Progress 09/14/15 to 09/10/20
Outputs
Progress Report Objectives (from AD-416): Objective 1: Investigate biological control and ecological interactions of whiteflies with their natural enemies using banker plant systems to promote environmentally sound control in vegetable and ornamental crops. Sub-objective 1b: Determine the compatibility of insecticide regimes with beneficial insects and natural enemies used in banker plant systems. Objective 2: Investigate structural, physiological, molecular and chemical aspects of the whitefly feeding process and identify inhibitor strategies/molecules such as but not limited to feeding disruptors and peptide inhibitors of disease transmission than can be used in the development of novel interdiction strategies envisioned to work either through production of transgenic plants or application of chemical treatments that block feeding/disease transmission. Sub-objective 2a: Develop transgenic tobacco expressing enzymatic inhibitors of whitefly salivary sheath formation test for resistance to Bemisia tabaci feeding. Sub-objective 2b: Test application of discovered small molecule inhibitors of sheath formation for their effect on whitefly feeding on tomato. Sub-objective 2c: Conduct proteomic analysis of salivary exudates to identify salivary sheath structural and biosynthetic proteins. Objective 3: Use molecular strategies to develop disease resistant banker plants to support large numbers of whitefly populations for production of biocontrol agents for use in the greenhouse. Approach (from AD-416): Research will focus on constructing a nonflowering papaya banker plant through biotechnology that is resistant to both papaya ringspot virus and powdery mildew. Strategically timed insecticide applications including neonicotinoids in the rotation regimes will be evaluated against MED (Q- biotype whitefly) for whitefly efficacy and compatibility with two of the natural enemies used in our banker plant systems: the predatory mite, Amblyseius swirskii, and the whitefly parasitoid, Encarsia sophia. Development and testing molecular inhibitors of whitefly feeding processes with specific emphasis on the processes that must occur for the whitefly to develop a successful feeding event. Two main objectives include: 1) Continued characterization of the whitefly salivary sheath biosynthesis and composition. We already have basic compositional data and some knowledge on structural arrangement. Molecular, biochemical and structural analyses will continue to identify key biosynthetic enzymes and sheath structural components. 2) Evaluation of inhibitors of sheath formation as control agents. This evaluation will be performed using artificial diet assays and development of transgenic tobacco expressing inhibitors and conducting bioassays where the whitefly feed on the artificial diet or the transgenic plants. This serves as the final report for Project No. 6034-22320-003-00D. Greenhouse chemical efficacy trials were conducted on salvia with new chemistries of non-neonicotinoids to improve existing management strategies for Bemisia tabaci (MEDiterranean, biotype Q whitefly) & stewardship of the neonicotinoid class of insecticide. The compatibility of insecticides used in rotation programs to control B. tabaci with the predatory mite, Amblyseius swirskii used in biological control programs was evaluated. Treatments included untreated control, insecticide control, predatory mite & insecticide plus predatory mite. Seven new chemistries in addition to the grower neonicotinoid standard (dinotefuran) were evaluated & each chemical greenhouse trial was repeated in 16 trials. Reports were published in Arthropod Management Tests in real-time for immediate release to stakeholders. Predatory mite compatibility trials were evaluated at the highest label rate either as drench or foliar applications. Products evaluated included: dinotefuran (Safari), cyantraniliprole (Mainspring), pyrifloquinazon (Rycar), flupryadifurone (Altus), spinetoram+sulfoxaflor (Xxpire), pymetrozine (Endeavor), & afidopyropen (Inscalis). All products except pymetrozine (Endeavor) significantly reduced MED whitefly populations during the majority of the study period (5 weeks). All products except spinetoram+sufoxaflor (Xxpire) were compatible with the predatory mite. The new insecticides for effective MED whitefly control & compatibility with biocontrol agents will greatly benefit our nursery & ornamental growers. Six non-neonicotinoid insecticide rotation regimes compatible with biological control agents were evaluated for whitefly control at the low rate on salvia in the greenhouse. Evaluations were done with moderate & high whitefly populations. Rotation regimes consisted of an application every 21 days (times 3) with an insecticide strategically placed in a rotation so that the mode of action was never replicated & the drench method of application was only used once in any rotation regime to manage insecticide resistance development. Rotation regimes with only non- neonicotinoid insecticides at the lowest label rate (abamectin, afidopyropen, cyantraniliprole, flupyradifurone, pyrifluquinazon) overall provided good to excellent control of MED whitefly season-long compared to the untreated control under both moderate & heavy whitefly populations. Drenches did not perform as well at the low rate compared to the high rate evaluated in previous compatibility studies. In the event the neonicotinoid class of insecticides are pulled from the market, growers are being provided with effective alternatives for whitefly management with application options to minimize the overall cost of the rotation. To determine order of application effect on whitefly control, three of the best performers in the low rate rotation trials were evaluated as foliar sprays in different orders in the rotation (pyrifloquinazon/ flypyradifurone/abamectin) under high whitefly pressure. All 3 rotations provided excellent control indicating a grower had flexibility in which product to use depending on the individual pest spectrum in their greenhouse or nursery. The chilli thrips, Scirtothrips dorsalis, is a cryptic species complex (group of morphologically indistinguishable species) of at least nine distinct species, two (South Asia 1 & East Asia 1) of which exist in the United States. To determine their distribution range & find the dominant member of this thrips complex in the United States, a nationwide survey program is underway. In FY17, we received samples from Florida (3), California (3), Texas (2), Georgia (1) & Massachusetts (1). Out of the 25 thrips samples received from different locations in the United States. 15 samples were confirmed as S. dorsalis. The S. dorsalis samples collected from Florida, Texas & California were South Asia 1. East Asia 1 was only found in samples collected from hydrangea in Massachusetts. Cooperative Agricultural Pest Survey previously indicated chilli thrips would not pose a threat to northern states that experience hard freezes but apparently East Asia 1 is capable of surviving winters in both New York (first reported 2012) & now in Massachusetts. In FY18, thrips were processed from Florida (3), Texas (3), California (2), Massachusetts (1) & New York (1). Out of the 28 thrips samples processed from different locations in the United States, 19 samples were confirmed as S. dorsalis. The S. dorsalis samples collected from Florida, Texas, & California were South Asia 1. East Asia 1 was only found in two northeastern states (New York & Massachusetts) indicating this species is overwintering and moving to neighboring states on hydrangea plants. In FY19, we processed 17 samples from Georgia (3), Tennessee (4), Rhode Island (1) & Florida (9). Roses were the primary host plant sampled except in Georgia where thrips were sampled from Dystylium, Aucuba & blueberry & from Hemp in Florida. All adult thrips samples that were sequenced were South Asia 1 which is the predominant cryptic species to date in the U.S. In FY20, we received samples from Alabama (3), & California (6) but have not been able to analyze them due to COVID. To determine the host range of the Scirtothrips dorsalis, South Asia 1, we conducted 11 greenhouse trials, where we evaluated 62 plant taxa (55 different species within 35 families & 27 plant orders) among vegetables, ornamentals, landscape plants, herbs & weeds as feeding and/or reproductive hosts of chilli thrips. Among all the tested plant taxa, 40 were true hosts (reproductive + feeding) & five were only feeding hosts (thrips will feed but avoid laying eggs). We also found 17 new true hosts of chilli thrips which were not earlier reported as reproductive hosts of this pest in the literature. In collaboration with University of Florida, ARS researchers are developing barcodes for predatory mites. Traditional taxonomy positively identified cohorts of each predatory mite population available from commercial biocontrol companies or occurring naturally using key morphological traits under stereomicroscope slide mount. After mite populations were validated for purity of species, DNA was isolated and genomic DNA libraries were constructed for 23 predatory mite populations representing 7 species: Phytoseiulus persimilis (5), Neoseiulus californicus (4), N. cucumeris (4), N. fallacis (2), Amblyseius swirskii (4), A. andersoni (3), Amblydromalus limonicus (1). Libraries were sequenced using Illumina platform (PacBio sequencing was performed on two samples for development of a more complete reference scaffold) resulting in large volumes of raw sequencing data that were assembled independently for all libraries. Initial analysis of phylogenetic relationships among or between species using a reference population with the highest quality sequence library for each species & genes suitable for phylogeny construction is currently being conducted. Whole genome comparisons are being conducted for intra- & inter- population diversity analysis. Evaluation is underway for potential cryptic species issues & purity of commercially available samples as well as identification of diagnostic genome regions within & among species to develop & validate reliable Polymerase Chain Reaction barcode strategies for quick evaluation of predator mite samples. This project was originally written with a focus on inhibition of salivary sheaths as a whitefly control strategy. As part of this work, a method of salivary sheath inhibition was developed, demonstrated & patented. This method was based on enzymatic or small molecule inhibition of salivary sheath polymerization. However, as the project advanced it became clear that delivery of any biological molecule (i.e. small peptides or enzymes) was a major roadblock to commercialization because: 1) biological molecules such as peptides are expensive to produce & unstable in the environment & 2) transgenic plant development (engineering plants to produce sheath inhibitors) requires costly & time- consuming regulatory approval process incompatible with delivering a strategy across many ornamental & horticultural crops. For this reason, development of a suitable alternative delivery strategy for peptides & other gene product defense molecules (i.e. Ribonucleic acid interference (RNAi) inducing double-stranded RNAs) became a primary focus. As a result, a new method of delivering genetic engineering solutions to ornamental & horticultural crops was developed & patented. This method is based on producing host plant cells that replicate autonomously & when transplanted back to the host can grow into an organ-like structure that can produce defense molecules that are secreted into the plant vascular tissue & move systemically. For whitefly control, this method is especially advantageous because the control molecules are secreted into the phloem, the whitefly feeding site. Advantages of this strategy include: 1) modified cells can be transplanted directly onto the host plant allowing use of non-transgenic hosts of virtually any variety; 2) cells grow into an organ that cannot survive away from the plant & cannot form whole plants, seeds or pollen; thus, gene flow into the environment is not an issue: 3) harvested commodity (fruit, nut etc.) is not genetically engineered. Replicated greenhouse studies showed that, in tomato, the attachment & growth of this organ-like structure did not reduce yield of greenhouse grown tomato. Furthermore, development of organs expressing a plant peptide that regulates plant growth, causes expected alteration in tomato growth structure, thus demonstrating that proteins produced by these organs can induce systemic effects. This delivery strategy is now being evaluated for expression of defense genes that can either kill whitefly or interdict the feeding process. Accomplishments 01 Producing plant biofactory organs that are transplanted to important ornamental and horticultural crops offers a new way of delivering genetic engineering solutions to crop pest/pathogen problems.. There is an urgent need to deliver solutions for the control of whitefly and their transmitted diseases. Delivery of genetic engineering solutions to problems in crop production have a costly and time-consuming regulatory approval process and consumer acceptance can be an issue. Regulatory and consumer concerns include: 1) mobility of recombinant genetic material through pollen/seed or escape of engineered plant; 2) creating a weedy/invasive engineered plant; and 3) consumption of genetically engineered food commodities. ARS researchers at Fort Pierce, Florida, in collaboration with private industry, developed a method of delivering genetic engineering solutions through engineering only a group of cells that can be attached to plants (essentially as a new organ) to produce desired molecules that are secreted into the plant vascular tissue and move throughout the plant. This ⿿new organ cannot survive away from the plant and does not move from the location where it is attached, thus the harvested commodity (i.e. fruit, nut etc. ) is not genetically engineered. It also cannot form whole plants, seed or pollen; thus, there is no escape of genetic material. ARS scientists in Ft. Pierce, Florida, are evaluating the ability of this strategy to cure Huanglongbing (HLB)-infected trees in existing groves by engineering these organs to produce natural peptides and Ribonucleid acid (RNAs) that kill the HLB-causing bacterium, and attaching these organs to ornamental and/or horticultural crops. Proof-of-concept has been completed in tomato. This strategy could be adapted as a means to rapidly deliver genetic engineering solutions in an environmentally sustainable and consumer acceptable method.
Impacts
(N/A)
Publications
- McKenzie, C.L., Sparks, A.N., Roberts, P., Oetting, R., Osborne, L. 2020. Survey of Bemisia tabaci (Hemiptera: Aleyrodidae) in agricultural ecosystems in Georgia. Journal of Entomological Science. 55(2):163-170.
- Colmar, S., McKenzie, C.L., Luo, W., Osborne, L. 2020. First report of Bemisia tabaci Mediterranean (biotype Q)(Hemiptera: Aleyrodidae) in the Dominican Republic. Florida Entomologist. 102(4):778-782.
- Kumar, V., Mehra, L., McKenzie, C.L., Osborne, L. 2020. Functional response and prey stage preference of Delphastus catalinae and D. pallidus (Coleoptera: Coccinellidae) on Bemisia tabaci (Hemiptera: Aleyrodidae). Biocontrol Science and Technology.
- Borovksy, D., Nauwelaers, S., Shatters, R.G. 2020. Biochemical and molecular characterization of Pichia pastoris cells expressing multiple TMOF genes (tmfA) for mosquito larval control. Frontiers in Physiology. 11:527-541.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): Objective 1: Investigate biological control and ecological interactions of whiteflies with their natural enemies using banker plant systems to promote environmentally sound control in vegetable and ornamental crops. Sub-objective 1b: Determine the compatibility of insecticide regimes with beneficial insects and natural enemies used in banker plant systems. Objective 2: Investigate structural, physiological, molecular and chemical aspects of the whitefly feeding process and identify inhibitor strategies/molecules such as but not limited to feeding disruptors and peptide inhibitors of disease transmission than can be used in the development of novel interdiction strategies envisioned to work either through production of transgenic plants or application of chemical treatments that block feeding/disease transmission. Sub-objective 2a: Develop transgenic tobacco expressing enzymatic inhibitors of whitefly salivary sheath formation test for resistance to Bemisia tabaci feeding. Sub-objective 2b: Test application of discovered small molecule inhibitors of sheath formation for their effect on whitefly feeding on tomato. Sub-objective 2c: Conduct proteomic analysis of salivary exudates to identify salivary sheath structural and biosynthetic proteins. Objective 3: Use molecular strategies to develop disease resistant banker plants to support large numbers of whitefly populations for production of biocontrol agents for use in the greenhouse. Approach (from AD-416): Research will focus on constructing a nonflowering papaya banker plant through biotechnology that is resistant to both papaya ringspot virus and powdery mildew. Strategically timed insecticide applications including neonicotinoids in the rotation regimes will be evaluated against MED (Q- biotype whitefly) for whitefly efficacy and compatibility with two of the natural enemies used in our banker plant systems: the predatory mite, Amblyseius swirskii, and the whitefly parasitoid, Encarsia sophia. Development and testing molecular inhibitors of whitefly feeding processes with specific emphasis on the processes that must occur for the whitefly to develop a successful feeding event. Two main objectives include: 1) Continued characterization of the whitefly salivary sheath biosynthesis and composition. We already have basic compositional data and some knowledge on structural arrangement. Molecular, biochemical and structural analyses will continue to identify key biosynthetic enzymes and sheath structural components. 2) Evaluation of inhibitors of sheath formation as control agents. This evaluation will be performed using artificial diet assays and development of transgenic tobacco expressing inhibitors and conducting bioassays where the whitefly feed on the artificial diet or the transgenic plants. The overall goal is to improve existing management strategies for Bemisia tabaci (B. tabaci) (MED or biotype Q) whitefly and stewardship of neonicotinoid class of insecticide (dinotefuran - grower standard). Currently, six non-neonicotinoid insecticide rotation regimes, based on efficacy of the high rate compatibility studies with biological control agents, were evaluated for whitefly control. Rotation regimes consisted of an application every 21 days (times 3) with an insecticide strategically placed in a rotation so that the mode of action was never replicated and the drench method of application was only used once in any rotation regime to manage insecticide resistance development. Rotation regimes with only non-neonicotinoid insecticides at the lowest label rate (abamectin, afidopyropen, cyantraniliprole, flupyradifurone pyrifluquinazon) overall provided good to excellent control of MED whitefly season-long compared to the untreated control. Drenches did not perform as well at the low rate compared to the high rate evaluated in previous studies. In the event the neonicotinoid class of insecticides are pulled from the market, growers are being provided with effective alternatives for whitefly management that are compatible with biological control agents and can be used at the lowest label rates applied as foliar sprays to minimize the overall cost of the rotation. In collaboration with University in Florida, ARS researchers are developing barcodes for predatory mites. Traditional taxonomy positively identified cohorts of each predatory mite population available from commercial biocontrol companies or occurring naturally using key morphological traits under stereomicroscope slide mount. After mite populations were validated for purity of species, DNA was isolated and genomic DNA libraries were constructed for 23 predatory mite populations representing 7 species: Phytoseiulus persimilis (5), Neoseiulus californicus (4), Neoseiulus cucumeris (4), Neoseiulus fallacis (2), Amblyseius swirskii (4), Amblyseius andersoni (3), Amblydromalus limonicus (1). Librairies have been sequenced using Illumina sequencing (PacBio sequencing was performed on one sample for development of a more complete reference scaffold) resulting in large volumes of raw sequencing data. Genome assembly is complete for 15 of the 23 libraries. Initial analysis of phylogenetic relationships among or between species using a reference population with the highest quality sequence library for each species and genes suitable for phylogeny construction has already identified taxonomic ambiguities among the populations and genera that are being evaluated is currently being conducted. Once all genomes are assembled, whole genome comparisons will be initiated so intra-population diversity can be evaluated to screen for potential cryptic species issues and purity of commercially available samples and for identification of diagnostic genome regions within and among species to develop and validate reliable Polymerase chain reaction (PCR) barcode strategies for quick evaluation of predator mite samples. The chilli thrips, Scirtothrips dorsalis, is a cryptic species complex (group of morphologically indistinguishable species) of at least nine distinct species, two (South Asia 1 and East Asia 1) of which exist in the United States. To determine their distribution range and find the dominant member of this thrips complex in the United States, a nationwide survey program is underway for the past four years. In FY19, we processed 17 samples (114 total thrips: 93 adults/21 larvae) from Georgia (3), Tennessee (4), Rhode Island (1) and Florida (9). Unforturnately thrips larvae are difficult to identify using either morphological or molecular techniques. Roses were the primary host plant sampled except in Georgia where thrips were sampled from Dystylium ⿿Emerald Heights⿿, Aucuba ⿿Goldstrike⿿ and blueberry and Florida where one sample was from Hemp. Five of the samples did not contain any Scirtothrips dorsalis and were mixtures of other thrips species including Thrips tabaci, Scolothrips sp., Frankliniella bispinosa, Selenothrips rubrocinctus, Aeolothripidas sp. and possible Franklinothrips. All adult thrips samples that were sequenced were South Asia 1 which is the predominant cryptic species to date. ARS researchers in collaboration with University in Florida and University in New York as part of a FARMBILL grant conducted a biological control study to compare the ability of Phytoseiulis macropilis and Neoseiulus californicus to manage the Glover mite (Tetranychus gloveri). Both predatory mites managed this mite but at the end of the experiment Chilli thrips populations were severely damaging the controls plants (Tetranychus gloveri and no predators) and the plants treated with P. macropilis. The plants treated with Neoseilus californicus had very little damage and almost no Chilli thrips. The mean number of immature thrips per 10 sampled leaves were 30, 1.6 and 41.1 for the untreated control, Neoseilus californicus and Phytoseiulis macropilis treatments, respectively. Based on these preliminary results, Neoseilus californicus is an excellent predator of chilli thrips and glover mite. ARS researchers in collaboration with APHIS, Florida Department of Agriculture and Consumer Services and the University of Florida initiated research on the phantasma scale, Fiorinia phantasma Cockerell & Robinson (Hemiptera: Diaspididae). Phantasma scale is a significant pest of at least 24 families and 44 genera of nursery and ornamental plants, particularly palms, as well as several fruit crops. The primary purpose of this FARMBILL grant is to support research for enhanced mitigation capabilities against the invasive, newly established armored scale by determining its current spread in south Florida as well as its potential plant host range, by improving our ability to properly identify this pest to species through the production of a taxonomic key, and by identifying effective biological and chemical control options. ARS researchers in an international collaboration with Brazilian scientists are evaluating new biological molecules for the control of the Bemisia whitefly. Together they demonstrated the effectiveness of a plant- derived peptide that is specifically toxic to insects and constructed transgenic plants expressing this peptide. Evaluation of these plants demonstrated that they induced mortality to several Lepidopteran insects that are economically important phytophagous pests in crops. Analysis of transgenic tobacco plants expressing this peptide are on-going. To support this work we have advanced two methods of delivering peptides directly to plant: 1) a direct vascular tissue infusion device was demonstrated to systemically delivery peptides to citrus trees where it induced a greater than 90% reduction in a phloem-limited bacterium after one week from a single application. This device is now being tested in other crop systems including tomato and potato; 2) The ARS scientists have developed a method of localized plant tissue transformation, where a specific tissue within the plant is transformed to produce desired molecules (peptides). In such plants only the modified tissue is transformed and we have demonstrated rapid systemic activity of peptides produced in this transformed ⿿biofactory tissue. This system is now available for use in evaluating potential biocontrol peptides of any type. Accomplishments 01 Georgia survey of Bemisia tabaci. Bemisia tabaci (Gennaduis) is a large complex of cryptic species whose members are particularly invasive pests of hundreds of economically important commodities including cotton, vegetables and ornamental crops. In 2016, MED (Mediterranean; biotype Q) was detected in the field and residential landscapes for the first time in the United States. In 2018, a new state detection on poinsettia from a commercial Wisconsin greenhouse brought the total positive MED states to 27 indicating that MED is still expanding both its geographical and environmental ranges in the United States. With high whitefly populations that were difficult to control and new viruses in high dollar agricultural crops appearing across the southeast in 2016, growers were very concerned if MED had also moved into the field in Georgia. In collaboration with Universities of Georgia and Florida, ARS scientists in Ft. Pierce, Florida, conducted a whitefly survey in Georgia of agricultural ecosystems from field, greenhouse and nursery plants. Seventy samples were collected from 19 crops across 23 counties in Georgia with the bulk of the samples taken in 2016 and 2017. Overall, cotton was the most heavily sampled commodity (27) followed by seven samples of bell or ornamental pepper and six samples each of poinsettia, peanut, and squash. Other crops sampled included verbena, lantana, cucumber, soybean, cowpea, corn, snap bean, zucchini, kale, tomato, sweet potato, eggplant, cantaloupe, and mum. MED whitefly was detected on verbena in 2011 and poinsettia in 2012 at commercial greenhouses. All MED whitefly sequenced were identical and considered to have an Eastern Mediterranean origin. Only MEAM1 (Middle Eastern Asia Minor 1; biotype B) was detected in all the field grown commodities sampled in Georgia regardless of the year. This survey serves as a baseline for Georgia in the event that MED whitefly are eventually detected in the field. 02 First report of Bemisia tabaci MED (Q biotype) in the Dominican Republic. The first reports of Bemisia tabaci in the Dominican Republic (DR) date back to 1975 when the New World (NW) member of the complex (biotype A) caused severe damage in beans in the Southwestern provinces primarily due to transmission of bean golden mosaic virus. In 1994, MEAM1 (Middle Eastern Asia Minor 1, biotype B) was detected and quickly moved across the country spreading plant viruses and causing physiological disorders. Recent reports of insecticide resistance and increasing whitefly populations in tomatoes indicated Mediterranean (MED or biotype Q) whitefly may be present. ARS scientists in Ft. Pierce, Florida, and University of Florida in collaboration with the Instituto Dominicano de Investigaciones Agropecuarios y Forestales (IDIAF) conducted a whitefly survey to determine the distribution and composition of Bemisia tabaci cryptic species populations in the Dominican Republic. Sixteen samples were collected from 11 crops across 11 Provinces in the DR. Provinces included Azua, Distrito Nacional (2), Espaillat, Independencia, La Vega, Monseñor Nouel, San Juan (2), Santiago (2), Santo Domingo, Samaná, and Peravia (3). Host plants sampled included squash, eggplant, Asian spiderflower, mint, tomato, bean, tobacco, sesame, spurge, turkey berry, and Mexican prickly poppy. MED was detected for the first time in the DR from two different Provinces (Santo Domingo and Santiago) and host plants (tomato and tobacco) and environments (greenhouse and open field). MEAM1 was detected in all but one sample which was 100% MED. NW was detected twice on eggplant in different geographical regions and once on Mexican prickly poppy in a native habitat, but always with MEAM1. Widespread difficulties managing whitefly populations efficiently make additional confirmatory cryptic species detections necessary to determine the extent of the MED invasion in the country, especially where control failures have been reported. 03 A new method of delivering systemically active peptides to the vascular tissue of plants has been developed and demonstrated in tomato. This method allows the engineering of specific plant tissue that grows as a ⿿biofactory producing desired peptides that are secreted into the plant vascular system. ARS scientists in Ft. Pierce, Florida, have demonstrated systemic activity of peptides produced by localized biofactory tissue with this method. Using this strategy, only the ⿿biofactory tissue is transformed while the rest of the plant is non- transgenic. Systemic activity of biofactory-produced peptides can be visualized in as early as 3 weeks after initiation and provides a rapid way of screening for in planta bioactive peptide activity. It would also be a rapid way to apply transgenic technology to emerging problems in diverse crop genotypes without having to produce whole transgenic plants.
Impacts
(N/A)
Publications
- Taravati, S., Mannion, C., McKenzie, C.L., Osborne, L. 2018. Oviposition preference of rugose spiraling whitefly (Hemiptera:Aleyrodidae) on five host plant species. Florida Entomologist. 101(4):611-616.
- Taravati, S., Mannion, C., Osborne, L., McKenzie, C.L. 2019. Lethal and sublethal effects of selected systemic and contact insecticides on the whitefly predator, Nephaspis oculata (Coleoptera: Coccinellidae), in a tri- trophic system. Journal of Economic Entomology. 112(2):543-548.
- Avery, P.B., Kumar, V., Skvarch, E.A., Mannion, C.M., Powell, C.A., McKenzie, C.L., Osborne, L.S. 2019. An ecological assessment of Isaria fumosorosea compared to a neonicotinoid treatment for regulating invasive ficus whitefly. The Journal of Fungi. 5:36
- Kumar, V., Kakkar, G., Palmer, C., Myers, W., McKenzie, C.L., Osborne, L.S. 2019. Chilli thrips, Scirtothrips dorsalis - small player with big damage. Acta Horticulturae. 1232:247-251.
- Sahayaraj, K., Subash, N., Allingham, R.W., Kumar, V., Avery, P., Mehra, L. , McKenzie, C.L., Osborne, L.S. 2018. Lethal and sub-lethal effects of three microbial biocontrol agents on Spodoptera litura and its natural predator Rhynocoris kumarii. Insects. 9(3):101.
- Sahavaraj, K., Kumar, V., Banu, N., Avery, P., Radhika, A., McKenzie, C., Osborne, L. 2018. Predation potential of Rhynocoris marginatus (Hemiptera: Reduviidae) against three mealybug species of agricultural importance. Applied Entomology and Zoology.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): Objective 1: Investigate biological control and ecological interactions of whiteflies with their natural enemies using banker plant systems to promote environmentally sound control in vegetable and ornamental crops. Sub-objective 1b: Determine the compatibility of insecticide regimes with beneficial insects and natural enemies used in banker plant systems. Objective 2: Investigate structural, physiological, molecular and chemical aspects of the whitefly feeding process and identify inhibitor strategies/molecules such as but not limited to feeding disruptors and peptide inhibitors of disease transmission than can be used in the development of novel interdiction strategies envisioned to work either through production of transgenic plants or application of chemical treatments that block feeding/disease transmission. Sub-objective 2a: Develop transgenic tobacco expressing enzymatic inhibitors of whitefly salivary sheath formation test for resistance to Bemisia tabaci feeding. Sub-objective 2b: Test application of discovered small molecule inhibitors of sheath formation for their effect on whitefly feeding on tomato. Sub-objective 2c: Conduct proteomic analysis of salivary exudates to identify salivary sheath structural and biosynthetic proteins. Objective 3: Use molecular strategies to develop disease resistant banker plants to support large numbers of whitefly populations for production of biocontrol agents for use in the greenhouse. Approach (from AD-416): Research will focus on constructing a nonflowering papaya banker plant through biotechnology that is resistant to both papaya ringspot virus and powdery mildew. Strategically timed insecticide applications including neonicotinoids in the rotation regimes will be evaluated against MED (Q- biotype whitefly) for whitefly efficacy and compatibility with two of the natural enemies used in our banker plant systems: the predatory mite, Amblyseius swirskii, and the whitefly parasitoid, Encarsia sophia. Development and testing molecular inhibitors of whitefly feeding processes with specific emphasis on the processes that must occur for the whitefly to develop a successful feeding event. Two main objectives include: 1) Continued characterization of the whitefly salivary sheath biosynthesis and composition. We already have basic compositional data and some knowledge on structural arrangement. Molecular, biochemical and structural analyses will continue to identify key biosynthetic enzymes and sheath structural components. 2) Evaluation of inhibitors of sheath formation as control agents. This evaluation will be performed using artificial diet assays and development of transgenic tobacco expressing inhibitors and conducting bioassays where the whitefly feed on the artificial diet or the transgenic plants. With the overall goal to improve existing management strategies for Bemisia tabaci (B. tabaci) (MED or biotype Q) whitefly and stewardship of neonicotinoid class of insecticide (dinotefuran - grower standard), eight greenhouse chemical efficacy trials were conducted on salvia. In these trials, the compatibility of non-neonic insecticides used in rotation programs to control Bemisia tabaci with the predatory mite, Amblyseius swirskii used in biological control programs was evaluated. Experiments testing flupyradifurone and afidopyropen as a drench application was compatible with the predatory mite, and were found significantly reducing whitefly population during the majority of the study period. Foliar application of spinetoram + sulfoxaflor, pyrifluquinazon and flupyradifurone was effective against whitefly; however, among these only flupyradifurone was found benign on the mite population. To complement the previous study, additional studies were conducted where the compatibility of insecticides used to control Bemisia tabaci with the parasitic wasp, Eretmocerus eremicus used in biological control programs was evaluated on mint in the greenhouse. Eretmocerus eremicus is among the parasitoids which utilize their prey for both food and site of reproduction resulting in suppression of the pest population on the plant. In this study, whitefly parasitization by wasps was insignificant, and thus parasitized immatures and emerged wasps were not observed. Results showed that the drench application of flupyradifurone, and the parasitic wasps applied alone or in combination can suppress whitefly population = 5 weeks. The chilli thrips, Scirtothrips dorsalis (S. dorsalis), is a cryptic species complex (group of morphologically indistinguishable species) of at least nine distinct species, two (South Asia 1 and East Asia 1) of which exist in the United States. To determine their distribution range and find the dominant member of this thrips complex in the United States, a nationwide survey program is underway for the past two years. In Fiscal Year 2018, thrips samples were processed from three counties of Florida (Hillsborough, Orange, and Indian River), two counties in Texas (Fort Bend, and Waller), two counties in California (Los Angeles, and Riverside) , and one county in Massachusetts (Barnstable) and New York (Suffolk). Out of the 28 thrips samples processed from different locations in the United States, 19 samples were confirmed as S. dorsalis. The S. dorsalis samples collected from Florida, Texas, and California were South Asia 1 species suggesting this species is more prevalent (dominant species) in the United States compared to another counterpart of S. dorsalis complex reported for the first time from the north eastern United States during 2012. East Asia 1 was only found on hydrangea samples collected from two eastern states (New York and Massachusetts) indicating movement of this species in the neighboring states on hydrangea plants. Identity of S. dorsalis samples collected from blueberry in Hillsborough County, Florida still needs to be verified. Apart from S. dorsalis, eight different thrips species were also identified from the collected samples that includes Frankliniella occidentalis Pergande, Frankliniella schultzei Trybom, Frankliniella bispinosa Morgan, Frankliniella cephalica Crawford, Echinothrips americanus Morgan, Neohydatothrips burungae (Hood) Pseudothrips inaequalis (Beach) and Thrips hawaiiensis Morgan. Additional thrips samples from ornamental nurseries in Alabama, North Carolina, Louisiana and Connecticut are expected during 2018. To determine the host range of the dominant member of chilli thrips, Scirtothrips dorsalis complex, South Asia 1, we conducted four greenhouse trials, where we evaluated 25 plant taxa (21 different species within 13 families and 11 plant orders) among ornamentals, landscape plant, herb and flowering shrubs as feeding and/or reproductive hosts of this pest. Among all the tested plant taxa, 16 were true hosts (reproductive plus feeding) and one was just feeding hosts (thrips will feed on these but avoid laying eggs). We also found eight new true hosts of chilli thrips which were not earlier reported as the reproductive hosts of this pest in the literature. Native to the Neotropical region, Aleurotrachelus trachoides Back, commonly known as the solanum or pepper whitefly, is an emerging pest of pepper and many other horticultural crops in the United States. Feeding by this pest can cause stress to the plant by removing nutrients and water and the sheer numbers of whitefly can bring down plants and cause death. It has been in the United States for more than five decades as an intermittent pest of pepper although, until recently, was never considered a key pest of economic importance. However, in the past few years, records of its spread and damage have been reported from private residences, nurseries and vegetable growers (including tomato) throughout Florida. Aleurotrachelus trachoides is not a known vector of any plant damaging viruses, but to reassure tomato growers that this whitefly does not have the ability to acquire and transmit tomato yellow leaf curl virus, experiments are currently in progress. Traditional taxonomy for mite identification is becoming a lost art and expertise is extremely difficult to acquire. In collaboration with University of Florida, ARS researchers are developing barcodes for predatory mites that are commercially available as well as those native to Florida. Traditional taxonomy is being used to identify mites morphologically and then directly correlate those populations with complementary (cDNA) library construction for deep sequencing data mining for identification of the best diagnostic genes for identification. Cryptic species analysis and intra and inter species variation for selected genes will be validated. Benefits to stakeholders include new tools to identify new predators and also help identify predators that are working in commercial settings, especially when growers are using multiple species and sources. Rugose spiraling whitefly (RSW) is an exotic invasive species which had an outbreak in south Florida that became economically damaging to numerous plant species in many urban areas. Oviposition preference was determined for the following five known plant hosts: coconut, gumbo limbo, avocado, black olive v. shady lady and giant white bird of paradise (GWBP). Gumbo limbo was the most preferred and also had the highest number of eggs per spiral. GWBP was the least preferred host plant species but no statistically significant difference was found among the survival of RSW on different host plant species tested. Also, no significant correlation was found between the leaf area and number of eggs on each host species. Nephaspis oculata is a whitefly beetle predator which has been reported feeding on several whitefly species including rugose spiraling whitefly (RSW) in south Florida. The management of RSW has been heavy reliant on the use of insecticides which may negatively impact biological control agents. We studied the effect of bifenthrin (spray) and imidacloprid (drench) application on survival, fecundity, and behavior of Nephaspis oculata in the laboratory. Generally, beetles avoided bifenthrin treatments and did not survive as long as imidacloprid treatments. The results from this study shows that systemic imidacloprid is not repellent to Nephaspis oculata and does not significantly affect mortality of adult beetles in the tri-trophic system tested. Therefore, using systemic imidacloprid and Nephaspis oculata for controlling RSW might be compatible or at least not significantly incompatible. Future studies in the field may help us to have a better understanding about such interactions in the landscape. Work on discovery of new molecules for the control of the Bemisia whitefly was advanced through international collaboration with scientists in Brazil. Together we have demonstrated the effectiveness of a plant- derived peptide that is specifically toxic to insects and constructed transgenic plants expressing this peptide. Evaluation of these plants demonstrated that they induced mortality to several Lepidopteran insects that are economically important phytophagous pests in crops. This research has led to the production of transgenic tobacco plants expressing this peptide that are now being used for feeding studies with Bemisia tabaci whiteflies. To support this work we have developed a method to deliver peptides directly into the phloem tissue and we are evaluating this strategy for delivery of the peptide to crop plants on which Bemisia tabaci is a pest. Because isolation and analysis of psyllid salivary sheaths was easier than that for the whitefly and because we previously demonstrated that the same sheath inhibitors worked for both insects, ARS researchers used the psyllid system as a surrogate for the whitefly. We have identified and purified a single highly abundant approximately 2.8kD peptide in salivary secretions of the psyllid and we hypothesis that this peptide is an important structural component of the salivary sheaths. Sequencing of this peptide is being done through a collaboration with a research molecular biologist, USDA-Agricultural Research Service, Ithaca, New York. This discovery has led to development of a research program to identify inhibitors of one of these proteins/enzymes. Accomplishments 01 Pre-shipping treatments of biopesticides mitigates trans-boundary movement of whitefly. Bemisia tabaci (B. tabaci) cryptic species complex represents a major threat to several important crops including vegetables, cereals, fruits, and ornamentals worldwide. One important mode of its dispersal is through the trans-boundary (domestic, international) movement of infested plant materials. To prevent the spread of this invasive whitefly pest on inter-state ornamental shipments, ARS researchers at Ft. Pierce, Florida, in collaboration with researchers from a university in Florida tested the efficacy of biopesticides through dip treatments of plant material pre-shipping. In several experiments, clean Mentha sp. (Lamiaceae) mint plants were exposed to adults of B. tabaci for 1 or 6 days. Adults were removed from plants and eggs, and immature stages were treated with one or a combination of the following: entomopathogenic fungi Beauveria bassiana (BotaniGard®), Isaria fumosorosea (Preferal®), the surfactant Naturl Oil¿, and soap (Publix®). After treatment application, plants were placed in commercial cardboard boxes and shipped from Apopka, Florida to Fort Pierce, Florida. Assessments for the treatment efficacies were conducted at 3, 7, and 14 days post arrival of the shipment. Fungal entomopathogens; B. bassiana, I. fumosorosea, and the surfactant Naturl Oil¿, showed significant reduction in the whitefly population compared to the untreated control (P < 0.05). Significantly higher corrected mortality of the whitefly populations was observed when Beauveria bassiana was applied alone (81%) or in combination with Naturl Oil¿ (86%), as well as Isaria fumosorosea applied in combination with Naturl Oil¿ (77%). Our results showed that pre- shipping dip treatment applications of either Naturl Oil¿ mixed with Beauveria bassiana or Isaria fumosorosea or Beauveria bassiana applied alone could help mitigate the inter-state spread of whitefly on ornamental shipments. 02 Horticultural oils compatible with entompathogenic fungi for Asian Citrus Psyllid Control. Horticultural oils are an important component of integrated management programs of several phytophagous arthropods and pathogens affecting fruit, ornamentals and vegetables in greenhouse and field production systems. Although effective against the target pest, their incompatibility with biological control agents can compromise efforts to develop eco-friendly management programs for important agricultural pests. ARS researchers at Ft. Pierce, Florida, in collaboration with University of Florida assessed the in-vitro effect of selected refined petroleum oils used in citrus and other horticultural crops with a biopesticide containing the entomopathogenic fungi, Isaria fumosorosea (PFR-97) under laboratory conditions. Leaf disk bioassays were used to evaluate the combined efficacy of petroleum oils and PFR-97 against the Asian citrus psyllid, Diaphorina citri, a major pest of citrus in the United States. All five petroleum oil treatments were compatible with PFR-97 blastospores, as none of them were found to affect colony-forming units and radial fungal growth measured up to 12 days post-inoculation. All mixed treatments performed better than PFR-97 alone against Diaphorina citri. Results indicated that horticultural oils in combination with PFR-97 could offer citrus growers an alternative treatment for integrating into their current management programs while battling against Diaphorina citri in citrus production systems. Due to their eco-friendly, broad-spectrum effect, it could provide control against various citrus pests, while also encouraging the retention of effective chemistries for a longer period in the marketplace. However promising, these combination treatments need to be tested further with PFR-97 under grove conditions to confirm their field efficacy. 03 New insect-toxic peptide (Jaburetox from Jack bean) shown to be effective at controlling insects when expressed in transgenic plants. Currently, the only commercial transgenic method for insect control involves the use of bacterial toxins called Bacillus thuringiensis (BT) endotoxin. Because of this limited diversity of control strategies, insect resistance to BT is becoming a major concern. Therefore new gene products that provide effective insect control are needed. Furthermore, no BT-toxin has been identified that works against hemipterans. Jaburetox is a plant derived insect toxin that has shown activity against some hemipterans. Therefore in collaboration with researchers from Brazil, ARS researchers at Ft. Pierce, Florida, have evaluated activity of this peptide in laboratory bioassays and in transgenic plants and demonstrated high insect mortality and developmental disruption in pest insects. They have now produced transgenic tobacco to study Jaburetox¿s effect on Bemisa tabaci. These experiments are currently underway and demonstration of their effectiveness will create a new strategy in the fight against this severe crop pest.
Impacts
(N/A)
Publications
- Aristizabal, L., Avery, P., Kumar, V., Caldwell, J., McKenzie, C.L., Osborne, L.S. 2018. Mitigating trans-boundary movement of Bemisia tabaci (Hemiptera: Aleyrodidae) on Mentha sp by pre-shipping treaments of biopesticides. Crop Protection. 107:71-78.
- Kumar, V., Kakkar, G., Seal, D.R., McKenzie, C.L., Osborne, L.S. 2017. Evaluation of insecticides for curative, preventative and rotational use on Scirtothrips dorsalis South Asia 1 (Thysanoptera: Thripidae). Florida Entomologist. 100(3):634-646.
- Kumar, V., Francis, A., Avery, P., McKenzie, C.L., Osborne, L.S. 2018. Assessing compatibility of Isaria fumosorosea and buprofezin for mitigation of Aleurodicus rugioperculatus (Hemiptera: Aleyrodidae) - an invasive pest in the Florida landscape. Journal of Economic Entomology. 111(3):1069-1079.
- Thomson, M., Sahavaraj, K., Kumar, V., Avery, P.B., McKenzie, C.L., Osborne, L.S. 2017. Mass rearing and augmentative biological control of Rhynocoris fuscipes (Hemiptera: Reduviidae) against multiple pests of cotton. Pest Management Science. 73(8):1743-1752.
- Kumar, V., Dickey, A.M., Seal, D.R., Shatters, R.G., Osborne, L.S., McKenzie, C.L. 2017. Unexpected high intragenomic variation in two of three major pest thrips species does not affect ribosomal transcribed spacer 2(ITS2) utility for thrips identification. International Journal of Molecular Sciences. 18(10):2100.
- Stover, E.W., Shatters, R.G., Gruber, B., Kumar, Moore, G.A. 2016. Influence of photoperiod duration and phloem disruption through scoring on growth, disease symptoms and bacterial titer in citrus graft-inoculated with Candidatus Liberibacter asiaticus. HortScience. 51:1215-1219.
- Ammar, E.D., Hall, D.G., Shatters, R.G. 2017. Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing-disease bacteria. Journal of Microscopy and Ultrastructure. 5(1):9-20.
- Zhang, S., Shi, Q., Albrecht, U., Shatters, R.G., Stange Jr, R.R., McCollum, T.G., Zhang, S., Fan, C., Stover, E.W. 2017. Comparative transcriptome analysis during early fruit development between three seedy citrus genotypes and their seedless mutants. Horticulture Research. 4:17041.
- Hall, D.G., Borovsky, D., Chauhan, K.R., Shatters, R.G. 2018. An evaluation of mosquito repellents and essential plant oils as deterrents of Asian citrus psyllid. Crop Protection. 108:87-94.
- Borovsky, D., Nauewelaers, S., Powell, C., Shatters, R.G. 2017. Cloning, genetic engineering and characterization of TMOF expressed in Saccharomyces cerevisiae to control larval mosquitoes. Journal of Insect Physiology. 106(2):134-146.
- Mann, M., Fattah-Hosseini, S., Ammar, E., Strange, R., Warrick, E., Sturgeon, K., Shatters, R.G., Heck, M.L. 2018. Diaphorina citri nymphs are resistant to morphological changes induced by ¿Candidatus Liberibacter asiaticus¿ in midgut epithelial cells. Infection and Immunity. 86(4) :e00889-17.
- Kruse, A., Fattah-Hosseini, S., Saha, S., Johnson, R., Warwick, E., Sturgeon, K., Mueller, L., Maccoss, M.J., Shatters, R.G., Cilia, M. 2017. Combining omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut. PLoS One.
- Saha, S., Hosmani, P.S., Billalobos-Ayala, K., Miller, S., Shippy, T., Flores, M., Rosendale, A., Cordola, C., Bell, T., Mann, H., DeAvila, G., DeAvila, D., Moore, Z., Buller, K., Ciolkevich, K., Nandyal, S., Mahoney, R., Van Voorhis, J., Dunlevy, M., Farrow, D., Hunter, D., Morgan, T., Shore, K., Guzman, V., Izsak, A., Dixon, D.E., Cridge, A., Cano, L., Cao, X., Jiang, H., Leng, N., Johnson, S., Cantarel, B.L., Richards, S., English, A., Shatters, R.G., Childers, C., Chen, M., Cilia, M., Mueller, L. , Munoz-Torres, M., Nelson, D., Poelchau, M.F., Benoit, J., Wiersma-Koch, Brown, S.J. 2017. Improved annotation of the insect vector of citrus greening disease: Biocuration by a diverse genomics community. bioRxiv.
- Kumar, V., Avery, P., Ahmed, J., Cave, R., McKenzie, C.L., Osborne, L.S. 2017. Compatibility and efficacy of Isaria fumosorosea with horticultural oils for mitigation of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). Insects. 8:119. doi.org/10.3390/insects.8040119.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): Objective 1: Investigate biological control and ecological interactions of whiteflies with their natural enemies using banker plant systems to promote environmentally sound control in vegetable and ornamental crops. Sub-objective 1b: Determine the compatibility of insecticide regimes with beneficial insects and natural enemies used in banker plant systems. Objective 2: Investigate structural, physiological, molecular and chemical aspects of the whitefly feeding process and identify inhibitor strategies/molecules such as but not limited to feeding disruptors and peptide inhibitors of disease transmission than can be used in the development of novel interdiction strategies envisioned to work either through production of transgenic plants or application of chemical treatments that block feeding/disease transmission. Sub-objective 2a: Develop transgenic tobacco expressing enzymatic inhibitors of whitefly salivary sheath formation test for resistance to Bemisia tabaci feeding. Sub-objective 2b: Test application of discovered small molecule inhibitors of sheath formation for their effect on whitefly feeding on tomato. Sub-objective 2c: Conduct proteomic analysis of salivary exudates to identify salivary sheath structural and biosynthetic proteins. Approach (from AD-416): Research will focus on constructing a nonflowering papaya banker plant through biotechnology that is resistant to both papaya ringspot virus and powdery mildew. Strategically timed insecticide applications including neonicotinoids in the rotation regimes will be evaluated against MED (Q- biotype whitefly) for whitefly efficacy and compatibility with two of the natural enemies used in our banker plant systems: the predatory mite, Amblyseius swirskii, and the whitefly parasitoid, Encarsia sophia. Development and testing molecular inhibitors of whitefly feeding processes with specific emphasis on the processes that must occur for the whitefly to develop a successful feeding event. Two main objectives include: 1) Continued characterization of the whitefly salivary sheath biosynthesis and composition. We already have basic compositional data and some knowledge on structural arrangement. Molecular, biochemical and structural analyses will continue to identify key biosynthetic enzymes and sheath structural components. 2) Evaluation of inhibitors of sheath formation as control agents. This evaluation will be performed using artificial diet assays and development of transgenic tobacco expressing inhibitors and conducting bioassays where the whitefly feed on the artificial diet or the transgenic plants. Wrote and submitted a USDA, NIFA, Specialty Crop Research Initiative grant entitled �Building A Better Banker-Plant Strategy To Manage Invasive Whiteflies: Using Precision Breeding For Improved Efficacy And Expanded Use In Greenhouse Grown Vegetables and Ornamentals�. This was done through a collaboration with University of Florida scientists with expertise in plant pathology, economics and entomology extension. The chilli thrips, Scirtothrips dorsalis, is a cryptic species complex (group of morphologically indistinguishable species) of at least nine distinct species, two (South Asia 1 and East Asia 1) of which exist in the United States. To determine their distribution range and find the dominant member of this thrips complex in the United States, a nationwide survey program is underway. To date, we received thrips samples from three counties in Florida (Hillsborough, Miami-Dade, and St. John), three counties in California (Los Angeles, Orange and Imperial), two counties in Texas (Harris, Brazos) and one county each in Georgia (McDuffie) and Massachusetts (Barnstable). Out of the 25 thrips samples received from different locations in the United States, 15 samples were confirmed as Scirtothrips dorsalis. The Scirtothrips dorsalis samples collected from Florida, Texas and California were South Asia 1 species suggesting this species is more prevalent (dominant species) in the United States compared to another counterpart of Scirtothrips dorsalis complex reported for the first time from the north eastern United States during 2012. East Asia 1 was only found in the samples collected from hydrangea in Massachusetts indicating movement of this species in the neighboring states on hydrangea. Cooperative Agricultural Pest Survey (CAPS) had previously indicated that chilli thrips would not pose a threat to northern states that experience hard freezes but apparently East Asia 1 is capable of surviving winters in both New York and now in Massachusetts. Identity of Scirtothirps dorsalis samples collected from roses in Riverside County, California, and Dallas County, Texas, areas still needs to be verified. Apart from Scirththips dorsalis, other thrips received during surveys were Frankliniella occidentalis, Frankliniella tritici and Leucothrips piercei. We expect to receive additional thrips samples from rose nurseries in Alabama, North Carolina, Louisiana and Texas during summer-fall 2017. To determine the host range of the Scirtothips dorsalis, South Asia 1, we conducted seven greenhouse trials, where we evaluated 37 plant taxa (34 different species within 22 families and 16 plant orders) among vegetables, ornamentals, landscape plants, herbs and weeds as feeding and/ or reproductive hosts of chilli thrips. Among all the tested plant taxa, 24 were true hosts (reproductive + feeding) and four were only feeding hosts (thrips will feed on these but avoid laying eggs). We also found nine new true hosts of chilli thrips which were not earlier reported as reproductive hosts of this pest in the literature. With the overall goal to improve existing management strategies for Bemisia tabaci (MEDiterranean or biotype Q whitefly) and stewardship of the neonicotinoid class of insecticide (dinotefuran - grower standard), seven greenhouse chemical efficacy trials were conducted on salvia. In these trials, the compatibility of insecticides used in rotation programs to control Bemisia tabaci with the predatory mite, Amblyseius swirskii used in biological control programs was evaluated. Experiments testing cyantraniliprole and flupyradifurone as a drench application was compatible with the predatory mite, and were found to significantly reduce whitefly populations during the majority of the study period. Foliar application of spinetoram, pyrifluquinazon and flupyradifurone was effective against whitefly, however among these only flupyradifurone was found benign on the mite population. Although foliar application of pymetrozine was compatible with the predaceous mite, it was not effective against MED whitefly by itself. To complement the previous study, additional studies were conducted where the compatibility of insecticides used to control Bemisia tabaci with the parasitic wasp, Eretmocerus eremicus, used in biological control programs was evaluated on mint in the greenhouse. Eretmocerus eremicus is among the parasitoids which utilize their prey for both food and sites of reproduction resulting in suppression of the pest population on the plant. In this study, whitefly parasitization by wasps was insignificant, and thus parasitized immatures and emerged wasps were not observed. Results showed that the drench application of cyantraniliprole and flupyradifurone, and the parasitic wasps applied alone or in combination can suppress whitefly population more than 5 weeks. In order to integrate biocontrol agents in the management program of Bemisia tabaci, predation potential of two naturally occurring predatory beetles (Delphastus catalinae and Delphastus pallidus) was evaluated against the two most notorious members (Mediterranean or MED, biotype Q and Middle Eastern Asia Minor 1 or MEAM1, biotype B) of Bemisia tabaci species complex. Under laboratory conditions, each of the two beetles was found to consume an average of 50-65 whitefly eggs per day, and feeding potential of Delphastus cataliane was higher than Delphastus pallidus. While comparing prey developmental stage preference between whitefly eggs and early immatures, both the beetle species preferred eggs over nymphs. Native to the Neotropical region, Aleurotrachelus trachoides commonly known as the solanum whitefly, is an emerging pest of pepper and many other horticultural crops in the United States. Lab bioassay trials are underway to evaluate the biocontrol potential of two generalist predatory beetles Delphatus catalinae and Delphastus pallidus against solanum whitefly on pepper as a host. For the studies, separate colonies of the two beetles and whitefly have been initiated. The pathogenicity of a naturally occurring entomopathogenic fungus, Isaria fumosorosea (PFR 97�) was evaluated under laboratory conditions against solanum whitefly on tomato as a host. In the preliminary studies, Isaria fumosorosea was found ineffective (mortality <50%) against the whitefly pest. Similar studies with another major whitefly plant host (pepper) is underway to determine the effect of plant hosts on the activity of entomopathogenic fungus for controlling this whitefly. In the wake of the potential threat of solanum whitefly to Florida tomato production, which is one of the important hosts of this pest, a greenhouse study is being conducted to assess the ability of solanum whitefly to vector Tomato Yellow Leaf Curl Virus disease. For the studies, whitefly colony on tomato has been initiated. Work on salivary sheath inhibition as a method of blocking Bemisa tabaci feeding was advanced. We have continued evaluations of topical application strategies for initial analysis instead of transgenic plant development. This decision was justified because of: 1) breakthroughs we have had in development of new topical applications strategies for the control of Asian citrus psyllid (ACP) sheath formation/sheath production; 2) our earlier demonstration that sheath inhibitors that work on ACP sheaths also work on Bemisia tabaci sheaths; and 3) completion of filing for patent to cover this technique that has resulted in initiation of a CRADA with a private company to advance this to a commercial product. Because isolation and analysis of psyllid salivary sheaths was easier than that for the whitefly and because we previously demonstrated that the same sheath inhibitors worked for both insects, ARS scientists used the psyllid system as a surrogate for the whitefly. We have conducted initial salivary sheath proteome analysis using the Asian citrus psyllid as a surrogate. This work was done in collaboration with a research molecular biologist, USDA-ARS, BioIPM Research Unit, Ithaca, New York, and resulted in the identification of two proteins presumed to be involved in sheath biosynthesis. This discovery has led to development of a research program to identify inhibitors of one of these proteins/ enzymes. Accomplishments 01 Urban invasion by serious whitefly pest spurs regulatory action. Bemisia tabaci MEDiterranean (Q biotype) is on the move in Florida to residential landscapes and may impact open field agriculture. For the first time in the United States, researchers from ARS in Fort Pierce, Florida and the University of Florida in Apopka, Florida have detected MED outside of greenhouse or nursery environments when it was collected from ten residential landscapes and two open field environments in Florida. MED was also detected in 11 wholesale nurseries from five counties and 19 retail nurseries from 8 counties in Florida resulting in stop sale actions of facilities with high whitefly populations. Hibiscus was the host plant driver for MED whitefly infestations in both retail and wholesale nurseries and in Florida residential landscapes. This situation has the potential to become a major agricultural problem if this whitefly known to be resistant to insect growth regulators and neonicotinoid insecticides spreads to vegetable field production. 02 Susceptibility of Bemisia tabaci to nicotinic insecticides in South Florida Tomato. Researchers from the University of Florida, Wimauma, Florida in collaboration with Saint Francis University, Loretto, Pennsylvania and ARS researchers at Fort Pierce, Florida, determined the insecticide susceptibility of populations of whitefly in vegetable fields in south Florida. Populations of Bemisia tabaci MEAM1 (Middle Eastern Asia Minor 1, biotype B) were established from nineteen locations in south Florida, primarily from commercial tomato fields, and were tested using a cotton leaf petiole systemic uptake method for susceptibility to the nicotinic acetylcholine agonist insecticides imidacloprid, thiamethoxam, dinotefuran and flupyradifurone. Eleven populations produced lethal concentration for 50% of the tested population (LC50s) for one or more chemicals that were not significantly different from the susceptible laboratory colony based on overlapping fiducial limits, indicating some degree of susceptibility. LC50s more than a 100-fold in the laboratory colony were measured in at least one population for each material tested, indicating tolerance. Based on overlapping fiducial limits, there were no significant differences in relative mean potency estimates for flupyradifurone and dinotefuran in relation to imidacloprid and thiamethoxam. Monitoring tolerance of Bemisia tabaci to neonicotinoid insecticides and establishing baseline information for new insecticides such as flupyradifurone are essential for the sustainable stewardship of crucial crop protection tools. 03 Identified proteins used by plant-feeding Hemipteran pest insects to develop a successful feeding event on host plants. ARS researchers at Ft. Pierce, Florida, in collaboration with ARS researchers at, USDA-ARS, BioIPM Research Unit, Ithaca, New York, identified proteins secreted during establishment of a successful feeding event by Hemipteran insects. Using psyllids as a surrogate for whiteflies because of their more prolific feeding and ease of salivary sheath isolation, we identified two proteins as major components of their saliva that is secreted during the piercing process that is used to establish a plant vascular tissue feeding event. These proteins are now being used to screen for inhibitors of their activity as a means of preventing salivary sheath formation and thus inhibiting the insect�s ability to feed on plants. Development of a commercially available feeding inhibitor of this process would offer a sustainable strategy to prevent crop damage resulting from feeding by this class of insects.
Impacts
(N/A)
Publications
- Dickey, A., Kumar, V., Morgan, J.K., Jara-Cavieres, A., Shatters, R.G., McKenzie, C.L., Osborne, L. 2015. A novel mitochondrial genome architecture in thrips (Insecta: Thysanoptera): extreme size asymmetry among chromosomes and possible recent control region duplication. BMC Genomics. 16:439. doi:10.1186/s12864-015-1672-4.
- Dickey, A.M., Trease, A.J., Jara-Cavieres, A., Kumar, V., Christenson, M.K. , Potluri, L.P., Morgan, J.K., Shatters, R.G., McKenzie, C.L., Davis, P.H., Osborne, L. 2014. Estimating bacterial diversity in Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) via next generation sequencing. Florida Entomologist. 97:362-366.
- Dickey, A.M., Kumar, V., Hoddle, M.S., Funderburk, J.E., Morgan, J.K., Jara-Cavieres, A., Shatters, R.G., Osborne, L.S., McKenzie, C.L. 2015. The Scirtothrips dorsalis species complex: Endemism and invasion in a global pest. PLoS One. 10(4):1-22. doi:10.1371/PO.2015-0123747.
- McKenzie, C.L., Osborne, L.S. 2017. Bemisia tabaci MED (Q biotype) (Hemiptera: Aleyrodidae) is on the move in Florida to residential landscapes and may impact open field agriculture. Florida Entomologist. 100(2):481-484.
- Smith, H.A., Nagle, C.A., MacVean, C.A., McKenzie, C.L. 2016. Susceptibility of Bemisia tabaci MEAM1 (Hemiptera: Aleyrodidae) to imidacloprid, thiamethoxam, dinotefuran and flupyradifurone in south Florida. Insects. 7(57). doi:10.3390/I.2016-7040057.
- Barr, N., Ruiz-Arce, R., Obregon, O., Shatters, R.G., Norrbom, A.L., Nolazco, A., Thomas, D.B. 2017. Diagnostic characters within ITS2 DNA support molecular identification of Anastrepha suspensa. Florida Entomologist. 100:182-185.
- Kumar, V., Xiao, Y., McKenzie, C.L., Osborne, L.S. 2015. Early establishment of the phytoseiid mite Amblyseius swirskii (Acari: Phytoseiidae) on pepper seedlings in a Predator-In-First approach. Experimental and Applied Acarology. 65(1):465-481.
- Taravati, S., Mannion, C., Osborne, L., McKenzie, C.L. 2016. Feeding and development of Nephaspis oculata (Coleoptera: Coccinellidae) on rugose spiraling whitefly. Florida Entomologist. 99(3):516-521.
- Dickey, A.M., Stocks, I.C., Smith, T., Osborne, L.S., McKenzie, C.L. 2015. DNA barcode development for three recent exotic whitefly (Hemiptera: Aleyrodidae) invaders in Florida. Florida Entomologist. 98(2):473-478.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Objective 1: Investigate biological control and ecological interactions of whiteflies with their natural enemies using banker plant systems to promote environmentally sound control in vegetable and ornamental crops. Sub-objective 1a: Build a better banker plant through biotechnology by genetically engineering papaya to contain attributes that make it more suitable for banker plant use: i.e. nonflowering, resistant to papaya ringspot virus and powdery mildew. Sub-objective 1b: Determine the compatibility of insecticide regimes with beneficial insects and natural enemies used in banker plant systems. Objective 2: Investigate structural, physiological, molecular and chemical aspects of the whitefly feeding process and identify inhibitor strategies/molecules such as but not limited to feeding disruptors and peptide inhibitors of disease transmission than can be used in the development of novel interdiction strategies envisioned to work either through production of transgenic plants or application of chemical treatments that block feeding/disease transmission. Sub-objective 2a: Develop transgenic tobacco expressing enzymatic inhibitors of whitefly salivary sheath formation test for resistance to Bemisia tabaci feeding. Sub-objective 2b: Test application of discovered small molecule inhibitors of sheath formation for their effect on whitefly feeding on tomato. Sub-objective 2c: Conduct proteomic analysis of salivary exudates to identify salivary sheath structural and biosynthetic proteins. Approach (from AD-416): Research will focus on constructing a nonflowering papaya banker plant through biotechnology that is resistant to both papaya ringspot virus and powdery mildew. Strategically timed insecticide applications including neonicotinoids in the rotation regimes will be evaluated against MED (Q- biotype whitefly) for whitefly efficacy and compatibility with two of the natural enemies used in our banker plant systems: the predatory mite, Amblyseius swirskii, and the whitefly parasitoid, Encarsia sophia. Development and testing molecular inhibitors of whitefly feeding processes with specific emphasis on the processes that must occur for the whitefly to develop a successful feeding event. Two main objectives include: 1) Continued characterization of the whitefly salivary sheath biosynthesis and composition. We already have basic compositional data and some knowledge on structural arrangement. Molecular, biochemical and structural analyses will continue to identify key biosynthetic enzymes and sheath structural components. 2) Evaluation of inhibitors of sheath formation as control agents. This evaluation will be performed using artificial diet assays and development of transgenic tobacco expressing inhibitors and conducting bioassays where the whitefly feed on the artificial diet or the transgenic plants. Developed collaboration to use a transgenic papaya line developed by the University of Florida that has been engineered to be Papaya ringspot virus resistant. The compatibility of insecticides used in rotation programs to control Bemisia tabaci (Mediterranean whitefly also known as biotype Q) with the predatory mite, Amblyseius swirskii used in biological control programs for whitefly control was evaluated on salvia ornamental plants in the greenhouse. Experiments testing dinotefuran (grower standard neonicotinoid) as a drench application was compatible with the predatory mite. No significant difference in mite life stages between mite treated and combination plots (mite + insecticide) were reported during the majority of study period. The mite was effective in suppressing MED whitefly life-stages throughout the study period and efficacy was overall comparable with plants treated with dinotefuran. Cyantraniliprole (anthanilic diamide) in rotation with pymetrozine (pyridine azomethines), both new classes of insecticide, were evaluated for predatory mite compatibility for control of whitefly. Drench application of cyantraniliprole at the low rate was compatible with the mite, whereas their number was significantly reduced post application of pymetrozine. The combination insecticide/predatory mite treatment was the most effective and the chemical rotation was the least effective in suppressing MED whitefly during the study period. To estimate the lethal concentration range of insecticides used in rotation greenhouse trials against swirskii mites used for biological control of whitefly pests, preliminary bioassays are underway where different solvent mixtures (benign to swirskii mite) for the preparation of insecticide concentrations have been tested and standardized. To ensure less than 5 percent control mortality, various bioassay arenas were designed and were tested for their suitability for the survival of swirskii mites. Solvent and solvent mixtures were optimized to ensure insecticide solubility and minimum residue accumulation in the Petri dishes. These parameters are critical to ensure the lowest control mortality. Work on salivary sheath inhibition as a method of blocking B. tabaci feeding was advanced. Due to lack of funds, we chose to focus on topical application strategies for initial analysis instead of transgenic plant development. This decision was justified because of: 1) breakthroughs we have had in development of new topical applications strategies for the control of Asian citrus psyllid (ACP) sheath formation/sheath production; and, 2) our earlier demonstration that sheath inhibitors that work on ACP sheaths also work on B. tabaci sheaths. Therefore ARS Scientists developed and tested a topical application assay for direct evaluation of sheath inhibitors on B. tabaci feeding. This system uses excised spearmint leaves that ARS Scientists demonstrated to support the complete B. tabaci life cycle. It can be used in both choice and no choice experiments to monitor feeding deterrence, adult feeding, egg laying, and nymph development. Because isolation and analysis of psyllid salivary sheaths was easier than that for the whitefly and because we previously demonstrated that the same sheath inhibitors worked for both insects, ARS scientists we used the psyllid system as a surrogate for the whitefly. Previous work has shown that the same salivary sheath inhibitors inhibit both Asian citrus psyllid and Bemisia tabaci whitefly salivary sheaths. Further, the availability of the whitefly genome and transcriptome will be used to identify the whitefly homologs to the proteins identified in the psyllid salivary proteome. We have conducted initial salivary sheath proteome analysis using the Asian citrus psyllid as a surrogate. This work was done in collaboration with a research molecular biologist, USDA-ARS, BioIPM Research Unit, Ithaca, New York. Also, a large batch of psyllid salivary sheaths was isolated for further compositional and structural analysis. The pathogenicity of a naturally occurring entomopathogenic fungus, Isaria fumosorosea (PFR 97�) alone and in combination with an insect growth regulator (Talus�) was evaluated against a new invasive pest, Rugose spiraling whitefly (RSW) under field conditions for the second season. This study complemented earlier work. Overall, Talus alone and its combination with PFR was found to be the most effective treatment for control of RSW eggs, early instars and late instar nymphs during the 10 week period. PFR alone was found to be least effective. Results suggest that Talus alone and in combination with PFR could be efficacious against RSW and would help mitigate the spread of this pest in Florida. Lab bioassay and greenhouse trials are underway to evaluate the biocontrol potential of two generalist predatory beetles Delphastus catalinae and Delphastus pallidus on two whitefly biotypes of Bemisia tabaci (MED and MEAM1). For the studies, separate colonies of the two beetles have been initiated. Accomplishments 01 Compositions and methods for control of hemipteran insect stylet sheath structure formation. Final patent submission US15/140,666. Hemipteran insects collectively represent one of the greatest limitations (either yield or economics) to crop production in the United States and worldwide. During the probing process used to establish a successful feeding event on a host plant, agriculturally important hemipteran pest insects, including whiteflies, aphids, scales, mealybugs, leafhoppers, planthoppers etc., secrete a liquid saliva that hardens into a continuous feeding tube leading from the surface of the plant tissue to the site of feeding (the plant vascular tissues). Through analysis of composition and structure of this sheath, Agricultural Research Service (ARS) researchers at Fort Pierce, Florida, identified inhibitors that block this process and demonstrated that when they are applied to the surface of the leaf, establishment of a successful feeding event is inhibited. This work presents a novel method of preventing these pest insects from feeding on important United States crop plants without the use of toxic pesticides. Furthermore, this, interdiction method does not kill the insect, but produces a plant that cannot be used as a food source by the insect and therefore represents a sustainable control method that is compatible with other biocontrol methods as part of any Integrated Pest Management strategy, and should not have a deleterious effect on other beneficial insects. As a result of this work, a CRADA with a private company has been initiated to support development of a commercializable product. 02 Geographical distribution and genetic analysis reveal recent global invasion of an agricultural pest whitefly, Bemisia tabaci primarily associated with three genetic types. The whitefly Bemisia tabaci is a cryptic species complex in which one member, Middle East-Asia Minor 1 (MEAM1) has invaded globally. After invading large countries like Australia, China and United States, MEAM1 spread rapidly to occupy most of the climatically suitable geography. In contrast, our analysis of MEAM1 in India showed a very different pattern. Despite the detection of MEAM1 being contemporaneous with invasions in Australia, United States and China, MEAM1 has not spread across the country and instead remains restricted to the southern and southwestern regions. An assessment of MEAM1 genetic diversity in India showed a level of diversity equivalent to that found in its presumed home range and much higher than what is observed across the invaded range. This high level of genetic diversity and restricted distribution of MEAM1 raises the prospect that its home range extends into India. To test this we explored diversity across the invaded range and observed that 84.5 percent of records are accounted for by 3 haplotypes of which one, Hap1, accounts for 79.8 percent. In 823 records pertaining to Australia, China and United States, Hap1 accounts for 88.6 percent whereas in India it remains restricted to only one part of one Indian State despite its first detection in 1999. This study not only unifies our understanding of B. tabaci diversity in India, it increases our understanding of species invasions. In particular, it shows clearly that the global invasion by MEAM1 has been driven primarily by a single haplotype rather than the entire species.
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