Location: Vegetable Research2012 Annual Report
1a. Objectives (from AD-416):
1. Develop sensitive diagnostic tools for the emerging viral diseases of greenhouse tomatoes and bacterial diseases on vegetable brassicas. 2. Characterize the etiology of emerging diseases of vegetables and identify critical factors affecting the epidemiology of those diseases. 3. Characterize and develop host resistance to effectively manage viral and Fusarium wilt diseases on watermelon.
1b. Approach (from AD-416):
Relative to Objective 1, several molecular detection techniques, including real-time RT-PCR, loop-mediated isothermal amplification (LAMP) and small RNA deep sequencing, will be used to study viral diseases of tomato, and develop strain-specific detection assays for Pepino mosaic virus (PepMV), pospiviroids, and the bacterium Liberibacter solanacearum. A combination of targeted and whole genome sequencing of select Pseudomonas syringae and P. cannabina strains will be conducted and analyzed using appropriate bioinformatics tools. Species-specific primer sets will be developed to detect P. cannabina pv. Alisalensis. Primer sensitivity will also be tested on related and nonrelated bacterial isolates from various sources. For Objective 2, the potential of transmitting tomato viroids on tomato seed and any synergistic interactions between the viroids and PepMV on tomato will be analyzed using newly developed, sensitive detection methods. If seed transmissibility of viroids on tomato is confirmed, a reliable seed health assay will be developed to select clean plants for the generation of viroid-free seed. The influence of environmental factors (e.g., humidity and temperature) on bacterial leaf blight of leafy green Brassicas will be studied under controlled conditions. Seed transmissibility, alternate hosts, and survivability of the bacterial pathogens will also be examined. Under Objective 3, molecular markers linked to resistance to viruses, including Papaya ringspot virus (PRSV) and Watermelon mosaic virus (WMV) in watermelon and its rootstock (bottle gourd), will be identified by analyzing candidate disease resistance genes (elongation factor sequences) associated with virus resistance in segregating populations (F2, F3, and BC1). PCR-based genetic analysis will also be applied to identify DNA markers linked to single or multiple virus (PRSV, WMV and ZYMV) resistance. Aided by marker-assisted selection (MAS), resistant lines of watermelon and bottle gourd will be developed. Inheritance of new sources of resistance to Fusarium wilt (FW) race 2 in watermelon will be elucidated using F2, F3, and recombinant inbred populations. Marker discovery will be conducted using Illumina Next-Generation sequencing, and markers for FW resistance will be mapped and tested for use in MAS.
3. Progress Report:
This is a new project as of May 1, 2012. An accurate and timely detection of the causal agent(s) is a prerequisite in designing an effective disease management strategy. Several molecular detection techniques, including real-time reverse transcriptase polymerase chain reaction (RT-PCR), loop mediated isothermal amplification (LAMP) and small Ribonucleic Acid (RNA) deep sequencing, were initiated in an effort to provide a sensitive detection of viruses, viroids, and Liberibacter on tomato. A combination of targeted and whole genome sequencing of select Pseudomonas syringae and P. cannabina strains were initiated in an effort to develop a species specific primer set for the effective detection of P. cannabina pv. alisalensis. To determine seed transmission potential of tomato viroids and characterization of synergistic interactions between tomato viroids and Pepino mosaic virus on tomato, seeds from viroid-infected tomato plants have been gathered and biological and molecular characterization are underway. The influence of environmental factors (e.g., humidity and temperature) on bacterial leaf blight of leafy green Brassicas were studied under controlled conditions. Experiments for seed transmissibility, host range, and survivability of the bacterial pathogens were also initiated. For determination of genetic inheritance of resistance, and development of molecular markers linked to virus resistance in watermelon and bottle gourd, segregating populations of bottle gourd have been generated and were evaluated. Efforts to identify new sources of resistance to Fusarium wilt race 2 in watermelon were initiated. Application of the technology of genotyping by sequencing has been established with collaborators in an effort to identity single nucleotide polymorphisms in association with virus or Fusarium resistance in watermelon and bottle gourd.
Ling, K., Wechter, W.P., Walcott, R.R., Keinath, A.P. 2011. Development of a real-time RT-PCR assay for squash mosaic virus useful for broad spectrum detection of various serotypes and its incorporation into a multiplex seed health assay. Journal of Phytopathology. 159:649-656.