2012 Annual Report
1a.Objectives (from AD-416):
1) Continuation of the breeding program to select and release germplasm with enhanced resistance, .
2)the development of new recombinant inbred line (RIL) mapping populations designed to identify and map new resistance genes, .
3)complete the development and begin the characterization of near-isogenic lines differing only in resistance, .
4)collect and characterize Macrophomina phaseolina (M. phaseolina) isolates across a wide geographic area,.
5)the characterization of the distribution and variation of M. phaseolina within a field and across years and.
6)the comparison of M. phaseolina isolates in the soil with those found in plants.
1b.Approach (from AD-416):
Obj.1: The current recombinant inbred line (RIL) population has exhibited transgressive segregation for charcoal rot resistance and we propose to continue the selection of lines with improved resistance. We are near the release of new germplasm "DS-880" which has improved resistance to charcoal rot, resistance to cyst nematode, and moderate resistance to reniform nematode, sudden death syndrome, and stem canker; all in a competitive yield background. We expect other releases as the program progresses.
Obj.2: In our 2008 field screening we identified lines that were more susceptible than the susceptible parent used in out current mapping population. This greater divergence in resistance may allow the identification of additional genetic loci involved in resistance. Using these lines, three more populations were initiated with the intent of developing new RIL mapping populations.
Obj.3: Over the last few years, we have had a program of developing near-isogenic lines (NIL) that differ only in their level of resistance. We propose the complete the development, confirm differences, and initiate molecular and phenotypic characterization of the NIL. Such characterization has the potential to determine the physiological basis of charcoal rot resistance and could indicate mechanisms for enhanced resistance.
Obj.4: Very little research has been conducted on the pathogen M. phaseolina. We propose to begin collecting and characterizing isolates from around the United States. Characterization will include both pathogenicity and molecular discrimination. Recently, we have completed the development of over 180 simple sequence repeat (SSR) markers specific to M. phaseolina, these will be used to fingerprint the isolates collected. This research is also critical to the breeding and molecular marker analysis described above. Currently we have no method of determining if the M. phaseolina isolates encountered in a particular field are the same or different from a field at another location, or even if isolates vary from one year to another. Characterization and molecular discrimination (fingerprinting) of isolates will allow the matching of resistance loci and resistant line/cultivars to specific isolates.
Obj.5: Almost no information is available regarding the distribution and variation of M. phaseolina within a given field, including variations in pathogen aggressiveness. We propose to spatially characterize the distribution and variation of isolates within a field and to determine what changes in distribution and variation occur from year to another. The SSR markers we have developed will be used to determine the variation and distribution of isolates. Coupled with the molecular characterization of field-based variation and distribution of M. phaseolina, we will determine the pathogen aggressiveness of the various isolates identified.
Obj.6: No information exists regarding the relationship between the M. phaseolina isolates that occur in the soil and those that actually infest the plant. We will use the SSR markers that we developed, to determine the relationship between isolate actually infesting plants with those isolates that occur in the soil.
An experiment was conducted to evaluate the relationship between seed yield and colony forming units (cfu) of Macrophomina (M.) phaseolina on an irrigated Sharkey clay soil, significant yield differences among five lines for seed yield were observed, ranging from 51 bu/a to 41 bu/a. Individual plot values for cfu ranged from zero to 6,000, while lines means for cfu ranged from 1,933 to 3,000. Unfortunately, the line with the highest mean cfu (Saline) had poor stands and so no yield data were taken for it. When analyzing bu/a and cfu both as random variables across all five lines, regression analysis found no relationship between yield and cfu when yield was evaluated as a function of cfu. However, when yield was evaluated as a function of cfu on an individual line basis, 1 out of 5 lines tested showed a significant regression with an F value of 5.98 and an R-squared of 0.43. This experiment is being repeated in 2012 and was planted on April 13th at Stoneville, MS. Individual plot values of cfu measured on a non-irrigated loam soil among breeding lines varied from zero to over 16,000 in 2011. Multiple breeding lines assayed for their reaction to charcoal rot had low mean levels of cfu’s (less than 400) and were advanced to yield trials. These will be assayed for yield and cfu’s in 2012 and were planted on April 13th in yield trials and April 9th in non-irrigated assay trials. Culturing charcoal rot isolates from soil and root and lower stem is essentially complete. In total 925 isolates have been cultured. Currently DNA is being isolated from the last approximately 100 isolates. Molecular marker characterization of the DNA is continuing with the marker set the ARS scientists optimized previously. Preliminary analysis of selected isolates indicates genetic variability.