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ARS Home Southeast Area Charleston, South Carolina Vegetable Research Research





- Ling, Kai-Shu
- 2023
- 2022
- Publications
- Plant Diseases (303)

Research Project: Harnessing Genomic Technologies Toward Improving Vegetable Health in Field and Controlled Environments **Location:
                            Vegetable Research** 2023 Annual Report **Objectives**
                                

1. Characterize biological and molecular properties of emerging and important plant pathogens to develop sensitive detection systems and accurate disease diagnostic protocols.
1.1. Develop a multiplex real-time PCR useful for seed health testing of tobamoviruses on tomato and engineer an infectious clone of tomato brown rugose fruit virus and its recombinants for functional characterization. 
1.2. Develop a bioassay for Fusarium oxysporum f. sp. niveum (Fon) race 1 and race 2 differentiation using genetically fixed Citrullus amarus differentials. 
1.3. Develop a PCR-based race determination assay to differentiate race 1 from race 2 of the watermelon Fusarium wilt pathogen Fusarium oxysporum f. sp. niveum (Fon). 

2. Identify and develop new germplasm or cultivars with resistance to emerging or important diseases or pathogen-vectors using molecular-based tools or biotechnology.
2.1. Develop plant genetic materials with resistance to emerging tobamoviruses, ToBRFV in tomato and CGMMV in watermelon.
2.2. Applying biotechnology to develop novel genetic materials with resistance to viruses or whitefly.
2.3. Develop bacterial blight resistant collard greens (Brassica oleracea var. viridis).
2.4. Develop bacterial blight resistant brassica leafy greens (Brassica rapa).

3. Design and implement integrated pest management to manage emerging and important diseases on vegetable crops under field and controlled environments.
3.1. Develop effective disinfectants and far UV light treatment for plant diseases and greenhouse whitefly on vegetable crops in controlled environment.
3.2. Develop Bacterial Leaf Blight resistant mustard and/or turnip-green lines suitable for controlled environment agriculture (CEA). **Approach**
                                

Relative to Objective 1, a multiplex real-time polymerase chain reqaction (PCR) useful for seed health testing and specific detection of three tomato infecting tobamoviruses: tomato brown rugose fruit virus (ToBRFV), tomato mottle mosaic virus (ToMMV) and tomato mosaic virus (ToMV) will be developed. We will engineer infectious clones of ToBRFV, ToMMV and ToMV and their respective recombinants for functional characterization, particularly on the resistance breaking by ToBRFV to the Tm-2^2 resistance gene. We will also conduct experiments to identify host tomato factors or receptors that resulted in the susceptibility by ToBRFV and characterize the gene-silencing suppressor and its interaction with host receptors.  In separate experiments, we will develop a bioassay for Fusarium oxysporum f. sp. niveum (Fon) race 1 and race 2 differentiation using genetically fixed Citrullus amarus differentials.  A PCR-based race determination assay will also be developed to differentiate race 1 from race 2 of the watermelon Fusarium wilt pathogen Fon. 

For Objective 2, we will identify and develop new germplasm or cultivars with resistance to emerging or important diseases or pathogen-vectors using molecular-based tools or biotechnology. First, on developing plant genetic materials with resistance to emerging tobamoviruses, ToBRFV in tomato and cucumber green mottle mosaic virus (CGMMV) in watermelon, segregating populations (F1, F2) of tomato to ToBRFV and watermelon to CGMMV have been developed using previously identified resistance germplasm. We will conduct phenotyping on the breeding populations for their resistance properties to the respective viruses and bulk segregant analysis (QTL-seq) on these tomato or watermelon genetic materials. Single nucleotide polymorphisms (SNPs) in association with the resistance traits will be identified and molecular markers developed. In a separate experiment, we will apply biotechnology to develop novel genetic materials with resistance to viruses or whitefly. Several candidate genes that are useful for genome editing to the target viruses and whitefly have been identified. Gene constructs will be used for transformation of appropriate plant materials. Transgenic plant materials with gene edited mutations will be used to evaluate for resistance to the target viruses. Once it is confirmed for their resistance, we will apply permit for large-scale field trials and deregulation of the gene-edited materials. In another experiment, we will develop bacterial blight resistant collard greens and brassica leafy greens through breeding, marker-assisted selection and field trial evaluations.  

Under Objective 3, we will design and implement integrated pest management to manage emerging and important diseases on vegetable crops under field and controlled environments. Developing effective disinfectants and far ultra violet (UV) light treatment that are useful to manage plant diseases and greenhouse whitefly on vegetable crops in controlled environment. In a seperate experiment, we will develop bacterial leaf blight resistant mustard and/or turnip-green lines suitable for controlled environment agriculture. **Progress Report**
                                

This portion of the progress report is related to Objective 1, Sub-objective 1.1: “Develop a multiplex real-time PCR useful for seed health testing of tobamoviruses on tomato and engineer an infectious clone of tomato brown rugose fruit virus and its recombinants for functional characterization”. A real-time PCR useful for seed health test ahs been developed for ToBRFV. In addition, respective real-time PCR for species-specific detection of ToMV and ToMMV have also been developed. We are optimizing the reagent concentration and reaction conditions to achieve sensitive detection of 3 tobamoviruses in a single reaction tube that are useful for seed health test. In another experiment, we have cloned a full genome of ToBRFV, engineered into an T-DNA binary vector, and are in the process of confirming its infectious nature through a bioassay using agroinfiltration on Nicotiana benthamiana and tomato ‘Moneymaker’.

This portion of the progress report is related to Objective 2, Sub-objective 2.1 “Develop plant genetic materials with resistance to emerging tobamoviruses, ToBRFV in tomato and CGMMV in watermelon”.  In our previous project, through screening the USDA germplasm collections, we have identified the Plant Introduction Accessions with resistance in a tomato wild relative (Solanum pimpinellifoliun) to ToBRFV and a watermelon wild relative (Citrullus colocynthis) to CGMMV.  These respective accessions were advanced through self-pollination for at least 3 generations to achieve genetic pure lines. Two families of segregating populations (F1, F2) of tomato to ToBRFV were developed. One family was generated between two S. pimpinellifolium and another family generated through an interspecies cross of S. pimpinellifolium and S. lycopersicum.  Similarly, a family of segregating populations (F1 and F2) were generated between two Citrullus colocynthis lines with resistance and susceptibility to CGMMV.  Through mechanical inoculation in a containment greenhouse, we evaluated the phenotype of their respective disease resistance using these segregating populations against respective viruses.  

This portion of the progress report is related to Objective 2, Sub-objective 2.2. “Applying biotechnology to develop novel genetic materials with resistance to viruses or whitefly”.  Through literature review and gene analog analysis, several candidate genes in the tomato genome with potential resistance to viruses or tolerance to insect were identified. Their respective guide RNAs to the target genes of interest or its promoter sequence were designed and engineered into a proper CRISPR-Cas9 construct.  Through agrobacterium transformation, about 20 lines of transgenic tomato plants per construct with gene-editing to 4 target genes were generated.  Two of these gene construct targeted a gene with a potential resistance to tobamoviruses (ToBRFV, ToMV) and another two constructs with a potential tolerance to whitefly. Bioassays to evaluate their resistance to tobamoviruses or tolerance to whitefly are underway. 

This portion of the progress report is related to Objective 3, Sub-objective 3.1. “Develop effective disinfectant and far UV light treatments to plant diseases and greenhouse whiteflies on vegetable crops in controlled environment”. ToBRFV is an emerging tobamovirus breaking the common resistance gene Tm-2^2. This highly contagious virus can be easily transmitted mechanically through many of plant handling.  Previously through extensive screening of chemical disinfectants, we have identified several disinfectants (Clorox bleach solution, Trisodium Phosphates, Virkon and Virocid) which are useful to deactivate ToBRFV infectivity. Some other disinfectants (Lactoferrin and SP2700) could be sprayed on plants to induce disease resistance to ToBRFV and CGMMV. Although Far UV light treatment has been shown to have some virucidal effect to viruses, it was not effective against ToBRFV infectivity when using leaf tissue extract for treatment. Therefore we shifted our efforts to use a cold plasma ozone treatment in contaminated water/nutrient solution with ToBRFV.  Preliminary results showed promising results in deactivate ToBRFV infectivity in contaminated water solution. **Accomplishments**
                                

1. Managing an Emerging Tomato Brown Rugose Fruit Virus Using Disinfectant and Disease Resistance. Tomato is one of the most important vegetable crops in the United States (U.S.) with over $2 billion farm gate value annually. A new emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has caused serious disease epidemics around the world, including the U.S. Since its first report in the U.S. by an ARS researcher in Charleston, South Carolina; in 2019, USDA-APHIS issued a federal order to inspect this quarantine pest from import tomato and pepper seeds and produces. In a recent outbreak in North Carolina in May 2023, half a million of tomato seedlings were destroyed by APHIS due to concerns from a contaminated seed lot. ARS researcher conducted research on two key areas to manage this emerging virus, a short term with sanitization using disinfectants and a long term with disease resistance using a novel source of resistance from a wild tomato. Several effective disinfectants against ToBRFV are being used by growers to manage this emerging virus. Through genome resequencing, molecular markers have been identified and will be developed to accelerate plant breeding process. **Review Publications**
                            *Abrahamian, P., Cai, W., Nunziata, S.O., Ling, K., Jaiswal, N., Mavrodieva, V.A., Rivera, Y., Nakhla, M.K. 2022. Genetic diversity and comparative analysis of tomato brown rugose fruit virus isolates. Viruses. 14:2816. https://doi.org/10.3390/v14122816.*
                                *Ling, K., Gilliard, A.C., Zia, B. 2022. Disinfectants useful to manage the emerging tomato brown rugose fruit virus in greenhouse tomato production. Horticulturae. 8: 1193. https://doi.org/10.3390/horticulturae8121193.*
                                *Padmanabhan, C., Zheng, Y., Shamimuzzaman, M., Fei, Z., Ling, K. 2022. The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato. PLOS ONE. 17(5):e0257936. https://doi.org/10.1371/journal.pone.0257936.*


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