Effects of Rate and Number of Applications on Residual Toxicity of Neem-Based Insecticides to the Leafminer, Liriomyza T...

MISSING IMAGE

Material Information

Title:
Effects of Rate and Number of Applications on Residual Toxicity of Neem-Based Insecticides to the Leafminer, Liriomyza Trifolii (Burgess) (Diptera Agromyzidae) on Snap Bean
Physical Description:
1 online resource (66 p.)
Language:
english
Creator:
Poudel, Manish
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
LEIBEE,GARY L
Committee Co-Chair:
WEBB,SUSAN E
Committee Members:
LEPPLA,NORMAN C

Subjects

Subjects / Keywords:
drench -- foliar -- liriomyza -- neem -- residual -- toxicity -- trifolii
Entomology and Nematology -- Dissertations, Academic -- UF
Genre:
Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The American serpentine leafminer, Liriomyza trifolii (Burgess)is a polyphytophagous pest of greenhouse crops and ornamentals that has developed resistance to most synthetic insecticides. Neem-based insecticides have shown promising attributes in management of leafminers. However, no attempt has been made to evaluate the effects of number of foliar applications on residual toxicity of neem products to leafminer. The toxicity of commercial neem (Azadirachta indica Juss)products, Aza-Direct (Gowan Company) and NimBioSys (The Ahimsa Alternative,Inc.) neem oil, was evaluated against different stages of the leafminer. Observations were done on number of mines, larval mortality and pupal mortality. Treatments consisted of one, two, and three foliar applications of each product at two rates. In addition to foliar application, drench application was evaluated for both products at two rates. Residual toxicity of both neem products was highly prevalent in snap beans using both foliar and drench application. Persistency was observed for at least 10 days from foliar application, while it lasted longer (14 days) in the drench application. In the foliar application experiment, numbers of mines were greatly reduced and larval mortality was very high with multiple applications compared to single application. Pupal mortality was 100% in all applications regardless of product type and different rates. Numbers of mines in drench application did not differ among neem treatments and water treated control. Larval and pupal mortality in drench application was higher in neem treatments compared to control, but lower than foliar application. Increasing the number of foliar applications of neem increased mortality of different stages of leafminer under greenhouse conditions.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Manish Poudel.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: LEIBEE,GARY L.
Local:
Co-adviser: WEBB,SUSAN E.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046423:00001


This item is only available as the following downloads:


Full Text

PAGE 1

1 EFFECTS OF RATE AND NUMBER OF APPLICATIONS ON RESIDUAL TOXICITY OF NEEM BASED INSECTICIDES TO THE LEAFMINER, Liriomyza trifolii (BURGESS) (DIPTERA: AGROMYZIDAE) ON SNAP BEAN By MANISH POUDEL A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

PAGE 2

2 2013 Manish Poudel

PAGE 3

3 To my parents and my wife for their love, support and encouragement

PAGE 4

4 ACKNOWLEDGMENTS I would like to express sincere gratitude to my supervisor Dr. Gary L. Leibee for the continuous support for my M.S. study and research with his knowledge and patience. I w ould also like to thank my committee members who were generous with their expertise and precious time. The good advice and support of Dr. Norman C. Leppla and Dr. Susan E. Webb has been invaluable during the course of my study. I would also like to thank m y parents, brother and sisters who have given me continuous support and encouraged me with their best wishes. Above all, I would like to thank my wife for her support and great patience at all times. I deeply appreciate her innumerable sacrifices cheering me up and standing by me through the ups and downs.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Snap Bean ................................ ................................ ................................ ........... 13 Snap Bean Product ion in Florida ................................ ................................ ......... 14 Insect Pests of Snap Beans ................................ ................................ ................. 14 Leafminer ................................ ................................ ................................ ............. 15 Biology ................................ ................................ ................................ .......... 16 Damage ................................ ................................ ................................ ......... 1 7 Control ................................ ................................ ................................ ........... 18 Neem ................................ ................................ ................................ ................... 20 Objectives ................................ ................................ ................................ ............ 24 2 MATERIALS AND METHODS ................................ ................................ ................ 25 General Background ................................ ................................ ............................ 25 Insect and Plant Sources ................................ ................................ .............. 25 Location and Conditions ................................ ................................ ................ 26 Neem Biopesticides ................................ ................................ ...................... 26 3 FOLIAR APPLICATION EXPERIMENT ................................ ................................ .. 27 Statistical Procedure ................................ ................................ ............................ 29 Results ................................ ................................ ................................ ................. 29 Number of Mines ................................ ................................ ........................... 29 Larval and Pupal Mortality ................................ ................................ ............. 31 Discussion ................................ ................................ ................................ ........... 32 Effect on Formation of Mines ................................ ................................ ........ 32 Mortality of Larval and Pupal Stages ................................ ............................. 34 4 DRENCH APPLICATION EXPERIMENT ................................ ................................ 41 Pre Drench Release of Liriomyza trifolii ................................ ............................... 41

PAGE 6

6 Post Drench Release of L. trifolii ................................ ................................ ......... 42 Statistical Procedure ................................ ................................ ............................ 43 Results ................................ ................................ ................................ ................. 43 Larval and Pupal Mo rtality from Pre Drench Release of L. trifolii .................. 43 Effect of Post Drench Release of L. trifolii ................................ ..................... 44 Number of mines ................................ ................................ .................... 44 Larval and pupal mortality ................................ ................................ ....... 44 Discussion ................................ ................................ ................................ ........... 46 Effect on Formation of Mines ................................ ................................ ........ 46 Mortality of Larval and Pupal Stages ................................ ............................. 47 5 CONCLUSION ................................ ................................ ................................ ........ 56 LIST OF REFERENC ES ................................ ................................ ............................... 59 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 66

PAGE 7

7 LIST OF TABLES Table page 3 1 Effect of different rates and number of applications of Aza Direct and NimBioSys neem oil on L. trifolii mines on snap beans. ................................ ..... 36 3 2 Effect of different rates and number of applications of Aza Direct and NimBioSys neem oil on larval mortality of L. trifolii ................................ ............ 36 3 3 Mean comparisons between different release of leafminer, rates and application frequency of Aza Direct and NimBioSys neem oil on L. trifolii mines using T ukey test. ................................ ................................ ...................... 37 3 4 Mean comparision between different release of leafminer, rates and application frequency of Aza Direct and NimBi oSys neem oil on larval mortality of L. trifolii using Tukey test. ................................ ................................ 37 4 1 Larval mortality (MeanSE) from different treatments whe n leafminer adults were released before drench application of neem. ................................ ............. 49 4 2 Pupal mortality per treatment when leafminer adults were released before drench application of neem. ................................ ................................ ................ 49 4 3 Mean number of mines when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys. ........................ 49 4 4 Mean number of mines from different treatments when leafminer adults were released after drench application of neem at different intervals. ......................... 50 4 5 Mean larval mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys. ........................ 50 4 6 Mean larval mortality from different treatments when leafminer adults were released after drench application of neem at di fferent intervals. ......................... 51 4 7 Mean pupal mortality from different treatments when leafminer adults were released after drench applicat ion of neem at different intervals. ......................... 51 4 8 Mean pupal mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys. ........................ 51

PAGE 8

8 LIST OF FIGURES Figure page 3 1 Mean ( SE) number of L. trifolii mines from various treatments of Aza Direct and NimBioSys neem oil from leafminer adults released 1 and 7 days after foliar application. ................................ ................................ ................................ 38 3 2 Mean ( SE) number of L. trifolii mines from different treatments of Aza Direct and NimBioSys using foliar application. ................................ ................... 38 3 3 Mean ( SE) number of mines developed from different number of applications (Aza Direct and NimBioSys data pooled). ................................ ...... 39 3 4 Mean ( SE) larval mortality (%) of L. trifolii from various treatments of Aza Direct and NimBioSys neem oil from leafminer adults released 1 and 7 days after f oliar application. ................................ ................................ ........................ 39 3 5 Mean ( SE) larval mortality of L. trifolii from different treatments of Aza Direct and NimBioSys using foliar application. ................................ ................... 40 3 6 Mean ( SE) larval mortality developed fro m different number of applications (Aza Direct and NimBioSys data pooled). ................................ .......................... 40 4 1 Larval Mortality (MeanSE) of L. trifolii from d ifferent treatments of Aza Direct and NimBioSys when leafminer adults were released before drench application. ................................ ................................ ................................ ......... 52 4 2 Pupal Mortality (MeanSE) of L. trifolii from different treatments of Aza Direct and NimBioSys when leafminer adults were released before drench application. ................................ ................................ ................................ ......... 52 4 3 Mean number of mines when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys. ........................ 53 4 4 Number of mines from different treatments when leafminer adults were released after drench application of neem at different intervals. ......................... 53 4 5 Larval mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys. ................................ ...... 54 4 6 Larval Mortality from different treatments when leafminer adults were released after drench application of neem at different intervals. ......................... 54 4 7 Pupal mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys. ................................ ...... 55

PAGE 9

9 4 8 Pupal Mortality from different treatments when leafminer adults were released after drench application of neem at different intervals. ......................... 55

PAGE 10

10 LIST OF ABBREVIATIONS ANOVA Analysis of variance cm Centimeters d.f Degree of freedom F Statistical F Value g/lw Grams per liter water L:D Relation of light to darkness ml/lw Milli liters per liter water oz/gal Ounce per gallon P Statistical probability value RH Relative humidity SAS Statistical Analysis Software SE Standard error

PAGE 11

11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF RATE AND NUMBER OF APPLICATION S ON RESIDUAL TOXICITY OF NEEM BASED INSECTICIDES TO THE LEAFMINER, Liriomyza trifolii (BURGESS) (DIPTERA: AGROMYZIDAE) ON SNAP BEAN By Manish Poudel December 2013 Chair: Gary L. Leibee Major: Entomolog y and Nematology The American serpentine leafminer, Liriomyza trifolii (Burgess) is a polyphytophagous pest of greenhouse crops and ornamentals that has developed resistance to most synthetic insecticides. Neem based insecticides have shown promising attri butes in management of leafminers. However, no attempt has been made to evaluate the effects of number of foliar applications on residual toxicity of neem products to leafminer. The toxicity of commercial neem ( Azadirachta indica Juss) products, Aza Direct (Gowan Company) and NimBioSys (The Ahimsa Alternative, Inc.) neem oil was evaluated against different stages of the leafminer. Observations were done on number of mines, larval mortality and pupal mortality. Treatments consisted of one, two, and three fol iar applications of each product at two rates. In addition to foliar application, drench application was evaluated for both products at two rates. Residual toxicity of both neem products was highly prevalent in snap beans using both foliar and d rench application. Persistency was observed for at least 10 days from foliar application, while it lasted longer (14 days) in the drench application. In the foliar

PAGE 12

12 application experiment, numbers of mines were greatly reduced and larval mortality was very high with multiple applications compared to single application. Pupal mortality was 100% in all application s regardless of product type and different rates Number s of mines in drench application did not differ among neem treatments and water treated control. Larval and pupal mortality in drench application was higher in neem treatments compared to control, but lower than foliar application. Increasing the number of foliar application s of neem increased mortality of different stages of leafminer under greenhou se conditions.

PAGE 13

13 CHAPTER 1 INTRODUCTION Snap Bean Snap bean ( Phaseolus vulgaris L) is an economically important vegetable crop in the family Fabaceae (Leguminosae). It is also commonly referred to as green bean, wax bean or string bean. Snap bean is believed to have originated in the Americas and was first described in Argen tina and Guatemala (Gentry 1969 ). It gradually spread to the southwestern United States and is now distributed worldwide Snap bean is an eco nomically important legume crop ; the fr esh immature pods are a mainstay for vegetable marketing while marketing is also done in processed form by canning and freezing. Snap bean is a warm season crop and can be successfully grown within the temperature range of 70 to 80 degrees Fahrenheit. Snap bean is categorized into t wo different groups, pole or bush beans, based on growth characteri s tics Bush bean form s short, erect and compact plants, 1 to 2 feet in height, and have a determinate growth habit. Pole bean produce s vines that can reach 8 to 1 0 feet and are trained on poles, fence s or string s Pole bean has indeterminate growth habit and s et s pod continuously. Bush bean is ready for harvest in 45 50 days after sowing while pole bean s are harvested after 50 70 days (Olson et al. 2012). Snap bean holds importance due to its wider geographica l distribution, high production as well as economic and nutritional role (Silbernagel et al. 1991). It is an important source of dietary fiber, vitamin C, folic acid and minerals like potassium and several micronutrients. In 2011, snap bean production in the world totaled 20.39 million tons (Food and Agriculture Organization, 2011). In the U.S., snap bean was planted in 99,600 acres of which 93,300 acres were harvested with a production of 274,434 tons

PAGE 14

14 for fresh market during 2012. Similarly, for processing, snap bean was planted in 179,675 acres and 169,555 acres was harvested with production of 733,430 tons (USDA, NASS, 2012). Florida, California and Georgia are the largest producers of fresh market sn ap bean while Wisconsin, Oregon, Michigan and New York lead the U.S. snap bean production for processing (USDA, NASS, 2012). Snap Bean Production in Florida F lorida has the highest production, acr eage, and total value of fresh market of snap bean in the U.S ( USDA, NASS, 2012) During the year 2010 2011, snap bean was planted in 46,000 acres and harvested in 40,000 acres with production of 121.92 tons. This represent s 43 percent of U.S. fresh market snap bean production worth 131.28 million dollars. Southeastern Florida is the principal snap bean production area with Miami Dade and Palm Beach counties leading in production. The major planting season ranges between August 15 and April 1 but it varies with region. Florida continuously produces snap bean throughout the year and is the only state that supplies snap beans during the winter months in the U.S. Both bush type and pole type snap beans are produced in Florida, but the ma jority grown is bush type. Pole bean is principally grown in Miami the most common varieties grown. Bush type varieties include Bronco, Caprice, Dusky, Frontier, Valentino and Prevail (Olson et al. 2013). Insect Pests of Snap Bean s Numerous insect pests, mites and diseases cause severe economic loss in snap bean production. The major pests in the U.S. include bean leaf beetle ( Cerotoma trifurcata Foster), corn earworm ( Helicoverpa zea Boddie), European corn borer ( Ostrinia nubilali s Hubner), two spotted spider mite ( Tetranych us urticae Koch), cowpea

PAGE 15

15 aphid ( Aphis craccivora Koch) and melon thrips ( Thrips palmi Karny). Silverleaf whitefly ( Bemisia argentifolii Bellows & Perring), melon thrips ( Thrips palmi Karny) leafminer ( Liriomyza trifolii and L. sativae ), bean leafroller ( Urbanus proteus Linnaeus), cabbage looper ( Trichoplusia ni Hubner) and southern green stink bug ( Nezara viridula Linnaeus) form the major pest complex infesting snap bean in Florida. Leafminer Leafminers were first described from Argentina and are considered endemic to South and North America. More than 300 species of Liriomyza (Diptera: Agromyzidae) are recorded, of which 24 species are considered of economic significance (Spencer, 1973). Lirio myza sativae Blanchard, L trifolii Burgess and L huidobrensis Blanchard are the most destructive phytophagous leafminers of vegetables and ornamental plants worldwide (Spencer 1973, Parella 1987) with L. trifolii and L. sativae being occasionally major pests of snap bean in Florida. L. sativae was recorded as an economically important pest of horticultural crops in Florida by 1940s (Spencer 1973). It established itself as a predominant leafminer species in Florida until 1970s but was gradually displaced by L. trifolii during late 1970s (Reitz and Trumble 2002). L. trifolii L. sativae population is attributed to its stronger ability to develop resistance to insecticides used for its management (Schuster and Everett 1982). L. trifolii is now considered a major pest of field grown and greenhouse vegetable crops in Florida (Seal et al. 2002, Waddill et al. 1986). It infests numerous crops, ornamentals and weeds distributed across different families including but not limited to Cucurbitac eae, Fabaceae, Solanaceae, Brassicaceae, Asteraceae, Compositae, Caryophyllaceae and others (Capinera 2001, Musgrave et al. 1975, Parkman et al. 1989).

PAGE 16

16 Biology L. trifolii has a tremendous reproductive potential can develop rapidly and has a short life cy cle that makes it a threatening pest at all times. It undergoes complete development from egg deposition to adult emergence in less than 19 days under optimal temperature (Leibee 1984, Lanzoni et al. 2002). Optimum temperature for development ranges from 2 5 C to 30 C and larval mortality increases as temperature increases above 30 C. As a result, it can have multiple, overlapping generations in a single cropping season. L. trifolii has four distinct life stages: egg, larva, pupa and adult. Studies have show n that different life stages of leafminer averages 2.7 days for egg hatch, 1.4, 1.4, and 1.8 days for three respective larval stages and 9.3 days for pupal development into adults at 25 C (Minkenberg, 1988). An adult leafminer female prefer s mature foliage for oviposition and usually lay s eggs on the upper leaf surface. The egg is oval and small in size (1.0 mm long and 0.2 mm wide) initially clear and eventually becomes creamy white in color. Eggs are laid singly, but in close proximity to each other i Leibee (1984) observed that a single leafminer can lay eggs at the rate of 35 to 39 per day totaling up to 400, on celery. Similar fecundity was reported by Parella (1987) in tomato with less total n umber of eggs due to lower preference of the host plant. Larval stage is comprise d of three distinct instars, usually distinguished by the length of their cephalopharyngeal skeleton. The l arva begins to feed soon after hatching until late third instar whe n it is ready to pupate. Both the eggs and larval stages are concealed internally within plant foliage. Often, a fourth instar (prepupal stage) is taken under consideration, and is a non feeding stage between puparium formation and pu pation. The l ate third instar exit s through the feeding mine, form s a pupa and drop s to

PAGE 17

17 ground or substrate for further development. The l eafminer pupa is initially golden brown in color and gradually become s darker brown. Pupal stage lasts for about 9 to 12 days depending upon temperature. The a dult L. trifolii is small, about 2 mm in body length and 1.25 to 1.9 mm in wing length. The head is yellow and black in color with red eyes The thorax and abdomen are gray, and the ventral surface and legs are black and yellowish Most of the females mate within 24 hours after emergence (Parella et al. 1983) M ultiple matings by female maximize s egg production. After mating, female puncture s the leaves with its ovipositor. These punctures are used for laying eggs as well as for feeding on exuding sap. Female feed s from all punctures whether or not they are used for oviposition. Feeding and oviposition activities of the female peak during mid day. Male leafminer solely depend s on oviposition punctures made by female for feeding ( Fagoonee and Toory, 1984). Male typically live s for about 2 3 days due to l imited food source while female live s for about a week. Damage L. trifolii cause s damage to crops in two ways. F irst, damage is caused by oviposition punctures made by adult female for laying eggs and for feeding (Bethke and Parella 1985). These stipples reduce the aesthetic appeal and subsequent marketability of the crop reduce photosynthesis in foliage (Trumble e t al. 1985, Johnson et al. 1983) and may even kill seedling plants. However, damage due to stippling is considered insignificant compared to the second mode of damage larval mining. Excessive mining reduces photosynthesis, plant vigor, growth and yie ld (Al Khateeb and Al Jabr, 2004 ). In addition, high number s of leafminer larvae cause mining of bean pods and premature leaf defoliation resulting in sunscald damage of fruits such

PAGE 18

18 as tomatoes The mines also serve as route for disease pathogens. Indirectl y, it creates quarantine restrictions and cause s huge economic loss. Despite the high number of leafminer infestation and mines, significant reduction on yield of crops are not reported (Kotze and Dennil, 1996). Crop damage is considered minimal as plants can tolerate significant level of mining without affecting the yield. Economic impact due to yield loss from mining has not been studied in detail but Schuster (1978) has recorded up to 90% loss in tomato foliage due to uncontrolled leafminer infestation Control C ontrol strategies for L. trifolii are mainly focused on application of synthetic insecticides since its establishment as the most dominant leafminer pest (Leibee 1981, Cox et al. 1995). The most commonly used insecticides for leafminer control in Florida include abamectin, cyromazine, spinosad, dimethoate, lambda cyhalothrin, rynaxypyr, spinetoram and azadirachtin During the early and mid nineties growers increased the frequency of application of these insecticide s to three t imes per week in an attempt to control leafminer but had little success (Waddill 1989). Reduction in effectiveness of insecticides occurred due to their indiscriminate use resulting insecticide resistance (Leibee 1981, Keil and Parrella 19 90 Ferguson 20 04). Ferguson (2004) documented cyromazine, a bamectin and spinosad resistance in L. trifolii but resistance was unstable. Parella et al (1984) also observed slight resistance of leafminers to methyl parathion and methamidophos (organo phosphates) and perme thrin (pyrethroids) when treated on chrysanthemum. In addition, insecticides used to control leafminer were ineffective because they failed to penetrate and affect protected life stages such as egg, lar vae and pupae (Parella 1987). O veruse of insecticides decimated population s of natural enemies that were successful in

PAGE 19

19 maintaining leafminer population below economically damaging levels (Johnson et al. 1980). The effectiveness of newly developed insecticides lasted only for two years in Florida (Leibee 1981) due to development of resistance. A limited number of insecticides such as abamectin, cyromazine and spinosad are successful ly used in management of leafminers (Civelek and Weintraub 2003, Leibee 1988, Seal et al. 2002). Alternative measures for leafmine r management are being increasingly sought in recent years. Some efforts have been made towards developi ng plant resistance (Jong and Ra demaker 1991), using selective translaminar insectici des (Weintraub and Horowitz 1997 ), using biological control (Liu et al. 2009) and use of botanical derivatives such as neem ( Azadirachta indica ) products (Stein and Parella 1985, Hossain et al. 2007). Integrated pest management (IPM) strategies provide effective and economical pest control with minimum disturbances to th e natural components of the farming system. Biological control by conserving natural enemies is an important IPM strategy to combat leafminer outbreak. About 40 hymenopteran parasitoids have been studied that parasitize Liriomyza spp. (Waterhouse and Norri s 1989) with 48.5 68.5% parasitism on crops and 83.7% parasitism on weeds of vegetables (Chen et al. 2003). Mujica and Kroschel (2011) observed about 29.5% parasitism of leafminers by 63 parasitoids including Eulophidae (41 sp ecies .), Braconidae (11 sp eci es .), Pteromalidae (8 sp ecies .), Fi gi ti dae (1 sp ecies .) and Mymaridae (2 sp ecies .). The most dominant parasitoids found in Florida belong to the families Braconidae ( Opius dimidiatus Ashmead and O. dissitus Muesebeck), Pteromalidae ( Halticoptera circulus Walker) and

PAGE 20

20 Eulophidae ( Diglyphus intermedius Girault) (Parkman et al. 1989). Predatory insects are also found to prey on leafminers but do not significantly contribute to leafminer control. There has been an increasing interest on use of biorational pes ticides as alternatives to conventional synthetic pesticides. Biopesticides such as neem products have been studied as potential products effective in controlling numerous insect pests such as leafminers (Hossain et al. 2008, Schmutterer 1990). Appropriate integration of biopesticides with natural enemies can effectively manage leafminer population. Such products should preferably have properties such as faster degradability in the environment low human toxicity, easy and cheap production, low impact on be neficial insects and low risk of selecting resistant pest biotypes. One possible alternative to synthetic pesticides can be the extracts from the neem tree, Azadirachta indica Juss. Neem The neem tree, Azadirachta indica Juss (Meliaceae) is an evergreen, fast growing plant highly recognized for its insecticidal properties and low toxicity to human s (Mordue and Nisbet 2000). It is believed to have originated in Southern Asia and gradually spread to Africa, Australia and Central and South America (Schmuttere r, 1990 ). Ingredients obtained from neem fruit kernels constitute the most important compounds for insect control, while ingredients from the leaves, barks, and roots are also toxic to insects in various ways. Neem oil, obtained by cold pressing seeds, and extracts of seed residue after removal of oil are two types of neem products that have been scientifically studied and widely recommended as a biorational pesticides (Isman 2006). Azadirachtin and its derivatives are the most effective active ingredient s used as insecticide s and primarily function as insect growth regulator s (Rembold et al. 1984)

PAGE 21

21 and anti feedant s (Schmutterer 1990). It is a complex tetranortriterpenoid limonoid that produces toxic effect s in treated insects (Mordue and Nisbet 2000). Oth er active compounds such as salanin, vilasinin, nimbinen and azadirodoid show strong anti feedant properties (Schwinger et al. 1984). Further effects of neem derivatives include olfactory repellency, anti oviposition, reduction in fecundity and egg fertili ty and reduction in vigor of adult insects. Neem products effectively control numerous pest species through different modes of action, resulting from systemic and contact activity in treated plants. They are non toxic to vertebrates and have low toxic ity t o beneficial insects. These properties contribute towards the use of neem as a potential bio rational pesticide. Schmutterer and Singh (1995 ) studied toxicity of neem products and found that about 400 insect species are susc eptible to neem extracts. Both h olometabolous and hemimetabolous insect orders such as Thysanura, Orthoptera, Hemiptera, Hymenoptera, Cole optera, Lepidoptera and Diptera are significantly affected. Neem causes insecticidal effect s in insects primarily by two modes of action: growth regul ation and feeding deterrence (Isman 2006). Studies on the physiological response of insects to neem show that azadirachtin prevents the synthesis and release of ecdysteroids or molting hormones from the prothoracic gland, thus interrupting ecdysis (Mordue and Blackwell 1993 ) Immature insects exposed to neem show reduced and delayed synthesis of juvenile hormones and ecdysone causing interference during molts (Rembold 1989). As a result, high mo rtality was observed during the larval pupal and pupal adult molts. Adult insects treated with neem show reduction in ecdysone levels,

PAGE 22

22 sterility caused by reduced ovarian development and no vitellogenin synthesis (Ascher 1993; Sieber and Rembold 1983). Sc hmutterer discovered antifeedant property of neem after observ ing desert locusts that avoided feeding on neem trees. Insect pests exposed to azadirachtin show different level s of sensitivity. Many lepidopterans are highly sensit ive to azadirachtin (<1 50 p pm); Coleoptera, Hemiptera and Diptera are less sensitive (100 600 ppm) while Orthoptera show a wide range in sensitivity (0.5 1000 ppm) (Schmutterer 1990; Mordue and Nisbet 2000). Insects feeding on neem exhibit reduced growth due to reduced trypsin act ivity, resulting in inhibition of digestion and utilization of proteins (Timmins and Reynolds 1992). Other influences of neem derivatives on insects include olfactory repellent effect on settling behavior, oviposition repellent, reduction in fecundity and egg sterility (Schmutterer 1990; Mordue and Blackwell 1993). Insect pests of medical and veterinary significance such as lice, mites, ticks, fleas, bugs, cockroaches and flies are also effectively controlled by neem treatments (Mehlhorn et al. 2011). In addition to insect pests, neem products possess deterrent effects resulting in significant mortality, reduced fecundity and retarded development on two spotted spider mites (Dimetry et al. 2009) and root knot nematodes (Javed et al. 2007), growth inhibitor y effect s on various fungi (Singh et al. 1980), as well as antibacterial properties (Mahfuzul et al. 2007). Phytophagous insects can be treated with neem either by foliar application or by soil drenching since neem functions both as a contact and a system ic insecticide (Holme s et al. 1999). Neem is system ic ally taken up by the plants and is present in the food source producing strong effects on insect pests (Larew et al. 1985, Schmutterer

PAGE 23

23 1990, Mordue et al. 1998). The systemic activity of neem is very imp ortant in management of piercing and sucking insects, stem and root feeding insects and leaf mining insects (Isman et al. 1991). Systemic effects of neem have been studied with different pests such as mountain pine beetle, Dendroctonus ponderosae Hopkins, (Nauman et al. 1994), two spotted spider mite, Tetranychus urticae Koch, (Sundaram et al. 1995), leafminer, L. huidobrensis Blanchard (Weintraub and Ho rowitz 1997), green peach aphid Myzus persicae Sulzer (Holmes et al. 1999) and on western flower thrips Frankliniella occidentalis (Thoeming et al. 2003). An important property of neem is its non toxicity to invertebrates which makes it a suitable alternative in integrated pest management and organic farming. Toxicological studies have shown that the rat oral acute LD50 is > 5000 mg/kg (Raizada et al. 2001) and that neem does no t produce any mutagenic or carcinogenic effects Neem derived insecticides do not produce skin irritations or organic alterations in mice and rats, even at higher concentrations (Mehlhorn et al. 2011). Studies have also shown non toxicity of neem to fish (Wan et al. 1996) and pollinators (Naumann and Isman 1996). However, t he timing of treatment can be important; parasitoid emergence is not highly affected if treatments are made b efore parasitism of the pests (Hohmann et al. 2010). Neem product s are highly degradable and do not leave residues in soil, having a half life of only one day (Kleeburg and Ruch 2006). Hence, no phytotoxicity is observed in plants nor any adverse effects a re seen on underground water (Mehlhorn et al. 2011). These properties make neem products a safe to use biopesticide for pest control and a viable option to replace synthetic pesticides.

PAGE 24

24 The insecticidal properties of neem have been investigated for a long time and have been shown to be useful where pesticide resistance is documented and management options are limited. Pesticide resistant agricultural pests such as leafminers are susceptible to neem treatments and have shown lower fecundity, reduced longev ity of adults (Parkman and Pienkowaski 1990), and increased mortality of the larval stages (Webb et al. 1983). Neem derivatives have proven to be effective through foliar spray (Seljasen and Meadow 2006) and drench application due to their systemic propert ies (Weintraub and Horowitz 1997) and have potential to be adopted in integrated pest management schemes. Objectives Neem extracts are developed as commercial insecticides and used for management of various pests. They have shown promising attributes in the management of leafminer and have a great significance in organic production systems. Neem based insecticides possess translaminar and systemic properties and so can be used as foliar and drench application. However, no attempt has been made to evaluate the residual effects of neem products applied at different frequencies. This study was conducted for the following purposes: 1. To evaluate the effects of different rates and number of applications of Aza Direct and NimBioSys neem oil on residual toxicity t o L. trifolii on snap beans using foliar application. 2. To evaluate the effects of different rates of Aza Direct and NimBioSys neem oil on residual toxicity to L. trifolii on snap bean using drench application.

PAGE 25

25 CHAPTER 2 MATERIALS AND METHODS General Backg round The experiments for this study were performed at Mid Florida Research and Education Center (MREC), University of Florida, Apopka, Fl orida All experiments were carried out in the greenhouse. Laboratory rearing and maintenance of the leafminer culture were done in an insectary at the same location. Insect and Plant Sources American serpentine l eafminer, Liriomyza trifolii Burgess, was chose n for the study for several reasons. It is a common pest of various crops as well as ornamentals, has a shorter life cycle, is highly prolific, and relatively easy to work with. The leafminer colony used in the experiments was established and maintained fr om infested carrot tops collected from Zellwood, Florida about 30 years ago. The insect culture was maintained in the insectary at a room temperature of 25 1C, 50 60 % RH and 15L: 9D photoperiod: scotoperiod. The leafminers were provided with cowpea see dlings in rearing cages for oviposition. Cowpea plants containing late third instar larvae were placed on a tray containing paper towel s as a substrate for pupation. The pupating larvae fell into the tray and were collected manually. This process was follo wed in order to obtain synchronized life stages. The collected pupae were stored in a refrigerator at about 4 5C for later use or allowed to develop depending on need. Two day old adults were used in all the experiments. The snap bean variety used in the experiment was Kentucky Wonder a pole bean type that exhibit s indeterminate growth. It was planted and maintained in the greenhouse throughout the experimental period.

PAGE 26

26 Location and Conditions Experiments were run in greenhouses and 45 day old snap beans w ere used. The day night temperature ranged from 24.4 34.6C with relative humidity from 50 70% throughout the experimental period. The pole beans were planted in 15 cm high 20 cm diameter pots, watered twice daily and fertilized once a week. Neem Biopest icide s Two neem based products, Aza Direct (Gowan Company LLC ), containing 1.2% azadirachtin, and NimBioSys (The Ahimsa Alternatives) 100% neem oil, were tested to evaluate the residual efficacy against leafminer, Liriomyza trifolii These p roducts were applied at label rates, using the lowest and the highest concentration commercially recommended. Aza Direct solution with dosage rates of 25 oz/100 gallons and 35 oz/100 gallons were used. Solution equivalent to 1.98 ml and 2.73 ml respectively were freshl y prepared by mixing thoroughly in a liter of deionized water For NimBioSys neem oil, 0.5% and 1% solution were prepared by dissolving 5 ml and 10 ml product in 1 liter of deionized water respectively. Prior to mixing the neem oil in water, a nonionic surfactant (Triton X 100) was added to the product to maximize mixing and reduce surface tension. 1 ml of surfactant for 0.5% and 2 ml of surfactant for 1.0% neem oil was used based on product label recommendations The solution was thoroughly mixed and freshly prepared before application.

PAGE 27

27 CHAPTER 3 FOLIAR APPLICATION EXPERIMENT Commercially available neem products Aza Direct (Gowan Company) containing 1.2% azadirachtin and NimBioSys neem oil (The Ahimsa Alternatives, Inc) containing 100% neem oil were used for the experiments. Two recommended high rates of Aza Direct (25 oz/100 gallons and 35 oz/100 gallons) and two recommended rates of NimBioSys neem oil (0.5% and 1 .0%) were used to prepare solutions for application. All product rates were prepared using the procedures mentioned earlier. Treatments consisted of one, two and three foliar applications of each product at two different rates and a water treated control (1 3 treatments). P lants receiving three applications were sprayed s t arting two weeks before exposure to leafminer adults, and were sprayed on a weekly interval. Similarly, plants receiving two applications were sprayed starting a week before leafminer rele ase Plants receiving single application of neem products were sprayed one day before exposure to leafminer adults Each plant was considered a single treatment and each treatment was replicated four times. Treatments were arranged on a randomized complete block design. Freshly prepared neem products were used for application and sprayed until runoff with a hand held mist sprayer. The plants were positioned in a horizontal orientation while spraying to avoid the spray dripping into the soil and to avoid upt ake by roots and systemic translocation. The plants were left to air dry for half an hour before righting and arranging in to randomized blocks. Leafminer adults were released on all plants simultaneously on the same day First sets of leafminers were released one day after all application s w ere complete while second sets of leafminers were released seven day s later A trifoliate leaf was randomly

PAGE 28

28 chosen and a micro perforated polypropylene bag was placed over it. Five pairs of leafminer adults (1 male: 1 female) were released into each perforated bread bag containing a trifoliate. The leaves were exposed to leafminer adults for 48 hours to ensure sufficient time for mating and oviposition. After 48 hours, the leafminer adults were removed manually by ki lling them in order to achieve synchronous development of immatures and adult emergence. T otal number of mines was recorded five days after leafminer adults were released The micro perforated polypropylene bag was left intact over the trifoliate, allowin g the late third instar larvae and/or prepupae to fall on to the bag for pupation. Pupae were then collected and placed in polyethylene cups until adult emergence. Observation s were made on the number of mines, larval mortality and pupal mortality. The tota l number of adult s that eclosed was used to determine pupal mortality. Larval mortality was calculated using the formula in Leibee (1988): % larval mortality = (m p)/m 100 Where, m denotes the number of mines or larvae in each treatment and p denotes the number of pupae reared from each treatment. Pupal mortality was assessed using the same formula with a little modification: % pupal mortality = (p a)/p 100 Where p was the number of pupae in each insecticide treatment and a was the number of adults emerged from each treatment. The observation from treatment receiving three applications of 1% NimBioSys neem oil was discarded for analysis, because of higher phytotoxicity to the plants. Very few mines, if any, were recorded on this treatment.

PAGE 29

29 Statistical Procedure The treatments were arranged i n a randomized complete block design (RCBD). SAS 9.3 (SAS Institute, 2011) was used for all statistical procedures. PROC FACTEX was used to randomize blocks and treatments within blocks The data were checked for normality using the UNIVARIATE procedure and Shappiro Wilk test was used as a measure of normality (Shappiro and Wilk 1965). In add ition, scatter plots of residuals versus predicted values were used to determine normality and homo geneity of variance s In case of non normality data were transformed. Data with numbers (count values) were transformed using square root and percentage data were t ransformed using arcsine square root before running an ANOVA. ANOVA was performed using th e MIXED procedure in SAS Four treatment (two neem products with two different rates each) means along with three application frequencies in two releases of leafminer were analyzed for difference s of means. (P < 0.05) as appropriate after a significant F test. In addition, contrast analysis was performed after ANOVA to compare between contro l versus rest of the treatments as well as the difference between two neem products and its rates Analysis between co ntrol and neem treatments was performed by treating each combination of number of application and rate as a unique treatment All tests were performed at 5% level of significance. Results Number of M ines Observation s w ere based on number s of mines formed by the first generation of leafminer larvae when adults were released one day and seven days after neem application T here was no significant difference in the number of mines when adult

PAGE 30

30 leafminers were released one day and seven days after applica tion of two neem products at different rates and frequencies (F = 1.22 ; df = 1 ; P = 0.2722 ). Therefore, data collected on number of mines were pooled for analysis. The number of mines formed from first and second release of leafminer adults ( seven days aft er neem application ) is depicted in Fig. 3 1. This indicated extended residual toxicity of two neem products against leafminer in treated plants. When number of mines between the different treatments (product and rates) was analyzed a significant differe nce was seen (F = 5. 68 ; df = 3; P = 0.001 4 ). So, contrast analysis was used to test difference in means between the two products and their rates. Significant difference was obtained when Aza Direct and NimBioSys product s were compared (t = 3.92 ; P = 0.0002). However, no significant differences existed between different rates o f Aza Direct (t = 1.24; P = 0.2200 ) and different rates of NimBioSys (t = 1.11; P = 0.2715 ). Out come based on number of mines show that effect of Aza Direct and NimBioSys w ere different (Fig. 3 2). Of particular significance was the result on number of mines when different number s of application were made. Highly s ignificant difference in number of mines was observed between the three ap plication frequencies (F = 22.81 ; df = 2; P < 0.0001). mean number of mines from different frequen cy of neem applications. Multiple application s resulted in significantly lower number s of mines compared to single application while the re was no significant difference in mine numbers when two and three applications were compared (Fig. 3 3). C ontrast analysis was used to compare number of mines between neem treatments and

PAGE 31

31 water treated control. Analysis showed a significant difference between control and neem treatments (t = 6.26; P < 0.0001) Mean number s of mines from various combinations are depicted in Table 3 1 Comparative significant differences on number of mines when adult leafminers were released one day and seven days after neem application, different treatments and number of applications are shown in Table 3 3. Larval and Pupal Mortality Observation s on larval mortality from two leafminer releases were pooled for analysis. Results showed that there was no significant difference in larval mortality when leafminer adult s were released one day and seven days after neem applicati on (F = 0.63; df = 1; P = 0.4290 ). Larval mortality was high in both releases indicating hi gh residual effect (Fig. 3 4). Contr ary to the number of mi nes, no significant difference i n larval mortality was observed among treatments (two p roduct and their rates) (F = 0.90 ; df = 3; P = 0. 4427 ) ( Fig 3 5 ). C ontrast analysis was used to compare larval mortality between water treated control and neem treatments A significant difference in larval mortality (t = 10.42; P < 0.0001) was observed indicating that difference in larval mortality was due to water treated control Both neem products applied at multiple frequencies showed signif icantly different larval mortality (F =24.54 ; df = 2; P < 0.0001). A single application resulted in a lower proportion of larval mortality as compared to multiple applications (Fig. 3 6 ). Mean larval mortality from various combinations are depicted in Tabl e 3 2. Comparative significant differences i n larval mortality between number of releases, treatments and number of app lications are shown in Table 3 4

PAGE 32

32 Both neem products were highly effective against pupal stage of leafminer irrespective of different rat e s and application frequency. All pupae developed from neem treated snap bean foliage were dead and no adult eclosion occurred. A t otal mortality of the generation was observed. On the other hand i n water treated control, all pupae except a few eclosed in to adults. Hence, no analysis of variance could be performed on observations from pupal mortality. Discussion This study presents the first attempt to evaluate the residual effects of different neem products with varying rates applied at different frequencies against leafminer under greenhouse conditions. It is clearly evident that multiple application s of neem derived insecticides are highly effective in controlling leafminer population compared to products applied once However, neem products applied two and three times did not result in significant differences in mean number of mines and larval mortality. The outcome indicate d anti oviposition al property of neem based on the number of mines formed Similarly, larval an d pupal mortality could be attributed to translaminar property of neem and high toxic ity against different immature stages of leafminer. Increasing applica tion frequency is negatively correlated with leafminer numbers and has extended residual effect for a t least 10 days. Effect on Formation of M ines The results obtained from the foliar application of different neem products at different rates and frequencies show ed that residual effect was highly prevalent for at least two weeks and pose d toxic effect to d ifferent de velopmental stages of leafminer It was observed that there was an inverse relationship between increasing the rate and frequency of application of neem products to the number of larval mines formed on the

PAGE 33

33 treated foliage. An explanation for thi s phenomenon could be due to anti oviposition propert y of neem products shown with different pests. Evidence o f anti oviposition propert y of neem are shown from studies on lepidopterous pests such as Sesamia calamistis Hampson and Eldana saccharina Walker (Bruce et al. 2004) and dipteran pests like melon fly, Bactrocera cucurbitae Coquillett and oriental fruit fly, Bactrocera dorsalis Hendel (Singh and Singh 1998). Bruce et al. conducted an experiment studying ovipositional behavior of noctuids on ma ize using 0, 0.075, 0.1 and 0.15 ml/plant neem oil and tested persistency at 0, 5 and 10 days after application. They observed that neem treatments resulted up to 88% reduction in numbers of eggs laid on maize leaves compared to control. T here was no diffe rence in oviposition between days after application of neem demonstrating long term persistency of the neem treatments Similarly, Singh and Singh (1998) experimented with different neem seed kernel extracts at different concentrations against fruit flies and observed oviposition deterrency Such anti ovipositional property could be one reason for reduction in number of larval mines in the experiment. Studies conducted on the e ffect s of neem on egg hatch have supported that neem insecticides do not r educe egg hatch. Hossain and Poehling (2006) studied effect of different rates of neem (1, 3, 5, 7 and 10 ml/lw) on egg mortality of L. sativae on tomatoes and found that nearly all eggs hatched irrespective of treatment. Similar studies on cabbage moth using 0 .5% concentration of commercial neem extract (NeemAzal T) also showed that number of egg hatch ed on cabbage plant were not affected (Seljasen and Meadow 2005). This study also reported inhibition of oviposition,

PAGE 34

34 reduced larval development, anti feeding and high mortality for at least two weeks providing evidence o f residual properties of neem. Mortality of Larval and Pupal Stages Results on l arval mortality indicated that neem products were highly toxic to leafminer larvae when multiple applications were made. This was due to translaminar property of Azadirachtin. A zadirachtin containing neem products sprayed as topical application successfully penetrated the foliage and affected larval stages of leafminer. Similar conclusions were found in studies c onducted by Hossain and Poehling (2006) who recorded up to 100% mortality of leafminer larvae through topical application of the commercial neem product NeemAzal T/S (Trifolio M GmbH, Germany). NeemAzal treatments at the r ates of 1, 3, 5, 7, and 10 ml/l w ere used to evaluate larval mortality in L. sativae and up to 100% larval mortality was observed in all larval stages. In similar studies, Webb et al. (1983) demonstrated that 100% larval mortality of L. trifolii was achieved through application of neem s eed extract (Vikwood Ltd, Sheboygan, Wisconsin). Aqueous solution s of neem seed extract (0.1 to 0.05%) used as dip on primary leaves of H enderson bush lima beans showed insecticidal properties that resulted in 91 to 100% larval mortality of L. trifolii La rew et al. (1986) also reported 98.1% larval mortality of L. trifolii on bean leaves painted on both si des with a 1% aqueous solution of neem extract The mortality of larvae hatching from eggs deposited 7 days after the neem treatment was still significant, and since about 3 days are required for larvae to hatch, the mortality recorded in 7 day leaves actually represented a 10 day residual effect. Weathersbee and McKenzie (2004) studied e ffects of neem products on immatures of citrus psyllids Ne emix 4.5 was used at a concentration range

PAGE 35

35 of 11 180 ppm where psyllid nymphs showed susceptibility even at very low concentration. In this study, l arvae that survived on neem treated snap beans were all killed during the pupal stage. Adult emergence was greatly influenced by all Aza Direct and NimBioSys neem oil concentrations in both direct and residual toxicities from foliar application. No adult eclosion occurred even with the lowest concentration of neem products used. This finding is similar with th e findings of several other studies Hossain and Poehling (2006) observed no adult emergence from plants treated with NeemAzal one, three or seven days before infestation indicating high residual effectiveness of the neem product. Similar report s were obta ined from Larew (1988 ) where pupal mortality was 100 percent when plants were treated with neem seed kernel extracts (1.6% Margosan O). Weintraub and Horowitz (1997) also obtained very low adult emergence ranging from 0.8 to 1.8% from pupae collected f rom plants sprayed with 15 ppm a zadirachtin (Neemix 45). It was observed from this study and several other studies that neem products are effective in controlling the leafminer population and have extended residual effects. In this study, insecticidal effect f rom neem residues lasted at least 10 days. Growth inhibitory effect s of neem were observed until two weeks, particularly during development of larva e and pupa e Multiple application of n eem exhibited greater toxicity to various stages of leafminer. Hence, foliar application of neem can be considered as a potential control measure for leafminer management under greenhouse condition s when multiple application s are used

PAGE 36

36 Table 3 1 Effect of different rates and number of a pplication s of Aza Direct and NimBioSys neem oil on L. trifolii mines o n snap beans Effect Categories Number of mines (Mean SE) Leafminer release F irst 26.0221 3.9 9 S econd 31.4085 3.9 9 Treatment Aza Direct 25 oz/100 gal 41.20834.8 5 Aza Direct 35 oz/100 gal 34.66674.8 5 Nimbiosy 0.5% 22.70834.8 5 Nimbiosys 1% 16.27785.9 3 Number of applications 1 49.28134.3 8 2 23.46884.3 8 3 13.39585.0 7 Note: Mean number of mines are the least square mean values Table 3 2 Effect of different rates and number of application s of Aza Direct and NimBioSys neem oil on larval mortality of L. trifolii Observation Categories Larval mortality (Mean SE) Leafminer release F irst 80.69 4.26 S econd 77.81 4.26 Treatment Aza Direct 25 oz/100 gal 75.01 5.23 Aza Direct 35 oz/100 gal 77.80 5.23 Nimbiosy 0.5% 77.42 5.23 Nimbiosys 1% 86.76 6.44 Number of applications 1 54.79 4.70 2 90.35 4.70 3 92.61 5.48 Note: Mean larval mortality are the least square mean values

PAGE 37

37 Table 3 3 Mean comparisons between different release of leafminer rates and application frequency of Aza Direct and NimBioSys neem oil on L. trifolii mines using Tukey test Observation Tukey Test P value Leafminer release First vs second 0.2722 Treatment Aza Direct 25 oz/100 gal vs Aza Direct 35 oz/100 gal 0.2200 Aza Direct 25 oz/100 gal vs NimBioSys 0.5% 0.0039 Aza Direct 25 oz/100 gal vs NimBioSys 1% 0.000 4 Aza Direct 35 oz/100 gal vs NimBioSys 0.5% 0.0863 Aza Direct 35 oz/100 gal vs NimBioSys 1% 0.0108 NimBioSys 0.5% vs NimBioSys 1% 0.2715 Number of applications 1 vs 2 0.0002 1 vs 3 <0.0001 2 vs 3 0.0909 Table 3 4 Mean comparision between different release of leafminer rates and application frequency of Aza Direct and NimBioSys neem oil on larval mortality of L. trifolii using Tukey test Observation Tukey test P value Leafminer release First vs second 0.4290 Treatment Aza Direct 25 oz/100 gal vs Aza Direct 35 oz/100 gal 0. 5113 Aza Direct 25 oz/100 gal vs NimBioSys 0.5% 0. 7282 Aza Direct 25 oz/100 gal vs NimBioSys 1% 0. 1140 Aza Direct 35 oz/100 gal vs NimBioSys 0.5% 0.7566 Aza Direct 35 oz /100 gal vs NimBioSys 1% 0.3076 NimBioSys 0.5% vs NimBioSys 1% 0.1986 Number of applications 1 vs 2 < 0.0001 1 vs 3 < 0.0001 2 vs 3 0. 8165

PAGE 38

38 Figure 3 1 Mean ( SE ) number of L. trifolii mines from various treatments of Aza Direct and NimBioSys neem oil from leafminer adults released 1 and 7 days after foliar application Figure 3 2 Mean ( SE ) number of L. trifolii mines from different treatments of Aza Direct and NimBioSys using foliar application 0 10 20 30 40 50 60 70 Number of Mines Leafminer Release First Second 0 5 10 15 20 25 30 35 40 45 50 Number of Mines Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%)

PAGE 39

39 Figure 3 3 Mean ( SE ) number of mines developed from different number of application s ( Aza Direct and NimBioSys data pooled). Figure 3 4 Mean ( SE ) larval mortality (%) of L. trifolii from various treatments of Aza Direct and NimBioSys neem oil from leafminer adults released 1 and 7 days after foliar application 0 20 40 60 80 100 120 Number of Applications One Two Three Larval Mortality 0 10 20 30 40 50 60 70 80 90 100 Larval Mortality (%) Release First Second

PAGE 40

40 Figure 3 5 Mean ( SE ) larval mortality of L. trifolii from different treatments of Aza Direct and NimBioSys using foliar application Figure 3 6 Mean ( SE ) larval mortality developed from diff erent number of applications ( Aza Direct and NimBioSys data pooled) 0 10 20 30 40 50 60 70 80 90 100 Larval Mortality (%) Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%) 0 20 40 60 80 100 120 Number of Applications One Two Three Larval Mortality (%)

PAGE 41

41 CHAPTER 4 DRENC H APPLICATION EXPERIMENT Pre Drench R elease of Liriomyza trifolii To evaluate the systemic effect of neem, commercially available neem products Aza Direct (Gowan Company) containing 1.2% azadirachtin and NimBioSys neem oil (The Ahimsa Alternatives, Inc.) containing 100% neem oil were used for the experiments. Neem products were freshly prepared using the same procedure as i n foliar application experiment. Two recommended rates of Aza Direct (25oz/100 gallons and 35 oz/100 gallons) and two rates of NimBioSys neem oil (0.5% and 1.0%) were used to study the effect of various rates. Additionally, a water treated control was used, adding up to five treatments. 30 snap beans (45 days old) were kept in a large cage and abo ut 200 leafminer adults (1male: 1female) were released into the cage. The plants were exposed to leafminer adults for 48 hours and taken out from the cages. 20 plants were randomly chosen and treatments were assigned to each plant. Each plant represented o ne treatment and one treatment was replicated four times. The drench application was done after the release of adult leafminers. FACTEX procedure in SAS was used to randomize the treatments within a block and to randomize block s as well. T reatments were ar ranged i n a randomized complete block design. Neem tr eatment s were applied by drenching 250 ml of each treatment into the potted plant. 250 ml of each product was used as it was just enough to meet the saturation capacity of the soil After drenching, the pot s were irrigated based on the water requirement only up to saturation capacity. In most cases 250 ml of water was used twice a day, usually during late morning and evening.

PAGE 42

42 A single trifoliate leaf was used for data collection. After 72 hours, the nu mber of mines was counted to determine the number of larvae. The total number of mines was recorded again after five days. The l arvae were allowed to develop on the trifoliate attached to the plant and a perforated bread bag was placed over the trifoliate leaf and secured Late third instar larvae and/or prepupae were allowed to pupate i n the bread bags. Pupae were then brought in the lab, transferred to a polythene cup and allowed to develop to adults Data collection was performed by recording number of m ines, larva e, p upa e and adults. Larval and pupal mortality was assessed using similar method as in the foliar experiment. Post D rench R elease of L. trifolii To evaluate the systemic and residual effects of Aza Direct and NimBioSys similar procedures as i n the pre drench experiment were followed for preparing product dilutions and product application. The treatment rates, treatment numbers and replication were similar to the pre drench release experiment. However, in this experiment, drench application of neem products was done prior to release of leafminers. Leafminers were released after drench ap plication of treatment products with a total of 5 releases made 1, 3, 5, 7 and 10 days after drenching. A trifoliate was randomly selected and 5 pair s (1male: 1 female) of leafminer adults were released for each trifoliate covered with a perforated bread bag. After exposure to leafminers for 48 hours the leafminers were killed to obtain synch ronous life stages. Data collection was done 72 hours after exposure of trifoliate to leafminer. The t otal n umber of mines w as recorded after five days ; and larvae were left to pupate in the bread bag and reared in

PAGE 43

43 the laboratory to record adult emergence. Larval and pupal mortality were assessed using the formula developed by Leibee (1988) as in other experiments. Statistical Procedure Treatments were arranged in randomized block design. PROC FACTEX in SAS was used for randomization of both blocks and treatments within a block. Observations on number of mines, larval and pupal mortality were pooled for analysis. Data with numbers (count values) were transformed using square root and percentage values were transformed using arcsine square root before running an ANOVA. ANOVA was performed using PROC GLM in SAS version 9.3 (SAS Institute, 2011). In tests where significant F values (P < 0.05) multiple range test. Results Larval and Pupal Mortality from Pre Drench R elease of L. trifolii A significant difference in larval mortality was observed between treatments (F = 11.32; df = 4; P = 0.0005) Using contrast analysis the control was shown to be si gnificantly different than all the neem treatments (t = 6.28; P < 0.0001) This indicat ed toxicity of the neem treatments on larval stages of leafminer (Table 4 1). In addition, t here was a significa nt difference in larval mortality between two products Aza Direct and NimBioSys (t = 2.32; P = 0.0390) However, there was no significant d ifferen ce between rates of Aza Direct (t = 0.63; P = 0.5386) and NimBioSys (t = 0.06; P = 0.9509 ) Larval mortality ranged from 1 percent to 22 percent (Figure 4 1). A significant difference in pupal mortality was observed between treatments and control (F = 183.87; df = 4; P < 0.0001) (Figure 4 2). Comparisons using contrast analysis indicated that control and neem treatments were significantly different (t =

PAGE 44

44 26.87; P < 0.0001). Pupal mortality ranged from 92 to 100 percent in neem treated population while in the control treatment, only 3% pupae were dead (Table 4 2). Similarly, comparisons using co ntrast analysis showed a significant difference in pupal mortality between Aza Direct and NimBioSys (t = 2.25; P = 0.0441) There was a significant difference between two rates of Aza Direct (t = 2.91; P = 0.0130) but NimBioSys rates were not significant ly different (t = 0.40; P = 0.6962) based on pupal mortality Adult eclosion was significantly affected by all azadirachtin treatments with v ery few ad ults eclosing in the neem treatments. Effect of Post Drench Release of L. trifolii Number of m ines Obse rvation on the number of mines was subjected to ANOVA using GLM procedure in SAS. Data on number of mines observed were pooled for analysis. At 5% level of significance, there was no significant difference in number of mines when leafminers were released a t different intervals (F = 1.84; df = 4; P = 0.1290 ) (Figure 4 3). The mean number of mines from first, second, third, fourth and fifth infestation were similar (Table 4 3). A significant difference in the number of mines was observed between treatments consisting of different neem products with different rates and a water treated control (F = 6.37; df = 4; P = 0.0001) (Figure 4 4). The mean number of mine s in neem treatments and water treated control are shown in Table 4 4 A contrast analysis between co ntrol and neem treatments showed significant differen ce in number of mines (t = 4.94; P< 0.0001). Larval and p upal m ortality Percentage data on larval mortality showed significant difference s at different infestation times (F = 4.64; df = 4; P = 0.0022) (Figure 4 5). Leafminer released three

PAGE 45

45 days after drench application showed highest larval mortality compared to other releases (Table 4 5). This could be an indication that systemic effect was prominent 5 7 days after drenching In addition, a significant d ifference in larval mortality was observed between treatments (F = 16.37; df = 4; P < 0.0001) (Figure 4 6). All neem treated population had higher larval mortality compared to control (Table 4 6 ). Larval mortality was observed up to 26 percent in nee m treatments while water treated control had lower than 2% larval mortality. Results on pupal mortality did not show significant difference when leafminers were released at different intervals after drench application of neem (F = 0.82; df = 4; P = 0.5133 ) (Figure 4 7). However, p upal mortality was high at all different release dates (Table 4 7). This indicated systemic persistency of neem in snap beans when applied as drench Pupal mortality was also significantly different between treatments ( neem products and water treated control ) (F = 296.85; df = 4; P < 0.0001). So, contrast analysis was used to compare differences between Aza Direct and NimBioSys and the rates used. Pupal mortality differed significantly between two neem products Aza Direct an d NimBioSys (t = 4.67; P < 0.0001). Similarly d ifferent rates of Aza Direct showed significant difference i n pupal mortality (t = 3.53; P = 0.0007) A significant difference in pupal mortality was also observed between two rates of NimB ioSys (t = 8.27; P < 0.0001). Contrast analysis showed significant difference in pupal mortality between neem treatments and water treated control (t = 32.94; P < 0.0001). H igher pupal mortality was seen on neem treated plants and very few, if any, pupae were dead on contr ol treatment (Figure 4 8). Pupal mortality ranged from 71 percent to 100 percent in neem treated plants while it was less than 4 percent on control (Table 4 8).

PAGE 46

46 Discussion The results from drench application studies clearly demonstrate systemic properties of azadrachtin and the varying levels of toxic effect on different life stages of L. trifolii. The studies ha ve shown that different neem based products (Aza Direct and NimBioSys) show similar effect on mortality o f leafminer. Azad irac htin used as a soil drench had little effect on larval mortality but a strong effect on pupal mortality as adult eclosion was greatly reduced in all applied treatment. Effect on Forma t ion of M ines Neem derived insecticides used in this experiment resulted in little or no effect on number of larval mines when applied as drench. The number of mines was similar in all treatment s including control. Increasing the rate of product used had very little effect on formation of mines compared to lower rates. Number of mines is a direct reflection of the number of eggs laid, as the hatched larvae cause mines by feeding. Thus, it can be inferred that neem products applied as a soil drench have very little, if any, effect on o viposition. Neem products d id not pos sess ovicidal properties as previously seen in the foliar stud y Hossain et al. (2007 ) demonstrated that soil drenching of 0.75, 1.5, 2.25 and 3.0 g/lw Neemaz al on tomato did not result sig nificant difference on L. sativae oviposition. However, s tudies eva luating the systemic properties of neem and its effect on oviposition or egg hatch are very limited. The p resent study does not show difference s in number of mines among treatments, except water treated control which mic action of neem on oviposition. Weintraub and Horowitz (1997) used azadirachtin (Neemix 45) at concentrations of 1, 5, 10 or 15 ppm a gainst L. huidobrensis but observe d no effects on oviposition Larew et al (1985) also used 0.4% crude neem oil as soil drench against L. trifolii and

PAGE 47

47 observed s imilar results Besides leafminer Naumann and Isman (1995) obtained a similar result on noctuid moths using different rates of neem seed oil These studies conclude that neem products used as soil drench does not deter oviposition significantly but produce toxic effects through systemic action. In addition to l ack of anti ovipositional properties neem products do not influence egg hatch. Hossain et al (2007 ) conclude d from his experiment on L. sativae that the neem product NeemAzal did not show any reduction i n egg hatch. He used NeemAzal concentrations at 0.21, 0.42, 0.63, and 0.83 g/kg of substrate for drench application but did not observe ovicidal effect Similar ly, Seljasen and Meadow (2005) reported similar results on a study on cabbage moth Failure of neem products to deter oviposition in leafminer and failure to affect egg hatch are possible explanation s for similar number of mines in different neem treatments and control in this study. Mortality of Larval and Pupal Stages Azadirachtin possesses systemic properties and is translocated to the larval mining sites. Larval mortality observed from different neem treatments compared to control provides evi dence as an eff ective insecticide for control of leafminer larvae. However, different product and different rate s do not seem to produce difference in larval mortality of L. trifolii Drench application of neem was seen to be most effective five days after application which is similar to results from Meisner et al. (1987). Systemic properties of neem showing toxic e ffect s against leafminer larva e are supported by studies from other authors. Hossain et al. ( 2007 ) demonstrated 100% larval mortality of L. sati vae by using different rates of neem (NeemAzal) as soil drench. Related studies using neem as drench showed toxic effects in pests other than leafminer. T hoeming et al (2003) and Meadow et al (2000) demonstrated systemic

PAGE 48

48 action of neem (NeemAzal T/S) tha t caused 90 percent larval mortality in western flower thrips and cabbage moth respectively. Larval mortality due to azadirachtin is primarily attributed to alterations in hormonal levels and interruption of physiological processes of development. Among all life stages of leafminer, pupal mortality was hig h ly affected by drench application of neem. The study show ed very low adult emerge nce from pupae developed on neem tre ated plants. Hossain et al (2007 ) studied effects of drench application of Neem Azal on leafminer pupae on tomato and observed 100 percent mortality of L. sativae pupae when higher rates were used (2.25 and 3 gram per liter of water). Studies by Weintraub and Horowitz (1997) also demonstrated 100% pupal mortality in L. huidobrensis us ing various concentrations of azadirachtin applied as drench in bean plants. Bean plants drench e d with 1, 5, 10 or 15 ppm azadirachtin resulted total pupa l mortality when the highest conc entration was used. A s imilar study using drench application of 1.0 a nd 2.0 ppm neem seed extract resulted 65.4% and 77.3% mortality of L. trifolii pupae on chrysanthemum (Parkman and Pienkowskii, 1990). It can thus be concluded that neem based insecticides possess systemic properties and toxicity resulting in mortality of leafminer pupae. Interference with development, primarily due to change in ecdysone level resulted in imbalanced hormonal regulation and high pupal mortality. Neem products are effective as soil drench t o control leafminer populati on and show extended persistency This study demonstrated extended residual effects of neem products applied as drench for at least two weeks However, the effect varied greatly based on the days after drenching. Larval mortality appeared to be highest at 5 to 7

PAGE 49

49 days after dre nch application. Higher pupal mortality was recorded where leafminer adults were released 10 days after drench application of neem. Results on larval mortality also suggest that neem products are effective as soil drench for prolonged time period. Persiste ncy of neem applied as soil drench could be attributed to protection of active ingredients in the soil and plant roots. Table 4 1 Larval mortality (MeanSE) from different treatment s when leafminer adults were released before drench application of neem Treatment Larval Mortality ( MeanSE ) Aza Direct (25oz/100gal) 15.782.19 A Aza Direct (35oz/100gal) 13.272.19 A NimBiosys (0.5%) 10.172.19 A NimBiosys (1%) 9.212.19 A Control 0.002.1 9 B Means with the same letter are not Table 4 2 Pupal mortality per treatment when leafminer adults were released before drench application of neem Treatment Pupal Mortality ( MeanSE ) Aza Direct (25oz/100gal) 92.131.5 B Aza Direct (35oz/100gal) 99.311.5 A NimBiosys (0.5%) 99.211.5 A NimBiosys (1%) 100 .00 1.5 A Control 3.021.5 C

PAGE 50

50 Table 4 3 Mean number of mines when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys Release Number of mines (MeanSE) Day 1 59.7 0 3.57 A Day 3 48.503.57 B Day 5 49.803.57 AB Day 7 47.653.57 B Day 10 51.653.57 AB Table 4 4 Mean number of mines from different treatments when leafminer adults were released after drench application of neem at different intervals Treatment Number of mines (MeanSE) Aza Direct (25oz/100gal) 49.203.57 B Aza Direct (35oz/100gal) 48.803.57 B NimBiosys (0.5%) 44.353.57 B NimBiosys (1%) 47.703.57 B Control 67.253.57A Table 4 5 Mean larval mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys Release Larval Mortality (MeanSE) Day 1 8.01.9 B Day 3 15.41.9 A Day 5 7.11.9 B Day 7 7.21.9 B Day 10 5.31.9 B Means with

PAGE 51

51 Table 4 6 Mean larval mortality from different treatments when leafminer adults were released after drench application of neem at different intervals Treatment Larval Mortality (MeanSE) Aza Direct (25oz/100gal) 14.41.9 A Aza Direct (35oz/100gal) 9.91.9 AB NimBiosys (0.5%) 11.31.9 AB NimBiosys (1%) 7.31.9 B Control 3.01.9 C Table 4 7 Mean pupal mortality from different treatments when leafminer adults were released after drench application of neem at different intervals Treatment Pupal Mortality (MeanSE) Aza Direct (25oz/100gal) 91.062.3 B Aza Direct (35oz/100gal) 99.132.3 A NimBiosys (0.5%) 72.382.3 C NimBiosys (1%) 97.582.3 A Control 2.462.3 D Table 4 8 Mean pupal mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys Release Pupal Mortality (MeanSE) Day 1 71.195.1 A Day 3 71.065.1 A Day 5 75.675.1 A Day 7 72.365.1 A Day 10 72.345.1 A Means with same letter are not significantly different

PAGE 52

52 Figure 4 1 Larval Mortality (MeanSE) of L. trifolii from different treatments of Aza Direct and NimBioSys when leafminer adults were released before drench application Figure 4 2 Pupal Mortality (MeanSE) of L. trifolii from different treatments of Aza Direct and NimBioSys when leafminer adults were released before drench application 0 5 10 15 20 25 Larval Mortality (%) Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%) Control 0 20 40 60 80 100 Pupal Mortality (%) Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%) Control

PAGE 53

53 Figure 4 3 Mean n umber of mines when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys Figure 4 4 Number of mines from different treatments when leafminer adults were released after drench application of neem at different intervals 0 10 20 30 40 50 60 70 Number of Mines Leafminer Release First Second Third Fourth Fifth 0 10 20 30 40 50 60 70 80 90 100 Number of Mines Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%) Control

PAGE 54

54 Figure 4 5 Larval mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys Figure 4 6 Larval Mortality from different treatments when leafminer adults were released after drench application of neem at different intervals 0 5 10 15 20 25 Larval Mortality (%) Leafminer Release First Second Third Fourth Fifth 0 5 10 15 20 25 Larval Mortality Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%) Control

PAGE 55

55 Figure 4 7 Pupal mortality when leafminer adults were released at different intervals after drench application of Aza Direct and NimBioSys Figure 4 8 Pupal Mortality from different treatments when leafminer adults were released after drench application of neem at different intervals 0 10 20 30 40 50 60 70 80 90 100 Pupal Mortality (%) Leafminer release First Second Third Fourth Fifth 0 10 20 30 40 50 60 70 80 90 100 Pupal Mortality (%) Treatments Aza-Direct (25oz/100gal) Aza-Direct (35oz/100gal) NimBioSys (0.5%) NimBioSys (1%) Control

PAGE 56

56 CHAPTER 5 CO NCLUSION Two neem derived insecticides, Aza Direct and NimBioSys were evaluated for control of leafminer, Liriomyza trifolii on snap beans under greenhouse conditions. Two different recommended rates were used in foliar and drench experiments. Foliar stud y consisted of one, two and three applications of two products on a weekly interval. Similarly drench experiments was conducted by evaluating systemic and residual effect of two products, two rates of each product and a water treated control applied befor e and after infestation From the experiments conducted in this study, it can be deduced that neem derived products are effective in management of leafminer population. This is particularly more important in organic production of vegetable and other crops under greenhouse conditions. Both neem derived insecticides produced significant toxic effect on different stages of leafmi n er Mortality effects were not immediate but ultimately resulted in high mortality in larva e and pupa e Mortality during immature stage s of leafminer is primarily due to growth regulatory effect of neem derivatives resulting in molting disruption. Various insec ts have shown such sensitivity to neem products Neem treated insects that developed into adults had malformed body parts suc h as crippled wings absence of proboscis and abnormal legs Such response s were studied extensively in Orthoptera ( Locusta migratoria migratoriodes ), Hemiptera ( Dysdercus spp., Rhodnius prolixus, Sogatella frucifera, Nephotettix virescens and Oncopeltus fasciatus ), Coleoptera ( Epilachna varivestis ) and L epidoptera ( Manduca sexta Lymantria dispar Helicoverpa zea and Heliothes virescens )

PAGE 57

57 In dipterans, sensitivity to growth regulator y effect of neem derivatives was more pronounced during the pupal stage compared to larval stage resulting in inhibition of adult emergence. Similar result s w ere obtained from this study where pupal mortality was higher compared to larval stages in both foliar and drench application methods. Physiological pro cess e s involving growth regulatory effect of neem derivatives on immature insects are not fully understood but are attributed primarily to hormonal control of molting. Studies have demonstrated that azadirachtin inhibits the release of ecdysone from hormone prod ucing brain/ring gland complex of insects and also delays the production of juvenile hormones. F oliar application of neem provides effective control of leafminer population compared to drench application. There was no difference between larval mortality and number of mines in leafminers released at weekly interval. This clearly demonstrated the extended residual effect of the neem products applied. Increasing th e number of applications resulted in higher mo rtality of leafminer larvae compared to single appli cation. However, there was no marked difference in formation of mines and larval mortality when two or three foliar applications were made. So, two foliar application s of neem derived insecticides at a we ek interval can be recommended for effective management of leafminer in snap beans grown under greenhouse conditions Different product s such as Aza Direct and NimBioSys and their different rates produce d similar toxic effect on leafminer pupae All pupae developed were dead regardless of number of applications and different rates used in foliar application Hence, foliar application of neem can be an effective measure for leafminer management under greenhouse conditions with extended residual effects.

PAGE 58

58 Drench application of neem products, on the other hand resulted in lower larval mortality compared to foliar application. Pupal mortality however, was greater from different neem treatments compared to control. One limitation on the use of botanicals lik e neem under field conditions is restricted residual effect due to effects of temperature, ultraviolet light and rainfall that exert negative influence on active ingredients. In addition, delayed effects of neem derivatives may lead to several repeated app lication of neem in a switching to synthetic insecticides for quick er knockdown of pests. It is thus importan t that growers be well informed about mode of action and delayed effect of neem to avoid dissatisfaction and wrong conclusions. Hence we can conclude that n eem products are a good fit in integrated pest management programs and their use must be encouraged to reduce dependency on synthetic chemicals They can play a significant role in manage ment of pests where insecticide resistance is a problem. However, s horter residual persistency of active ingredients pose limitation s but such products can b e mixed with other bio products to obtain higher efficacy. Further studies should be aimed at integ rating bio insecticides such as neem with biological control for increasing efficacy in pest management Possible impacts of bio insecticides on natural enemies could be another area of study that needs to be further explored.

PAGE 59

59 LIST OF REFERENCES Agricu ltural Statistics Annual U.S. Department of Agriculture. National Agricultural Statistics Service 2012. http://www.nass.usda.gov/Publications/Ag_Statistics/2011/index.asp Al Khateeb, S. A., and A. M. Al Jabr. 2004. Effect of leafminer Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) on gas exchange capacity of cucumber, Cucumis sativus L. grown under greenhouse conditions. In International Symposium on Greenhouses, Env ironmental Controls and In house Mechanization for Crop Production in the Tropics. 710: 423 428. Ascher, K. R. S. (1993). Nonconventional insecticidal effects of pesticides available from the Neem tree, Azadirachta indica Arch. Insect Biochem. Physiol., 22: 433 449. Bethke, J. A. & M. P. Parrella. 1985. Leaf puncturing, feeding and oviposition behavior of Liriomyza trifolii. Entomol Exp. App. 39: 149 154. Bruce, Y. A., G. Saka., A. Chabi Olaye., H. Smith., and F. Schulthess. 2004. The effect of neem (A zadirachta indica A. Juss) oil on oviposition, development and reproductive potentials of Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and Eldana saccharina Walker (Lepidoptera: Pyralidae). Agric. Forest. Entomol. 6: 223 232. Capinera, J. L. 2001. Entomology and Nematology. University of Florida. http://entnemdept.ufl.edu/creatures/veg/leaf/a_serpentine_leafminer.htm Chen, X., F. Lang., Z. Xu., J. He., and M. A. Yun. 2003. The occurrence of leafminers and their parasitoids on vegetables and weeds in Hangzhou area, Southeast China. Biocontrol 48: 515 527. Civelek, H. S., and P. G. Weintraub. 2003. Effects of bensultap on larval serpentine leafminers, Liriomyza tr ifolii (Burgess) (Diptera: Agromyzidae), in tomatoes. Crop Protection 22: 479 483. Cox, D. L., M. D. Remick., J. A. Lasota, and R. A. Dybas. 1995. Toxicity of avermectins to Liriomyza trifolii (Diptera: Agromyzidae) larvae and adults. J. Econ. Entomol. 88: 1415 1419. Dimetry. N. Z., S. A. Amer, and A.S. Reda. 2009. Biological activity of two neem seed kernel extracts against the two spotted spider mite Tetranychus urticae Koch. J. App. Entomol. 116: 308 312.

PAGE 60

60 Fagoonee I., and V. Toory. 1984. Contributio n to the study of the biology and ecology of the leaf miner Liriomyza trifolii and its control by neem. Insect Science Appl. 5:23 30. Ferguson, J. S. 2004. Development and stability of insecticide resistance in the leafmine r Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad. J. Econ. Entomol. 97: 112 119. Florida Department of Agriculture and Consumer Services (FDACS), Florida Agricultural Crop Facts and Statistics Overview (Tallahassee, FL: FDACS, 2011), http://www.florida agriculture.com/brochures/P 01304.pdf Gentry, H. S. 1969. Origin of the common bean, Phaseolus vulgaris. Econ. Bot. 23: 55 69. Hohmann, C. L., A. C. Flavia, F. A. C. Si lva, and T. G. de Novaes. 2010. Selectivity of neem to Trichogramma pretiosum Riley and Trichogrammatoidea annulata De Santis (Hymenoptera: trichogrammatidae). Neoptrop. Entomol. 39: 985 990. Hossain, M. B., H. M. Poehling., G. Tho eming, and C. Borgemeist er. 2008 Effects of soil application of neem (NeemAzal U) on different life stages of Liriomyza sativae (Diptera: Agromyzidae) on tomato in the humid tropics. J. Plant. Dis. Protect. 115 (2): 80 87. Hossain, M. B., and H. M. Poehling. 2006. Effects of a neem based insecticide on different immature life stages of the leafminer Liriomyza sativae on tomato. Phytoparasitica.34 (4): 360 369. Holmes, M. S., E. Hassan, R. P. Singh, and R. C. Saxena. 1999. The contact and systemic action of neem seed extract against green peach aphid Myzus persicae Sulzer (Hemiptera: Aphididae). Azadirachta indica A. Juss. 93 101. Isman, M. B. 2006. Botanical in secticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. Annu. Rev. Entomol. 51 : 45 66. Isman, M. B., O. Koul, J. T. Arnason, J. Stewart, and G. S. Salloum. 1991. Developing a neem based insecticide for Canada. Mem Entomol. Soc. Can.159: 39 46. Javed, N., S. R. Gowen, M. Inam ul Haq, K. Abdullah and F. Shahina. 2007. Systemic and persistent effect of neem ( Azadirachta indica ) formulations against root knot nematodes, Meloidogyne javanica and their storage life. Cr op Protection. 26: 911 916. Johnson, M. W., E. R. Oatman, and J. A. Wyman. 1980. Effects of insecticides on population of the vegetable leafminer and associated parasitoids on fall pole potatoes. J. Econ. Entomol. 73: 61 71.

PAGE 61

61 Johnson, M. W., S. W. Welter, N. C. Toscano, I. P. Ting, and J. T. Trumble. 1983. Reduction of tomato leaflet photosynthesis rates by mining activity of Liriomyza sativae (Diptera: Agromyzidae). J. Econ. Entomol. 76: 1061 1063. Jong J. D., and W. Ra demaker. 1991. Life history studies of the leafminer Liriomyza trifolii on susceptible and resistant cultivars of Dendranthema grandiflora Euphytica 56: 47 53. Keil, C. B., and M. P. Parella. 1990. Characterization of insecticide resistance in two colonies of Liriomyza trifolii (Diptera: Agromyzidae). J. Econ. Entomol. 83: 18 26. Kleeberg, H., and B. Ruch. (2006, November). Standardization of neem extracts. In Proceedings of International Neem Confe rence, Kunming, China. 11 15. Kotze, D.J. and G. B. Dennill. 1996. The effect of Liriomyza trifolii (Burgess) (Diptera, Agromyzidae) on fruit production and growth of tomatoes, Lycopersicon esculentum (Mill) (Solanaceae). J. Appl. Entomol. 120(4):231 235. Lanzoni A., G. G. Bazzocchi., G. Burgio, and M. R. Fiacconi. 2002. Comparative life history of Liriomyza trifolii and Liriomyza huidobrensis (Diptera: Agromyzidae) onbeans: Effect of temperature on development. Environ. Entomol 31:797 803. L arew H. G. 1988. Limited occurrence of foliar, root, and seed applied neem seed extract toxin in untreated plant parts. J. Econ. Entomol 81: 593 598 Larew, H. G., J. J. Knodel Montz., R. E. Webb, and J. D. Warthen. 1985. Liriomyza trifolii (Burgess) (Dipera: Agrom yzidae) control on chrysanthemum by neem seed extract applied to soil. J..Econ. Entomol. 78: 80 84. Leibee, G. L. 1981. Insecticidal control of Liriomyza spp on vegetables. In D. J. Schuster Proceedings of the Institute of Food and Agricultural Sciences Industry Conference on Biology and Control of Liriomyza Leafminers 2:216 220. University of Florida Gainesville, FL. Leibee, G. L. 1984. Influence of temperature on development and fecundity of Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) on celery Environ. Entomol. 13: 497 501. Leibee, G. L. 1988. Toxicity of abamectin to Liriomyza sativae (Burgess) (Diptera: Agromyzidae). J. Econ. Entomol. 81:738 740. Liu, T. X., L. Kang., K. M. Heinz, and J. Trumble. 2009. Biological control of Liriomyza leafm iners: progress and perspective. Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 4: 1 16.

PAGE 62

62 Mahfuzul, H. M. D., M. L. Bari., Y. Inatsu, V. K. Juneja, and S. Kawamoto. 2007. Antibacterial activity of Guava ( Psidium guajava L .) and neem ( Azadirachta indica A. Juss.) extracts against foodborne pathogens and spoilage bacteria. Foodborne Pathogens and Disease. 4: 481 488. Meadow, R., R. Seljasen., and P. Brynildsen. 2000. The effect of neem extracts on the turnip root fly and ca bbage moth. In: Kleeberg, H., C. P. W. Zebitz. Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones: Proceedings of the 9 th workshop, Hohensolms, Germany, March 13 15, 2000, pp. 55 60. Mehlhorn, H., K. A. S. Al Rasheid, and F Abdel Ghaffar. 2011. The neem tree story: extracts that really work. In: Nature helps, Parasitology Research monographs 1, Mehlhorn, H., (Ed.). Springer Verlag, Berlin. 77 108. Meisner, J., V. Melamed Madjar, S. Yathom, and K. R. S. Ascher. 1987. The in fluence of neem on the European corn borer ( Ostrinia nubilalis ) and the serpentine leafminer ( Liriomyza trifolii ). In Natural pesticides from the neem tree ( Azadirachta indica A. Juss) and other tropical plants. Proceedings of the 3 rd International Neem Conference, Nairobi, Kenya. 10 15 July 1986 461 477. Minkenberg, O. P. J. M. 1988. Life history of the agromyzid fly Liriomyza trifolii on tomato at different temperatures. Entomol. Exp. App. 48: 73 84. Mordue (Luntz) A. Jennifer, and A. Blackwell. 1993. Azadirachtin, an update. J. Insect. Physiol. 39: 903 924. Mordue (Luntz), A. Jennifer, and A. J. Nisbet. 2000. Azadirachtin from the neem tree Azadirachta indica : its action against insects. An. Soc. Entomol. Bras. 29: 615 632. Mo rdue, A. J., M. S. J. Simmonds., S. V. Ley., W. M. Blaney., W. Mordue., M. Nasiruddin, and A. J. Nisbet. 1998. Action of Azadirachtin, a plant allelochemical against insects. Pesticides Science. 54: 277 284. Mujica, N., and J. Kroschel. 2011. Leafminer fl y (Diptera: Agromyzidae) occurrence, distribution, and parasitoid associatons in field and vegetable crops in the Peruvian coast. Env. Entomol. 40: 217 230. Musgrave, C., S. L. Poe, and H. V. Weems. 1975. The vegetable leafminer Liriomyza sativae Blanchar d. Entomology circular, Florida Department of Agriculture and Consumer Services, Division of Plant Industry No. 162. 1 4. Na umann, K. and Isman, M. B. (1996 ), Evaluation of neem Azadirachta indica seed extracts and oils as oviposition deterrents to noctuid moths. Entomol. Exp. App. 76: 115 120

PAGE 63

63 Naumann, K., & Isman, M. B. 1996. Toxicity of a neem ( Azadirachta indica A. Juss) insecticide to larval honey bees. Am Bee J 136 Naumann, K., L. J. Rankin, and M. B. Isman, (1994). Systemic action of neem seed extract on mountain pine beetle (Coleoptera: Scolytidae) in lodgepole pine. J. Econ. Entomol. 87(6): 1580 1585. Olson, S. M., P. J. Dittmar., S. E. Webb., S. Zhang., S. A. Smith., E. J. McA voy, and M. Ozores Hampton. 2013 Legume Production in Florid a: Snap Bean, Lima Bean, Southern Pea, Snowpea HS 727, (Gainesville, FL: University of Florida Institute of Food and Agricultural Sciences, 2012). http://edis.ifas.ufl.edu/cv125 Parella, M. P. 1987. Biology of Liriomyza Ann. Rev. Entomol. 32: 201 224. Parrella, M. P., K. L. Robb, and J. Bethke. 1983. Influence of selected host plant on the biology of Liriomyza trifolii (Diptera: Agromyzidae). Ann. Entomol. Soc. Am 76: 112 115. Parella, M. P., C. B. Keil, and J. G. Morse. 1984. Insecticide resistance in Liriomyza trifolii. Ca. Agric. 38: 22 23. Parkman, P., J. A. Dusky, and V. H. Waddill. 1989. Leafminer and leafminer parasitoid incidence on selected weeds in South Florida. Fl. Entomol. 72: 559 561. Parkman, P., and R. L. Pienkowski. 1990. Sublethal effects of neem seed extract on adults of Liriomyza trifolii (Diptera: Agromyzidae). J. Econ. Entomol. 83: 1246 1249. Reitz S.R., and J.T. Trumble. 2002a. Competitive displacement among insects and arachnids. Ann. Rev. Entomol. 47: 435 465. Rembold, H. 1989. Azadirachtins. Their structure and mode of action. In J. T. Arnason, b. J. R. Philogene, and P. Morand (eds). ACS Symp. Ser. 387, Washington, D.C. Rembold, H., H. Forster, C. H. Czoppelt, P. J. Rao, and K. P. Sieber. 1984. The azadirachtins, a group of insect growth regulators from the neem tree. In Natural pesticides from the neem tree and other tropical plants proceedin gs of the second international neem conference, pp. 25 28. SAS. 2011. Version 9.3. SAS Institute, Cary, NC. Schmutterer, H. 1990. Properties and potential of natural pesticides from the neem tree, Azadirachta indica Ann. Rev. Entomol. 35: 271 297.

PAGE 64

64 Schmutterer, H., and R. P. Singh. 1995. Lists of insect pests susceptible to neem products. Pp. 326 365. In: Schmutterer., H. The neem tree: Azadirachta indica A. Juss and other Meliaceae plants. Schwinger, M., B. Ehhammer, and W. Kraus. 1984. Methodolog y of the Epilachna varivestis bioassay of antifeedants demonstrated with some compounds from Azadirachta indica and Melia azedarach. In Natural Pesticides from the Neem Tree and Other Tropical Plants. Proc. 2nd Int. Neem Conf. 587. Schuster, D. J. 1978. V egetable leafminer control on tomato, 1977. Insectic. Acaric. Tests 3:108. Schuster, D. J., and P. H. Everett. 1982. Laboratory and field evaluation of insecticides for control of Liriomyza spp. on tomatoes. Proc. 3 rd Annu. Ind. Conf. Leafminer, San Diego Calif. Alexandria, Va.: Soc. Am. Florists. P.20 30. Seal, D. R., R. Betancourt, and C. M. Sabines. 2002. Control of Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) using various insecticides. Proc. Florida Hort. Soc. 115: 308 314. Seljasen, R., and R. Meadow. 2006. Effects of neem on oviposition and egg and larval development of Mamestra brassicae L: dose response, residual activity, repellent effect and systemic activity in cabbage plants. Crop Protection 25: 338 345 Shappiro, S. S., and M. B. Wil k. 1965. An analysis of variance test for normality. Biometrika. 52: 591 611. Sieber, K., and H. Rembold. 1983. The effects of azadirachtin on the endocrine control of moltingin Locusta migratoria. J. Insect. Physiol. 29: 523 527. Silbernagel, M. J., W. Janssen., J. H. C. Davis, and G. Mondes de Oca. 1991. Snap bean production in the tropics: implications for genetic improvement. In: Common Beans: Research for Crop Improvement. A. Van Schoonhoven, and O. Voyset, eds., CAB Intern. Wallingford, Oxon, U.K. pp 835 862. Singh, S., and R. P. Singh. 1998. Neem (Azadirachta indica) seed kernel extracts and azadirachtin as oviposition deterrents against melon fruit fly ( Bactrocera cucurbitae) and the oriental fruit fly (Bactrocera dorsalis) Phytoparasitica 26 (3 ): 191 197. Singh, U. P., H. B. Singh, and R. B. Singh. 1980. The fungicidal effect of neem (Azadirachta indica) extracts on some soil borne pathogens of gram ( Cicer arietinum ). Mycologia. 72: 1077 1093. Spencer, K. A. 1973. Agromyzidae (Diptera) of econ omic importance. Series Entomologica. 9: 1 418.

PAGE 65

65 Stein, U., and M. P. Parrella. 1985. Seed extract shows promise in leafminer control. California Agriculture 4: 19 20. Sundaram, K. M. S., R. Campbell., L. Sloane, and J. Studens. 1995. Uptake, translocation, persistence and fate of azadirachtin in aspen plants ( Populus tremuloides Michx.) and its effects on pestiferous two spotted spider mite ( Tetranychus urticae Koch.). Crop Prot. 24: 415 421. Thoeming, G., C. S. Borgemeister., and M. Setamou. and H M. Poehling. 2003. Systemic effects of neem on western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae). J. Econ. Entomol 96: 817 825. Timmins, W. A. and S. E. Reynolds. (1992), Azadirachtin inhibits secretion of trypsin in midg ut of Manduca sexta caterpillars: reduced growth due to impaired protein digestion. Entomol. Exp. Appl. 63: 47 54. Trumble, J. T., I. P. Ting., and L. Bates. 1985. Analysis of physiological, growth, and yield response of celery to Liriomyza trifolii Ent omol. Exp. Appl. 38:15 21. Waddill V., D. Schuster, and R. Sonoda. 1986. Integrated pest management for Florida tomatoes. Plant Disease 70: 96 102. Wan, M. T., R. G. Watts, M. B. Isman, and R. Straub. 1996. An evaluation of the acute toxicity to juvenile Pacific Northwest Salmon of azadirachtin, neem extract and neem based products. Bull. Environ. Contam. Toxicol. 56: 432 439. Waterhouse, D. F., and K. R. Norris. 1987. Biological control: Pacific Prospects (Inaka Press), pp. 454. Weathersbee, A. A., and C. L. McKenzie. 2005. Effect of a neem biopesticide on repellency, mortality, oviposition, and development of Diaphorina citri (Homoptera: Psyllidae). Fl. Entomol. 88(4): 401 407 Webb, R. E., M. A. Hinebaugh., R. K. Lindquist, and M. Jacobson. 1983. Eval uation of aqueous solution of neem seed extract against Liriomyza sativae and Liriomyza trifolii (Diptera: Agromyzidae). J. Econ. Entomol. 76: 357 362. Weintraub, P. G., and A. R. Horowitz. 1998. Effects of translaminar versus conventional insecticides on Liriomyza huidobrensis (Diptera: Agromyzidae) and Diglyphus isaea (Hymenoptera: Eulophidae) populations in celery. J. Econ. Entomol. 91: 1180 1185. Weintraub, P. G., and A. R. Horowitz. 1997. Systemic effects of a neem insecticide on Liriomyza huidobrens is larvae. Phytoparasitica 2 5: 283 289.

PAGE 66

66 BIOGRAPHICAL SKETCH Manish Poudel was born in 1983 in Chitwan Nepal. He graduated from Institute of Agriculture and Animal Sciences, Tribhuvan University, Nepal with a Bachelor of Science in Agriculture in 2008. H Nepal for a brief period in the field of agriculture, women empowerment and education. In August 2010, he joined a doctorate degree in Plant M edicine at the University of Florida and later switched to M.S in interest is in integrated pest management of vegetables and fruits. He graduated with a Master of Science in 2013. His long term career is to c ontinue research and extension i n crop protection tow ards development and dissemination of novel pest control strategies.