi isolates from Gino were Penicillium chrysogenum and Fusarium oxysporum) while the high crude protein in Terra tin products is as a result more fungi contaminants (Cotran et al. 1999, Diane 2004). Atteh (2002) reported a similar increase in the level of crude protein and ash of dried tomato. The result of the proximate analysis showed that, there was significant difference (p