Location: Vegetable Research
2018 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
Using a commercial sample originally collected in Germany, we were able to obtain the full genome sequences of two cucumber green mottle mosaic virus (CGMMV) isolates belonging to the European genotype. In an effort to develop an infectious clone, the full genome sequence of a Canadian CGMMV isolate was amplified by polymerase chain reaction (PCR) and cloned into a plant expression vector. Initial evaluation through agro-infection or by mechanical inoculation using in-vitro transcripts on watermelon seedlings did not result in infectivity, additional evaluations will be necessary in the coming year. However, in a separate experiment, using seeds collected from watermelon infected by CGMMV, we were able to evaluate the mechanism of seed transmission through seedling grow-out and mechanical inoculation. Although we did not observe seed transmission in germinating seedlings in the limited number of seeds evaluated, a high incidence of virus transmission was observed through mechanical transmission to seedlings. Various molecular and serological methods were evaluated in seed health assays for CGMMV. In collaboration with a commercial company, serological methods using a new antibody was shown to achieve the sensitivity and reliability for seed health assay on commercial seed samples.
Using two distinct genotypes of Tomato planta macho viroid (also known as Mexican papita viroid) that produce differing effects on disease symptoms (stunting or not stunting on infected tomato plants), infectious clones were generated from a series of viroid mutants consisting of recombinant sequences from both genotypes and shown to be able to incite various levels of severity in disease symptoms on infected tomato plants. Seeds from TPMV-infected tomato plants have been generated and will be used to study the mechanism of seed transmission in tomato.
With the recent successful studies on sequencing the whitefly (B. tabaci) genome and transcriptome analysis on whiteflies, a series of differentially expressed genes were identified and selected for evaluation of their effects on whitefly control using RNA interference (RNAi) technology. A number of RNAi constructs were shown through a ring test in three separate laboratories to produce consistent effects with higher rates of whitefly mortality than controls.
Although our previous study through in-vitro analysis using dsRNAs in an artificial diet generated promising results, there is a serious limitation in delivering such dsRNA in field conditions, particularly for its application to control cassava whiteflies in Sub-Saharan Africa. For a proof of concept, we were interested in evaluating the RNA interference (RNAi) effects on whiteflies using stable transgenic tomato plants. Gene constructs containing selected promising sequences were engineered and used for tomato transformation. A number of transgenic tomato plants containing various RNAi constructs have been generated and will be used to evaluate their effects against whitefly survivability and virus transmission.
Tomato mottle mosaic virus (ToMMV) is an emerging new virus first identified by ARS scientists in Charleston, South Carolina and has been shown to have broad geographic distribution in the world affecting tomato, pepper and possibly other crops. In screening tomato cultivars, a resistance breaking phenomenon to ToMMV was found on some tomato cultivars with a general resistance to other tobamoviruses, particularly when plants were grown under high temperature conditions. These materials will be useful to determine the mechanism of resistance breaking in tomato against ToMMV.
We have used an F2:3 population derived from a cross with USVL246-FR2, a wild watermelon line with resistance to Fusarium wilt race 1 and 2 developed and released from our group, and a highly susceptible watermelon USVL114 to identify quantitative trait loci (QTL) associated with Fusarium race 1 and 2 resistance. These single nucleotide polymorphic (SNP) markers are being converted to markers useful for breeders using marker assisted selection techniques. We have recently finished generating a recombinant inbred population of over 200 families at the F8 generation. This RIL population is being used to further elucidate genes involved in disease resistance.
Laboratory has screened nearly 200 of the 300 remaining Brassica rapa accessions from the USDA Brassica collection. The remaining 100 accessions have a tendency to bolt early, this confounds the assay and are now being tested in a growth chamber assay. From the 200 accessions already tested, we have found two with significant levels of resistance and are vernalizing these plants in preparation for self-pollinations.
We used several new types of carbon source in our first trial of Anaerobic Soil Disinfestation (ASD) during the spring of 2018. We used sweetpotato, rice hulls, cotton seed meal, and the control molasses/pelletized chicken manure. We determined that cotton seed meal work as well as the control for reducing numbers of the bacterial wilt pathogen Ralstonia solanacearum. This treatment is half the cost of the control treatment, and will be further tested during FY 2019.
Accomplishments
1. The genome of bottle gourd (Lagenaria siceraria), a vegetable and an important rootstock for watermelon, was sequenced. In collaboration with a team of international scientists, ARS researchers at Charleston, South Carolina, were the first in the world to sequence the genome of bottle gourd, an ethnic vegetable and an important rootstock for watermelon and other cucurbits in the U.S. and around the world. The genome sequence provides fundamental knowledge and novel insights into broad spectrum disease resistance against multiple viruses and other horticultural traits, including formation of unique fruit shapes. The molecular markers developed should be useful in marker-assisted selection to facilitate bottle gourd breeding by public and private scientists.
Review Publications
Sui, X., Zhang, S., Wu, Z., Ling, K. 2018. Reverse transcription loop-mediated isothermal amplification for species-specific detection of tomato chlorotic spot orthotospovirus. Journal of Virological Methods. 253:56-60. https://doi.org/10.1016/j.jviromet.2018.01.002.
Ling, K. 2017. Decontamination measures to prevent mechanical transmission of viroids. In: Hadidi, A., Flores, R., Randles, J., Palukaitis, P., editors. Viroids and Satellites. San Diego, CA: Academic Press. p. 437-446. https://doi.org/10.1016/B978-0-12-801498-1.00041-3
Zhao, R., Wang, N., Liu, S., Ling, K., Fan, Z., Zhou, T. 2016. p22 of tomato chlorosis virus, an ribonucleic acid silencing suppressor, is naturally expressed in the infected plant. Acta Virologica. 60:423-425.
Peng, D., Xle, J., Qiang, W., Ling, K., Gui, L., Fan, Z., Zhou, T. 2017. One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus. Journal of Virological Methods. 248:154-158.
Wu, S., Shamimuzzaman, M., Sun, H., Salse, J., Sui, X., Wilder, A.J., Wu, Z., Levi, A., Xu, Y., Ling, K., Fei, Z. 2017. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ringspot virus resistance locus. Plant Journal. 92:963-975. https://doi.org/10.1111/tpj.13722.
Daley, J., Branham, S., Levi, A., Hassell, R., Wechter, W.P. 2017. Mapping resistance to alternaria cucumerina in cucumis melo. Phytopathology. 107(4):427-432. https://doi.org/10.1094/PHYTO-06-16-0246-R.
Branham, S., Farnham, M.W., Robinson, S.M., Wechter, W.P. 2018. Identification of resistance to bacterial leaf blight in the U.S. Department of Agriculture collard collection. HortScience. 53:838-841. https://doi:10.21273/HORTSCI12347-17.
Branham, S., Levi, A., Katawczik, M.L., Fei, Z., Wechter, W.P. 2018. Construction of a genome-anchored, high-density genetic map for melon (Cucumis melo L.) and identification of Fusarium oxysporum f. sp. melonis race 1 resistance QTL. Journal of Theoretical and Applied Genetics. 131:829-837. https://doi:10.1007/s00122-017-3039-5.
Gundersen, D.E., Adrianos, S.L., Allen, M.L., Becnel, J.J., Chen, Y., Choi, M.Y., Estep, A., Evans, J.D., Garczynski, S.F., Geib, S.M., Ghosh, S.B., Handler, A.M., Hasegawa, D.K., Heerman, M.C., Hull, J.J., Hunter, W.B., Kaur, N., Li, J., Li, W., Ling, K., Nayduch, D., Oppert, B.S., Perera, O.P., Perkin, L.C., Sanscrainte, N.D., Sim, S.B., Sparks, M., Temeyer, K.B., Vander Meer, R.K., Wintermantel, W.M., James, R.R., Hackett, K.J., Coates, B.S. 2017. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Applications of RNA interference and CRISPR gene editing technologies in pest control. Trends in Entomology. 13:109-137.
Hasegawa, D.K., Chen, W., Zheng, Y., Kaur, N., Wintermantel, W.M., Simmons, A.M., Fei, Z., Ling, K. 2018. Comparative transcriptome analysis reveals networks of genes activated in the whitefly, Bemisia tabaci when fed on tomato plants infected with Tomato yellow leaf curl virus. Virology. 513:52-64. https://doi.org/10.1016/j.virol.2017.10.008.
Levi, A., Ling, K. 2017. USVL-380, A zucchini yellow mosaic virus resistant watermelon breeding line. HortScience. 52(10):1448-1450. https://doi:10.21273/HORTSCI12292-17.
