Identification Of Pathogen Causing Charcoal Rot And Screening For Resistance In Soybean

The soybean charcoal rot is the most important disease that can reduce the yield of soybean in the world. Recently, it also has occurred severely in some regions of Northern China. The objectives of this study were to identify the pathogen causing charcoal rot in soybean and its biological characteristics, and screen for resistance to charcoal rot in soybean and wild soybean germplasm. The main results as follows:1. In late August 2005 and 2007, symptoms of charcoal rot-like were observed on soybean plants in one field in Tianjin and Beijing city, respectively. Fungal isolates were made from the diseased stem tissue in order to identify the causal agent. Five resulting isolates were cultured on potato dextrose agar at 25°C, and were identified as Macrophomina phaseolina based on the gray color of the colony and the presence of microsclerotia 49.8 to 111.4μm in size. Pathogenicity was performed by inoculation of 2-week-old seedlings of cv. Hefeng 25 using hypocotyl inoculation and cut stem techniques. For each isolate tested,M. phaseolina was reisolated from inoculated plants. To confirm the morphological identification, the rDNA-ITS region of the five isolates was amplified with universal primer ITS4 / ITS5. The obtained ITS sequences (one sequence GenBank accession No. FJ175382) were aligned with GenBank and showed 97～98% similarity with 14 isolates of M. phaseolina. PCR specific primer MpKF1/ MpKR1 for M. phaseolina was used to detect genomic DNA of the five isolates,the primer yielded single amplified product of 350 bp with the five isolates, and the result confirmed them as M. phaseolina. In the study,it is the first report of charcoal rot of soybean in Northern China.2. It is important to confirm the biological characteristics of M. phaseolina. The 5 isolates were inocubated on PDA, easily producing abundant microsclerotia. Its optimum temperature for growth in culture is 30～35℃, and the optimum pH for growth in culture is about 5. The influence of osmotic potential on growth of 5 isolates in vitro was studied using the PDA adjusted to differential osmotic potentials with sucrose, PEG 6000 and NaCl. The growth of the mycelium in culture was promoted by 100～500 mM concentrations of sucrose, 10%～30% concentrations of PEG 6000 and 100～300 mM concentrations of NaCl. However, the concentration of NaCl about 200 mM increased the growth of the 5 isolates. In conclusion, low acid, high temperature and dry environment conditions were in favor of growth of M. phaseolina cuasing charcoal rot in soybean.3. To screen the resistant cultivars or lines, 100 soybean germplasms and 50 wild soybeans to charcoal rot were identified for resistance by using the cut stem inoculation technique or the detached leaf inoculation technique respectively. A cultured variety, Xiaoheidou(ZDD09279)was resistant to the isolate Mp5, 18 other soybean germplasms had middle resistance to the isolate. Of 50 wild soybean, only 13 were resistant to the isolate. The use of resistant resources could provide the most practical means of controlling the disease. It could be conductive for the resistant breeding.