Location: Vegetable Crops Research2012 Annual Report
1a. Objectives (from AD-416):
1) Identification of sources of PVYNO resistance genes and determination of whether taxonomic or biogeographic data predict the distribution of resistance genes in wild Solanum species. 2) Determination of whether wild Solanum species that exhibit resistance to PVYO also express resistance to PVYNO. 3) Initiation of genetic studies that will elucidate the inheritance pattern of PVYNO resistance in selected wild Solanum species. 4) Characterization of the mechanisms of PVYNO and PVYO resistance (e.g. extreme resistance, hypersensitive response, inhibition of phloem transport) in new germplasm sources. 5) Determination of whether existing molecular markers for PVY resistance co-segregate with resistance in new germplasm sources. 6) Characterization of in-plant distribution of PVY throughout the growing season in selected resistant germplasm. 7) Initiation of efforts to introgress new PVYNO and PVYO resistance genes into the cultivated potato.
1b. Approach (from AD-416):
A set of true potato seeds of 160 accessions of 40 wild Solanum species (4 accessions per species) has been obtained from the NRSP-6 Potato Gene Bank. Seeds will be sown in a greenhouse and transplanted to individual pots 3 weeks later. One week after transplanting, the seedlings will be mechanically inoculated with the PVYO strain. Leaves from each asymptomatic plant will be evaluated for PVY titer using ELISA. Beginning 4 weeks after inoculation, individual plants will be scored weekly for symptom expression. Tubers will be collected from each plant and evaluated for PVY titer using ELISA. The proposed research project will repeat this study using PVYNO instead of PVYO. Diploid wild species clones selected for resistance to PVYNO will be crossed with susceptible diploid clones of the cultivated potato to initiate genetic and introgression studies. F1 hybrids will be created in the first year of the study. We will compare responses of each interaction type (susceptible, extreme res., local hypersensitive and systemic hypersensitive) using non-inoculated individuals (from cuttings of Solanum species taken before PVY inoculations). Plants from two different accessions (from two different species, if possible) from each interaction category will be grown to 6 weeks of age and inoculated with PVY on a lower leaf. One cm2 leaf samples will be taken from the inoculated leaf and a non-inoculated upper leaf 0 hours, 1 day, 5 days, and 7 days after inoculation. These samples will be used for RNA extraction. Real-time reverse transcription PCR will be used to assay the transcription of known pathogenesis-related genes (e.g. PR1a, PDF1, PR3, and PR5). Results will give us a better understanding of the temporal responses of the host during different resistance responses.
3. Progress Report:
Identification of sources of Potato Virus Y (PVY) NO recombinant genes and determination of whether taxonomic or biogeographic data predict the distribution of resistance genes in wild Solanum species. PVY resistance was found to be associated with elevation. Determination of whether wild Solanum species that exhibit resistance to PVYO also express resistance to PVYNO. Clones that were resistant to PVYo were inoculated with PVYNO and found to retain their resistance. Clones are being multiplied for field trials to confirm these results. Initiation of genetic studies that will elucidate the inheritance pattern of PVYNO resistance in selected wild Solanum species. Genetic studies have been carried out and a major gene for resistance has been identified. Characterization of the mechanisms of PVYNO and PVYO resistance (e.g. extreme resistance, hypersensitive response, inhibition of phloem transport) in new germplasm sources. The mechanism of resistance is extreme resistance. It has been found to be heat stable, unlike other sources of resistance. Determination of whether existing molecular markers for PVY resistance co-segregate with resistance in new germplasm sources. Existing markers do not co-segregate. However, single-nucleotide polymorphism (SNP) markers have localized the gene on chromosome 9. Candidate markers are being evaluated. Characterization of in-plant distribution of PVY throughout the growing season in selected resistant germplasm. Seed multiplication and preliminary testing is underway. To investigate how the timing of PVY infection within the growing season affected the efficiency of tuber infection or distribution of infection within developing daughter tubers, replicated field experiments were conducted at locations in central Wisconsin corresponding to the commercial potato production regions within the state. Experimental plots consisted of the potato varieties Russet Norkotah, Silverton Russet, Dark Red Norland, and Goldrush replicated in a randomized complete block design (RCBD) to equalize the exposure of each susceptible variety to immigrating populations of potential aphid vectors. Each experimental replicate consisted of 40 plants arranged in 2, 20-plant rows spaced 91 cm between rows. A within row plant spacing of 35.6 cm was used for the four cultivars. In an effort to minimize the possibility of PVY interference between replicates, a double guard row of PVY-resistant, var. ‘Eva’ was planted between varieties and surrounding the experimental plot. Experimental plots were established in Hancock, Wisconsin and all varieties in rows received 45.5 kg N ha-1 applied in-furrow at planting. To ensure a source of PVY inoculum, all plants in each variety-replicate were mechanically sap-inoculated with a PVYO isolate at the 2-3 true-leaf developmental stage. Beginning two weeks after inoculation and continuing twice monthly throughout the growing season, leaflet samples were inoculated from all plants in each variety / replicate combination and tested using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) to determine if and when each plant was infected with PVY. At the end of the season the virus in a subset of the infected plants in replicated plots was examined using ELISA. Field experiments were terminated in September 2011 using two successive weekly applications of Reglone® 240 at a rate of 2 L ha-1. Soon after the second application of foliar desiccant, all plants determined to be infected with PVY were individually hand harvested from each experimental location and all tubers handled and labeled. In addition to tuber weight and size data, specific gravity (SG) was determined. Plants were considered uninfected if bi-monthly serological testing consistently yielded a negative result throughout the growing season. To examine rates of tuber infection and within tuber distribution of PVY from plants infected at different intervals over the course of the growing season in field experiments, dormancy was chemically broken on one half of all harvested tubers as described previously. One portion of tuber halves were then placed in the greenhouse to initiate meristem activity and shoot elongation and the remainder placed in storage. After 12-20 days of “green-sprouting” in the greenhouse, = 5 meristem sections were cut from each tuber region, transplanted into soil mix, and developing shoots serologically tested for PVY infection. To comparatively determine how periods of cold storage might affect PVY distribution within tubers, longitudinal half-sections were removed from storage after 98 days and allowed to complete dormancy break through bench-top, green-sprouting. Half sections were then sub-sectioned into rose, middle and stem-end regions and = 5 meristems were excised, transplanted, and tested serologically for PVY infection after plants grew to a height of approximately 15-20 cm. The efficiency of tuber infection among developing tubers under plants naturally infected with PVY in the field remained relatively unchanged in selected varieties as plants became infected during later developmental stages. With increasing plant age at the time of infection, reductions in the mean proportion of PVY-infected shoots only occurred within the varieties R. Norkotah and Silverton Russet. Mean proportions of infected tubers varied significantly in the proportion of infected shoots in the varieties Dark Red Norland and Goldrush. This pattern of tuber infection with increasing age of infection was most consistent among the two varieties tested when compared to R. Norkotah and Silverton Russet. The estimated within tuber distribution of PVY varied among rose, middle, and stem regions. In each case, the highest proportion of PVY infected shoots was detected from tuber meristems, or that classified as the ‘rose-end’ section when compared to either middle or stem-end tuber sections. Moreover, PVY infection frequencies among shoots produced on complementary tuber half sections did not appear to be influenced by post-harvest storage compared to chemical dormancy break. Initiation of efforts to introgress new PVYNO and PVYO resistance genes into the cultivated potato. Crosses have been made to cultivated germplasm and families have been created at the diploid and tetraploid levels. Seedling families are in the field for evaluation of adaptation and agronomic quality. This research relates to Objective 1, Develop adapted potato clones with enhanced resistance to major potato diseases.