Incorporating Natural, Non-toxic Arthropod Resistant Tomato Varieties into Southern Production Systems

Objective 1: Determine if acylsugar producing tomato lines provide sufficient resistance to twospotted spider mites and select the most spider mite-resistant line.

Acylsugar tomato lines that produce different types and/or levels of acylsugars were provided by the tomato breeding program at Cornell University led by Mutschler. Since the initial lines with CU071026 background suffered from reduced fruit set, seed set and germination, a new set of lines with greatly improved seed production and germination was created with CU17NBL background. These improved seeds were provided to co-PIs. The new acylsugar lines in CU17NBL background were developed by backcross breeding transferring acylsugar QTL from the lines in CU071026 background to CU17NBL using marker assisted selection. Seed production was performed in greenhouses, for maximum control of plant conditions and to protect against possible seed borne pathogens.

A series of laboratory, greenhouse and field experiments were conducted at facilities of the University of Georgia and Clemson University to evaluate the resistance of selected acylsugar-producing tomato lines against sweetpotato whiteflies and twospotted spider mites from 2019 to 2022.

Research team at Clemson University, under Chong and Gill, compared the susceptibility of acylsugar tomato lines (CU071026, CU17NBL, QTL6/CU17 and QTL6/AS) to a commercial cultivar (Amelia) for twospotted spider mite and sweetpotato whiteflies in a series of laboratory and greenhouse bioassays. In no-choice tests, a scintillation vial filled with water was glued to the bottom of a plastic cup and a hole was drilled in its lid to insert the petiole of fully expanded leaf (third from the top) from each line. A total of 12 leaves from each line were used in this experiment. Five pairs of whiteflies were introduced into each arena to lay eggs, and after 72 hours the whiteflies (adults, nymphs and eggs) were counted under microscopes.  In two-choice tests, the five tomato lines were paired and exposed to twospotted spider mite. Each tomato line was paired with itself or another line, resulting in 10 comparisons. Ten adult female spider mites were introduced to the plastic platform connecting both leaves. Each pair was replicated eight times. The numbers of spider mite motiles (i.e., adults and nymphs) and eggs on each leaf were recorded after 72 hours. In all-choice tests, the acceptance of the five tomato lines to twospotted spider mites and sweetpotato whiteflies was evaluated in the greenhouse. To test for spider mite preference, potted plants from each experimental line were randomly placed together in a block, resulting in a total eight blocks. Spider mites dispersing from infested plants maintained in the same greenhouse were allowed to infest the experimental plants for 12 weeks. After that, three fully expanded leaves (one each from top, middle and bottom thirds) were randomly collected from each plant weekly for three weeks, and the numbers of spider mites (eggs, nymphs and adults) were counted under microscopes. For the no-choice test for whiteflies, one potted plant of each acylsugar and commercial line was isolated in each cage covered with fine mesh. A total of 40 plants (eight from each line) were used. Five pairs of whiteflies were introduced to the plant from each line. Three fully expanded leaves (one each from top, middle and bottom thirds) were randomly collected from each plant biweekly for 8 weeks, and the numbers of whiteflies (eggs, nymphs and adults) were counted under the microscope. Leaf samples of all acylsugar and commercial lines used this these experiments were collected and dried at PDREC, and sent to Cornell University for acylsugar analysis using standard assay developed in the Mutschler lab. The analysis will enable co-PIs to correlated different levels ad types of acylsugars with the observed experimental data on the susceptibility of these selected lines.

Research team at the University of Georgia, under Schmidt and Pandey, conducted a series of greenhouse and laboratory experiments to further evaluate the resistance of selected acylsugar-producing tomato lines against sweetpotato whiteflies. The tomato lines used in the choice and no-choice tests were five acylsugar lines (CU071026, CU17NBL, QTL6/sw5/AS, QTL6/CU17, QTL6/sw5/CU17NBL) and a commercial tomato cultivar (Amelia F1 Hybrid). Whiteflies were obtained from a greenhouse colony maintained on “Red Snapper” tomato plants at the University of Georgia-Tifton Campus. The tomato plants with whitefly colonies were isolated in bugdorms (60x60x60 cm WxDxH, and mesh size 150x150ǀ160 µm aperture). The no-choice test was conducted in a greenhouse by placing a tomato plant from each line in a bugdorm, and then isolating two leaflets per plant with a leaf cage. Ten whitefly adults were introduced into each leaf cage. The leaflets were carefully removed six days after infestation and the number of whitefly eggs and nymphs on each leaflet were recorded. Each line was repeated five times, with ten leaflet samples per tomato line. The choice test was conducted with detached leaflets in a temperature-controlled environmental chamber (30°C, 100 µml photoperiod 16L:8D hours). Petioles of a fully expanded leaflet of all six lines were obtained from tomato plants maintained in a greenhouse. The leaflets were checked for the presence of arthropods or infestation. Only clean leaflets were used in the experiment. A transparent cylindrical cage (20 cm long and 13.7 cm in diameter) was made from a transparent plastic sheet, within which a leaflet from a tomato line was inserted (kept vigorous by inserting the petiole into a scintillation vial with water). Twenty whitefly adults were introduced per cage and allowed the whiteflies six days to interact with the leaflets. The experiment was repeated three times. Hence, we obtained 15 replicates per line. At the end of six days, we removed the cages from the environmental chamber and collected the adult whiteflies and leaflets. Each leaflet was observed under a stereo microscope. We counted and recorded the number of eggs and nymphs present on a leaflet.

Objective 2: Determine the compatibility of acylsugar producing lines with biological control of thrips and whiteflies by four known biological control agents.

Research team at the University of Georgia (UGA), under Schmidt, conducted a greenhouse study to evaluate the efficacy of suppressing whitefly abundance on selected acylsugar tomato lines with the predatory mite, Amblyseius swirskii. (Amblyseius swirskii is also a predator of thrips.) The tomato varieties used in this experiment were three acylsugar lines (CU071026, FA7/AS and QTL6/AS) and two commercial varieties (Amelia and Florida 47). Tomato plants were grown inside the bug dorms (W60 x D60 x H60 cm; mesh size = 150 x 150 | 160 µm aperture). Tomato seedlings at five-leaf stage were infested with whiteflies that were obtained from a clean non-virus colony maintained on the cotton seedlings at Riley’s lab at UGA. Whiteflies were allowed to colonize the plants in a choice test, all varieties in same enclosed area, for a period of three weeks. Each of the six bug dorms contained five plants, one plant of each variety (total of 30 plants). After release of whitefly adults into the dorms, the team sampled whitefly populations weekly by removing one leaflet and counting all eggs, nymphs and adults for three weeks. Each plant received a treatment of one mite sachet, which hung on each plant. After predatory mite introduction, weekly samples of whitefly populations were taken by removing one leaflet per plant variety from each of the cages, for a total of 30 leaves per time period over four weeks. The leaflet samples were transported to the lab for the count of whitefly eggs and nymphs, and count of A. swirskii eggs and adults under stereomicroscopes.

Research team at Clemson University, under Chong and Gill, conducted a series of laboratory bioassays to detect potential reduction in biological control efficacy of selected tomato lines (CU071026, CU17NBL, QTL6/CU17, QTL6/AS and Amelia) to three natural enemies—Delphatus catalinae (whitefly predator), Orius insidiosus (thrips predator) and Eretmocerus eremicus (whitefly parasitoids). The biological control agents were purchased from commercial suppliers. For the Delphastusbioassays, leaflets of each tomato lines were collected and placed in an arena (described for whitefly preference bioassay in Objective 1). The leaflets were infested with adult whiteflies for three days, after which the adult whiteflies were removed, leaving only whitefly eggs on the leaf. One predator was released into each arena and the numbers of consumed whitefly eggs were recorded after 24 hours. For Orius and Eretmocerus bioassays, whole tomato plants were infested with adult whiteflies and leaflets were collected from the tomato plants after whitefly nymphs had reached the third instar. The leaflets were placed individually in arena (described in Objective 1) and one Orius and female Eretmocerus was introduced into the arena for 24 hours. After Orius removal, the leaflets were examined to count the numbers of nymphs consumed. After Eretmocerusremoval, the leaflets were kept in the arena until whitefly nymphs reached the “pupal” stage. The numbers of parasitized pupae were recorded.

Objective 3: Determine the compatibility of acylsugar producing lines with biological control of spider mites by three predatory mites.

Research team at Clemson University, under Chong and Brown, conducted a series of laboratory and greenhouse experiments to determine the compatibility between acylsugar producing tomato lines and three predatory mite species—Phytoseiulus persimilis, Neoseiulus californicus and Neoseiulus andersoni. The predatory mites were purchased from commercial suppliers. Because laboratory and greenhouse bioassay in Objective 1 demonstrated that only CU071026 suffered some level of infestation, and therefore, will require additional intervention in the field to prevent damage and yield loss, this team decided to focus assessments in this objective on CU071026 and Amelia. The laboratory bioassays were conducted as two-choice tests where a pair of tomato leaflets, one of a selected tomato line, infested with twospotted spider mite adults and eggs were placed in an arena described spider mite preference bioassays in Objective 1. The numbers of predatory mites and consumed spider mites or spider mite eggs on each leaflet were recorded after 24 hours. The greenhouse bioassays were conducted as a no-choice test on whole tomato plants of CU071026 (because this is the only variety that had suffered some level of spider mite infestation) and Amelia (the commercial cultivar) to assess if the addition of predatory mites can complement suppression of spider mite population provided by acylsugars. Whole tomato plants were grown in greenhouse and infested with spider mites for 4 weeks before the bioassays. The infested tomato plants were placed individually in plastic tubs filled with water so that the spider mites and predatory mites could not leave the tomato plants and interfered with the biological control activities on the other plants. The spider mite and predatory mite population were sampled weekly over 6 weeks to detect potential interference or complementation of acylsugar-producing tomato line with biological control.

Objective 4: Examine impacts of acylsugar producing lines on pest and natural enemy abundance, and virus incidence in southern commercial tomato fields.

Initially we had secured three commercial growers (Bill Brim of Lewis Taylor Farms, Will Harris of White Oak Pastures, and Matthew Horry of Kindlewood Farms) to participate in field trials in Georgia and South Carolina. The COVID pandemic prevented field research and outreach activities planned for 2020 and 2021. Once travel and field work restrictions were lifted in 2021, we reached out to ten growers in Georgia and South Carolina to plan for implementation of our research results in commercial farms. However, discoveries from the breeding program and our greenhouse trials suggested that the pest resistant varieties are still not producing flowers, fruits and taste at a comparable level to current commercial varieties, and they do not prevent whitefly-transmitted diseases. After reviewing these discoveries, our grower, Extension specialist and agent partners voiced their concerns about the commercial value of these pest resistant varieties at this time and advised the project team to wait until the varieties have been further improved to rival commercial cultivars before conducting the field trials. Paraphrasing of one participating growers: Nothing kills a variety faster than not having the desirable quality even if the variety is resistant to all pests and diseases in the world. Instead of generating poor reputation for an incomplete product (in this case, the tomato lines), the project team decided to suspend the field demonstration of the project based on cooperators’ inputs.

Resistance of the tomato lines to sweetpotato whiteflies were evaluated in the field at the University of Georgia in 2019 to 2022. During these times, insufficient infestation by spider mites limited our ability to assess the resistance of acylsugar-producing lines against spider mites in the field. For whiteflies, five acylsugar-producing lines (CU071026, CU17NBL, QTL6/sw5/AS, QTL6/CU17 and QTL6/sw5/CU17NBL) and a commercial cultivar (Grand Marshall) were evaluated. Seedlings at 5-6 leaf stage were transplanted to raised bed plots covered with white plastic mulch. Each plot represented a replicate of the tomato line and received 10 plants each. Four replicates of each line and the plots were randomly assigned for each line. Whiteflies were allowed to infest the tomato plants naturally after transplant. Radiant® @ 0.7L/ha insecticide was applied once and hand-picking was conducted twice to manage tobacco hornworms and fall armyworms. Weekly sampling was conducted two weeks after transplanting for ten weeks. Three plants from a plot were selected and the number of adult whiteflies were counted. A leaflet sample was taken by selecting three plants randomly in a plot and taking a leaflet from the middle portion of the selected plants. The leaflets were then observed under a stereomicroscope to count whitefly eggs, nymphs and adults. Visual inspections were also conducted to assess the presence of Tomato Yellow Leaf Curl Virus (TYLCV) weekly. However, only two plants out of ten plants in a plot with the experimental acylsugar line QTL6/sw5/CU17NBL displayed symptoms of TYLCV. Disease ratings from bacterial leaf spots caused by Xanthomonas spp. were conducted on all plants at the end of the sampling period.

 The diversity of arthropods (pest and non-pest) in the tomato field was sampled to understand the effect of the acylsugar-producing tomato lines on non-pest arthropods. A 30-second suction sample from the canopy of the middle three tomato plants in each plot using a modified reverse-flow leaf blower into a fine mesh bag at the end of the field experiment period. The contents were transported to the laboratory, where each was sieved, individuals sorted, and categorized to taxa (order level or higher) to provide estimates of arthropod counts in each tomato line.

The abundances of whiteflies and natural enemies on selected acylsugar and commercial lines had been evaluated in an organic field at UGA’s Tifton Campus. In addition to the target predatory mite, A. swirskii, the research team at UGA (led by Schmidt and Dutta) also monitored for other endemic natural enemies colonizing the tomato plants. The tomato varieties used for this experiment were two acylsugar lines (CU071026 and FA7/AS) and two commercial lines (SV7631TD, which is susceptible to Tomato Yellow Leaf Curl Virus, and Skyway 687, which is resistant to TYLCV). The seeds were sown in the greenhouse and planted to the fields at four- to five-leaf stage. Whiteflies were allowed to infest the plants naturally and the mite treatments were applied to the plants right after transplant. In this field experiment the research team tried three different mite application types, i.e. dusting the mites on the top of the plants (top), dusting the mites on the base of the plants (basal) and hanging the mite sachets on the plants (sachet), and prepared a treatment without biological control. The team documented the abundances of whiteflies and A. swirskii over time by removing leaflets from plants then assessing the numbers of arthropods colonizing the plants. The presence of TYLCV was quantified using an immunostrip test.

We have very compelling data on the “deterrent” effects of acylsugar-producing tomato lines on multiple pests in the laboratory bioassays and greenhouse experiments. In lieu of a trial in commercial fields, this project team developed a semi-field trial where tomato lines were planted in fields at experiment stations so that pest management of the experimental tomato lines can be assessed in semi-field conditions. Our results are promising for deterring whiteflies and spider mites, and therefore, our stakeholders and Extension cooperators are very excited by the possibilities of these new tomatoes lines with resistance/ deterrence to whiteflies and spider mites. Once fruit set and quality reaches current commercial levels, and disease is bred into the lines, these will be great for future field trials with cooperators.