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1 SEASONAL ABUNDANCE AND DISTRIBUTION OF LEAFMINER, LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDAE) AND ITS PARASITOIDS ON BEAN CROP IN SOUTH FLORIDA By JIAN LI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011
2 2011 Jian Li
3 To my parents and my friends
4 ACKNOWLEDGMENTS I sincerely thank my advisor Dr. Dakshina Seal for his academic guidan ce and support during my graduate study and research. I also extend my gratitude to my co chair advisor Dr. Gary Leibee and my graduate committee Dr. Oscar Liburd. I thank them for their unending instruction and assistance in promoting my research project. I thank all the technicians, C. Sabines, C. Carter, E. Arias and J. Betancourt in Vegetable Insect Pest Management Laboratory for their hard work in planting and maintaining the bean crops for my research. I would like to extend my appreciation to Dr. Gar y Steck (D ivision of Plant Industry, Gainesville, Florida ) for the leafminer identification and Dr. Sonja Sch e ffer ( Systematic Entomology Laboratory, US D epartment of Agriculture, Maryland ) for the assistance of parasitoid identification. I appreciate the opportunity studying at University of Florida. I really enjoyed my graduate study in the Entomology and Nematology Department, and I thank all of the faculties who ever instructed me in the courses etc. I thank my great parents Pizeng Li and Yulan Feng for their tremendous love and support overseas. I thank Charles Stuhl for his help in my study and my life. I also thank all of my friends both in China and USA for their help and company.
5 TABLE OF CONTENTS page ACKNOWL EDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW OF THE LEAFMINER LIRIOMYZA TRIFOLII ( B URGESS ) ................................ ................................ ................................ ............ 12 Biology and Life Cycle ................................ ................................ ............................ 12 Economic Importance ................................ ................................ ............................. 13 Management ................................ ................................ ................................ ........... 14 Chemical Control and Insecticide Resistance ................................ .................. 14 Physical Control ................................ ................................ ............................... 15 Biological Control ................................ ................................ ............................. 16 Ecological Study ................................ ................................ ............................... 18 Research Objectives ................................ ................................ ............................... 19 2 SEASONAL ABUNDANCE AND SPATIAL DISTRIBUTION OF LEAFMINER LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDAE) AND ITS PARASITOI D OPIUS DISSITUS (HYMENOPTERA: BRACONIDAE) IN SOUTH FLORIDA ........ 21 Materials and Methods ................................ ................................ ............................ 23 Study Sites and Bean Production ................................ ................................ ..... 23 Seasonal Density of Leafminer and Parasitoid ................................ ................. 24 Spatial Distribution of Leafminer and Parasitoid ................................ ............... 25 Parasitism and Host Density ................................ ................................ ............ 26 ................................ .......................... 27 Results ................................ ................................ ................................ .................... 27 Seasonal Density of Leafminer and Parasitoid ................................ ................. 27 Spatial Distribution of Leafminer ................................ ................................ ....... 28 Spatial Distribution of Parasitoid ................................ ................................ ....... 30 Parasitism and Host Density ................................ ................................ ............ 31 ................................ .......................... 31 Discussion ................................ ................................ ................................ .............. 31 3 DIEL DENSITY PATTERN OF LEAFMINER, LIRIOMYZA TRIFOLII AND THE PARASITOIDS, OPIUS DISSITUS (HYMENOPTERA: BRACONIDAE) AND DIGLYPHUS SPP. (HYMENOPTERA: EULOPHIDAE) ................................ .......... 45
6 Materials and Methods ................................ ................................ ............................ 46 Study Site and Beans Preparation ................................ ................................ ... 46 Plot Design and Diel Activity ................................ ................................ ............. 47 Statistical Analysis ................................ ................................ ............................ 48 Results ................................ ................................ ................................ .................... 48 Discussion ................................ ................................ ................................ .............. 51 4 THE COMPOSITION AND SEASONAL ABUNDANCE OF HYMENOPTERAN PARASITOIDS OF LIRIOMYZA TRIFOLII ON BEANS IN SOUTH FLORIDA ........ 62 Materials and Methods ................................ ................................ ............................ 63 Study Site ................................ ................................ ................................ ......... 63 Leaf Sampling and Insect Rearing ................................ ................................ ... 63 Results and Discussion ................................ ................................ ........................... 64 5 CONCLUSION ................................ ................................ ................................ ........ 79 LIST OF REFERENCES ................................ ................................ ............................... 82 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 88
7 LIST OF TABLES Table page 2 1 s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in September 2010 ....... 40 2 2 s pertaining to the distribution of L. trifolii and O. dissitus on beans in November 2010 ........ 40 2 3 to the distribution o f L. trifolii and O. dissitus on beans in December 2010 ........ 41 2 4 to the distribution of L. trifolii and O. dissitus on b eans in January 2011 ............ 41 2 5 to the distribution of L. trifolii and O. dissitus on beans in February 2011 ........... 42 4 1 Number of leafminer L. trifolii and its parasitoids (%) reared from the bean foliages (300 leaves / month) from September 2010 to February 2011 in south Florida ................................ ................................ ................................ ....... 78
8 LIST OF FIGURES Figure page 2 1 Density (mean SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 1, from September to October 2010. ................................ .. 35 2 2 Density (mean SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 2, from October to December 2010. ................................ ... 3 6 2 3 Density (mean SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 3, from December 2010 to February 2011. ........................ 37 2 4 Density (mean SE / 5 leaves) of leafm iner pupae, emerged L. trifolii and O. dissitus in each month, from September 2010 to February 2011. ...................... 38 2 5 Density (mean SE / yellow sticky card / 24 h) of L. trifolii and O. dissitus adult s in each month, from September 2010 to February 2011. ......................... 39 2 6 Bean crop field (50 m 30 m) was divided into 15 equal plots (10 m 10 m). ... 43 2 7 Bean foliages were sampled and placed in the laboratory. ................................ 43 2 8 Leafminer and the parasitoids were reared in the laboratory. ............................. 44 3 1 Bean site 1, Nov 09 2010. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / yellow stick y trap during each 2 h interval ........................ 54 3 2 Bean site 1, Nov 16 2010. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / yellow sticky trap durin g each 2 h ................................ .... 55 3 3 Bean site 1, Nov 23 2010. Mean ( SE) number of the L. trifolii O. dis situs and Diglyphus spp. / yellow sticky trap durin g each 2 h ................................ .... 56 3 4 Bean site 1, Dec 01 2010. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / yellow sticky trap durin g each 2 h ................................ .... 57 3 5 Bean site 1. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / 15 yellow sticky traps during each 2 h interval after 8:00 ES T ................. 58 3 6 Bean site 2. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / 15 yellow sticky traps during each 2 h interval after 8:00 EST ................. 59 3 7 Seasonal density: mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. per 15 yellow sticky traps / 10 h at bean site 1, 2010 and site 2, 2011 ................................ ................................ ................................ .............. 60 3 8 Fifteen yello w sticky traps were set in bean field from 8:00 18:00 EST. ............. 61
9 3 9 The caught insects during different 2 h interval were marked on the yellow sticky card. ................................ ................................ ................................ .......... 61 4 1 Liriomyza trifolii (Agromyzidae). ................................ ................................ .......... 70 4 2 Opius dissitus (Braconidae). ................................ ................................ ............... 70 4 3 Euopius sp (Braco nidae). ................................ ................................ .................. 71 4 4 Diaulinopsis callichroma (Eulophidae). ................................ ............................... 71 4 5 Diglyphus begini (Eulophidae). ................................ ................................ ........... 72 4 6 D. intermedius (Eulophidae). ................................ ................................ .............. 72 4 7 D. isaea (Eulophidae). ................................ ................................ ........................ 73 4 8 Neochrysocharis sp. (Euloph idae) ................................ ................................ ...... 73 4 9 Closterocerus sp. (Eulophidae). ................................ ................................ ......... 74 4 10 Zagrammosoma lineaticeps (Eulophidae). ................................ ......................... 74 4 11 Z. muitilineatum (Eulophidae). ................................ ................................ ............ 75 4 12 Pnigalio sp. (Eulophidae). ................................ ................................ ................... 75 4 13 Chrysocharis sp. ( Eulophidae). ................................ ................................ ........... 76 4 14 Halticoptera sp. (Pteromalidae). ................................ ................................ ......... 76 4 15 Seasonal abundance of parasitoids, O. dissitus D. callichroma and Diglyphus spp. on snap bean crop from September 2010 to February 2011. ..... 77
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements f or the Degree of Master of Science SEASONAL ABUNDANCE A ND DISTRIBUTION OF L EAFMINER, LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDA E) AND ITS PARASITOI DS ON BEAN CROP IN SOUTH FLORIDA By Jian Li August 2011 Chair: Dakshina R. Seal Major: Entomology and Nemato logy The leafminer, Liriomyza trifolii (Burgess) is a phytophagous fly infesting a wide range of vegetable and ornamental plants Knowledge of the biology of this pest is essential to develop an effective management program. Various aspects of i ts biology and parasitoids were studied in snap bean ( Phaseolus vulgaris ) fields in Miami Dade County from 2010 to 2011. L. trifolii showed a seasonal preference having high population density during December 2010 (17.9 1.5 adults / 5 leaves) and January 2011 (30.3 2.7 adults / 5 leaves) when the temperature was relatively low. Opius dissitus the major parasitoid of leafminer, showed a pattern of population density similar to L. trifolii abundant during December 2010 (4.5 0.45 adults / 5 leaves) and January 2011 (5.4 0.73 adults / 5 leaves). Both L. trifolii and O. dissitus tended to show an aggregated pattern of distribution when their densities were higher during December 2010 and January 2011, but a regular pattern when densities were lower in S eptember 2010 and February 2011. Diel density patterns of L. trifolii and its parasitoids, O dissitus and Diglyphus spp. were evaluated by using yellow sticky traps in bean fields. L. trifolii density was found to
11 be more abundant from 8:00 to 10: 00 EST than any other time throughout the day during fall 2010, but did not show any clear pattern of diel abundance in spring 2011. There was no significant difference in diel density pattern of O. dissitus in this study. Diglyphus spp. was the most abundant fro m 10:01 to 12: 00 EST in fall 2010 and from 12:01 to 14: 00 in spring 2011. The composition and seasonal abundance of hymenopteran parasitoids of L. trifolii was surveyed on snap beans in south Florida. Thirteen species or genera of parasitoids were collec ted from the bean foliages. O. dissitus was the most abundant larval pupal endoparasitoid during all the bean seasons, from September 2010 to February 2011. Diaulinopsis callichroma (Crawford) was the most abundant larval ectoparasitoid, and it was prevale nt during fall 2010. Other parasitoids reared from bean foliages include Euopius sp., Diglyphus begini (Ashmead) D. intermedius (Girault) D. isaea (Walker) Neochrysocharis s p Closterocerus sp., Chrysocharis sp., Zagrammosoma lineaticeps (Girault) Z. muitilineatum (Ashmead) Pnigalio sp., and Halt i coptera sp Among these parasitoids, the D. isaea reared from Liriomyza leafminer is the first record in Florida. The morphological characteristics of the leafminer parasitoids were described, and the photos containing key characters were presented for identification.
12 CHAPTER 1 LITERATURE REVIEW OF THE LEAFMINER LIRIOMYZA TRIFOLII ( BURGESS ) Biology and Life Cycle The leafminer, Liriomyza trifolii ( Burgess ) is a worldwide pest feeding on vegetable and ornamen tal plants. The adult female causes leaf puncturing by its ovipositor and feeds on the exudates from the injuries ( Musgrave et al 1975) The Liriomyza adult female s can live 15 20 days and male s can live 10 15 days depending on temperature and food sup ply (Parrella and Bethke 1984). Adults mate soon after the emergence, and their mating activity can be affected by temperature (Dimetry 1971). Adult L. trifolii is about 2 mm long, and the wing is 1.25 to 1.9 mm long. L. trifolii head is yellow, eyes are red, thorax and abdomen are mostly gray and black, ventral surface and legs are yellow. L. trifolii has a grayish black mesonotum which differs from the closely related species, L. sativae (Blanchard) with shiny black mesonotum. The hind margins of L. tri folii eyes are yellow, while those of L. sativae are black. L. trifolii differs from L. huidobrensis (Blanchard) in having yellow femora while L. huidob rensis has dark femora ( Cap inera 2001 ). Liriomyza trifolii has a relatively short life cycle. It require s about 19 days from egg deposition to adult emergence at 25C D evelopment rate increases with the increase of temperature up to 30 C (Leibee 1984). Adult females deposit eggs in the feeding punctures. The total fecundity of a L trifolii female can be 20 0 to 400 eggs on celery ( Leibee 1984 ) The egg is white in color and deposited in the plant tissue below the epidermis through the adaxial or abaxial leaf surface. The egg is oval shape d, about 0.1 mm long and 0.2 mm wide (Capinera 2001)
13 The larva of L. trifolii i s yellow in color and cylindrical in shape Larva goes through four instars. Capinera (2001) described different instars of L. trifolii The body length and mo uth size of the first instar are ~ 0.39 mm and ~ 0.10 mm, the second instar are ~ 1.00 mm and ~ 0.17 mm, and the third instar are ~ 1.99 mm and ~ 0.25 mm, respectively The fourth instar occurs between the pupariation and pupation The period of pupariation is about 2 4 hours depending on temperature. Leibee (1984 ) indicated that mature t hird instar larva of L. trifolii exits the leaf mine in the morn ing and pupates on the ground. H e also found the pupariation of L. trifolii larva could be delayed for a short time by continuous lighting condition (Leibee 1986). The pupa e show a golden brow n color at the early stage and turn to be a dark brown color during the late stage. P upa l development time is about 8 11 days under greenhouse or field conditions (Parrella 1987). The L. trifolii pupa has been repor ted to exhibit a diapause at 16 C (Suss 1984). Economic I mportanc e Twenty three species of Liriomyza leafminer are economically import ant pest of agricultural and ornamental plants (Spencer 1973 ). Among these, L trifolii is a worldwide pest with a broad range of host plants. There were 55 hos ts reported from Florida including bean, pepper, potato, squash, beet, carrot, celery, cucumber, eggplant, lettuce, melon, onion, pea and tomato ( Ste gmaier 1966). Besides the crop hosts, several genera of weeds were found as the alternative host of Liriomy za leafminers in tomato field (Schuster 1991). Liriomyza leafminers can cause serious economic damage to its host crops. In the instance of severe infestation, it can cause total defoliation. Schuster (1978) reported that 90% of the tomato foliages might b e lost in absence of an effective control method. In Miami Dade Co., leafminer is the
14 predominant pest infesting foliages of snap beans, and causes serious economic loss annually. Both larva e and adult s cause damage to the host plants. The larva begins fe eding on the host immediately after the eclosion. The larva e live inside the foliage and form mine s i n the mesophyll layer. The rate of mine diameter and formation increases as larval development progresses, and Fagoonee (1984) found that leaf material con sumption and feeding rate by the L. trifolii third instar is 643 and 50 time s greater than the first instar, respectively. Female adult s injure mesophyll cells by its ovipositor and feeds on the punctures Bethke (1985) indicated that all punctures should be considered as injuries because females feed at all of these sites. The stipples and mines caused by L. trifolii activity significantly reduce the photosynthesis parameters on plant hosts. Parrella et al. (1985) repor ted that the leaf stipples reduce d t he photosynthesis parameters up to 75%. It was reported that the injuries caused by L. trifolii activities allow the entry of plant pathogens, such as bacteria of Pseudomonas chichorii ( Broadbent 1990) Management Chemical Control and Insecticide R esistanc e Liriomyza trifolii has been a pest of vegetable crops in Florida since 1945 (Wolfenbarger 1947). The most commonly used method of leafminer control is insecticid e application. Application of nicotine sulphate was first used to control leafminer in Florid a when leafminer was present (Lei bee and Capinera 1995) Chlordane was recommended for controlling leafminer on potato crops in south Flori da (Wolfenbarger 1947). Genung et at (1979) indicated that the use of diazinon, naled, and azi nphosmethyl reduced bo th vegetable seedlings mortality and yield reductions by
15 leafminer before 1974, but they were not effective after 1974. In Florida, methamidophos and permethrin were used to control leafminer on celery, but permethrin became ineffective in less than two ye ars (Leibee and Capinera 1995). Poe and Strandberg (1979) reported that oxamyl was effective for controlling leafminer. Cyromazine, an insect growth regulator (IGR), was used to control leafmin er in celery industry in 1982. Leibee and Capinera (1995 ) confi rmed the presence of a strain that was high ly resistant to cyromazine. They found that the resistant strain survived 300 ppm of cyromazine which was the highest lab el of concentration in the field. Abamectin, a GABA agonist, was applied to control leafmin er on celery in early 1990, and c yromazine was used as the rotation. Spinosad and abamectin were highly effective for control ling leafminer on vegetable crops (Seal and Betancourt 2002). Ferguson (2004) found that all strains of L. trifolii resistant to c yromazine, abamectin and spinosa d, reverted to susceptible after five generations in the absence of i nsecticide selection pressure. Webb (2002) reported that spinosad and emamectin benzoate could be applied to control L. trifolii and they are relatively c ompati ble with the natural parasites. It was reported that Coragen (rynaxypyr) and Venom (dinotefuran) were used to control leafminer in rec ent years (Webb and Stansly 2008 ). In Turkey, Civelek and Weintraub (2003) demonstrated bensultap was effective in controlling L. trifolii larvae under the high larval density. They found all insecticide treatments (1.5, 2.0 2.5 and 3.0 kg / ha) had significant ly fewer alive larvae than control ( P < 0.01) within one day of application and the number of live larvae dec reased after 10 days of treatm ent. Physical C ontrol The yellow sticky cards can be used to monitor le afminer adult abundance, adult movement and field dispersion Yellow sticky traps used for monitoring L. trifolii on
16 chrysanthemum in greenhouse situation provided consistent information (Parrella and Jones 1985). Aluminum foil mulch was also found to effectively repel Liriomyza adults on tomato and squash and reduce the crop in festation (Wolfenbarger 1968). In addition, various cultural practices can be he lpful in reducing L. trifolii on various hosts. Destruction of weed hosts and soil deep ploughing can effectively reduce leafminer alternative hosts and the abundance of pupae in the soil. Biological C ontrol Biological control plays an important role in th e in tegrated pest management (IPM) of a pest. Several p arasitoid wasps are used in the biological control of Liriomyza leafminer s. Valladares and Salvo (2001) indicated that species richness and density of leafminer and the parasitoid community were positi vely correla ted in the forest of central Argentina. They found that parasitism was greater when parasitoid community has a higher species number and lower do minance. Capinera (2001) reported that at least 14 species of L. trifolii para sitoids occur in Flor ida. Among all the parasitoid families, Eulophidae, Braconidae and Pteromalidae are the most studied for contro lling leafminer. The eulophids, Diglyphus isaea (Walker) D. begini (Ashmead) D. intermedius (Girault) and D. carlylei (Girault) are solitary e ctoparasitoids (larval parasites) of dipteran leafminers occurring in North American (Lasalle an d Parrella 1991). The female Diglyphus adult lays one or more eggs attached to the leafminer late instar larvae (Minkenberg 1987). The parasitoid larvae hatch o ut of egg s and feed on t he leafminer larva externally, eventually killing the leafminer larvae. The parasitoid larva develops through three instars and pupates in the mine before emerging as an adult. Development time is temperature dependent. D. isaea is o ne of the most effective
17 biological control agents of Liriomyza leafminer in greenhouse (Minkenberg 1987; Boot et al. 1992). D. isaea takes about 10 days to complete egg to adult development on L. trifolii and L. huidobrensis at 25 C (Bazzocchi et al. 200 3). Endosymbiont s, such as Wolbachia have been studied for improving the effectiveness of leafminer biological control. Tagami et al (2006 a ) reported that Wolbachia showed strong cytoplasmic incompatibility (IC) and perfect vertical transmission in L. t rifolii and it may be applied in leafminer control. In another study, Tagami et al (2006 b ) indicated that endosymbiont, Rickettsia might cause thelytokous reproduction i n the leafminer parasitoids, Neochrysocharis formosa (Westwood) (Hymeno ptera: Euloph idae), and it may increase the effectiveness of leafminer biological control. Nematodes have also been studied as a biological control agent for controlling leafminer L. trifolii LeB eck et al (1993) found that all L. trifolii larval stages were susceptib le to the entomopathogenic nematode, Steinernema carpocapsae and the second instar was the most susceptible to S. carpocapsae They indicated the mines produced by L. trifolii larvae were suitable for S. carpocapsae to survive. The nematodes enter the min es through the oviposition punctures on the leaf and penetrate the leafminer larva v ia the anus, mouth or spiracle. It was found using nematodes and parasitoid wasps D. begini together resulted in a higher effective result in controlling leafminer L. trifo lii than using either biological control agent alone ( Sher et al. 2000) T he importance of biological control and insecticide application in leafminer control promotes further studies on compatibility between the biological control agent s and common insec ticides. Weintraub (2001) found that cyromazine and abamecti n
1 8 significantly reduced the population of parasitoid D. isaea compared to non treated control. Para sitoid population from abamecti n treatment recovered sooner than in cyromazine treatment. Kaspi ( 2005) found that the percentage emergence of D. isaea from the mine and lon gevity of the emerged adult were not affected by the abamectin treatment when ap plying to chrysanthemum plants. The use of reduced risk insecticides decreases the impact on the biol ogical agents in an IPM program. Ecological Study Knowledge of seasonal population dynamics and spatial distribution of pests and natural enemies are essential for developing IPM strategies. Few studies on seasonal abundance of leafminer and parasitoids ha ve been conducted on bean crops in south Florida Valladares (2001) reported that the parasitism of leafminer was higher in summer but lower i n winter in Central Argentina. Shepard et al (1998) determined that leafminer L. huidobrensis has a seasonal inci dence in various crops in Indonesia. They found that the abundance of L. huidobrensis was very low on potato during the dry season. The abundance of parasitoid Hemiptarsenus varicornis (Hymenoptera: Eulophidae) was always high when L. huidobrensis number w as low. Temperature has a direct effect on its abundance. Abou Fakhr Hammad (2000) reported that the population density of L. huidobrensis reduced in September and October 1994 in Lebanon due to the high da ily average temperature of 31.2 C and 29.7 C respe ctively. Various studies indicated cy increased when their host had an aggr egative distribution, and it lead to a dire ct density dependent parasitism (Hassell and May 1974; Head and Lawton 1983). The leafminer, Ophiomyia maura showed a Poisson distribution in the early season but later became weakly clumped (Ayabe and Shibata 2008).
19 Both biotic and abiotic factors affect the distribution of leafminer and the parasitoids. The distribution of leafminer O. maura showed a P ois son distribution in the early growth season but later became weakly clumped in Okazaki City, Japan (Ayabe and Shibata 2008). Saito et al. (2008) found leafminer Chromatomyia horticola (Diptera: Agromyzidae) and parasitoids of D. isaea and D. minoeus were a bundant in the cool season from December to May, in Japan. However, the parasitoid Chrysocharis pentheus was abundant in the warm season from May to June. T he natural enemy should coincide with their host distribution and have a similar thermal requirement (Kang Le at el., 2009). Tantowijoyo (2010) determined that altitude could affect the distribution of leafminer L. huidobrensis and L. sativae They found L. huidobrensis was more abundant at 700 m above sea level and L. sativae was more abundance below 60 0 m. T emperature was the overriding influence on their altitudinal distribution. In addition, b oth plant leaf age and life history can affect female leafminer a oviposition preference. Facknath (2005) demonstrated that the infestation by L. trifolii starts from the lower leaves and proceeds to middle and upper l eaves on both bean and potato. They found the larval survival was significantly lower in the smaller upper leaves th an the lower and older leaves. Parrella (1983) determined that intraspecific competition of L. trifolii larvae leads to small larvae, fewer pupa e and less vigorous adults. The older and thick leaves have more mesophyll and can supply sufficient space and food. In another study it was reported that some insects could increase thei r nitrogen utilization in the old plan t parts (Williams et al. 1998). Research O bjectives This research study was conducted to develop an effective IPM strategy for controlling L. trifolii Information about pest density levels at different times of the ye ar is
20 essential to monitor, initiate management programs and select appropriate management tools. The spatial distribution of L. trifolii and its parasitoids indicates their ecological behavior and interactions with host plants. This information is essenti al for developing a successful leafminer management programs. In addition, studying the composition and seasonal abundance of leafminer hymenopteran parasitoids leads to exploration of potentially effective bio control agent s. My specific research objective s are : 1) Determine s easonal abundance and spatial distributions of leafminer, L trifolii and its parasitoid, O. dissitus on bean crops in south Florida 2) Determine die l density pattern of leafminer, L. trifolii and its two parasitoids of O. dissitus and Digl yphus spp on beans. 3) Determine the composition and seasonal abundance of hymenopteran parasitoid s of L. trifolii on beans in south Florida.
21 CHAPTER 2 SEASONAL ABUNDANCE A ND SP ATIAL DISTRIBUTION O F LEAFMINER LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDA E) AND ITS PARASITOID OPIUS DISSITUS (HYMENOPTERA: BRACON IDAE) IN SOUTH FLORI DA The leafminer, L iriomyza trifolii ( Burgess ) is a phytophag o us fly feeding on a wide range of or namental and vegetable plants. The species is distributed in the temperate and tropical regions worldwide. The adult female injures the plant tissue s with its oviposi tor, and adult female feeds on the punctures. The eggs are deposited in the punctures and hatch into larvae. The mining act ivity of larvae causes damage to the mesophyll layer o f the leaf. Leaf consumption and feeding r ate increases rapidly as the larvae develop ed (Fagoonee 1984). L. trifolii is one of the main pests of vegetable crops in south Florida (Seal and Betancourt 2002) The mature larvae exit the mine, drop to the groun d and pupate. The Opius dissitus wasp is one solitary larva pupal endo parasitoid of L. trifolii O. dissitus female deposit s its egg directly inside the leafminer larva, and the host larva continues to develop unti l pupation (Bordat et al. 1995 a). O. diss itus develops inside the host pupa and f inally emerges out of the pupa. G enera lly, only one parasitoid emerges from one pupa. Several studies reported that O dissitus was reared from L. trifolii from infested celery leaves, tom ato leaves and weeds in Flor ida (Stegmaier 1972 ; Schuster and Wharton 1993 ; Schuster and Gilreath 1991). O. dissitus was the most abundant parasitoid of L. trifolii on the snap bean crop in our preliminary study. Effective monitoring of leafminer and parasitoid density is essential for making management decisions in biological control. Recent studies showed that rapid increase in leafminer population is primarily due to intensive insecticide applications leading to the development of resistance (Saito 2004) and a reduction in natural enemy densities
22 (Minckenberg and van Lenteren 1986). Some other abiotic factors also contribute to leafminer seasonal abundance. Temperature is an important factor affect ing leafminer and parasitoid densities ( Shepard et al 1998; Saito et al. 2008 ) High daily temperatures C ) reduce d density ( Abou Fakhr Hammad 2000) For parasitoid O. dissitus Bordat et al. (1995 b) reported that 20 C was optimal for both adults male and female, and the optimum temperature for female reproduction was at 25 C Light c ondition could also affect leafminer density level, and t he pupariation of emergent L. trifolii larva could be delayed for a short time by continuous lig hting condition (Leibee 1986). In addition, Shepard et al. (1998) reported that m oisture level might be another factor affecting l eafmine r density. They found that the infestation on potato by L. huidobrensis was more severe during the wet season possibly indicating Insect spatial distribution allows us to understand insect ecological behavior in the fiel d and the ir interactions with parasitoid s Some authors believe foraging efficiency increases when their host is in an aggr egative distribution pattern, w hich leads to a direct density dependent parasitism and better biological control effectiveness (Hassell and May 1974; Head and Lawton 1983). In general, insect spatial distribution patte rn might be affected by several factors The leafminer adult female p refers the high quality foliages of host plants for oviposition (Faeth 1991), so that the larvae can have better quality food and a better performance. In addition, leafminer oitberg 1995).
23 The objectives of this study were (1) to determine the seasonal abundance of the leafminer L. trifolii and its parasitoid O. dissitus (2) to characterize their spatial distribution patterns on bean growth season (September 2010 to Februar y 2011) (3) to determine the relationship between parasitism and host density, and (4) to assess t he relationship between insect s patial distribution pattern and density. Materials and Methods Study Sites and Bean Production The study was conduct ed in Hom estead, Dade County, FL. In this area, bean crops are grown commercially in open fie ld conditions. There are two growing seasons per year, extending from October to February. In this study, three bean sites (each 50 m 30 m as shown Figure 3 6), were pre pared at the Tropical Research and Education Center (TREC), University of Florida. S ite 1 was planted with bean from Se ptember to October 2010, site 2 from October to Decemb er 2010, and site 3 from December 2010 to February 2011. The soil type of the study area is Krome gravelly loam soil, which consists of about 33% soil and 67% pebbles. The snap bean ( Phaseolus vulgaris ) seeds were supplied from Harris Mor an seed Company Modesta, California Pre plant herbicide, halosulfuron methyl ( 51.9 g / ha, Sandea Gowan Company LLC., Yuma, Arizona) was app l ied to control nutsedge and broad leaf weeds. At the time of bean planting, granular fertilizer 6: 12: 12 (N : P : K) was applied at 1345 kg / ha Liquid fertilizer ( 4: 0: 8 ) was applied at the rate of 0.56 kg N / ha / day in fur row by the side of the seed row 3, 4 and 5 weeks after planting. Plants were irrigated once a day delivering one inch (2.54 cm) / each time to maintain optional soil moisture. The fungicide c hlorothalonil ( 2.81 L / ha, Bravo Syngenta Cro p Protection, Inc.,
24 Greensboro, North Carolina) was used during early plant growth to prevent fungal diseases. No insecticide was used at these study sites. Season al Density of Leafminer and Parasitoid For the purpose of sampl ing, each bean site was divide d into 15 equal plots (10 m 10 m). S ampling was initiated when the bean plant s had tw o primary leaves fully unfolded, and continued once a week until the beans were harvested. Five bean leaves one leaf / plant, were randomly collected in each plot (tota l 75 leaves) per week. When the bean plants produced new leaves and the primary leaves dropped off, the older bottom leaves from the plants were always collected as samples because of L. trifolii feeding preference to the older mature leaf (Facknath 2005). The sampled leaves from each plot were placed separately into a Petri dish (10 cm diameter) Each Petri dish was labeled with plot number and sampling date (Figure 2 7). All samples were transported to the IPM laboratory, TREC and were placed in growth ch amber at 25 C 70% RH and 14: 10 (L: D) h for further development of L. trifolii and O. dissitus The samples were checked every day for leafminer pupae in each Petri dish (10 cm diameter). Pupae were carefully separated from the leaves and transferred in to one new Petri dish and marked with the same information to detect their origin. The number of pupae from each plot was record ed. The pupae were placed in the same growth chamber for further development into adults The number s of emerged adults of L. tr ifolii and parasitoid O. dissitus from the pupae were recorded (Figure 2 8) As another monitoring method 15 yellow sticky traps ( 7.6 cm 12.7 cm ), as a nother monitoring method, were set in the center of each experimental plot (one trap / plot) for 24 ho urs. At the time of collection, each trap was wrapped with transparent
25 polyethylene sheets to avoid inclusion of dusts. This method also facilitated transportation and storing of the traps in the laboratory for further study. The traps were checked in the laboratory using a binocular microscope at 10 30 to identify and record number of leafminer and various species of parasitoids. The traps were placed once per week until the beans were harvested The density of L. trifolii was calculated based on the num ber of emerged pupae and adults from the leaf samples and the number of adults caught on sticky traps. The density of O. dissitus was calculated as the number of emerged adults from leafminer pupae and the adults caught by yellow sticky traps. The density of L. trifolii and O. dissitus was analyzed by analysis of variance (ANOVA, PROC GLM, SAS institute Inc. 2003) to determine the difference by growing month and by bean sites a nd the means were separated by Least Significance D ifference (LSD) ( P < 0.05). Spatia l Distribution of Leafminer and Parasitoid This study was conducted in the same field in the seasonal abundance study. Plot design, sample collection and sample preparation were as described in the previous study. The spatial distribution was determ ined based on data collected from two different sized plots: a) 10 m 2 of 15 plots; and b) 30 m 2 of 5 plots. Spatial distribution patterns of L. trifolii and O. dissitus regression model is: log s 2 = b log +log a
26 Where the slope b is the index of aggregation, and a is the factor related to sample size relation and to evaluate the distribution patterns. The linear regression model is: x = + Where x is the mean crowding (Lloyd 1967) expressed as x = + s 2 / 1, slope is the index of regression, and is the sampling factor. In both of the models, when the slope ( b and ) value is not significantly different from 1, it indicates a random distribution pattern; slope significantly > 1 indicates an aggregated distribution pattern; and slope significantly < 1 indicates a regular distribution pattern ( P < 0.05). The fitness of each data set to the linear regression model was evaluated by r 2 value. The student t test was used to determine the significance of the slopes in both of the models ( P < 0.05). Parasitism and Host Density The relationship between O. dissitus parasitism proportion and leafminer density was analyzed by transformed log linear regression analysis (PROC GLM, SAS I nstitute Inc. 2003 ) y = b log + a The fit of da ta set to the linear regression was evaluated by r 2 value. The parasitism efficiency was expressed as parasitism proportion in each sampling plot calculated as: y = number of emerged O. dissitus / number of the leafminer pupae. means the density of leafminer pupae. The slope b values were used to determine the relationship between the parasitism proportion and host density ( P < 0.05). When slope b is significantly > 0, it indicates a direct density dependence; significantly < 0 indicates
27 a reverse density dependence ( P < 0.05). The regression analyses were carried out on D Den sity The L. trifolii and O. dissitus distribution patterns on snap beans were determined in each month. The densities of L. trifolii and O. dissitus were assessed by two methods discussed before. T he relationship between nd their population density of was evaluated. Results Season al Density of Leafminer and Parasitoid T he density of L. trifolii (0.4 ~ 4.9 pupae / 5 leaves) and O. dissitus (0.0 ~ 1.9 adults / 5 leaves) was low in the bean field at site 1 from September to October 2010 (Figure 2 1) At this site density of L. trifolii (4.9 0.7 pupae / 5 leaves) and O. dissitus (1.9 0.3 adults / 5 leaves) were significantly higher ( F = 12.88, df = 7, 112, P < 0.0001) ( F = 5.38, df = 7, 112, P < 0.0001) in the middle of t he bean growth period than the beginning and end of the bean growth periods (less than 1.0 / 5 leaves ) At the bean site 2 the density of l eafminer (2.2 ~ 3.3 pupae / 5 leaves) and the parasitoid (0.0 ~ 2.1 adults / 5 leaves) was low in early growth perio d during October 2010, but it increased rapidly at the end of November 2010 (16.2 2.3 pupae / 5 leaves; 3.1 0.5 parasitoids / 5 leaves). The density of L. trifolii (17.9 1.5 pupae / 5 leaves) and O. dissitus (4.5 0.45 adults / 5 leaves) were both s ignificantly higher ( F = 51.99, df = 9, 140, P < 0.0001) ( F =21.7, df = 9, 140, P < 0.0001) during the late growth period during December 2010 than earlier growth period at this site (Figure 2 2). At the bean site 3 the density of L. trifolii (53.1 5.8 pupae / 5 leaves) and O. dissitus (14.4 1.8 adults / 5 leaves) reached the highest density level in early January
28 2011 and significantly higher ( F = 61.1, df = 8, 126, P < 0.0001) ( F = 35.6, df = 8, 126, P < 0.0001) than the rest growth period (Figure 2 3) The density level of L. trifolii and O. dissitus decreased at the end of January 2011, and it was in a low level (0.87 ~ 4.1 pupae / 5 leaves; 0.2 ~ 0.67 parasitoids / 5 leaves) in February 2011. Overall, the seasonal abundance of L. trifolii was low in September and October 2010 and February 2011 The leafminer density level started to increase in the middle of November 2010 and reached the highest average density level (30.3 2.7 pupae / 5 leaves) ( F = 61.5, df = 5, 354, P < 0.0001) in January 2011 (Figure 2 4) The parasitoid density level was the highest (5.4 0.73 adults / 5 leaves) ( F = 30.95, df = 5, 354, P < 0.0001) during January 2011, and O. dissitus density was always high when leafminer population was abundant The yellow sticky traps data showed a similar pattern of seasonal density level of L. trifolii with the foliage sampling method (Figure 2 5). The abundance of leafminer was low in the September 2010 (1.6 0.4 adults / trap) October 2010 (1.4 0.5 adults / trap) and February 2011 ( 1.7 0.9 adults / trap) and it was high in November 2010 (10.6 0.98 adults / trap), December 2010 (25.7 2.7 adults / traps) and January 2011 (4.6 0.9 adults / trap) The number of caught parasitoid O. dissitus adults was relatively low when compare d with the result of emerged parasitoid from the samples. The average temperature was relatively lower (mostly less than 20 C ) during November 2010 to January 2011 than other months during the bean growth season, shown from Fig ure 2 4 and Figure 2 5. Spati al Distribution of Leafminer In September 2010, t he distribution pattern o f L. trifolii (Table 2 1), when plot size was 10 m 2 was aggregated ( b = 1.22, P = 0.006, r 2 =
29 0.49) but a regular distribution pattern ( =0.60, P = 0. 164, r 2 = 0.23) based patchiness regression When plot size was 30 m 2 L. trifolii showed a regular distribution pattern b = 0.98 P < 0.0001, r 2 patchiness regression ( b = 0.74, P =0.035, r 2 = 0.44). I n November 2010 L trifolii showed an aggregated distribution pattern in both sizes of plots, 10 m 2 plot ( b = 1.20, P = 0.0038, r 2 = 0.38) ( =1.20, P < 0.0001, r 2 = 0.8) and 30 m 2 plot ( b = 1.58, P = 0.008, r 2 = 0.52) ( = 1.45, P = 0.001, r 2 = 0.67) plots (Table 2 2 ) In December 2010, L. trifolii had an aggregated distribution pattern ( b = 1.63, P = 0.0045, r 2 = 0.47) ( = 1.10, P < 0.0001, r 2 = 0.95) when plot size was 10 m 2 based on both analy zing methods. When plot was 30 m 2 it showed a regular distribution pattern ( b = 0.98, P = 0.0352, r 2 = 0.13) ( = 1.05, P = 0.0004, r 2 = 0.86) (Table 2 3) I n January 2011 L. trifolii presented an aggregated distribution pattern (Table 2 4 ) based on two analyzin g methods in both plot sizes of 10 m 2 ( b = 1.74, P = 0.0019, r 2 = 0.42) ( = 1.12, P < 0.0001, r 2 = 0.92) and 3 0 m 2 ( b = 1.41, P = 0.0009, r 2 = 0.69) ( = 1.05, P < 0.0001, r 2 = 0.99) The results in February 2011 (Table 2 5) indicated L. trifolii in both plot sizes of 10 m 2 and 30 m 2 showed a regular distribution pattern ( b = 0.81, P = 0.038, r 2 = 0.23) ( b = 0.98, P = 0.054, r 2 = 0.32) based on Taylo but an aggregated distribution pattern ( = 1.23, P = 0.0002, r 2 = 0.54) ( = 1.20, P = 0.0012, r 2 = 0.67) based on
30 Spatial Distribution of Parasitoid r egression, p arasitoid O. dissitus had an ag gregated distribution pattern ( b = 1.37, P = 0.033, r 2 = 0.56) ( = 2.74, P = 0.069, r 2 = 0.45) when plot size was 10 m 2 and a regular distribution pattern ( b = 0.32, P = 0.77, r 2 = 0.023) ( = 0.38, P = 0.88, r 2 = 0.0066) when plot size was 30 m 2 (Table 2 1) In November 2010 (Table 2 2), when plot size was 10 m 2 O. dissitus had a regular distribution pattern ( b = 0.97, P = 0.0004, r 2 = 0.53) law, but an aggregated distribution pattern ( = 1.11, P < 0.0001, r 2 = 0.70) based on O. dissitus showed an aggregated distribution pattern when the plot size was 30 m 2 based on both methods ( b = 1.10, P = 0.0013, r 2 = 0.66) ( = 1.15, P = 0.0004, r 2 = 0.73) In Decemb er 2010, O. dissitus presented an aggregated distribution pattern in both of plot sizes, 10 m 2 ( b = 1.90, P = 0.0034, r 2 = 0.50) ( = 1.42, P < 0.0001, r 2 = 0.81) and 30 m 2 ( b = 1.51, P = 0.082, r 2 = 0.37) ( = 1.19, P = 0.0013, r 2 = 0.79), based on both a nalyzing methods (Table 2 3). In January 2011, O. dissitus showed an aggregated distribution ( b = 1.13, P = 0.005, r 2 = 0.38) ( = 1.09, P < 0.0001, r 2 = 0.88) when plot size was 10 m 2 based on both methods. When plot size was 30 m 2 O. dissitus presented an aggregated distribution ( b = 1.10, P = 0.024, r 2 = 0.41) ( = 0.99, P < 0.0001, r 2 = 0.89) (Table 2 4) In February 2011 (Table 2 law, parasitoid O. dissitus showed a r andom dis tribution pattern ( b = 1.00, P < 0.0001, r 2 = 0.72) when plot size was 10 m 2 and a regular distribution pattern ( b = 0.87, P = 0.001, r 2 = 0.86) when plot
31 size was 3 0 m 2 O. dissitus presented a regular distribution pattern ( = 0.63, P = 0.11, r 2 = 0.17) ( = 0.47, P = 0.189, r 2 = 0.23) in both of 1 0 m 2 and 3 0 m 2 size plots. Parasitism and Host Density The results of emerged leafminer pupae and emerged O. dissitus adults from e ach week sampling were used to analyze the relationship between O. dissitus parasitism and L. trifolii density A direct density dependent relationship between O. dissitus parasitism proportion and L. trifolii density was observed based on the combin ed data of three bean sites ( b = 0.095, df = 1, 21, r 2 = 0.17, P = 0.049). Distribution Pattern In comparing the results on density and distribution of L. trifolii and O. dissitus it is distinct that distribution pattern of O. dissitu s followed the distribution pattern of L. trifolii T he spatial distribution of L. trifolii tended to be an aggregated pattern when their density was high (4.2 ~ 30.3 pupae / 5 leaves) from November 2010 to January 2011. L. trifolii tended to have a relat ively regular distribution pattern when their abundance w as relatively low (1.7 ~ 2.6 pupae / 5 leaves) in September 2010 and February 2011. O. dissitus showed a similar pattern of abundance and distribution as its host during the above mentioned period of study. Discussion Based on the results from 3 different bean sites, the present study demonstrated that density of L. trifolii and O. dissitus did not have any preference during a single bean site season N o specific pattern of population density of L. tr ifolii and O. dissitus was found at any specific growth period of bean crop (Fig ure 2 1, Figure 2 2, Figure 2 3 ) The density of L. trifolii and O. dissitus was high from late November 2010 to January
32 2011, but low during other growing season. Therefore, i t indicated the abundance of leafminer and the parasitoid had a seasonal preference on bean crop. Temperature is an important factor affecting the leafminer and the parasitoid abundance during different growing seasons. Abou fakhr Hammad (2000) reported th at population density of L. huidobrensis was reduced by the high daily average temperature. Saito et al. (2008) found leafminer Chromatomyia horticola (Diptera: Agromyzidae) were abundant whe n it was cool season in Japan. Homestead is located within a trop ical area, and the leafminer L. trifolii was more abundant when the average monthly temperature was relatively low (< 20 C ) The seasonal density level of the parasitoid O. dissitus had a similar trend to the leafminer. O. dissitus was much more abundant when leafminer density was high, and less abundant when leafminer density was low. The present findings on the density level of L. trifolii and its parasitoid O. dissitus agreed with Valla dares and Salvo ( 2001) who stated that abundance of the parasitoid, O. dissitus was positively correlated with leafminer host density The distribution of L. trifolii and O. dissitus showed an aggregated pattern when their abundance was high, but regular pattern when their abundance was low. It was observed that L. trifoli i distribution pattern was affected by the plant leaf size, and smaller size of the leaf caused an aggregated distribution pattern in L. trifolii (Ayabe and Shibata 2008). In this study, the number of total leaves and total leaf area / bean plant increased with the progression of the plant growing period, which did not increase in population density and aggregation level. Therefore, I conclude that leafminer and the distribution pattern might be affected by the density but not by the p lant host foliage size I n addition, both L. trifolii and O. dissitus tended to have an
33 aggregated distribution pattern during the cool season, but a regular pattern during the warm season. Therefore, temperature might be another factor affecting L. trifol ii and O. dissitus distribution pattern. The plot size also affe cted their distribution pattern. Population of L. trifolii and O. dissitus tended to be aggregated distribution in the plot size of 10 m 2 and regular distribution when the plot size was 30 m 2 The regression slope of transformed equation showed a density dependent parasitism by O. dissitus. However, the density dependent relationship between the host density and parasitism proportion was weak in the present study. Nelson and Roitberg (1995) re ported that leafminer parasitoid, O dimidiatus tend ed to have a density dependent behavio r when host mine density increased. For spatial distribution, some authors indicate d that host aggregated distribution pattern would increase the parasi toid foraging efficiency, and this may lead to a density dependent parasitism (Hassell and May 1974; Heads and Lawton 1983). However, our study did not show this aggregate distribution pattern for L. trifolii and O. dissitus There was no significant higher parasitism r ate found when leafminer had an aggregated distribution pattern. This result supported that spatial aggregation pattern of parasitism was not the condition for increasing biological control efficiency ( Reeve and Murdoch 1985 ). The density of leafminer L. trifolii and parasitoid O. dissitus had a strong seasonal preference. In our study, yellow sticky traps showed an effective monitoring result for L. trifolii but not for O. dissitus There was not a significant density level of O. dissitus adult caught by yellow sticky trap through the whole bean growth period (September 2010 to February 2011) and the open field environment might impact the parasitoid survival and activity. The parasitoid O. dissitus was found to be the most abundant larval pupal
34 endopar asitoid of leafminer during the whole bean growth season, and it is a high potential leafminer biological control agent in south Florida.
35 Fig ure 2 1. Density (mean SE / 5 leaves) of leafminer pupae emerged L. trifolii and O. dissitus at bean site 1 from September to October 2010.
36 Figure 2 2. Density (mean SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 2 from October to December 2010.
37 Figure 2 3. Density (mean SE / 5 leaves) of leafminer pupae, emerg ed L. trifolii and O. dissitus at bean site 3 from December 2010 to February 2011.
38 Figure 2 4. Density (mean SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus in each month, from September 2010 to February 2011
39 Figure 2 5 Density (mean SE / yellow sticky card / 24 h) of L. trifolii and O. dissitus adults in each month, from September 2010 to February 2011
40 Table 2 1 ion parameters pertaining to the d istribution of L. trifolii and O. dissitus on beans in September 2010 Plot size (m 2 ) r 2 a b r 2 L. trifolii 10 30 0.49 0.23 0.013 0.46 1.22AGG 0.60REG 0.72 0.44 0.64 2.06 0.98REG 0.74REG O. dissitus 10 30 0.56 0.023 0.027 0.40 1.37AGG 0.32REG 0.45 0.0066 2.27 3.77 2.74AGG 0.38REG AGG, aggregated distribution, slop b is sign ificantly >1. REG, regular distribution, b is significantly < 1 ( P 2 and 30 m 2 respectively. Table 2 2. ion parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in November 20 10 Plot size (m 2 ) r 2 a b r 2 L. trifolii 10 30 0.38 0.52 0.052 0.22 1.2 0 AGG 1.58AGG 0.8 0.6 7 0.24 1.02 1.20AGG 1.45AGG O. dissitus 10 30 0.53 0.66 0.084 0.0065 0.97 REG 1.10AGG 0.7 0.73 0.19 0.114 1.11AGG 1.15A GG AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 ( P 2 and 30 m 2 respectively.
41 Table 2 3. hiness regressio n parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in December 2010 Plot size (m 2 ) r 2 a b r 2 L. trifolii 10 30 0.47 0.13 0.63 0.16 1.63AGG 0.98REG 0.95 0.86 0.76 0.76 1.10AGG 1.05AGG O. dissitus 10 30 0.5 0.37 0 .73 0.25 1.90 AGG 1.51AGG 0.81 0.79 0.16 0.33 1.42AGG 1.19AGG AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 ( P and 5 for plot sized at 10 m 2 and 30 m 2 resp ectively. Table 2 4. sion parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in January 2011 Plot size (m 2 ) r 2 a b r 2 L. trifolii 10 30 0.42 0.69 0.80 0.06 1.74 AGG 1.41AGG 0.54 0.67 0.31 0.33 1.23AGG 1.20AGG O. dissitus 10 30 0.38 0.41 0.013 0.06 1.13AGG 1.10AGG 0.88 0.89 0.69 1.27 1.09AGG 0.99REG AGG, aggregated distribution, slop b is significantly >1. REG, regula r distribution, b is significantly <1 ( P 2 and 30 m 2 respectively.
42 Table 2 5. ssion parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in February 201 1 Plot size (m 2 ) r 2 a b r 2 L. trifolii 10 30 0.23 0.32 0.11 0.11 0.81REG 0.98REG 0.54 0.67 0.31 0.33 1.23 AGG 1.20AGG O. dissitus 10 30 0.72 0.86 0.00 0.13 1.00RAD 0.87REG 0.17 0.23 0.16 0.05 0.63REG 0.47REG RAN, random distribution, b is not significantly different from 1 aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 ( P The division number of 15 and 5 for plot sized at 10 m 2 and 30 m 2 respectively.
43 Figure 2 6. Bean crop field ( 50 m 30 m) was divided into 15 equal plots (1 0 m 10 m). Figure 2 7. Bean foliages were sampled and placed in the laboratory.
44 Figure 2 8. Leafminer and the parasitoids were reared in the laboratory.
45 CHAPTER 3 DIEL DENSITY PATTER N OF LEAFMINER, LIRIOMYZA TRIFOLII AND THE PARASITOIDS, OPIUS DISSITUS (HYMENOPTERA: BRACON IDAE) AND DIGLYPHUS SPP. (HYMENOPTERA: E ULOPHIDAE) Agromyzid leafminer is an important phytophag o us fly feeding on a wide range of ornamental and vegetable plants. T he American serpentine leafminer, Liriomyza trifolii (Burgess) is a serious pest of snap bean crop in south Florida. Both adults and lar vae can cause economic damage to the plants. The females puncture the leaves with their ovipositors and insert their mou thparts at the punctures to feed on cell contents The larvae consume mesophyll tissues of the leaf ( Musgrave et al 1975; Fagoonee 1984 ). The feeding of larvae and egg laying by adults can significantly impact plant physiology (Parrella 1985) and create a proper environment for pathogens ( Broadbent 1990 ). Chemical control is the main method to manage leafminer s on various commercial crops Frequent use of these insecticides enhances development of resistance in leafminers, resurgence of secondary pests and elimination of natural enemies. Thus insec ticide resistance development limits the control of leafminers (Leibee and Capiner a 1995). To reduce sole dependence on insecticides, various studies have been conducted to manage insect pests using biocontrol ag ents alone or in combination or alteration with insecticides and other control techniques. Several h ymenopteran parasitoids were found to be effective for contro lling agromyzid leafminer s There are at least 14 species of L. trifolii parasitoids in Florida (Capinera 2001). The parasitoid wasp s O. dissitus and Diglyphus spp. were found abundant on bean crops in south Florida (unpublished report, Li. 2011). It was reported that Opius sp. and Diglyphus spp. were reared from L.
46 trifolii from celery and tom ato l eaves in Florida (Stegmaier 1972; Schuster and Wharton 1993). In developing an integrated pest management program, knowledge of the biology of the pest and its natural enemies is essential. Negative response of natural enemies to the commonly used insectic (2005) and Weintraub (2001) found that parasitoid D. isaea was more compatible with abamectin than cyromazine. Various studies are available on the biology of L. trifolii The activity of L. trifoli i and its parasitoid s is affected by various factors. The leafminer L. trifolii activity is affected by temperature, light, moisture and host plants type. Temperature is an important factor affecting the activity of Agromyzidae leafminer and their parasito ids in the natural environment ( Abou Fakhr Hammad 2000; Bordat et al 1995) Temperature tends to vary throughout the day. W e assumed the activity or density level s of L. trifolii and its parasitoids will be different throughout various period s of the dayt ime. Few studies ever assessed the diel density pattern of L. trifolii and its parasitoids. Understanding the diel density pattern of L. trifolii parasitoids is crucial in reducing the insecticide impact on these natural enemies in integrated pest manageme nt program To create more effective IPM decisions for controlling L. trifolii the objective of this study was to determine th e die l density pattern of leafminer, L. trifolii and its parasitoids, O. dissitus and Diglyphus spp. Materials and M ethods Study Site and Beans P reparation The study was carried out in the TREC research plots, Homestead FL Two bean fields (site 1 and site 2), each 0.4 ha, were selected for the present study (Figure 3 8)
47 The soil type of the study sites was Krome gravelly loam so il, which consists of about 33% soil and 67% pebbles. Both fields (site 1 and site 2) were planted with snap bean ( Phaseolus vulgaris supplied from Harris Mor an seed Company Modesta, CA, USA ) on 3, Oct. and 10, Dec. 2010, respectively. Pre plant herbicid e halosulfuron methyl ( 51.9 g / ha Sandea, Gowan Company LLC., Yuma, Arizona), was applied 3 weeks before planting for controlling nutsedge and broad leaf weeds the seeds. The beans were seeded directly in row s at the rate of three seeds / foot. The bean seeds were spaced 3 feet between two adjacent rows. Granular fertilizer 8: 16: 16 at the rate of 1345 kg / ha was used at planting in a band 1 m apart from the seed rows. Additionally, liquid fertilizer (4: 0: 8) at the rate of 0.56 kg / ha / day was used five weeks after planting at each site. The plants were irrigated through drip tubes twice every day delivering 1.0 inch (2.54 cm) each time. D uring the early bean growth period, fungicide c hlorothalonil ( 2.81 L / ha, Bravo Syngenta Crop Protection, Inc ., Greensboro, NC) was used to prevent fungal disease Both sites were managed for conducting present study until 15, Dec. 2010 and 22 Feb. 2011, respectively. No insecticide was used during the study period. Plot Design and Diel Activity Each study site was divided into 15 equal plots each (10 m 10 m) One yellow sticky trap (7.6 cm 12.7 cm) was set in the center of each plot from 08:00 to 18:00 EST within a day. For the purpose of studying diel activity pattern of leafminer and its parasitoids, stic ky taps were checked at 2h intervals (08:00 10:00, 10:01 12:00, 12:01 14:00, 14:01 16:00 and 16:01 18:00 EST) once in every week at the 4 th 5 th 6 th and 7 th week after the bean planting in each site. The cards were checked 20 min
48 before each int erval. At the time of checking sticky cards, each insect on the card deemed to be leafminer and its parasitoids was marked with colored pen (Figure 3 9), different marks for different intervals. At the end of the day, all traps were collected and wrapped i ndividually with transparent polyethylene sheet to avoid any trapping of unwanted insects. Each trap was marked with plot number, date, and period. All traps were transported to the IPM laboratory, TREC, Homestead, FL. The numbers of leafminer and various parasitoids were recorded using a binocular microscope. The leafminers we re sent to Division of Plant Industry (DPI) for identification. The parasitoids were sent to Systematic Entomology Laboratory ( USDA, MD) for confirmation of identification to genus an d species levels. Each week, a new set of sticky traps was used on the day of study. Statistical Analysis The diel densities of insects were calculated as the trapped ones during each 2h interval. The mean number of trapped L. trifolii O. dissitus and Dig lyphus spp. from each different period was compared by analysis of variance (ANOVA, PROC GLM, SAS Institute Inc. 2003). The means were separated by the Least Significance Difference (LSD) ( P < 0.05). Results S ite 1. The population abundance of L. trifolii O. dissitus and Diglyphus spp. in each two hours interval within the day was assessed on November 09, 2010 (F igure 3 1 ). T he abundance of leafminer was significantly higher ( F = 10.58; df = 4,74; P < 0.0001) in the first (8:00 10:00 EST) and second 2h i nterval (10:01 12:00 EST) than other intervals. Population abundance of Leafminer decreased as day light hours increased. Lowest activity of leafminer was observed during the fifth interval.
49 Population abundance of Diglyphus spp. was significantly highe r ( F = 3.33; df = 4,74; P = 0.0148 ) in the second, third and fourth intervals than in the first and fifth intervals. Diglyphus spp. was almost absent during the first 2h interval (8:00 10:00 EST). Abundance of O. dissitus did not show any significant dif ference ( F = 2.04; df = 4 ,74; P = 0.0979) among the intervals within the day (Figure 3 1). Overall, O. dissitus poplulation was low during this study. On November 16, 2010, the density of L. trifolii was significantly higher ( F = 4.24; df = 4,74; P = 0.004 ) in the first 2h interval than in the second and fifth intervals (Figure 3 2). However, population abundance of leafminers in the first two hours did not differ from the third and fourth intervals within the day. The highest peak of leafminers populations was observed at 8:00 10:00 followed by 14:01 16:00; 12:01 14:00; 16:01 18:00 and 10:01 12:00 EST. Diglyphus spp. was significantly more abundant ( F = 5.06; df = 4,74; P = 0.0012) in the second two hours interval than in the other intervals (F igure 3 2). Diglyphus spp. was almost absent in the first two hours interval. Like Diglyphus spp., O. dissitus was absent in the first interval (08:00 10:00 EST); and increased insignificantly in the rest of the intervals ( F = 0.88; df = 4,74; P = 0.4778 ). On November 23, 2010, the L. trifolii density was significantly higher ( F = 17.8; df = 4,74; P < 0.0001) in the first 2h interval (08:00 10:00 EST) than the rest of the intervals within a day (Figure 3 3). Diel density of leafminer decreased as the d ay time progressed. The lowest activity of leafminer was observed at the end of the day (16:01 18:00 EST). The peak increase in diel activity of Diglyphus population was observed during the second interval (10:01 12:00) ( F = 4.28; df = 4,74; P = 0.0037 ) (Figure 3 3).
50 Decrease in activity of Diglyphus sp. was observed before and after the second interval. Population abundance of O. dissitus was low in all intervals ( F = 1.5; df = 4,74; P = 0.2128 ) (Figure 3 3). No clear peak in the diel pattern of O. dis situs was observed during this study ( F = 1.5; df = 4,74; P = 0.2128). On December 01, 2010, the L. trifolii population abundance was still significantly higher in the first 2h interval ( F = 12.77; df = 4,74; P = 0.0002) (Figure 3 4) followed by the fourt h, third, second and fifth intervals. Population activity of leafminers was minimum at the end of the day. Population of Diglyphus spp. peaked at the second two hours interval ( F = 6.46; df = 4,74; P < 0.0001) (Figure 3 4). Activity of Diglyphus decreased thereafter with the increase or decrease of day light hours. However, Diglyphus populations were present all across the day. Population of O. dissitus was significantly lower during this study (Figure 3 4). No significant difference ( F = 0.2; df = 4,74; P = 0.9394) of the O. dissitus density was found among all the intervals. When data across all intervals of a day were combined, a distinct pattern in the diel activity of leafminers was observed (Figure 3 5). Peak activity of leafminers was observed during the first 2h interval, which was significantly different ( F = 7.22; df = 4,19; P = 0.0019) from all other intervals The abundance of leafminers did not differ among the rest of intervals. Unlike leafminer, t he density of Diglyphus spp. was highest ( F = 7. 6; df = 4,19; P = 0.0015 ) during the second two hours interval than other intervals (Figure 3 5). The lowest density level of Diglyphus was observed during the first and last 2h intervals. No significant difference ( F = 1.44; df = 4,19; P = 0.2696) was fou nd in O. dissitus density level among all the intervals within the day
51 Site 2. The die l activity pattern of L. trifolii was studied based on the combined four ata from 15 yellow sticky traps. The leafminer density did not show any significant diff erence ( F = 0.7; df = 4,19; P = 0.6049) (Figure 3 6) within the daytime. Unlike site 1, t he Diglyphus spp. had a significantly higher density level ( F = 6.43; df = 4,19; P = 0.0032) (Figure 3 6) in the third two hours interval (12:01 14:00 EST). The O. d issitus density presented a significant difference ( F = 3.8; df = 4,19; P = 0.0251) during the daytime, and its density was highest in the second two hours (10:01 12:00 EST) among all the intervals Discussion Temperature was a major factor for L. trifol ii and its parasitoid abundance. H igh daily temperature s C ) could reduce density ( Abou Fakhr Hammad 2000). At the bean site 1 (November to December 2010), the average temperature within the daytime changed greatly (22 to 27 C ), and it was relatively low (< 25 C ) in the morning and high (> 25 C ) during the rest of the day. The die l density of leafminer, L. trifolii was significantly higher in the first two hours interval when the temperature w as lowe r (< 25 C ) than other periods of the day The leafminer population started to decrease when the temp erature increased above 25C after the second two hours interval. Therefore, the low temperature in the first 2h (8:00 10:00 EST) might be the main reason for higher density of leafminer in the m orning than other periods of a day. The parasitoid, O. dissitus did not show any significant density difference among the periods within the day It was reported that the optimum temperature for both of male and female adults was 20 C, and female had a hi gher reproductio n at 25C (Bordat et al. 1995b). The Diglyphus spp. density was always higher in the second two
52 h ou r s interval than any other periods when the temperature was above 25 C within the day. There were three different species in Diglyphus spp. found in our study, indicating D. begini (Ashmead) D. intermedius (Girault) and D. isaea (Walker). The Diglyphus spp., is a larval ectoparasitoid, which has been used in leaf miner biological control program in greenhouse (Minkenberg 1987). T he optimum tem perature for rearing D isaea from L. trifolii ranged between 32.3 C and 32.6C (Bazzocchi et al. 2003). The relatively low temp erature (< 25C) in the first two h ou r s interval in the day time might reduce the Diglyphus spp. density level. The density of Di glyphus increased to a higher level at the second two hours interval when the temperature increased (> 25 C). in site 2 in spring 2011, shown in the figure 3 6 did not provide a similar diel density pattern as in bean site 1. The average daily temperature was relatively high during late February and early March. The temperature in the morning was low but not significantly different from the temperature at other periods within the day, and it may not affect the diel density level o f L. trifolii O. dissitus density is relatively higher in the second two hours interval. The highest density level of Diglyphus spp. was in the third two hours (12:01 14:00 EST) interval These results agree d with the study of temperature influence on O dissitus and D iglyphus spp. (Bordat et al. 1995b; Bazzocchi et al. 2003) abundance of L. trifolii was higher in the bean site 1 than in bean site 2 (Figure 7), which migh t be a reason that the leafminer showed a significant density level in the first two h ou r s interval (8:00 12:00 EST) within a day in the site 1. However, there was no t a
53 significant diel density level of leafminer during daytime at the site 2 where the l eafminer density was very low. Yellow sticky traps were used for monitoring leafminer and its parasitoids diel density pattern in the study, and it showed an effective monitoring result. The yellow sticky traps had more chance to catch leafminer when the d ensity was high, but less chance when leafminer density was low. Parrella and Jones (1985) revealed yellow sticky trap was a useful and rapid tool to estimate L. trifolii density on Chrysanthemum in greenhouse. In our study, the results indicated that the yellow sticky trap was also an effective tool for monitoring the seasonal abundance of Diglyphus parasitoids density. Therefore, yellow sticky traps can be used as an effective tool in the research on leafminer and some parasitoid ecology. The parasitoids of O. dissitus and D iglyphus spp. were found to be the most abundan t parasitoid wasps of leafminer L. trifolii on bean crops O. dissitus is a larval pupa endoparasitoid of Liriomyza leafminer and adults emerge from the leafminer pupae. Diglyphus parasit oids were ectoparasitoid of leafminer larvae and adults emerge from the mines on foliages. Understanding the diel biological behavior of leafminer and parasitoid could improve IPM strategies in the open field crops. Releasing the leafminer parasitoids duri ng the appropriate time and season could enhance the biological control effectiveness in the open field environment In addition, insecticides application during the selective time period could relatively reduce the impact on the parasitoids.
54 Figure 3 1 Bean site 1, Nov 09 2010. Mean ( SE) number of the L. trifolii O dissitus and Diglyphus spp. / yell ow sticky trap during each 2 h interval after 8:00 EST Means with the same letter are not significantly different ( P < 0.05, LSD test).
55 Figure 3 2. Bean site 1, Nov 16 2010. Mean ( SE) number of the L. trifolii O dissitus and Diglyphus spp. / yell ow sticky trap during each 2 h interval after 8:00 EST Means with the same letter are not significantly different ( P < 0.05, LSD test).
56 Figure 3 3. Bean site 1, Nov 23 2010. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / yell ow sticky trap during each 2 h interval after 8:00 EST Means with the same letter are not significantly different ( P < 0.05, LSD test).
57 Figure 3 4. Bean site 1, Dec 01 2010. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / yell ow sticky trap during each 2 h interval after 8:00 EST Means with the same letter are not significantly different ( P < 0.05, LSD test).
58 Figure 3 5. Bean site 1. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / 15 yello w sticky traps during each 2 h interval after 8:00 EST (based on combined data of Nov 09, Nov 16, Nov 23, Dec 01, 2010). Means with the same letter are not s ignificantly different ( P < 0.05, LSD test).
59 Figure 3 6. Bean site 2. Mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. / 15 yello w sticky traps during each 2 h interval after 8:00 EST (based on combined of Jan 31, Feb 17, Feb 24, Mar 01, 2011). Means with the same letter are not significantly different ( P < 0.05, LSD test).
60 Figure 3 7. Seasonal density: mean ( SE) number of the L. trifolii O. dissitus and Diglyphus spp. per 15 yello w sticky traps / 10 h at bean site 1, 2010 and site 2, 2011. The mean temperature was in November 2010 to February 2011.
61 Figure 3 8. Fifteen yellow sticky traps were set in b ean field from 8:00 18:00 EST Figure 3 9. The caught insects duri ng different 2 h interval were marked on the y ellow sticky card.
62 CHAPTER 4 THE COMPOSITION AND SEASONAL ABUNDANCE O F HYMENOPTERAN PARASITOIDS OF LIRIOMYZA TRIFOLII ON BEANS IN SOUTH FL ORIDA The phytophagous leafminer (agromyzidae: Liriomyza spp.) has a significant economic impact on vege table and or namental production worldwide. The l eafminer Liriomyza trifolii (Burgess), L. sativae (Riley) and L. huidobrensis (Blanchard) are the important species threatening the agriculture in the USA (Parrella 1987 ; Capinera 2001 ; Schuster and Wharton 1993 ). The tr a ditional control of leafminer is using broad spectrum ins ecticides. This has a high potential for develop ing leafminer r esistance (Leibee and Capinera 1995) and it also impact ed the natural enemies density (Johnson et al. 1980). B iological control is an important component in integrated pest management (IPM) of leafminer. Research has been conducted to evaluate the biological control effectiveness by using parasitoid wasps in leafminer management (Johnson et al 1980; Patel et al 2003). For minimizing t he impact of insecticides on parasitoids, studies have been performed to evaluate the compatibility of traditional and new insecticides with leafminer parasitoids (Ferguson 2004; Weintraub 2001; Kaspi 2005). Several studies have been conducted to identify leafminer parasitoids in various regions and on different plant hosts in North America (Lasalle and Parrella 1991; Stegmaier 1966; Stegmaier 1972; Schuster and Wharton 1993). Schuster and Wharton (1993) reported 4 families and 15 species of leafminer para sitoid on tomato in Florida. Snap bean is an important agricultural crop in Florida, and it valued $138.4 million during 2000 2001 (Mossler and Nesheim 1999). Leafminer L. trifolii is the most significant pest on beans. The leafminer hymenopteran parasit oids were found in the bean crop
63 status is the primary step for investigating new potential biological control agents. It is also important to reveal the leafminer and its parasitoid interactions, and community structure in the crop field in management. For identifying the leafminer parasitoids group on snap bean crops and exploring potential effective biological control agents the objective of this study research w as to study the co mplex and seasonal abundance of leafminer parasitoids on beans in south Florida. Materials and Methods Stud y Site The leafminer parasitoids complex was investigated in the same bean crop fields mentioned in chapter 2 in the Tropical Research and Education Center (TREC), Unive rsity of Florida. The site 1 was planted to bean on September and maintained until November 2010, site 2 was from October to December 2010, and site 3 was from December 2010 to February 2011. The snapb ean ( Phaseolus vulgaris ) seed s wer e supplied from Harris Mor an seed Company Modesta, CA, USA The bean fields were prepared with cultural practices as the same as discussed in chapter 2 and chapter 3. No insecticide was used during the study. Leaf Sampling and Insect Rearing The parasit oids were obtained from the bean foliages. The sampling began when the bean plant had two primary leaves fully unfolded. To address all variability in the sampling, the field was divided into 15 equal plots. Five leaves, one leaf / plant, were randomly col lected from each plot weekly (75 leaves / week) When the bean plants became mature and their primary leaves dropped off, the older bottom leaves were always collected from the plants because of L. trifolii feeding preference to the older mature leaf (Fack nath 2005; Williams et al 1998). The sampled leaves from each plot
64 were placed into a 15 cm diam eter Petri dishes (5 leaves per Petri dish) and were marked with date and plot number. The samples were then transported to the laboratory and placed into a gro wth at 25 C 70% RH and 14:10 (L: D) h for the development of leafminer and its parasitoids The bean leaves were checked every day to collect leafminer pupae. The pupae in each Petri dish were separated from the leaves and transferred into one new Petri d ish labeled with t he sampli ng date. The pupae were checked every day to record larval pupal endoparasitoid emergence. The leaves were also checked every day to collect larval ectoparasitoid from leaf mines. All the emerged parasitoids were collected and p reserved in the 75% alcohol for identification. The leafminer s were identified following morphological characters described by Capinera (2001). For further confirmation, the leafminers were sent for identification to the D ivision of Plant Industry (DPI) G ainesville, FL. The parasitoids were identified based on the external characters used in previous studies (Lasalle and Parrella 1991; Gordh and Hendrickson 1979; Asadi et al 2006) and further verified by Systematic Entomology Laboratory, USDA, MD. Results and Discussion In the present study, about 99% of the leafminer collected from the beans was identified as L. trifolii (Figure 4 1). The L. trifolii adult is about 2 mm long and the wing length is 1.25 to 1.9 mm (Capinera 2001). The red eyes, grayish black mesonotum and yellow hind margins of the eyes are the key characters of L. trifolii The L. trifolii abundance was high in Decemb er 2010 and January 2011 (Table 4 1). The average temp erature of these two months was lower (15 to 19 C ) than other planting m onths (20 to 26 C ) Saito et al. (2008) reported that leafminer Chromatomyia horticola ( Diptera: Agromyzidae) was abundant in the cool season on the pea crop Abou fakhr Hammad
65 (2000) reported that population density of L. huidobrensis was reduced by the h igh daily average temperature s Opius dissitus (Figure 4 2). Two different Braconidae parasitoid s, O dissitu s and Euopius sp. were observed on beans. O. dissitus is a solitary larval pupal endoparasitoid of L. trifolii (Nelson and Roiterg 1995; Bordat et al. 1995 a; Bordat et al. 1995 b). O. dissitus adult is small in size (about 1.50 mm). It is black in color with long antennae, which are thin and black (Figure 4 2 ) O. dissitus was found to be the most abundant leafminer parasitoid on snap beans in the Miami Dade Co. (Table 4 1). The parasitoid females deposit their eggs inside the leafminer larvae. The parasitized host larvae continue to develop until pupae stage. This observation is supported by Bordat et al. (1995 a) reported that the parasitoid femal es lay their eggs directly inside their host larvae. The development of O. dissitus takes place inside the host pupae. At the end of the development, adult emerged out from the pupae. One O. dissitus adult was found to emerge from a single leafminer pupa i n our study. The density of O. dissitus was positively correlated with leafminer density within the whole growth season. The density of O dissitus was high when the temperature was cool (15 ~ 19 C ) in December 2010 and January 2011 (Figure 4 15) The opti mum temperature for both of O. dissitus male and female was 20 C, and female had a higher reproduction at 25 C (Bordat et al 1995 b ). The O. dissitus was ever reported from celery and infested tomato leaves in Florida (Stegmaier 1972; Schuster and Wharton 1993). Euopius sp. (Figure 4 3). Eu opius sp. is also a larval pupal endoparasitoid. One Euopius sp. adult emerged from a single host pupa. Euopiu s sp. has almost the same body size and antennae as O. dissitus It has an overall yellow color body, which i s
66 clearly different from O. dissitus being black in color. The abundance of Euopius sp. (Table 4 1) was low on the bean foliage in the study during fall 2010, and no Euopius sp. was found after December 2010. The Euopius sp. was reared from Liriomyza leaf miner infested weeds on Bidens alba (Schuster and Gilreath 1991). Diaulinopsis callichroma (Figure 4 4 ) There were ten different species of parasitoids found in the family of Eulophidae. The D callichroma was found to be the second largest group of le afminer parasitoid in this study (Table 4 1 ) Its abundance was high in October and November 2010, h owever, was low in January and February 2011 (Figure 4 15). The adult hind femora are basal dusky, fore and middle femora are pale (Gordon and Hendrickson 1 black and enlarged. The body size of adults is about 1.10 1.30 mm. This parasitoid is a larval ecto parasitoid of leafminer. The D. callichroma larvae feed on the host larva directly and kill the host even tually. The parasitoid larvae pupate inside the mine and emerged from the mine. The abundance of D. callichroma might be affected by both of the host density and temperature condition. Diglyphus spp. (Figure 4 5; Figure 4 6; Figure 4 7). Three different sp ecies of Diglyphus parasitoids ( D. begini D. intermedius and D. isa e a ) were reared from the bean foliages They were not present until November 2010 and became abundant in February 2011 (Figure 4 15) The D. begini abundance was relatively higher than D. intermedius and D. isaea (Table 4 1) The D. intermedius and D. isaea had a relatively the similar density to each other. Diglyphus spp. parasitoids were the third largest group in the study. Diglyphus spp. is a larval ectoparasitoid, and female adult gene rally lays more than one egg beside of the host larva ( Minkenberg 1987 ) Diglyphus sp. is
67 characterized by their antennae with two funicular segments. The forewings of these three species are dense setose. The basal cell is uniformly dense setose. These th ree species can be differentiated from each other based on the dark area on their hind tibia: D. begini basal hind tibia has a short and less than 25% metallic dark area (Figure 4 5). D. intermedius basal tibia has a relatively larger area of 25 35% dark color and with extended dusky color (Figure 4 6); D. isaea basal hind tibia with over 75% metallic dark color proportion (Figure 4 7) ( Lasalle and Parrella 1991; Gordh and Hendrickson 1979). Schuster and Wharton (1993) reported D. intermedius and D. begin i were reared from tomato crop in Florida. The Diglyphus parasitoids were observed the most abundant larval ectoparasitoid on tomato crop (Schuster and Wharton 1993). However, in our study, the most abundance larval ectoparasitoid on bean crop was D calli chroma The D. isaea was used as an effective biological control agent in greenhouse for controlling Liriomyza leafminer ( Minkenberg 1987; Boot et al. 1992 ) Several studies were conducted on Diglyphus parasitoid and its compatibility with common insectici des (Bazzocchi et al 2003; Kaspi and Parrela 2005 ; Weintraub 2001). Neochrysocharis sp. (Figure 4 8) was reported as an endoparasitoid of leafminer larvae, and the adults emerge from the mines on the leaves. The adult body is overall metallic green and e yes are red. The fore and middle legs, and hind tibia are pale. The species of N punctiventris was ever reported as the fourth abundant leafminer parasitoid on tomato crops in Florida (Schuster and Wharton 1993). Closterocerus sp. (Figure 4 9). The popul ation abundance of Closterocerus sp. was low on bean foliage during this study. It is an endoparasitoid of leafminer young stage larvae (Asadi et al. 2006). The adult body length is about 1.0 mm. The forewings
68 of Closterocerus are with characteristic dark bands. Stegmaier (1966) reported C. cinctipennis was a parasitoid of leafminer L. trifolii in Florida. Zagrammosoma spp. (Figure 4 10; Figure 4 11). Zagrammosoma lineaticeps and Z. muitilineatum are the larval ectoparasitoid of Liriomyza leafminer. Both of Z lineaticeps and Z. muitilineatum were found in Florida (Stegmaier 1972; Stegmaier 1972; Schuster and Wharton 1993 ). The Z lineaticeps is black or very dark color and the forewing has a dark line along the apical margin (Figure 4 10), while Z. muitilin eatum is predominately yellow and mesoscutum with dark stripes (Figure 4 11). The abundance of both species was high in October and November 2010. In this study, Z. muitilineatum was relatively more abundant (20 adults) than Z lineaticeps (10 adults) (T ab le 4 1 ) Pnigalio sp. (Figure 4 12) is a larval ectoparasitoid of Liriomyza leafminer and adult emerges from the mine on the bean foliage. This genus of parasitoid was also reported to parasitize on Diptera, Lepidoptera, Hymenoptera and Coleoptera (Asadi et al. 2006). The body length of adult females is 1.7 ~ 1.9 mm and male is 1.5 ~ 1.7 mm. Its antennae are with 4 funicular segments, and male has a laterally branched structure antenna (Asadi et al. 2006). Lasalle and Parrella (1991) indicated that only on e Nearctic species of P. flavipes attacks Liriomyza However, Schuster and Wharton (1993) reported species of P. maculipes was reared from the tomato foliage in Florida. The species of Pnigalio in this study was unknown. Chrysocharis sp. (Figure 4 13) was the larval pupal endoparasitoid of leafminer, and it was the second most abundant larval pupal endoparasitoid (Table 4 1). In our study, Chrysocharis adults emerged from leafminer pupae. It only appeared in spring
69 season in January and February 2011. Chrys ocharis sp. post marginal vein is always longer than the stigma vein, and petiole is present and distinct. Halticoptera sp. (Figure 4 14) was found in the family of Pteromalidae. Halticoptera sp. petiole is present and relatively long, and antenna has 6 funicular segments. This species is another larval pupal endoparasitoid of leafminer in beans. The abundance of this species was very low and only present in October and November 2010. Schuster and Wharton (1993) reported a high abundance of Halticoptera s p. on tomato. The H circulus was reported to be the only one Nearitic species of Halticoptera known to parasite Liri o myza leafminer (Lasalle and Parrella1991). Many biotic and abiotic factors, as temperature, rainfall, host crops, alternative plant hosts, and intra or inter competition, may affect the leafminer parasitoids population. In our study, 13 species in three families of leafminer parasitoid were reared from the bean foliages in south Florida. Four species were leafminer larval pupal endoparasito id, two were larval endoparasitoid, and seven were larval ectoparasitoid. The O. dissitus was the most abundant larval pupal endoparasitoid and D. callichroma was the most abundant larval ectoparasitoid on snap beans. Our study is the first report on D. is aea in Florida We assumed the nursery industry in Miami Dade Co. might bring and release this species of parasitoid for controlling leafminer on the ornamental plants. The recent study on parasitoid complex on bean crop will provide significant informatio n of potential biological agents of leafminer for IPM. In addition, the pictures in this study w ill benefit leafminer hymenopteran parasitoids identification in the future work.
70 Figure 4 1. L iriomyza trifolii (Agromyzidae). Figure 4 2. Opius dissi tus (Braconidae).
71 Figure 4 3 Euopius sp (Braconidae). Figure 4 4. Diaulinopsis callichroma (Eulophidae).
72 Figure 4 5. Diglyphus begini (Eulophidae). Figure 4 6. D intermedius (Eulophidae).
73 Figure 4 7. D. isaea (Eulophidae). Figure 4 8. Neochrysocharis s p. (Eulophidae)
74 Figure 4 9. Closterocerus sp. (Eulophidae). Figure 4 10. Z agrammosoma lineaticeps (Eulophidae).
75 Figure 4 11. Z. muitilineatum (Eulophidae). Figure 4 12 Pnigalio sp. (Eulophidae).
76 Figure 4 13. Chrysocharis sp. (Eulophidae). Fig ure 4 14 Halticoptera sp. (Pteromalidae).
77 Figure 4 15. Seasonal abundance of parasitoids, O. dissitus D. callich roma and Diglyphus spp. on snap bean crop from September 2010 to February 2011.
78 Table 4 1. Number of leafminer L. trifolii and its parasitoids (%) reared from the bean foliages (300 leaves / month) from Septe mber 2010 to February 2011 in south Florida Family and species Sep Oct Nov Dec Jan Feb Total Agromyzidae L. trifolii (Burgess) 101 18 132 583 1805 66 2805 Braconidae Opius dissitus (Muesebeck) 41 (73%) 12 (11%) 91 (44%) 202 (79%) 401(83%) 31 (26%) 778 (63%) Euopius sp 3 (5.0%) 3 (3.0%) 2 (1.0%) 2 (0.7%) 0 0 10 (0.8%) Eulophidae Diaulinopsis callichrom a (Crawford) 8 (14%) 73 (69%) 78 (38%) 18 (7.2%) 27 (5.6%) 9 (7.0%) 213 (18%) Diglyphus begini (Ashmead) 0 0 4 (2.0%) 10 (4.0%) 17 (3.5%) 21 (17%) 52 (4.2%) D. intermedius (Girault) 0 0 1 (0.5%) 4 (1.6%) 5 (1.0%) 20 (16%) 30 (2.4%) D. isaea (Wa lker) 0 0 1 (0.5%) 2 (0.7%) 9 (1.9%) 14 (12%) 26 (2.1%) Neochrysocharis sp. 2 (4.0%) 7 (7.0%) 7 (3.5%) 8 (3.2%) 2 (0.4%) 14 (12%) 40 (3.3%) Closterocerus sp. 2 (4.0%) 1 (1.0%) 2 (1.0%) 4 (1.6%) 1 (0.2%) 0 10 (0.8%) Zagrammosoma lineaticeps (Gir ault) 0 3 (3.0%) 2 (1.0%) 0 0 0 5 (0.4%) Z. muitilineatum (Ashmead) 0 6 (5.0%) 13 (6.0%) 0 1 (0.2%) 1 (0.8%) 20 (1.6%) Pnigalio sp. 0 0 4 (2.0%) 5 (2.0%) 1 (0.2%) 0 10 (0.8%) Chrysocharis sp. 0 0 0 0 19 (4.0%) 11 (9.2%) 30 (2.4%) Pteromalidae Halticoptera sp. 0 1 (1.0%) 1 (0.5%) 0 0 0 2 (0.2%)
79 CHAPTER 5 CONCLUSION Leafminer, Liriomyza trifolii is an important pest infesting vegetable and ornamental plants all over the world. It became a problem on celery and other vegetables sinc e 1945 in Florida (Wolfenbarger 1947). Managements have been established to control this pest on economic crops, including chemical control, biological control and cultural practice The importance of integrated pest management (IPM) of leafminer has been realized due to the problems caused by chemical controls. The development of insecticides resistance and impact on the natural enemies in the traditional chemical control reduce the effectiveness of leafminer management. L. trifolii is one of the serious p ests on bean production in south Florida. For enhancing the IPM control of leafminer, several studies have been conducted for assessing leafminer L. trifolii seasonal abundance, spatial distribution and its hymenopteran parasitoids complex on bean crops in south Florida. Liriomyza trifolii abundance presented a seasonal preference, and its density was high during the cool season, but low in the warm season. The parasitoid, O. dissitus had a similar seasonal density trend as L. trifolii density level. Both L trifolii and O. dissitus showed an aggregated distribution in the bean field when the abundance was high and a regular pattern when their abundance was low. This information will be helpful for monitoring leafminer density and determining leafminer econo mic threshold in bean production. The diel density of L. trifolii and its two parasitoids was studied in snap bean field based on five divided 2h intervals within a day. L. trifolii presented a higher density level during the first 2h (8:00 10:00 EST) th an any other time within a day in cool season
80 during November and October 2010. There was no significant difference in parasitoid O. dissitus density throughout the day. Diglyphus spp. presented a peak density level in the second 2h (10:01 12:00) within a day in the cool season and the third 2h (12:01 14:00) in the warm season. Information of their diel activity can guide a chemical control strategy, which reduces the direct impact on these parasitoids. Chemical insecticides can be applied when leafmine r density is high and parasitoids density is low. This information also benefits the biological control application, and releasing leafminer parasitoids during its preferred period within a day can enhance the control effectiveness. Hymenopteran parasitoid s have been used to control Liriomyza leafminers. In my study, thirteen different genera or species of parasitoids belonging to three families were collected from beans. The Braconidae parasitoids include O. dissitus Euopius sp The Eulophidae parasitoids include Diaulinopsi s callich roma Diglyphus begini D. intermedius D. isaea ., Neochrysocharis sp., Closterocerus sp., Zagrammosoma lineaticeps Z. muitilineatum Pnigalio sp. and Chrysocharis sp. One genus of Halt i coptera sp. was found in family Pteromal idae. O pius dissitus was found the most abundant leafminer parasitoid, and it is a larval pupal endoparasitoid. D. callich roma was the second abundant parasitoid, and it is a larval ectoparasitoid. Diglyphus spp. was the third abundant parasitoid group, an d they are also larval ectoparasitoid. The importance of surveying the composition and seasonal abundance of leafminer parasitoids is to provide information for developing management program incorporating effective biocontrol agents. It will also guide
81 gro wers to properly apply insecticides and to reduce their harmful impact on the natural enemies. This research was established to assess leafminer L. trifolii biology characters, including its seasonal abundance and field spatial distribution. This study al so investigated the composition and seasonal abundance of its natural hymenopteran parasitoids. O. dissitus was found to be the most abundant parasitoid of L. trifolii on snap bean crop. This study will help make pest management strategies and enhance the effectiveness of IPM in leafminer control.
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88 BIOGRAPHICAL SKETCH Jian Li was born in Shenyang, Liaoning Province, China, in 1986 He graduated from Shenyang No. 27 high s chool in 2005, and began his undergraduate study at 2009. His undergraduate education included plant pathology, entomology, and plant chemical control. His undergradua te research was focused on integrated pest management of root knot nematode, Meloidogyne incognita under the supervision of Professor Duan Yuxi. His undergraduate thesis was ranked first place in the Plant Liriomyza leafminer seasonal abundance, spatial distribution, and leafminer He participated in extension activities to help growers and crop management advisors. He also shared his research information with the local growers and industry research Liriomyza trifolii and its parasitoid Opius meeting of Entomology Society of America, Southeastern branch, Puerto Rico, 2011. He won th e 3 rd place in the Student Oral Presentation Competition in Florida State Horticulture Society Annual Meeting, St. Petersburg, 2011. Jian will start his PhD study in Horticultural Science at University of Florida in fall 2011. His prospective research will focus on the tomato breeding with molecular biology techniques and enhance the tomato resistance to pathogens. His career goal is to earn a position as a professor with a university or researcher with a corporation.