Location: Vegetable Research2020 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.” As a tobamovirus, Cucumber green mottle mosaic virus (CGMMV) is seed-borne, but its mechanism in seed transmission was not clear. To study the mechanism of seed transmissibility of CGMMV on watermelon, we focused our efforts in using naturally infected seeds through bioassays using seedling growth out or by mechanical inoculation on seedlings using tissue extract of contaminated seeds to simulating natural situation. Although there was no seedling infected through natural seedling grow-out, melon seedlings were infected by contaminated seed extract through mechanical inoculation. Understanding the mechanism of seed transmission is important as it is necessary to plant seeds that have been tested to be free from CGMMV. In addition, it is important to minimize the chances in touching plants as well as to practice hygiene and use disinfectants to prevent virus transmission. 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.” Tomato planta macho viroid (TPMVd, also known as Mexican papita viroid) is an emerging viroid. Previously, we have developed a series of infectious clones of TPMVd recombinants with chimeric sequences from both genotypes of TPMV inciting respective mild or severe phenotypes to map the virulence determinant. TPMVd-infected tomato seeds have been collected and will be used to study the mechanism of seed transmission of TPMVd on tomato. Bioassays to evaluate seed transmission of TPMVd has been delayed due to the COVID-19 pandemic. 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 of the sweet potato whitefly (MEAM1) and African cassava whitely (SSA1), functional genomic analysis through transcriptome evaluation and small RNA analysis of whiteflies upon acquisition of Tomato yellow leaf curl virus (TYLCV), we have selected a number of target genes for evaluation for their mortality effect on whitefly using RNA interference (RNAi). Several RNAi constructs tested through ring tests in three labs had 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.” Using tomato as a model system, we were interested in developing transgenic plants expressing those sequences with RNAi effects against whiteflies from in vitro and/or in planta assays. 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. The majority of the work has been completed, but additional experiments for verification have been delayed due to the Covid pandemic. However, transgenic T0 plant materials and T1 seeds have been maintained and can be continued as soon as the facility is back to normal operation. This portion of the progress report is related to Objective 3, Sub-objective 3.1. “Genotyping-by-sequencing will be used to identify single nucleotide polymorphisms (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, causes a partial resistance breaking on certain tomato cultivar with Tm2^2 gene. In genotyping by sequencing (GBS) experiments, we could not identify SNPs associated with the resistance breaking. Therefore, we shifted our focus to understand global gene expression differences between resistance and susceptible plants. Transcriptomic analysis identified 42 differentially expressed genes, with major classes of function in pathogenesis related genes, transcription factor genes and some proteases. These genes and their associated pathways may be responsible in causing Tm-2^2 resistance breaking in tomato by ToMMV. 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 regions associated with Fusarium wilt race 1 and race 2. 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 lines have been vernalized, then self-pollinated to generate S2 lines. The S2 lines were tested in a large field assay this Spring. The largest growers and processors of brassica leafy greens in the U.S. were invited to the field trial and, with their input, additional selections made from the most resistant individuals. The selected lines (four lines) have been vernalized, self-pollinated and crossed into a commercial turnip green cultivar “Topper.” Seeds from these crosses and self-pollinations have been collected. 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.” Several filed tests were conducted this year, in collaboration with researchers at Clemson University using cotton seed meal as a carbon source, and the control carbon source molasses/pelletized chicken manure. These studies have shown that cotton seed meal work as well as the control for reducing or eliminating the bacterial wilt pathogen from the soil down to more than 12 inches deep, but also appears to have reduced weeds by more than 90% in the anaerobic soil disinfestation (ASD) treatments compared to non-treated soils. Unfortunately, pepper plants transplanted into the cottonseed meal ASD treatments were negatively impacted in terms of growth. We will be looking at reducing the amount of cottonseed meal, as well as aeration of the soil post treatment to reduce the impact on the plants.
1. Genome sequencing, functional genomic and RNA interference to manage whitefly and its transmitted viruses on tomato and African cassava. Whitefly (Bemisia tabaci), a major vector transmitting hundreds of viruses causing diseases to many economically important crops worldwide, resulting in more than a billion dollars of crop losses. The genome of whitefly (B. tabaci) biotype B (=MEAM1) was sequenced for the first time using an isogenic colony of whitefly developed by ARS researchers in Charleston, South Carolina (Chen et al., 2016). Subsequently, through USAID support, the genome of African cassava whitefly was also sequenced using field-collected whiteflies in Africa (Chen et al., 2019). Functional genomic analysis using comparative transcriptome (Hasegawa et al., 2018) or small RNA profiling (Shamimuzzaman et al., 2019) revealed candidate genes potentially associated with virus transmission. Some selected genes are used to evaluate RNA interference effects against whiteflies on tomato which could offer a promising and environment friendly means of management to whitefly and its transmitted viruses.
2. Genome sequencing of bottle gourd and identification of makers associated with resistance to Papaya ringspot virus. The genome of bottle gourd (Lagenaria siceraria) cv. USVL1VR-Ls was sequenced for the first time using an inbred line (cv. USVL1VR-Ls) developed by ARS researchers in Charleston, South Carolina. Comparative genomic analysis between bottle gourd and other cucurbits identified strong chromosome level syntenic relationships. A dominant monogenic resistance locus, Prs, conferring resistance to papaya ringspot virus (PRSV) was mapped and a DNA marker tightly linked to the Prs locus was developed and could be useful for marker-assisted selection.
5. Record of Any Impact of Maximized Teleworking Requirement:
The maximized teleworking requirement has caused some delays in timely carrying out some bioassays in greenhouse to evaluation seed transmission of TPMVd on tomato in the Objective 1.2 as well as for bioassays of transgenic tomato plants with RNA interference effect against whiteflies in the Objective 2.2. However, plant materials are maintained or seeds collected which could be quickly used to carry out the planned experiments once the facility is open.
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