1a. Objectives (from AD-416):
1) Develop breeding/mapping populations and select high-yielding resistant breeding lines from the new sources of resistance identified in the first phase of this project; 2) Determine the genetics of resistance of these new sources of resistance and develop molecular markers for these traits; 3) Determine pathogen diversity, with emphasis on how this affects the expression of resistance to PSD in soybean.
1b. Approach (from AD-416):
Field screening and laboratory assays will be conducted to determine: 1) The resistance of soybean lines to PSD; and 2) The genetic and pathogenic diversity of PSD isolates collected and purified from different genotypes of soybean in different geographic origins.
3. Progress Report:
Development of breeding and genetic mapping populations Soybean cultivars and elite breeding lines from the Midwest or from the University of Arkansas were crossed with the following soybean germplasm accessions purported to be resistant to Phomopsis seed decay (PSD): PI 80837, PI 417479, PI 567381B, PI 416834, PI 417089B, PI 567046A, PI 567238, PI 506947, PI 567129, and PI 567139B. Midwestern lines were also crossed with the PSD-resistant accessions PI 209908, PI 279088, and PI 360841. Although only a low percentage of those were successful, a few pods with putative F1 seeds have been found. Additional crosses are being made in the field in Urbana this summer. PSD-resistant accessions from maturity groups (MGs) II through IV were planted in the field and PIs from higher MGs were planted in the greenhouse. Thirteen F1 seeds derived from crosses to PSD-resistant accessions were planted in the greenhouse, but several were eaten or fatally damaged by mice. Additional putative F1 seeds that should have PSD resistance were planted to replace the destroyed and displaced seeds to the extent that this is possible. Tissue has been collected from the surviving plants for DNA extraction to confirm their hybrid genotype. Phomopsis longicolla isolates Cultures of P. longicolla isolates were initiated from seeds with PSD-like symptoms that had been harvested in Urbana in 2012. These isolates were subsequently purified by subculturing mycelia or spores, and the isolates were used to inoculate plants in greenhouse assays described below. Another putative P. longicolla isolate that has novel morphological characteristics has been isolated from F2 seeds of the cross LD00-3309 × (PI 628932 × NE3001 F6:8); it seemingly combines features commonly found in both P. longicolla and Diaporthe phaseolorum var. sojae. PCR assays using primers that anneal to unique internal transcribed spacer (ITS) sequences are being used to confirm the identity of the pathogen isolates. PSD Resistance Assays To evaluate a novel greenhouse assay for PSD resistance, elite and exotic PI parents of breeding populations that segregated for PSD symptoms in Florida (as part of the USB-supported soybean rust research there) were planted in flats (trays) containing 3 inches of pure sand. Some of the PIs reported to be resistant to PSD in the cooperator's 2011 review chapter were planted as resistant checks. Each line was planted in several replications so that tests of statistical significance could be made. Growing the plants at a relatively high density in sand and under a short photoperiod makes it possible to keep the stature of the plants short, and to test hundreds of plants in a few square feet of greenhouse bench space. These plants were inoculated with conidia (asexual spores) from the first of the putative Phomopsis longicolla isolates above, starting when seeds were just beginning to develop in pods on the earliest-maturing plants (i.e., at the R5 stage of development). Due to the variable maturity of the accessions tested, and the randomized planting design, all plants were inoculated twice. Although we have not yet begun to harvest and assay the seeds, rows of pycnidia were observed on the stems of some plants that recently senesced, indicating that the microenvironment among plants in the sand flats was favorable for the pathogen, and that the inoculations were successful. This “sand flat” greenhouse assay is also being tested with a mapping population from a cross of a PSD-resistant line developed by the University of Illinois (LD02-4485) and a PSD-susceptible, but soybean rust resistant germplasm accession, PI 417208. Segregation for PSD resistance had initially been observed in seed from lines grown in Florida, and the resistance of LD02-4485 was subsequently confirmed with help from the cooperator. Lines from this segregating population were inoculated twice with conidia from the first putative Phomopsis longicolla isolate mentioned above: once when the majority of the plants reached the R5 stage (beginning of seed fill), and another time when the majority of the plants had reached the R6 (late seed development) stage. Although the plants have not yet senesced in this population, abscised petioles were found to have rows of pycnidia, indicating successful inoculation in these sand flats as well. This mapping population is also segregating for powdery mildew resistance, which infected the population in the greenhouse. Fungicides could not be used for risk of affecting inoculations with P. longicolla. Seed from the sand flat assays have been or will be harvested, examined, and cultured to obtain data on PSD infection, and the data will be analyzed to determine whether the observed differences are significant and repeatable. Data will also be analyzed to determine whether powdery mildew infection suppresses, enhances, or plays no role in pod infection and subsequent seed infection by P. longicolla.