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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

2019 Annual Report


1a. Objectives (from AD-416):
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.


1b. Approach (from AD-416):
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.


3. 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”. To study the mechanism of seed transmissibility of CGMMV on watermelon, our initial efforts to develop a traceable infectious clone was not successful. Consequenctly, we focused our efforts in using naturally infected watermelon seeds for this purpose. Bioassays were conducted in a greenhouse through seedling grow-out and by mechanical inoculation. Through natural seedling grow-out, we did not observe seed transmission of CGMMV to germinating seedlings. However, efficient transmission of CGMMV was observed using bioassays on melon plants through mechanical inoculation of seed extract prepared from CGMMV-contaminated seeds. Understanding its seed-borne nature and the ease of mechanical transmission of CGMMV from a contaminated seed to seedling is an important finding. A new infection might occur through accidental touching of seedlings by hands or tools in handling of contaminated seeds. Therefore, a sensitive seed health test is necessary to ensure CGMMV-free seed lots are used for planting. 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”. A series of five infectious clones of TPMVd recombinants with chimera sequences from both genotypes were engineered and demonstrated to incite various levels of disease symptoms on infected tomato plants. TPMVd-infected tomato seeds will be used to study the mechanism of seed transmission of TPMVd on tomato. 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”. Following the successful genome sequencing and transcriptome analysis of whitefly (Bemisia tabacci MEAM1), we were able to extend the genome sequencing of the African cassava whitefly B. tabaci, Sub-Saharan Africa - East and Central Africa (SSA-ECA) and characterization of the genetic diversity of cassava whiteflies in Africa. To evaluate the RNA interference (RNAi) effect against whitefly mortality, double-stranded ribonucleic acid (dsRNA) molecules targeting relevant crucial whitefly functional genes were generated and tested in vitro on artificial diet or in planta through topical application for their effects on whiteflies. Several dsRNA constructs were shown through a ring test to generate a significant higher mortality rate against two types of whiteflies tested. 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”. Using those sequences with RNAi effects against whiteflies from in vitro and/or in planta assays, transformation vectors expressing these RNAi gene sequences were generated and used in Agrobacterium-mediated transformation of tomato (cv. MoneyMaker). Twenty seven transgenic tomato lines (cv. Moneymaker) expressing four different RNAi constructs were generated and maintained in greenhouse. These transgenic lines with various levels of expression of the target sequences were used for bioassays for their effects aginst whieflies, through choice and no-choice tests. Furthermore, T1 seeds of these transgenic lines have been collected and will be used to evaliuate their resistance to whiteflies. 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”. Tomato mottle mosaic virus (ToMMV), a new tomato-infecting tobamovirus, was capable of causing a resistance breaking on certain tomato cultivar that containing Tm2^2 gene, which is used in tomato for resistant to Tomato mosaic virus (ToMV) for half a century. There is no sequence variation in the Tm-2^2 gene between the susceptible (S) and resistant (R) plants. In Genotyping by sequencing (GBS) experiment, we could not identify SNPs associated with the resistance breaking. To understand global expression differences between resistance and susceptible plants, transcriptomic analysis resulted 42 differentially expressed genes, with major classes of function in pathogenesis related genes, transcription factor genes and some proteases. 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 have developed a recombinant inbred line (RIL) population at the F8 or more advanced generation from USVL246-FR2, a wild watermelon line with resistance to Fusarium wilt race 1 and 2 developed and released from our group, and the highly susceptible watermelon USVL114 to identify quantitative trait loci (QTL) associated with Fusarium race 1 and 2 resistance. The RIL population allows us to have more than 200 “immortal, genetically-fixed” lines derived from a single cross. Thus, an unlimited number of assays, whether for Fusarium wilt resistance or other disease or pest resistance associated with the original parents, can be performed. Working with the RIL population has allowed our laboratory to narrow the QTL region described last year. The current data has been used to develop Kompetetive Amplified Sequence Polymorphism (KASP) markers that will be used for high-throughput marker assisted breeding programs for incorporation of resistance into new watermelon cultivars. These KASP markers are currently being validated in more advanced populations derived from USVL246-FR2. This portion of the progress report is related to Objective 4, Sub-objective 4.1: “Develop bacterial blight resistant germplasm in Brassica rapa”. Our laboratory has identified several sources of resistance to the bacterial leaf blight pathogen, Pseudomonas cannabina pv. alisalensis from the USDA Brassica rapa collection. These line have been vernalized, then self-pollinated to generate S2 lines. The S2 lines will be tested in both greenhouse and field assays for resistance this Fall and additional selections made from the most resistant individuals. We currently have 7 lines with differing, but significant, levels of resistance to the pathogen. 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 repeated the field trials from last year in which we tested several new types of carbon source to achieve Anaerobic Soil Disinfestation (ASD) of R. solanacearum. We used sweetpotato, rice hulls, cotton seed meal, and the control molasses/pelletized chicken manure. The present study confirmed our previous study in that cotton seed meal work as well as the control for reducing numbers of the bacterial wilt pathogen. This treatment is half the cost of the currently utilized ASD amendment system that used both molasses and chicken manure. A larger trial planned for this year to focus on optimizing the ASD conditions using cotton seed meal, as well as to determine how pepper and tomato plants will grow in the ASD treated soils.


4. Accomplishments
1. Release of a watermelon breeding line resistance to Cucumber green mottle mosaic virus. In screening the USDA watermelon germplasm collections, ARS scientists at Charleston, South Carolina, were the first to identify a desert watermelon (Citrullus colocynthis) line with resistance to an emerging seed-borne and highly contagious virus, Cucumber green mottle mosaic virus (CGMMV). Through multiple generations of self-pollination and screening, a CGMMV-resistant breeding line ‘USVL18-157VR’ was released by ARS. A limited number of seeds have been distributed to private seed companies for breeding to incorporate the CGMMV resistance trait into their elite breeding materials.

2. New rootstock for watermelon production with resistance to Fusarium wilt and nematodes. Fusarium wilt and the Root Knot nematode are the top two pathogens/pests of watermelon production throughout the world. With the loss of methyl bromide fumigant, grafting of a susceptible scion onto a resistant rootstock, has been used for nearly 100 years in many Asian countries as a way to grow watermelon on infested land. However, current commercial rootstocks are susceptible to one or both of those pathogens. A new rootstock ‘Carolina Strongback’ was developed at the U. S. Vegetable Laboratory to protect the plant against both Fusarium wilt and nematodes. An evaluation license was negotiated between Syngenta Seed Company and USDA, ARS for a large-scale increase in seed production of the new Fusarium wilt and nematode resistance watermelon rootstock ‘Carolina Strongback’ by the seed company. Last year Syngenta Seed Company evaluated over 1 million seeds in different locations in North America. The current seed increase under this license agreement will generate up to ten million seeds. Syngenta Seed Company is currently negotiating a license for ‘Carolina Strongback with the USDA, ARS. Trials over the past several years with Carolina Strongback (Plant Variety Protection pending) have shown that this rootstock, derived from the wild watermelon Citrullus amarus, provides significant protection against the two main races of the Fusarium wilt fungus, as well as to the Root Knot nematode and the Reniform nematode. Yields of seedless watermelon scions grafted to Carolina Strongback are as good or better than other rootstocks. This is the first Fusarium wilt and nematode resistant Citrullus rootstock to date.


Review Publications
Keinath, A.P., Ling, K., Adkins, S.T., Hasegawa, D.K., Simmons, A.M., Hoak, S., Mellinger, C., Kousik, C.S. 2018. First report of cucurbit leaf crumple virus infecting three cucurbit crops in South Carolina. Plant Health Progress. 19:322-323. https://doi.org/10.1094/PHP-07-18-0039-BR.
Sombart, S., Reanwarakorn, K., Ling, K. 2018. Developing a multiplex real-time RT-PCR for simultaneous detection of Pepper chat fruit viroid and Columnea latent viroid. Australasian Plant Pathology. 47:615-621. https://doi.org/10.1007/s13313-018-0597-1.
Sui, X., Li, R., Shamimuzzaman, M., Wu, Z., Ling, K. 2018. Understanding the transmissibility of cucumber green mottle mosaic virus in watermelon seeds and seed health assays. Plant Disease. https://doi.org/10.1094/pdis-10-18-1787-re.
Shamimuzzaman, M., Hasegawa, D.K., Chen, W., Simmons, A.M., Fei, Z., Ling, K. 2019. Genome-wide profiling of piRNAs in the whitefly, Bemisia tabaci reveals cluster distribution and potential association with begomovirus transmission. PLoS One. 14(3):e0213149. https://doi.org/10.1371/Journal.pone.0213149.
Ling, K., Tian, T., Gurung, S., Salati, R., Gilliard, A.C. 2019. First report of Tomato brown rugose fruit virus infecting greenhouse tomato in the U.S. Plant Disease. 103:1439. https://doi.org/10.1094/PDIS-11-18-1959-PDN.
Smith, C., Freeman, J., Burelle, N.K., Wechter, W.P. 2019. Screening cucurbit rootstocks for varietal resistance to Meloidogyne spp. and Rotylenchulus reniformis. HortScience. 54(1):125-128. https://doi.org/10.21273/HORTSCI13094-18.
Branham, S., Levi, A., Wechter, W.P. 2019. QTL mapping identifies novel source of resistance to Fusarium wilt race 1 in Citrullus amarus. Plant Disease. https://doi.org/353289.
Zheng, Y., Wu, S., Bai, Y., Sun, H., Jiao, C., Guo, S., Zhao, K., Blanca, J., Zhang, Z., Huang, S., Xu, Y., Weng, Y., Mazourek, M., Reddy, U., Ando, K., McCreight, J.D., Schaffer, A.A., Burger, J., Tadmor, Y., Katzir, N., Tang, X., Liu, Y., Giovannoni, J.J., Ling, K., Wechter, W.P., Levi, A., Garcia-Mas, J., Grumet, R., Fei, Z. 2018. Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops. Nucleic Acids Research. 47(D1):1128-1136. https://doi.org/10.1093/nar/gky944.
Branham, S., Levi, A., Katawczik, M.L., Wechter, W.P. 2019. QTL mapping of resistance to bacterial fruit blotch in Citrullus amarus. Theoretical and Applied Genetics. https://doi.org/359214.
Wu, S., Wang, X., Reddy, U., Sun, H., Bao, K., Patel, T., Oritz, C., Abburi, L., Nimmakayala, P., Branham, S., Wechter, W.P., Massey, L.M., Ling, K., Kousik, C.S., Hammar, S.A., Tadmor, Y., Portnoy, V., Gur, A., Katzir, N., Guner, N., Davis, A., Hernandez, A.G., Wright, C.L., McGregor, C., Jarret, R.L., Xu, Y., Zhang, X., Wehner, T.C., Grumet, R., Levi, A., Fei, Z. 2019. Genome of ‘Charleston Gray’, the principal American watermelon cultivar, and genetic characterization of 1,365 accessions in the U.S. National Plant Germplasm System watermelon collection. Plant Biotechnology Journal. https://doi.org/10.1111/pbi.13136.
Branham, S., Wechter, W.P., Lambel, S., Massey, L.M., Ma, M., Fauve, J., Farnham, M.W., Levi, A. 2018. QTL-seq and marker development for resistance to Fusarium oxysporum f. sp. niveum race 1 in cultivated watermelon. Molecular Breeding. 38:139.
Chen, W., Wosula, E.N., Hasegawa, D.K., Casinga, C., Shirima, R.R., Fiaboe, K.K., Hanna, R., Fosto, A., Goergen, G., Tamò, M., Mahuku, G., Tripathi, L., Mware, B., Kumar, L.P., Ntawuruhunga, P., Moyo, C., Yomeni, M., Boahen, S., Edet, M., Awoyale, W., Wintermantel, W.M., Ling, K., Legg, J.P., Fei, Z. 2019. Genome of the African cassava whitefly Bemisia tabaci and distribution and genetic diversity of cassava-colonizing whiteflies in Africa. Insect Biochemistry and Molecular Biology. 110:112-120. https://doi.org/10.1016/J.Ibmb.2019.05.003.