Location: Vegetable Research
2021 Annual Report
Objectives
1. Develop sensitive and reliable serological and molecular based pathogen detection methods for the emerging and endemic viral diseases of vegetable crops.
• Sub-Objective 1.1. Develop a traceable clone of Cucumber green mottle mosaic virus to study the mechanism of seed transmission and to improve seed health assay on watermelon seeds.
• Sub-Objective 1.2. Develop traceable clones of pospiviroids (Tomato planta macho viroid and Potato spindle tuber viroid) that can be used to study the mechanism of seed transmission and to develop a reliable seed health assay on tomato.
2. Apply RNAi technology to reduce whitefly vector transmission of plant viruses, including Tomato yellow leaf curl virus in tomato and other viruses in cassava.
• Sub-Objective 2.1. Develop dsRNA constructs to evaluate their RNAi effect on whitefly (Bemisia tabaci) as a sprayable insecticide.
• Sub-Objective 2.2. Develop transgenic tomato plants with RNAi effect against whitefly as a proof of concept to control whitefly-transmitted viruses.
3. Develop molecular markers associated with host resistance to viral diseases in vegetables and Fusarium wilt on watermelon.
• Sub-Objective 3.1. Genotyping-by-sequencing to identify SNPs in association with disease resistance breaking of tomato by the emerging Tomato mottle mosaic virus.
• Sub-Objective 3.2 Develop molecular markers associated with fusarium wilt resistance in watermelon.
4. Develop environmentally sustainable disease management strategies against diseases of vegetable crops.
• Sub-Objective 4.1. Develop bacterial blight resistant germplasm in Brassica rapa.
• Sub-Objective 4.2. Develop an anaerobic soil disinfestation system effective in reduction or elimination of Ralstonia solanacearum in solanaceous crops.
Approach
Relative to Objective 1, an infectious clone of Cucumber green mottle mosaic virus will be developed to study the mechanism of seed transmission in watermelon. A sensitive bioassay will be developed to improve the seed health assay on watermelon seeds for CGMMV. Infectious clones of Tomato planta macho viroid and Potato spindle tuber viroid will be developed and used to study the mechanism of seed transmission of pospiviroids on tomato. Sensitive bioassays will be developed to allow a reliable seed health assay on tomato using seedling growout or through mechanical inoculation of seed extract, depending on the mechanism of seed transmission.
For Objective 2, based on the results from whitefly genome and transcriptome analysis, double-stranded ribonucleotide acid (dsRNA) constructs will be developed to evaluate the RNA interference (RNAi) effect on whitefly survival through topical spray application on plants. Transgenic tomato plants will be developed to evaluate the RNAi effect against whitefly as a proof of concept to control whitefly-transmitted viruses on crop plants.
Under Objective 3, genome sequencing technologies will be used to identify single nucleotide polymorphisms (SNPs) associated with disease resistance breaking of tomato by the emerging Tomato mottle mosaic virus. In other experiments, sequencing will be used to identify SNPs associated with genes that confer resistance against Fusarium oxysporum using populations generated from the USVL246-FR2 breeding line demonstrated to have resistance to Fusarium oxysporum f. sp. niveum (Fon) races 1 and 2.
For Objective 4. Using traditional breeding techniques, bacterial blight resistant germplasm in Brassica rapa with a Chinese cabbage-like phenotype, will be advanced through back-crosses and additional crosses to the locally-preferred, genetically-related turnip green cultivars. In separate experiments, an anaerobic soil disinfestation system effective in reducing or eliminating Ralstonia solanacearum in solanaceous crops will be developed. An anaerobic soil disinfestation strategy can be implemented to reduce or eliminate the bacterial wilt pathogen in infested soils.
Progress Report
This portion of the progress report is related to Objective 1, Sub-objective 1.1: “Develop a traceable clone of Cucumber green mottle mosaic virus to study the mechanism of seed transmission and to improve seed health assay on watermelon seeds”. ARS researchers in Charleston, South Carolina, observed efficient transmission of CGMMV to test cucurbit plants (melon) through mechanical transmission using CGMMV-contaminated seed extract, suggesting CGMMV-contaminated seed can initiate a new virus infection. Since CGMMV can spread easily through mechanical transmission, to prevent virus spread, we evaluated 16 disinfectants and identified at least four disinfectants that are effective in deactivating CGMMV infectivity. Furthermore, in collaboration with collaborators in Alberta, Canada, we conducted field trials using disinfectants and resistance cultivars to manage CGMMV in greenhouse cucumber. This study has advanced our knowledge on the route of virus transmission from contaminated seeds to seedlings and identification of disinfectants useful to manage virus spread under greenhouse cucumber production.
This portion of the progress report is related to Objective 1, Sub-objective 1.2 “Develop traceable clones of pospiviroids (Tomato planta macho viroid and Potato spindle tuber viroid) that can be used to study the mechanism of seed transmission and to develop a reliable seed health assay on tomato”. We developed a series of infectious clones of tomato planta macho viroid (TPMVd) recombinants with chimeric sequences to map the virulence determinant. In collaboration with scientists in Thailand, we developed a multiplex real-time RT-PCR for simultaneous detection of two other related viroids: Pepper chat fruit viroid and Columnea latent viroid, which are being used to improve seed health testing of viroids in tomato and pepper seeds.
This portion of the progress report is related to Objective 2, Sub-objective 2.1 “Develop dsRNA constructs to evaluate their RNAi effect on whitefly (Bemisia tabaci) as a sprayable insecticide”. Follow-up with our previous success in sequencing the whitefly genome, transcriptome analysis and small RNA profiling, a number of target genes were selected to develop transgenic tomato plants expressing the double-stranded RNAs (dsRNAs). Evaluation on insecticidal effect of those dsRNA constructs through in vitro feeding assays demonstrated a wide range effect on whitefly survival rate. In addition, several of these RNA interference (RNAi) constructs were demonstrated through a ring test in three collaboration labs in the U.S. and in Africa with some promising results against whiteflies through in vitro bioassay on artificial diet.
This portion of the progress report is related to Objective 2, Sub-objective 2.2: “Develop transgenic tomato plants with RNAi effect against whitefly as a proof of concept to control whitefly-transmitted viruses”. Twenty-seven transgenic tomato lines (cv. Moneymaker) expressing four different RNAi constructs were generated and maintained in a greenhouse. These transgenic lines with various levels of expression of the target sequences were used for bioassays for their mortality effects against whiteflies. Bioassays are underway to evaluate through in planta analysis to assess whitefly mortality due to RNAi effect on the feeding whiteflies. These results demonstrated the possibility in using RNAi to control whiteflies through topical application or by transgenic plants.
This portion of the progress report is related to Objective 3, Sub-objective 3.1. “Genotyping-by-sequencing will be used to identify SNPs in association with disease resistance breaking of tomato by the emerging tomato mottle mosaic virus”. Following the first identification of tomato mottle mosaic virus (ToMMV) in 2013 and molecular and biological characterization on its host range and resistance breaking, we developed a sensitive molecular detection for species-specific identification. During the course of this study, we made a first report of another more aggressive and resistance breaking tobamovirus, Tomato brown rugose fruit virus (ToBRFV) in the United States. Following our first report, the USDA-Animal and Plant Health Inspection Service (APHIS) issued a Federal Order to inspect imported tomato and pepper. We characterized the ToBRFV U.S. isolates for its host range, resistance breaking and developed a sensitive real-time PCR detection system, which is useful for seed health testing. In addition, several effective disinfectants were selected and recommended to greenhouse tomato growers in the U.S. and around the world. Finally, a genetic source of resistance to ToBRFV has been identified and genetic and genomic analyses are underway.
This portion of the progress report is related to Objective 3, Sub-objective 3.2: “Develop molecular markers associated with fusarium wilt resistance in watermelon”. We identified several quantitative trait loci (QTL) in watermelon associated with both race 1 and race 2 Fusarium wilt resistance, as well as developed molecular-based markers for use in a marker assisted breeding. Additionally, we identified QTL associated with Downy mildew resistance. KASP markers were developed and validated for resistance in Cucumis melo for Fusarium oxysporum f. sp. melonis race 1 and race 2, Downy mildew, Alternaria Leaf Blight, Powdery mildew, and sulfur tolerance. All Citrullus amarus plant introductions from the USDA have been completely sequenced and sequencing data made available to collaborating researchers from several U.S. universities through the Cucurbit Genomics Database (CuGenDB). QTL attributed to watermelon root morphology and development have been identified in a project related to our work on Fusarium wilt and nematode resistance in watermelon.
This portion of the progress report is related to Objective 4, Sub-objective 4.1: “Develop bacterial blight resistant germplasm in Brassica rapa”. We have identified and developed two uniform B. rapa lines, USVL588 and USVL1000 that exhibit very high resistance to Pseudomonas cannabina pv. alisalensis (Pca). We have made repeated selections and self-pollinations of both lines to ensure selected lines exhibit a uniform resistant response and uniform plant morphotype. Additionally, we have been able to select away from the Chinese cabbage leaf shape in USVL588 to one more resembling a turnip green. We have made selections from a segregating F2 population derived from the cultivar “Topper”, a popular turnip green hybrid, selecting for leaf phenotype (Topper is susceptible to Pca) and have taken a desired selection to the S6 generation through controlled self-pollinations and identified as USVL-TOP. We have made an F1 hybrid of USVL-TOP x USVL588, followed by field selection for Pca resistance and leaf shape in three generations (S5).
This portion of the progress report is related to Objective 4, Sub-objective 4.2: “Develop an anaerobic soil disinfestation system effective in reduction or elimination of Ralstonia solanacearum in solanaceous crops”. We have identified a new carbon source, cotton seed meal (CSM), a low-cost alternative to the standards of either molasses and composted chicken manure or rice bran for use in anaerobic soil disinfestation (ASD). At less than half the cost of the standard carbon sources, CSM-based ASD was found to be effective in reducing Ralstonia solanacearum, causing bacterial wilt of solanaceous crops, by more than 99% in the soil. Additionally, CSM-based ASD has been found to significantly reduce crop-limiting weeds, including yellow nut sedge and Palmer amaranth in treated areas.
Accomplishments
1. Managing Tomato brown rugose fruit virus, an emerging virus infecting tomato and pepper. Tomato brown rugose fruit virus (ToBRFV), an emerging tobamovirus, causes serious disease outbreaks on greenhouse tomatoes around the world in recent years. This seed-borne and mechanically transmitted virus poses a serious threat to the $2.5 billion tomato and pepper industries in the U.S. In 2019, ARS researchers in Charleston, South Carolina, made a first report on an outbreak of ToBRFV on tomato in the U.S. Due to the potential devastation of ToBRFV to the U.S. tomato industry, the USDA-APHIS issued a Federal Order in 2019 to inspect imported tomato and pepper for ToBRFV infection. We conducted molecular and biological characterizations of ToBRFV isolates in the U.S. Using genome sequence information, we developed a highly sensitive real-time PCR detection system for ToBRFV, which is useful for seed health testing. Several disinfectants with effect to deactivate virus infectivity and prevent disease spread on tomato were selected and recommended to growers. Finally, in screening tomato germplasm, new sources of resistance have been identified which could be used in breeding for resistance. The achievements obtained in the present study will provide fundamental knowledge and practical solutions to prevent and protect tomato and pepper crops in the U.S. and around the world from potential devastation by this emerging virus.
2. A new disease-resistance rootstock for watermelon and cucumber is in commercial production. ARS researchers in Charleston, South Carolina, licensed Carolina Strongback, a new rootstock for watermelon and cucumber, with resistance to Fusarium wilt race 1 and 2, as well as resistance to several plant parasitic nematodes licensed to Syngenta Seed Company. More than 2 million seeds in 3 countries were sold by Syngenta in 2020-2021. Seed increase this year is more than 12 million, for which most are already committed to growers. During 2021, working with USDA office of technology transfer, product protection and licensing approval has been granted or are in the final stages of completion for Australia, Mexico, Chile, Israel, Canada and the European Union This accomplishment is the first in developing a rootstock for watermelon and cucumber that is resistant to major soil-borne pathogens.
Review Publications
Ellouze, O., Mishra, V., Howard, R.J., Ling, K., Zhang, W. 2020. Preliminary study on the control of cucumber green mottle mosaic virus in commercial greenhouses using agricultural disinfectants and resistant cucumber varieties. Agronomy. 10:1879. https://doi.org/10.3390/agronomy10121879.
Andreason, S.J., Olaniyi, O.G., Gilliard, A.C., Wadl, P.A., Williams Iii, L.H., Jackson, M.D., Simmons, A.M., Ling, K. 2021. Large scale seedling grow-out experiments do not support seed transmission of sweet potato leaf curl virus in sweetpotato. Plants. 10(1):139. https://doi.org/10.3390/plants10010139.
Chanda, B., Shamimuzzaman, M., Gilliard, A.C., Ling, K. 2021. Effectiveness of disinfectants against the spread of tobamoviruses: tomato brown rugose fruit virus and cucumber green mottle mosaic virus. Virology Journal. 18:7. https://doi.org/10.1186/s12985-020-01479-8.
Hasegawa, D.K., Shamimuzzaman, M., Chen, W., Simmons, A.M., Fei, Z., Ling, K. 2020. Deep sequencing of small RNAs in the whitefly, Bemisia tabaci revealed novel microRNAs potentially associated with begomovirus acquisition and transmission. Insects. 11(9):562. https://doi.org/10.3390/insects11090562.
Branham, S., Daley, J., Levi, A., Hassell, R., Wechter, W.P. 2020. QTL mapping and marker development for tolerance to sulfur phytotoxicity in melon (Cucumis melo). Frontiers in Plant Science. 11:1097-1105. https://doi.org/10.3389/fpls.2020.01097.
Keinath, A.P., Dubose, V.B., Katawczik, M.L., Wechter, W.P. 2020. Identifying races of Fusarium oxysporum f. sp. niveum in South Carolina recovered from watermelon seedlings, plants, and field soil. Plant Disease. 104:2481-2488. https://doi.org/10.1094/PDIS-11-19-2385-RE.
Katuuramu, D.N., Wechter, W.P., Washington, M., Horry, M.I., Cutulle, M.A., Jarret, R.L., Levi, A. 2020. Phenotypic diversity for root traits andiIdentification of superior germplasm for root breeding in watermelon. HortScience. 55(8):12-72-1279. https://doi.org/10.21273/HORTSCI15093-20.
