The Insecticidal Property of Pansit-Pansitan Leaf Crude Extract Against Green Leaf Hoppers

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THE INSECTICIDAL PROPERTY OF PANSIT-PANSITAN (Peperomia pellucida (Linn.) (HBK)) LEAF CRUDE EXTRACT AGAINST GREEN LEAFHOPPER (Nephotettix malayanus (Ishihara et Kawase))

A Thesis Presented to the College of Arts and Sciences University of Perpetual Help System DALTA Pamplona, Las Piñas City

In Partial Fulfillment of the Requirements for the Degree in Bachelor of Science in Biology

Gerard Majello B. Dalisay March 2010

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APPROVAL SHEET The thesis entitled “The Insecticidal Property of Pansit-pansitan (Peperomia pellucida (Linn.) (HBK)) Leaf Crude Extract Against Green Leafhopper (Nephotetix malayanus (Ishihara et Kawase))”, prepared by Mr. Gerard Majello B. Dalisay in partial fulfillment of the requirements for the Degree in Bachelor of Science in Biology, is hereby recommended for oral examination. MARIA LOURDES C. AGUIRRE, MSc. Adviser Approved in completion of the requirements for the Degree of Bachelor of Science in Biology by the Oral Examination Committee.

MIRIAM B. RAÑOLA, M.A. Member

RAQUEL MARI-ANN O. PAJE, M.A. Member

MARY GRACE B. MARIBAO, M. Ed. Chairman Accepted in partial fulfillment of the requirements for the Degree in Bachelor of Science in Biology.

FLORENCIA C. MARFIL,Ph.D. Dean, College of Arts and Sciences March 2010 ACKNOWLEDGEMENT

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First of all I would like to express my gratitude to the One God for the many blessings that He has given. He is always there taking good care of me and always comforts me whenever I was in trouble, pain or lonely. He guided me in all the activities of my life including the construction of this study and He never lets me go both in good times and in the bad. I would also like to thank the following persons who helped me in making this study. To my parents, Mr. Honorio and Victoria Dalisay who greatly supported me in physical, mental, emotional, spiritual and especially financial terms. I thank them for the guidance, encouragement and love that they gave to me. To my uncle, Mr. Teodoro Dalisay for the information about my test organism. Mrs. Irenea Dalisay for assisting the construction of the insect cages. Mr. Gregorio Gomez Jr., Mrs. Editha Roldan and the rest of the employees of Dalisay Poultry Supply and Dalisay Piggery Farm for the construction of the insect cages and assisting me whenever I go to the site of the study. To all the rice farmers of the town of Lian, Batangas for looking after my experimental set ups whenever I am unable to do so. To the thesis adviser, Mrs. Maria Lourdes Aguirre for her extreme patience, assistance and support. Truly the unmatched and ultimate “Mother of Perpetual Help” for always listening to our complaints and problems and comforting us when we are in need. To Ms. Miriam Rañola, for the patience and endurance in dealing with ones papers and giving suggestions that can contribute to the betterment of the study.

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To Mrs. Mary Grace Maribao, for the assistance and life-inspiring words which has proven to be a valuable asset in achieving ones goal. To Ms. Raquel Paje, for the wise and practical examples which enables one to efficiently tackle the problems of life. To Mr. Eduardo Jimmy Arma, for the humorous statements and antics that never fails to enlighten anyone. Truly a walking piece of pure joy that makes you feel happy and refreshed anytime, anywhere. To Mr. Allan Alvarez, Dr. Vendivil and the rest of the staff of the Botany Division of the National Museum for the plant identification. To Dr. Sinohin, Mrs. Fe Alzona and the rest of the staff of the Crop Protection Cluster of the University of the Philippines Los Baños, Laguna for the identification of test organism. Dr. Timbang and the rest of the staff of the IHS Laboratory of the University of Perpetual Help System DALTA for lending us laboratory apparatuses required for the study. Also to Mr. Arturo Tobias Calizon for the statistical analysis. To Mr. Paolo Louis Almario for allowing me to be patient, kind and enduring, I thank you for teaching me all of these. To Mrs. Kristina Ezra SalcedaDumdum for the tremendous amount of humor that she has given me. I thank you for the huge amount of food that you are giving to us whenever we are at your house. Ms. Monique Moldez for teaching me how to be strong, to be able to fight back from other people’s insults whether in a casual or formal manner. I am very grateful for changing me from my doormat and submissive self to the strong willed and firm individual that I am today. To Ms. Joanna Marie Valloso for

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always reminding me of assignments, quizzes and other academic tasks that are needed to accomplish. You truly are a genuine secretary in training. Thank you also for sharing your food with me whenever you are unable to finish it. To Mr. Gene LemuelAbanto, Olivier Thoenen and the rest of the B.S. Biology students of the University of Perpetual Help System DALTA. I humbly thank you all for the comedy, drama, horror, romance and all the emotions that you let me experience. Truly I never have encountered such people and the experiences that they gave to me are simply life changing and priceless. Lastly to the people whom I failed to mention I thank you for your generosity and effort in aiding me in this study. Once more I gratefully thank you and God bless us all.

Gerard Majello Bulao Dalisay

ABSTRACT This study entittled “The Insecticidal Property of Pansit-pansitan (Peperomia pellucida (Linn.)(HBK)) Leaf Crude Extract Against Green

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Leafhopper (Nephotettix malayanus (Ishihara et Kawase))” aims to confirm the active components present in Pansit-pansitan leaf crude extract using phytochemical analysis. The study also aimed to determine the insecticidal property of Pansit-pansitan based on the mortality rate of Green Leafhopper and compare the different concentrations of Pansit-pansitan leaf crude extract with Sevin ® 85 WP (positive control). The leaf crude extract of the plant material was obtained through decoction method. Phytochemical analysis was done to confirm the different constituents present in Pansit-pansitan leaf crude extract. In each treatment three trials have been carried out. These were Sevin ® 85 WP (positive control) and the varying concentrations of the leaf crude extract of Pansit-pansitan (50%, 75% and 100%) have been prepared by dissolving 5g, 7.5g and 10g of leaf crude extract in 10ml of distilled water, respectively. Insecticidal property was determined based on the mortality rate of Green Leafhopper by administering 10ml of each treatment and the positive control through spray method. Results showed that the highest mortality rate of Green Leafhoppers was obtained for Sevin ® 85 WP (positive control) followed by 100% concentration of Pansit-pansitan leaf crude extract and the least mortality rate has been observed at 50% concentration. Statistical analysis showed that the computed F value of 3.303303 is greater than the critical value of F of 3.24 at 0.05 level of significance thus, the null hypothesis was rejected. There is a significant difference in the

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insecticidal property between Pansit-pansitan leaf crude extract and the positive control, Sevin ® 85 WP.

TABLE OF CONTENTS TITLE APPROVAL SHEET

i ii

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ACKNOWLEDGEMENT ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF APPENDICES

iii vi viii x xi xii xiii

CHAPTERS I

THE PROBLEM AND ITS BACKGROUND Introduction Theoretical Framework Conceptual Framework Statement of the Problem Hypothesis Significance of the Study Scope and Delimitation Definition of Terms

1 2 3 4 4 5 5 7

II REVIEW OF RELATED LITERATURE Conceptual Literature Scientific Classification of Pansit-pansitan Botanical Description Chemical Content of Botanical Extract Brief Background about Green Leafhopper Classification Taxonomy Life Cycle Sevin 85 ® WP Research Literature

8 8 9 10 12 12 13 14 15 15

III RESEARCH METHODOLOGY AND PROCEDURE Research Design Research Setting Research Procedure Collection and Preparation of Plant Specimen Identification of Plant Material Identification of Test Organism Extraction Procedure Decoction Method Preparation of Different Concentrations of Leaf Crude Extract

18 18 19 19 19 20 20 20 20

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Preparation of Sevin ® 85 WP (control) Preparation of the Test Organism Preparation of Green Leafhopper Cage Collection of Green Leafhopper Preparation of Green Leafhopper for Mating Application of Leaf Extract to Green Leafhopper Statistical Treatment IV PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

20 21 21 21 21 22 22 23

V SUMMARY, CONCLUSION AND RECOMMENDATION Summary Conclusion Recommendation BIBLIOGRAPHY PLATES APPENDICES CURRICULUM VITAE

28 29 30

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CHAPTER 1 THE PROBLEM AND ITS BACKGROUND Introduction Rice is the staple food of the Filipino people. This particular crop has been in the Philippine table for thousands of years from its early inhabitants up to the modern age. It is an annual plant that is cultivated in warm and wet areas in which the seeds are cooked and used for food. The said crop is widely cultivated throughout the country, carefully planted in well irrigated areas, tended by the most skilled farmers and efficiently processed before going out to the market. However, rice continues to decrease in yield due to pest infestations thus plant cultivation and harvesting is severely affected. The most effective method to get rid of pest infestations nowadays is to use commercial insecticides however they may cause unwanted effects such as land poisoning and may endanger human health if not properly handled. Today’s farmers are running low on funds to buy insecticides which are too expensive. Thus the use of botanical extracts have been encouraged as possible substitute for commercially available insecticides. It is interesting to note that certain plants have insecticidal properties. Among these plants that have shown such properties is Pansit-pansitan (Peperomia pellucida (Linn.) (HBK)) . The said herbaceous annual plant contains

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chemical substances such as alkaloids that is proven to cause irritation and death to most types of insects. Theoretical Framework Pansit-pansitan has long been used by people from all parts of the world in a variety of ways. In the old days infusion of the leaves and stems are used for gout and arthritis. The plant has a history of ethnomedicinal use. Anti-inflammatory, chemotherapeutic, and analgesic properties have been found in crude extracts of Pansit-pansitan. Data in Bolivia documents the whole plant being crushed, mixed with water, heated, and then administered to stop hemorrhage (www.drugs.com). Externally it is used as a facial rinse for complexion problems. Pounded whole plant used as warm poultice for boils, pustules and pimples. It is also used for headaches and rheumatic pains (http://www.stuartxchange.org/Pansit.html).In Southeast Asia, Pansit-pansitan is reported to have analgesic, antibacterial and antifungal activity. According to a recent study, the plant contains alkaloid which is a plant-derived compound that is toxic or physiologically active. It has many uses depending on its type such as quinine which has anti-malarial compound or tropane alkaloids which have been used throughout history as poisons (Murray G, Frank M, 1973). Phytochemical studies have also revealed the presence of alkaloids and tannin in abundant amounts (De Padua LS, Pancho JV. 1983).

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Conceptual Framework Green Leafhoppers are a common pest problem throughout the Philippines. Millions of pesos are spent to control them every year. Pansit-pansitan has been used as an alternative to commercial insecticides that can be used to exterminate such rice pests.

Independent variable

Dependent variable

Varying concentrations

Insecticidal property of

of Pansit-pansitan leaf

the leaf crude extract

crude extract (50%, 75%, 100%) and Sevin ® 85 WP (positive

Mortality rate of Green Leafhoppers

control)

Figure 1. A schematic diagram showing the insecticidal property of Pansitpansitan against Green Leafhopper (N. malayanus (Ishihara et Kawase))

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Statement of the Problem The study was conducted in order to determine the insecticidal property of Pansit-pansitan (Peperomia pellucida (Linn.)(HBK)) leaf crude extract against Green Leafhopper (Nephotettix malayanus (Ishihara et Kawase)). Specifically it aimed to: 1. confirm the active components present in Pansit-pansitan (Peperomia pellucida (Linn.)(HBK)) leaf crude extract using phytochemical analysis; 2. determine the insecticidal property of Pansit-pansitan (Peperomia pellucida (Linn.)(HBK)) against Green Leafhoppers (Nephotettix malayanus (Ishihara et Kawase)); and 3. compare the different concentrations of Pansit-pansitan (Peperomia pellucida (Linn.)(HBK)) leaf crude extract with Sevin ® 85 WP (positive control) against Green Leafhoppers (Nephotettix malayanus (Ishihara et Kawase)). Hypothesis There is no significant difference in the insecticidal property between Pansit-pansitan (Peperomia pellucida (Linn.)(HBK)) leaf crude extract and the positive control, Sevin ® 85 WP against Green Leafhoppers (Nephotettix malayanus (Ishihara et Kawase)). Significance of the Study

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The main purpose of this study is to determine the insecticidal property of Pansit-pansitan leaf crude extract against Green Leafhopper. The result of the study will encourage researchers to formulate more plant based products which are organic, environmental friendly and cost efficient. It could also be used to help minimize the occurrence of pests on rice and on other crops, thus pest control would be stabilized. Ultimately, farmers will be financially benefited because of the inexpensiveness of the plant extract thus they could focus more on increasing rice production. Also farmer’s consumption of inorganic insecticides will be reduced which are harmful to the environment and health. Rice production will increase and the Philippine economy will eventually improve. Scope and Delimitation This study focused on the collection and preparation of the plant material. The plant extraction procedure done for Pansit-pansitan leaves was through decoction method by which varying concentrations have been derived such as 50%, 75% and 100%. The extraction method was performed in the IHS Laboratory, University of Perpetual Help System DALTA located in Pamplona, Las Piñas City. Phytochemical analysis was also conducted in order to confirm the chemical components of the Pansit-pansitan leaf. Three treatments were performed in the study. In each treatment three trials have been carried out. These were Sevin ® 85 WP (positive control) and the varying concentrations of Pansit-pansitan leaf crude extract. The varying concentrations of Pansit-pansitan leaf crude extract was prepared by dissolving 5g, 7.5g and 10g of leaf crude extract in 10ml of distilled water in order to obtain

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50%, 75% and 100% concentrations, respectively. The study used adult forms of Green Leafhopper which were about 11 to 13 days upon hatching. The insects have been cultured in a natural habitat by placing a fine net on a site in the rice field to ensure that the specimen will not escape and were allowed to reproduce. Green Leafhopper eggs were then collected after 6 days upon laying by gently brushing the rice plants located on the site with a soft brush to remove the adult insect thus the insect eggs remained in the rice plant. The rice plants along with the Green Leafhopper eggs were then carefully transferred to another site in the rice field covered with a fine net and allowed to reach adulthood after 11 to 13 days upon hatching (Hou, 1987 modified). The number of mortality of Green Leafhopper was counted after the leaf crude extract has been applied through spray method every 6 minutes for a total of 30 minutes. The insects were sprayed with the leaf crude extract between 2:00-3:00 PM. The study was conducted from December, 2009 to January, 2010.

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Definition of Terms The following terms are defined for better understanding of the study: Decoction method

It is an extract of herbs produced by boiling the plant on water.

Extract

It is a mean to obtain from a substance by chemical or mechanical action.

Extraction

It is an act of extracting.

Insecticidal

It is a chemical substance to kill insects.

Mortality

It means death of large numbers.

Mortality rate

It is the whole number of death in a given time or community.

Oviposition

It is the process of laying eggs in insects.

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CHAPTER 2 REVIEW OF RELATED LITERATURE This chapter presents previous studies and information related to the study. Conceptual Literature Pansit-pansitan, from the family Piperaceae is an annual herb that is found in many South American and Asian countries, shallow rooted, that reach 40 cm high, with succulent stems. Scientific Classification Kingdom Plantae – Plants Class Magnoliopsida – Dicotyledons Subclass Magnoliidae Order Piperales Family Piperaceae – Pepper family Genus Peperomia Ruiz & Pav. – peperomia Species Peperomia pellucida (L. HBK) Common Names: Pansit-pansitan, Ulasimang-bato (Tag.), Shiny bush , silver bush (Engl.)

Botanical Description

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Pansit-pansitan (Peperomia pellucida (Linn.) (HBK)) It belongs to the family Piperaceae. It is an herbaceous annual plant with succulent stems found in many South American and Asian countries. The plant grows to a height of 15 to 45 cm. Its leaves are

alternate,

heart-shaped

and

turgid, as transparent and smooth as candle wax. Tiny dot like flowers

Figure 2. Peperomia pellucida (Linn. HBK)

scattered along solitary and leafopposed stalk maturing gradually from the base to the tip and turning brown when ripe.

It

propagates

by

seeds.

Numerous

tiny

seeds

drop

off

when mature and grow easily in clumps. The species develops during rainy periods and thrives in loose, humid soils under the shade of trees (www.drugs.com). The decoction of leaves is used for gout and arthritis (http://www.stuartxchange.org/Pansit.html). Also the pounded stems are applied on the face for boils and pimples and have said to have anti bacterial property against Bacillus subtilis and Staphylococcus aureus. (Lans, Journal of Ethnobiology and Ethnomedicine, 2007)

Chemical Content of Botanical Extract Pansit-pansitan (Peperomia pellucida (Linn.) (HBK))

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It was found out that the plant leaf contains ascorbic acid, beta-carotene, calcium, carbohydrates, fat, fiber, iron, niacin, phosphorus, potassium, protein, riboflavin,

sodium,

thiamine,

water

(http://www.ars-grin.gov/cgi-

bin/duke/farmacy2.pl). Other substances are benzenoid, steroids and apiol found throughout the plant (Manalo JB, et al. 1983). Alkaloids and tannin are mainly abundant in leaves (De Padua LS, Pancho JV. 1983). Alkaloids are plant-derived compound that is toxic or physiologically active. It has a complex structure and is of limited distribution in the plant kingdom. Alkaloids have many uses such as poisons which build up nerve impulses transmitted by the brain which results in restlessness, convulsions, nausea, vomiting and death (Dales MJ, 1996). Quinine which is a type of alkaloid has anti-malarial compound. Another form of alkaloids is atropine and is used to dilate the pupils of the eye. Atropine is also a CNS stimulant and is used as a treatment for nerve gas poisoning (Murray G, Frank M, 1973). Apiol (C12H14O4) is an essential oil usually found in some succulent plant species. It is green, non-viscous, oily liquid with a peculiar odor and a disagreeable acrid taste (United States National Library of Medicine). In humans extremely high doses of the chemical may produce effects such as ringing in the ears, headache and vertigo. In pregnant women it can cause an accumulation of blood in the pelvic-uterus area and liver damage. High doses of some oils, especially

containing

apiol

can

have

narcotic

(http://www.juniperandsage.com/oils/toxicity.php).

or

paralyzing

effects

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Tannins are polyphenols that are obtained from various parts of different plants belonging to multiple species. It is found in abundance in the tree bark, wood, fruit, leaves, and roots and also in plant gall. Interestingly, tannins are found almost in all plants and in all climates all over the world however its percentage varies depending on plant species. Tannins are usually found in large quantities in the bark of trees where they act as a barrier for micro-organisms like bacteria and fungi and protect the tree. It is also used in dyeing, photography, astringent, stops infection, and refining beer and wine As tannins often lower the absorption of some materials into the body, tannins are also often known as anti-nutrients. Tannins can be classified into two broad groups - hydrolysable tannins and condensed tannins. Hydrolysable tannins are basically derived from simple phenolic acids like gallic acid that when heated they give away pyrogallol. Pyrogallol is also known as hepatotoxic and has antiseptic as well as caustic properties. Condensed tannins do not split easily and hence it is difficult to analyze these. When condensed tannins are heated, catechol emerges as the final product. Unlike hydrolysable tannins, condensed tannins do not possess any trace of hepatotoxicity or any adverse side effects and hence are favorable for use (Harborne Jeffery, H. Baxter. 1983).

Brief Background about Green Leafhopper (Nephotettix malayanus (Ishihara et Kawase)) Classification of Green Leafhopper

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Domain Eukaryota- eukaryotes Kingdom Animalia – animals Subkingdom Bilateria- bilaterians, triploblastic animals Branch Protostomia- protosomes Infrakingdom Ecdysozoa- ecdysozomes Superphylum Panarthropoda Phylum Arthropoda- arthropods Subphylum Mandibulata Class Insecta -insects Subclass Dicondylia Order Coleoptera Suborder Auchenorrhyncha Superfamily Cicadelloidea Family Cicadellidae Genus Nephotettix Species N. malayanus (Ishihara et Kawase)

Taxonomy

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Green Leafhopper belongs to the Phylum Arthropoda. It constitutes the suborder Polyphaga within the class Insecta. The insect is said to be dioecious and have paired reproductive organs. Green Leafhoppers are serious pests in rice. They damage the plants directly by sucking the plant sap resulting in complete plant drying.

Figure 3. Nephotettix malayanus (Ishihara et Kawase)

Green Leafhoppers are slender, green and may have black markings on the head or wings. Eggs are white and elongate or cigar-shaped. Individual eggs are arranged neatly and lie parallel to each other in each egg batch. A single egg measures 0.9 - 0.8 mm. Upon maturation, the egg turns brownish and develops red eyes. As the nymph matures, it turns to green. (Pathak MD, 1994). As the insect matures, blackish markings on the abdomen become more prominent as well as the blackish band on the last abdominal segment. (Reissig WH, 1986). They are less than 13mm long and have an average life span of 3 weeks. They disperse rapidly when disturbed. Both adults and nymphs run sideways and are good jumpers or hoppers. They are generally found in small numbers at the leaf blade and feed on the upper portion of the rice canopy. A female hopper can lay as many as 200-300 eggs (Shepard BM, 1995). Green Leafhoppers are common in rain fed and irrigated wetland environments. They

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are not prevalent in upland rice. Both the nymphs and adults feed on the dorsal surface of the leaf blades rather than the ventral surface. They prefer to feed on the lateral leaves rather than the leaf sheaths and the middle leaves. They also prefer rice plants that have been fertilized with large amount of nitrogen (http://everest.ento.vt.edu/~idlab/vegpests/vegi/LH.jpeg). Life Cycle Green Leafhoppers begin as an egg laid on a plant leaf. The eggs are cylindrical, whitish or pale-yellow

and

later

become brown with red eyespots

when

the

embryos develop. The

Figure 4. Life Cycle of Green Leafhopper

eggs are laid in masses of 8-16 eggs and hatch within about 4-8 days. Upon hatching the nymph measures 0.9 mm long with whitish and shiny appearance and as it matures, it turns yellowish. The shape of the nymph is similar to that of the adult except that the nymph is smaller and is wingless (Estoy A, 2000). After about 10 to 14 days after hatching the leafhopper turns to adult which has green appearance with black markings on the head, thorax, and abdomen. A pair of black spots is either present or absent on the forewings. The adult insect is 4.2-4.3mm long and the total life cycle from egg to adult is between 22-26 days (Joshi R, 2001). Green

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Leafhoppers are most destructive at adult stage due to increased nutrient consumption from rice plant in preparation for mating and reproduction (Shepard BM, et al. 1995) Sevin ® 85 WP Sevin ® 85 WP is a wide-spectrum insecticide which controls over 100 species of insects on fruit, cotton and other crops as well as on poultry and pets. Its active ingredient is carbaryl (C12H11NO2) from the chemical group carbamate (1-naphthyl-N-methyl) (Instant EPA's Pesticide Facts.1996). It kills insects by disrupting the normal function of their nervous system. Nerve impulses are transmitted from one nerve to another by a chemical called acetylcholine. Under normal conditions an enzyme called acetylcholinesterase destroys acetylcholine so that another nerve impulse may be transmitted. Carbaryl stops the functioning of the enzyme so acetylcholine builds up resulting in restlessness, paralysis and death. Carbaryl affects human nerves in the same way as in insect nerves. However in insects it affects the central nervous system while in humans it affects the nerve and muscle interaction. Thus carbaryl poisoning symptoms are different in humans and in insects (Ware G, 2000). Research Literature In a study conducted by Dasmariñas, Jover and Pasig (2001) Pansitpansitan was used as insecticide for termites. According to the study the plant contains considerable amounts of monoterpenes, flavonoids, amides and alkaloids. The chemicals mentioned are known to have insecticidal property in which it disrupts the nerve and motor functioning of insects thus causing death. In

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the experiment mentioned, the researchers prepared four treatments: Treatment A consisting of 100% Pansit-pansitan extract, Treatment B with 75% Pansitpansitan extract, Treatment C with 50% Pansit-pansitan extract, Treatment D with 25% Pansit-pansitan extract and Treatment E as positive control consisting of unknown commercial pesticide. The treatments were tested on termites randomly collected and placed in containers with 20 termites in each and three replicates are made per treatment. Number of mortality was counted every 3min until 15min was achieved. Results showed that Treatment A (100% Pansit-pansitan extract) showed the highest mortality rate of 100% compared with the other plant extracts and is comparable with Treatment E which is the positive control which also showed 100% mortality rate. Pansit-pansitan however has lethal activity according to Lim (2006). The researcher assessed the plant’s lethal activity using the Brine Shrimp (Artemia salina) Lethality Bioassay. The testing involved the exposure of the organism to different aqueous extract concentrations of Pansit-pansitan (10%, 30% and 50%) in three serial dilutions. Mortality among organisms was observed for 48 hours in experimental and control groups in three replications and LC50 value was computed using the Spearman-Karber Method. Significant differences among sample observations were determined by employing One-Way Analysis of Variance. Lethal activity of the test plant on the invertebrate model was proven with 9.18 LC50 value and 50 percent extract concentration as the most lethal treatment.

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Another study by Cambe, Delfin and Sales, (2003) is aimed to find out if oleander (Thevetia peruviana) fruit extract can be used as insecticide against rice bugs. The experimental design was Completely Randomized Block Design (CRBD) with the following treatments: Treatment A - (25 ml extract + 25 ml water), Treatment B - (50 ml extract + 25 ml water), Treatment C - (75 ml extract + 25 ml water), Treatment D - (100 ml extract), Controlled set-up (100% water) and replicated three times. The crude extract was obtained by pounding, squeezing, and filtering. Phytochemical analysis revealed that extract of the young fruit contains alkaloid, thevetin, peruvoside and cyanogenic glycosides. The chemicals have influenced the effectiveness of extract and using greater concentrations can hasten and increase mortality to 100%. Using the One-way Analysis of Variance and Duncan’s Test, results led to the conclusion that oleander can be used as an insecticide against rice bugs.

CHAPTER 3

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RESEARCH METHODOLOGY AND PROCEDURE This chapter presents the research design, methods and adapted procedures in the study. Research Design An experimental method was used in the study to determine the insecticidal property of different concentrations (50%, 75% and 100%) of Pansitpansitan leaf crude extract against Green Leafhopper. This experiment used three treatments. Different concentrations (50%, 75% and 100%) of the plant’s leaf material have been prepared by dissolving 5g, 7.5g and 10g of Pansit-pansitan leaf crude extract in 10ml of distilled water, respectively. Sevin ® 85 WP was used as positive control. The positive control was prepared by dissolving 10g of the insecticide to 10ml of distilled water. Research Setting This study along with the collection of plant material and the culturing of the test organism was conducted at the Dalisay Piggery Farm Compound located in Lian, Batangas. Phytochemical analysis was done at the IHS Laboratory, University of Perpetual Help System DALTA located in Las Piñas City. The leaf crude extract was tested for the following:

Test for Tannin

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A few drops of Ferric Chloride T.S. was added to 2ml plant extract. Test for Alkaloids The addition of 1% HCL was used to modify the aqueous extract when one or two drops of Mayer’s reagent was added. Grease Spot Test The plant material was pressed on a bond paper. A temporary presence of grease spot indicates that the plant material contains volatile oil while permanent spot indicates the presence of fixed oil. The study was conducted from December, 2009 to January, 2010. Research Procedure Collection and Preparation of Plant Specimen The plant material was collected at the Dalisay Piggery Farm Compound in Lian, Batangas. The collected plant material was washed with distilled water to remove dust and insects and air-dried for three days. Afterwards, it was preweighed prior to submission for phytochemical analysis. Identification of Plant Material The plant material was initially identified by the researcher and verified by Mr. Wilfredo F. Vendivil Ph.D, Senior Researcher of Botany Division, National Museum located at P. Burgos St. Manila.

Identification of Test Organism

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The test organism was identified by Mrs. Fe D. Alzona, Researcher of the College of Agriculture, Crop Protection Cluster, University of the Philippines Los Baños located at Los Baños, Laguna. Extraction Procedure Decoction Method The researcher used decoction method in order to obtain the leaf crude extract. Five hundred ml of water was boiled first before adding the leaves. The chopped leaves which weighed 500g was added. The heat was lowered to 5060 °C in order to maintain a slow simmer for 10 to 15 minutes. It was removed from heat and strained before using. Preparation of Different Concentrations of Leaf Crude Extract The preparation of varying concentrations (50%, 75% and 100%) of Pansit-pansitan leaf crude extract were as follows: for 50%, 5g of leaf extract was dissolved in 10ml of distilled water, for 75%, 7.5g of leaf extract was dissolved in 10ml of distilled water and for 100%, 10g of leaf extract was dissolved in 10ml of distilled water. Preparation of Sevin® 85 WP (control) Ten grams (10g) of the powdered form of the control (Sevin 85 WP) was dissolved in 10ml of distilled water.

Preparation of the Test Organism

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Preparation of Green Leafhopper Cage Sixteen cages were prepared on the natural habitat of the test organism using a fine net firmly paced on the site by supporting wooden pegs. The first two cages were used to house the insects in the wild which measured 1m x 1m x 0.46m. The second two cages were used to house the insect eggs laid by the previous generation which have the same dimension as the cages that were used previously. The remaining twelve cages which measured 0.30m x 0.30m x 0.15m were then used to house the cultured adult forms of Green Leafhopper in accordance to the number of treatments, trials and control that were used (Hou, 1987 modified). Collection of Green Leafhopper Random wild Green Leafhoppers were collected at Dalisay Piggery Farm Compound in Lian, Batangas. Insects were collected by constructing cages on the natural habitat and were cultured to ensure uniformity. Preparation of Green Leafhopper for Mating Random Green Leafhopper adults were collected from the wild by constructing 2 cages which measured 1m x 1m x 0.46m on the natural environment of the insects. Green Leafhopper eggs were collected after 6 days by gently brushing the rice plants located on the site with a soft brush to remove the adult insect thus the insect eggs remained in the rice plant. The rice plants along with the Green Leafhopper eggs were carefully transferred to another 2 cages in the natural habitat which measured the same as the other 2 cages that were used previously and was allowed to reach adulthood. After 11

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to 13 days mature insects were collected and distributed to the remaining 12 cages which measured 0.30m x 0.30m x 0.15m in accordance with the number of treatments, trials and control that were used (Hou, 1987 modified). Application of Leaf Extract to Green Leafhopper Spray method was done to apply the different treatments using atomizer. About 10ml of each treatment was sprayed to the respective cages containing 10 cultured Green Leafhoppers. Each treatment had three replications, thus the total number of cultured Green Leafhoppers used was 120 including the control. Higher mortality rate of the insect indicates the effectiveness of the plant extract. The mortality rate of Green Leafhopper was determined after the extract was applied through spray method at intervals of every 6 minutes for a total of 30 minutes. Statistical Treatment One way analysis of variance (ANOVA) was used to determine the significant difference in the insecticidal property between Pansit-pansitan (Peperomia pellucida (Linn.HBK)) leaf crude extract and the positive control Sevin ®85 WP.

CHAPTER 4 PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

23

This chapter presents the data gathered, the analysis of data and the different findings together with their respective analysis and interpretation of results. In this study the insecticidal property of Pansi-pansitan leaf crude extract was tested against Green Leafhopper through spray method. The presentation of data includes the results of the phytochemical analysis, mortality rate of Green Leafhopper using different concentrations of Pansit-pansitan leaf crude extract and Sevin ®85 WP (control) and the result of the ANOVA test. Table 1. Results of Phytochemical Analysis of Pansit-pansitan (Peperomia pellucida (Linn.HBK)) Leaf Crude Extract Tests

Theoretical Result

Actual Result

Tannins

+

+

Alkaloids

+

+

Grease Spot

+ (volatile oil)

+ (volatile oil)

Table 1 shows the different constituents present in Pansit-pansitan leaf extract based on phytochemical analysis. Active components present were tannins which were indicated by blue to black precipitate, alkaloids which was indicated by the presence of precipitate and volatile oil which was indicated by a temporary grease spot. The result justifies the research by De Padua LS and Pancho JV (1983) on the chemical components of Pansit-pansitan in which the results also showed the same active components.

Table 2. Mortality Rate of Green Leafhopper Using Different Concentrations of Pansit-pansitan Leaf Crude Extract and Sevin ® 85 WP (Control)

24

Treatments

Time in Minutes 6

12

18

24

30

Sevin 85 WP (control)

10%

26.67%

33.33%

16.67%

6.67%

100%

6.67%

13.33%

20%

6.67%

0%

75%

3.33%

10%

16.67%

3.33%

0%

50%

0%

3.33%

10%

6.67%

0%

Table 2 shows the mortality rate of Green Leafhoppers using varying concentrations of Pansit-pansitan leaf crude extract and Sevin ® 85 WP (control). Based on the results, the positive control Sevin ® 85 WP showed a mortality rate of 10% after 6 minutes of application, 26.67% after 12 minutes, 33.33% after 18 minutes, 16.67% after 24 minutes and 6.67% after 30 minutes of application. The 100% concentration of the leaf crude extract showed a mortality rate of 6.67% after 6 minutes of application, 13.33% after 12 minutes, 20% after 18 minutes, 6.67% after 24 minutes and 0% after 30 minutes of application. The 75% concentration of the leaf crude extract showed a mortality rate of 3.33% after 6 minutes of application, 10% after 12 minutes, 16.67% after 18 minutes, 3.33% after 24 minutes and 0% after 30 minutes of application. The 50% concentration of the leaf crude extract showed a mortality rate of 0% after 6 minutes of application, 3.33% after 12 minutes, 10% after 18 minutes, 6.67% after 24 minutes and 0% after 30 minutes of application. It was observed that there is an increasing trend of mortality rate of Green Leafhopper up to 18 minutes, a decrease in mortality rate after 24 minutes of application and at 30 minutes, all the different concentrations of Pansit-pansitan

25

leaf crude extract showed 0% mortality rate. The positive control Sevin ® 85 WP, showed 6.67% mortality rate from 6minutes onwards to 30 minutes after application. It can be inferred from the data that both the positive control, Sevin ® 85 WP and the different concentrations of Pansit-pansitan leaf crude extract provide optimum mortality rate at 18 minutes after application of Pansit-pansitan leaf crude extract on Green Leafhoppers.

26

35

30

25

20

Sevin ® 85 WP 100% 75% 50%

Mortality Rate 15

10

5

0 6 minutes

12 minutes

18 minutes 24 minutes Time Interval

30 minutes

Figure 5. Mortality rate of Green Leafhopper using varying concentrations of Pansit-pansitan leaf crude extract and Sevin 85 ® WP (control)

Figure 5 shows the mortality rate of Green Leafhoppers using varying concentrations of Pansit-pansitan leaf crude extract and Sevin ® 85 WP (positive control). The red column represents Sevin 85 ® WP (positive control), the yellow column represents 100% concentration of Pansit-pansitan leaf crude extract, the green column represents 75% concentration of Pansit-pansitan leaf crude extract and the blue column represents 50% concentration of Pansit-pansitan leaf crude extract.

27

Based on the figure, the positive control Sevin 85 ® WP and the varying concentrations (100%, 75% and 50%) of Pansit-pansitan leaf crude extract showed the highest mortality rate of 33.33%, 20%, 16.67% and 10% respectively after 18 minutes of application. The lowest mortality rate of 6.67%, 0%, 0% and 0% was attained for the positive control Sevin 85 WP and the varying concentrations (100%, 75% and 50%) of Pansit-pansitan leaf crude extract respectively which occurred after 30 minutes of application. Table 3. Summary of Computation of ANOVA Test to Determine the Significant Difference of Pansit-pansitan Leaf Extract and Sevin 85 WP (Control) Source

df

SS

MS

F

Treatments

3

55

18.333

Error

16

88.8

5.55

Total

19 143.8

3.303303

Critical Decision Conclusion value of F

3.24

Reject Ho

Significant

Table 3 shows the computed value of Pansit-pansitan leaf crude extract and Sevin 85 ® WP (control). Since the computed F value of 3.303303 is greater than the critical F value which is 3.24 at 0.05 level of significance, the null hypothesis is rejected. This means that there is a significant difference in the insecticidal property between Pansit-pansitan leaf crude extract and the positive control, Sevin ® 85 WP.

CHAPTER 5

28

SUMMARY, CONCLUSION AND RECOMMENDATION This chapter discusses the summary, conclusions and recommendations of the study. Summary of Findings This study entitled “The Insecticidal Property of Pansit-pansitan (Peperomia pellucida Linn. HBK) Leaf Crude Extract against Green Leafhopper (Nephotettix malayanus Ishihara et Kawase)” was conducted to confirm the active components present in Pansit-pansitan leaf crude extract using phytochemical analysis, determine the insecticidal property of Pansit-pansitan based on the mortality rate of Green Leafhopper, determine the insecticidal property of Pansitpansitan based on the mortality rate of Green Leafhopper and lastly to compare the different concentration of Pansit-pansitan leaf crude extract with Sevin ® 85 WP (positive control). Pansit-pansitan leaves were collected at the Dalisay Piggery Farm Compound in Lian, Batangas. The collected plant material was washed with distilled water to remove dust and insects and air-dried for three days. Phytochemical analysis was conducted at the IHS Laboratory, University of Perpetual Help System DALTA located in Las Piñas City. This experiment has used three treatments. Different concentrations (50%, 75% and 100%) of the plant’s leaf material have been prepared by dissolving 5g, 7.5g and 10g of Pansitpansitan leaf crude extract in 10ml of distilled water, respectively. Sevin ® 85 WP had been used as positive control by dissolving 10g of the powdered form of the insecticide to 10ml of distilled water.

29

Based on the results of the phytochemical analysis, Pansit-pansitan leaf crude extract contains alkaloids, tannins and volatile oils. The effects of the varying concentrations of Pansit-pansitan leaf crude extract on mortality rate of Green Leafhopper showed that the highest mortality rate obtained was that of the control (Sevin ® 85 WP) which has 28 deaths or 93.33% followed by the 100% concentration of Pansit-pansitan leaf crude extract which has 14 deaths or 46.67%. The least mortality rate of Green Leafhoppers has been observed at 50% concentration of Pansit-pansitan leaf crude extract which only showed 6 deaths or 20%. The ANOVA test reveals that the null hypothesis was rejected. There is a significant difference in the insecticidal property between Pansit-pansitan leaf crude extract and the control, Sevin 85 ® WP. Conclusions Based on the above findings, the following conclusions were drawn: 1. active components present in Pansit-pansitan leaf crude extract are alkaloids, tannins and volatile oil; 2. varying concentrations of Pansit-pansitan leaf crude extract on mortality rate of Green Leafhoppers showed that the highest mortality rate obtained was that of the control, Sevin ® 85 WP which showed 28 deaths or 93.33% followed by the 100% concentration of Pansit-pansitan leaf crude extract which has 14 deaths or 46.67%. The least mortality rate of Green Leafhoppers has been observed at 50% concentration of Pansit-pansitan leaf extract which only showed 6 deaths or 20%; and

30

3. there is a significant difference in the insecticidal property between Pansit-pansitan leaf crude extract and the positive control, Sevin 85 ® WP. Recommendations Based on the experiment results, the researcher would like to put forward the following recommendations: 1. a study regarding the insecticidal property of Pansit-pansitan on the different stages in the life cycle of Green Leafhoppers must be conducted; 2. test other parts of Pansit-pansitan plant for insecticidal property; 3. test other local plants that may have insecticidal property; 4. try other extensive methods of plant extraction; and 5. a thorough study on the physiological responses of Green Leafhopper at different time of application.

1

BIBLIOGRAPHY Cambe R, Delfin NC,Sales LO. 2003. Insecticidal Effect of Yellow Oleander Fruit Extract on Rice Bugs. Capiz National High School Dales MJ. 1996. A Review of Plant Materials Used for Controlling Insect Pests. U.J Natural Resources Institute, Kent U.K p.84. Dasmariñas JT, Jover EM, Pasig KC. 2001. Peperomia pellucida for Termite Control. University of Southern Mindanao Kabacan, Cotabato De Padua LS, Pancho JV. 1983. Handbook on Philippine medicinal plants, Volume IV. Los Baños: UPLB Documentation and Information Section, Office of the Director of Research. Estoy AB., LB Flor, EM Valdez et al. 2000. Occurance of Major Rice Insect Pest in PhilRice Stations. Philippine Rice Research Institute (PhilRice). Harborne Jeffery, H. Baxter. 1983. Phytochemical Dictionary: A Handbook of Bioactive Compounds from Plants. Taylor & Frost, London. 791 pp. Hou R. F. 1987. Mass Rearing of Green Rice Hopper (Nephotettix cincticeps). International Rice Research Institute Joshi R, Estoy A, Arida G et al. 2001. Rice Technology Bulletin No: 37. Philippine Rice Research Institute (PhilRice). Latorre P-Jay D. 2005. Insecticidal Property of Acacia Seeds and Bark Against Termites. Bureau of Soils Lipa City Manalo JB, Byung HH, Yong NH, et al. 1983. Studies on Ether-Soluble Neutral Compounds of Peperomia pellucida. Annual Report of Natural Products Research Institute, Seoul National University; 22: 16-9.

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Manalo JB, Recaña MP, Coronel VQ et al. 1984. Potassium Nitrate from Peperomia pellucida (L) HBK. NIST J. 1:25-31 Murray G. and Frank Morehouse. 1973. Organic Molecules in Action. Gordon and Breach. Pathak MD, Khan ZR. 1994. Insect Pests of Rice. Manila (Philippines): International Rice Research Institute. 89 p. Pathak MD, Vea E, John VT. 1967. Control of Insect Vectors to Prevent Virus Infection of Rice Plants. J. Econ. Entomol. 60:218-225. Reissig WH, Heinrichs EA, Litsinger JA, Moody K, Fiedler L, Mew TW, Barrion AT. 1986. Illustrated Guide to Integrated Pest Management in Rice in Tropical Asia. Manila (Philippines): International Rice Research Institute. 411 p. Shepard BM, Barrion AT, Litsinger JA. 1995. Rice-Feeding Insects of Tropical Asia. Manila (Philippines): International Rice Research Institute. 228 p Ware, G.W. 2000. The Pesticide Book. Fresno, CA: Thomson Publications. p. 57, 83. Unpublished sources Manalo JB. 1983. A Phytochemical Investigation of Pepercmia pellucida (L) HBK. UP Manila Internet sources Bayer CropScience Chile (http://www.bayercropscience.cl/soluciones/fichaproducto.asp?id=123) DOST Investigatory Projects (http://www.investigatoryprojectexample.com/search/) Dr. Dukes Phytochemical and Ethnobotanical Databases

1

(http://www.ars-grin.gov/cgi-bin/duke/farmacy2.pl) Instant EPA's Pesticide Facts (http://www.instantref.com/carbaryl.htm) Juniper and Sage Essential Oils (http://www.juniperandsage.com/oils/toxicity.php) Lans. 2007. Journal of Ethnobiology and Ethnomedicine 2007 3:3 doi: 10.1186/1746-4269-3-3 (http://www.ethnobiomed.com/content/3/1/3/table/T4) Pesticide Management Education Program (PMEP), Cornel University Ithaca, NY (http://pmep.cce.cornell.edu/profiles/extoxnet/carbaryl-dicrotophos/carbarylext.html#top) Sri Lanka Department of Agriculture (http://www.agridept.gov.lk/institutes_more_sub_links.php? id=184&sMenu=National+Botanic+Gardens+(NBG+) United States Department of Agriculture Natural Resource Conservation Service (http://plants.usda.gov/java/profile?symbol=PEPE5) United States National Library of Medicine (http://chem.sis.nlm.nih.gov/chemidplus/ProxyServlet? objectHandle=DBMaint&actionHandle=default&nextPage=jsp/chemidheavy/Res ultScreen.jsp&ROW_NUM=0&TXTSUPERLISTID=000523808) Virginia Tech's Entomology Department. Species of leafhoppers. (http://everest.ento.vt.edu/~idlab/vegpests/vegi/LH.jpeg) http://www.herbs2000.com/h_menu/tannins.htm http://www.oisat.org/pests/insect_pests/hoppers/glh.html

2

1

PLATES

2

1

Plate 1. Phytochemical Analysis of Plant Material

2

1

Plate 2. Materials Used in the Conduct of the Experiment

2

1

1. fresh Pansit-pansitan leaves

2. dried Pansit-pansitan leaves

4. filtrate of Pansit-pansitan being evaporated

3. dried Pansit-pansitan leaves boiled in water

5. Pansit-pansitan leaf crude extract

Plate 3. Extraction Procedure Done for Pansit-pansitan Leaves

1

1. cages for containing wild Green Leafhoppers

2. cages for containing insect eggs

3. cages for containing cultured insects

Plate 4. Green Leafhopper Cages in the Rice Field

2

1

Plate 5. Application of Varying Concentrations of Pansit-pansitan Leaf Crude Extract to Cultured Insects

2

1

APPENDICES

1

Appendix A Data showing the number of Green Leafhoppers died in varying concentrations of Pansit-pansitan leaf crude extract and the control (Sevin ® 85 WP) at different time intervals.

Treatments Number of Number of Dead Total Total Test Leafhoppers Based on Time Number Number Organisms (minutes) of of Deaths Deaths in 6 12 18 24 30 Percent Sevin ® 85 WP (control)

30

3

8

10

5

2

28

93.33%

50%

30

0

1

3

2

0

6

20%

75%

30

1

3

5

1

0

10

33.33%

100%

30

2

4

6

2

0

14

46.67%

Appendix B

2

Outline of Research Procedure

R e s e a rc C o ll e c t io n o f P la n t M a te r i a l Appendix C

3

Certification of Borrowing of Laboratory Equipment

University of Perpetual Help – Las Piñas Pamplona, Las Piñas City

TO:

DR. VALERIANO V. TIMBANG JR. Director, IHS Laboratories This University

THRU:

MRS. CLARISSE ROMULO Laboratory Coordinator

SUBJECT:

As stated

DATE:

November 21, 2009

Greetings! We are fourth year BS Biology students from this University and we are currently enrolled in Thesis Writing. It is a requirement in this subject to conduct an experiment/analysis about biological problems. With this regard, may we ask your good office to allow us to use/borrow some materials and equipments needed for the conduct of our methodology. Attached herewith is the list of materials to be used for the different experiments with the planned schedule. Hoping for your kind consideration. Thank you so much.

Truly yours DALISAY, GERARD MAJELL MOLDEZ, MONIQUE VALLOSO, JOANNA MARIE Noted: MRS. MA.LOURDES C. AGUIRRE Adviser

4

DATE December 9, 2009 7:30-10:30 (Class of Mr. RetardoMorphoanatomy)

LIST OF MATERIALS Centrifuge

QUANTITY 1

CHEMICALS Trisodium (anhydrous)

citrate

Micropipettor Test tube

1 1

Mortar and pestle Water bath Bunsen burner Erlenmeyer flask Filter paper Beaker(50 ml and 100ml) Litmus paper (blue and red)

1 1 1 1 2

Ferric chloride Lead acetate HCl Mayer’s reagent Sodium Carbonate 10% NaOH

Blender Funnel Filter paper Water bath Bunsen burner Beaker(100ml) Erlenmeyer flask Evaporating dish Test tubes (big) Pipette aspirator Test tube rack

1 1

Ethyl Alcohol (95%) Distilled water (100mL)

1 1 2 2 1 5 5 2 1

5

Appendix D Certification of Statistical Analysis of Data

February 1, 2010

CERTIFICATION

This is to certify that the study entitled “The Insecticidal Property of Pansitpansitan (Peperomia pellucida (Linn.) (HBK)) Leaf Crude Extract Against Green Leafhopper (Nephotettix malayanus (Ishihara et Kawase))” conducted by Mr. Gerard Majello B. Dalisay was statistically analyzed by the undersigned:

MR. ARTURO TOBIAS CALIZON Faculty, Mathematics Department

Noted:

MRS. AIDA REGACHO Chairman, Mathematics Department Appendix E

6

Certification of Identification of Plant Material

7

Certification of Identification of Test Organism

CURRICULUM VITAE

8

Personal Background Name:

Gerard Majello B. Dalisay

Age:

19 years old

Birthday:

April 26, 1990

Address:

Herrera Subdivision Lian, Batangas

Name of Father:

Honorio L. Dalisay

Name of Mother:

Victoria B. Dalisay

Educational Background School

Year

Elementary

Nasugbu East Central School

1996-2002

High School

Blessed Christ Child Montessori Foundation

2002- 2006

College

University of Perpetual Help System DALTA

2006- present