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ARS Home » Southeast Area » Charleston, South Carolina » Vegetable Research » Research » Research Project #431518

Research Project: Biological, Genetic and Genomic Based Disease Management for Vegetable Crops

Location: Vegetable Research

2022 Annual Report

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.

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 is the final report for this project. Refer to project 6080-22000-031-000D, "Harnessing Genomic Technologies Toward Improving Vegetable Health in Field and Controlled Environments" for additional information. To study the mechanism of seed transmissibility of CGMMV on watermelon, we focused our efforts in using naturally infected seeds through bioassays. We compared seedling growth out and bioassay through mechanical inoculation on seedlings using tissue extract of contaminated seeds. Although no seed transmission of CGMMV through seedling grow-out of limited seedlings, the virus was able to cause disease on test plants through mechanical inoculation. To prevent virus spread, we evaluated 16 disinfectants and identified four of them to be the most effective in deactivating CGMMV infectivity. Furthermore, we conducted field trials using disinfectants and resistance cultivars to manage CGMMV in greenhouse cucumber. Results from these studies advanced our knowledge on seed transmission of CGMMV and application of disinfectants to manage CGMMV. We developed a series of infectious clones of tomato planta macho viroid (TPMVd) recombinants with chimeric sequences from both genotypes of TPMVd inciting respective mild or severe phenotypes to map the virulence determinant. In a separate study, recognizing the current real-time polymerase chain reaction (PCR) for pospiviroid detection had some limitation for emerging viroids, in collaboration with scientists in Thailand, we developed a multiplex real-time PCR for simultaneous detection of Pepper chat fruit viroid and Columnea latent viroid, useful for seed health testing. After functional genomic analysis differentially expressed genes in whiteflies upon virus acquisition, a number of target genes were selected to develop transgenic tomato plants for RNA interference (RNAi) against whiteflies. Selected RNAi constructs were demonstrated promising results through a ring test in three labs in the U.S. and Tanzania in Africa through in vitro bioassay against whiteflies. Those promising ones were selected for stable expression through transgenic tomato plants. 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 on their mortality effects against whiteflies. Bioassays are underway to evaluate through in planta analysis to assess whitefly mortality due to the RNAi effect. Tobamoviruses have been managed quite well using resistance cultivars. However, the emerging Tomato brown rugose fruit virus (ToBRFV) is able to infect tomato cultivars with Tm-2^2 gene. In this study, we characterized the molecular and biological properties of ToBRFV isolates in the U.S. and developed a real-time PCR for ToBRFV detection. To prevent virus transmission, we screened over 16 chemicals and identified at least four effective disinfectants to prevent the spread of the disease. In an effort to develop tomato with resistance to ToBRFV, we screened approximately 500 accessions of tomato germplasm and identified several accessions of Solanum pimpinelliforium with resistance to ToBRFV. Corresponding segregating populations have been generated. We used high throughput sequencing to identify SNPs that are associated with the ToBRFV resistance loci. 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. 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 were 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 (S6). Additionally, we have developed a mustard green from the USDA line “Carolina Broadleaf” and a cross with a selection from “Topper” that has been designated as “Charleston Broadleaf” that has significant resistance to Pca, an improved leaf shape and a slower bolting characteristic. 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.

1. Identification and implementation of effective disinfectants against the emerging tomato brown rugose fruit virus. Tomato is one of the most important vegetable crops in the world. U.S. is the third largest tomato producer with over $2 billion farm gate value. Tomato is especially susceptible to many viral diseases, which are difficult to manage. An emerging virus, tomato brown rugose fruit virus (ToBRFV), has caused serious disease epidemics around the world, including the U.S. ARS researchers in Charleton, South Carolina, made the first report of ToBRFV outbreak in the U.S. in 2019. Following that report, USDA-APHIS issued a Federal Order to quarantine and control this pest. In the present study, we conducted a comprehensive evaluation on two key biological properties for ToBRFV, host range and disease resistance breaking. We also developed a sensitive and species-specific detection method that could be useful for seed health testing. For disease management, several disinfectants effective against the emerging ToBRFV on tomato were identified, which have been implemented by greenhouse tomato growers in the U.S. Identification of several common disinfectants with similar efficacies against two tobamovirus species on different plants suggest their broader effects against other viruses.

Review Publications
Schlub, R.L., Li, R., Bamba, J.P., Marutani, M., Ling, K. 2021. Complete genome sequence of tomato leaf curl Guam virus, a novel tomato-infecting begomovirus from Guam, USA. Microbiology Resource Announcements. 10(49):e00954-24.
Kousik, C.S., Ikerd, J.L., Wechter, W.P., Barnham, S., Turechek, W. 2022. Broad resistance to fruit rot in USVL watermelon germplasm lines to isolates of Phytophthora capsici from across USA. Plant Disease. 106:711-719.
Branham, S.E., Kousik, C.S., Mandal, M., Wechter, W.P. 2021. QTL mapping of resistance to powdery mildew race 1 in a recombinant inbred line population of melon. Plant Disease.
Katuuramu, D.N., Branham, S.E., Levi, A., Wechter, W.P. 2022. Genome-wide association analysis of resistance to Pseudoperonospora cubensis in citron watermelon. Plant Disease. 106:1952-1958.