2012 Annual Report
Five different genotypes each of commercial cultivars of peas, lentils, and chickpeas, will be inoculated with each isolate of R. leguminosarum or M. ciceri. Replicated plants will be grown individually in sterile “conetainers” using a low-nutrient sand-based potting mix. Each conetainer will be fertilized with 10 ml 2 M ammonium sulfate with 10% isotopic 15-N at 1 wk and 2 wk to isotopically “label” the plant biomass. At 3 wk and 6 wk after planting, each conetainer will receive 5 ml total volume of a broth culture of a single rhizobium isolate at 105 – 106 cells per ml. Plants will be harvested at 12 wk after planting. The number of nodules per plant will be determined. Biomass will be dried, weighed, ground, and analyzed for both total N and 15-N. The proportion and total amount of plant N that was fixed by the rhizobial symbiont will be determined with this information.
Real-time (TaqMan) PCR assays will be developed for quantifying the amount of R. leguminosarum or M. ciceri present in root nodules. Briefly, isolates will be amplified using SRAP markers to identify amplicons that are present in all examined isolates of R . leguminosarum or M. ciceri. Species-specific amplicons will be cloned and sequenced and the sequence will be analyzed using Primer Express software to identify candidate Taqman probes and primers.
Pea and lentil plants will be inoculated with R. leguminosarum and grown in greenhouse flats. Approximately 5 d after emergence plants will be inoculated with a zoospore suspension of the soilborne pathogen Aphanomyces euteiches. Control plants will comprise two treatments, one in which the plants are inoculated with only R. leguminosarum and the other being plants inoculated only with A. euteiches. Plants will be scored for disease severity 14 d after inoculation using a scale of 1 (healthy plant) to 5 (dead plant) that is well accepted for scoring Aphanomyces root rot in pea and lentil. The number of nodules per plant will be determined. DNA will be extracted from the entire root system of each plant. The amount of R. leguminosarum and A. euteiches DNA present in the root system will be determined using real-time PCR. Correlations between the amount of pathogen DNA and R. leguminosarum DNA present in root systems will be calculated, as will correlations between the number of nodules in root systems and the amount of A. euteiches and R. leguminosarum DNA present in roots.