2013 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.
Sensitive virus detection is particularly necessary against emerging diseases where current virus detection methods may not be available. In the present study, several molecular-based detection methods were developed for the emerging virus tomato necrotic stunt virus and emerging viroid Mexican papita viroid. A simple sample preparation was developed which allows us to effectively detect these pathogens. In the Brassica bacterial pathogen study, numerous isolates of both bacterial pathogens responsible for bacterial leaf blight of leafy green Brassicas were collected from several affected states. These isolates were genetically and biochemically characterized. A combination of targeted and whole genome sequencing of select leaf blighting and black rotting Xanthomonas campestris pv. campestris strains were initiated for use in development of a species-specific primer set for the effective detection of the leaf-blighting form of this pathogen.
A genotype shift of PepMV in North America to a more aggressive genotype (CH2) was determined and linked to commercial tomato seed lots. Thus, a more rigorous seed health test was recommended to manage pepino mosaic from spreading. We also determined the seed-transmission potential of Potato spindle tuber viroid (PSTVd) in tomato and developed a sensitive molecular detection for virioids on tomato seeds. In an effort to understand the global virus distribution on tomato, we initiated a global survey of tomato viruses and viroids using previously developed small RNAs (sRNA) deep sequencing technology. Over 500 samples from all over the world were collected and nearly 100 sRNA libraries were sequenced using next generation sequencing. Numerous new viruses and virus strains were discovered from these libraries. In studies on the influence of environmental factors (e.g., humidity and temperature), leaf blight on leafy green Brassicas was studied under controlled conditions. The first year of experiments for host range and field survivability of the bacterial pathogens responsible for leaf blight of leafy brassica greens were completed.
In evaluating genetic inheritance of resistance to three cucurbit potyviruses in bottle gourd, we determined that the identified multiple virus resistance in bottle gourd, a common watermelon rootstock, was likely controlled by a single dominant gene. Using recently developed high throughput genotyping technology, several single nucleotide polymorphisms (SNPs) that were closely associated with the potyvirus resistance were identified. The identification of these molecular markers could facilitate efficient selection of breeding materials. For determination of genetic inheritance of resistance to Fusarium wilt, and development of molecular markers linked to resistance in watermelon, segregating populations of resistant and susceptible watermelon have been generated and were evaluated. Efforts to identify new sources of resistance to Fusarium wilt race 2 in dessert-type watermelon were initiated. A service agreement to apply the technology of genotyping by sequencing has been established to identify SNPs in association with Fusarium resistance in watermelon.
Multi-virus resistant bottle gourd (Lagenaria siceraria) lines with potential as rootstocks for watermelon grafting. Viral diseases are one of the major factors limiting watermelon production. Several insect-transmitted viruses in the family Potyviridae, including Papaya ringspot virus watermelon strain, Watermelon mosaic virus, Zucchini yellow mosaic virus, and Squash vein yellowing virus, can cause serious damage to watermelon production. In recent years, grafting watermelon to a rootstock is gaining popularity in the U.S. However, many current commercial watermelon rootstocks are susceptible to infection by one or more of these viruses. In the present study, ARS scientists in Charleston, South Carolina, evaluated several breeding lines of bottle gourd for resistance to the four above-mentioned viruses in greenhouse trials, and confirmed their field performance for multi-virus resistance in two consecutive years at two locations in South Carolina. These breeding lines with broad spectrum virus resistance are useful materials for plant breeders focused on developing superior bottle gourd varieties that can be deployed as watermelon rootstocks. These materials will be made publically available through an ARS germplasm release.
Ling, K., Li, R., Panthee, D.R., Gardner, R.G. 2013. First report of potato spindle tuber viroid naturally infecting greenhouse tomatoes in North Carolina. Plant Disease. 97:148.
Ling, K., Li, R., Bledsoe, M. 2013. Pepino mosaic virus genotype shift in North America and development of a loop-mediated isothermal amplification for rapid genotype identification. Virology Journal. 10:117.
Ling, K., Levi, A., Adkins, S.T., Kousik, C.S., Miller, G., Hassell, R.L., Keinath, A.P. 2013. Development and field evaluation of multiple virus-resistant bottle gourd (Lagenaria siceraria). Plant Disease. 97:1057-1062.
Sarris, P.F., Trantas, E.A., Baltras, D.A., Bull, C.T., Wechter, W.P., Yan, S., Ververidis, F., Almeida, N.F., Jones, C.D., Dangl, J.L., Panopoulos, N.J., Vinatzer, B.A., Goumas, D.E. 2013. Comparative genomics of multiple strains of Pseudomonas cannabina pv. alisalensis, a potential model pathogen of both Monocots and Dicots. PLoS One. 8(3):1-16.
Nyczepir, A.P., Kluepfel, D.A., Waldrop, V., Wechter, W.P. 2012. Soil solarization and biological control for managing Mesocriconema xenoplax and Short Life in a newly established peach orchard. Plant Disease. 96:1309-1314.
Schofield, D., Bull, C.T., Rubio-Salazar, I., Wechter, W.P., Westwater, C., Molineux, I.R. 2013. "Light-tagged" bacteriophage as a diagnostic tool for the detection of phytopathogens. Bioengineered. 4:1-5.