Location: Subtropical Insects and Horticulture Research
2020 Annual Report
Objectives
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
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.
Progress Report
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
1. 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.
Review 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. https://doi.org/10.18474/0749-8004-55.2.163.
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. https://doi.org/10.1653/024.102.0417.
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. https://doi.org/10.1080/09583157.2020.1749833.
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. https://doi.org/10.3389/fphys.2020.00527.
