MOLECULAR DIFFERENTIATION OF FUNGI ASSOCIATED WITH BROWN STEM ROT ANDDETECTION OF PHIALOPHORA-GREGATA IN RESISTANT AND SUSCEPTIBLE SOYBEANCULTIVARS

A collection of 79 isolates of Phialophora gregata from soybean, mung bean, and adzuki bean obtained from several midwestern states, Brazil, and Japan was studied for intraspecific genetic variation in the nucl ear ribosomal DNA (rDNA). The Phialophora isolates also were compared with 16 isolates of Acremonium spp. isolated from soybean. All the iso lates of P. gregata shared one unique banding pattern after restrictio n enzyme digestion of the polymerase chain reaction (PCR)-amplified in ternal transcribed spacer (ITS) and the 5' end of the large subunit rD NA. Isolates of Acremonium spp. from soybean were clearly differentiat ed from P. gregata isolates. The ITS region of isolates representing v arious DNA groups, based on restriction digestion, were sequenced comp letely on both strands. The isolates of P. gregata from soybean from t he United States and Brazil had identical ITS sequences. The ITS seque nce of P. gregata isolated from adzuki bean from Japan was 98% similar to that of P. gregata from soybean. At least two groups of Acremonium spp. were associated with soybean brown stem rot, and one of the grou ps could be a Plectosproium sp., based on ITS sequence comparisons. Tw o PCR primers, BSR1 and BSR2, based on the ITS sequence, were designed specifically for P. gregata from soybean to detect the pathogen in in fected plants. The specific primers were used in PCR to amplify a 483- bp DNA fragment in isolates of P. gregata from soybean and mung bean b ut not from P. gregata from adzuki bean at a specified annealing tempe rature. PCR with the specific primers did not detect the DNA fragment in Acremonium spp. or any other fungi tested, nor in soybean DNA. PCR experiments with mixed DNAs of P. gregata and Acremonium sp. showed th at the specific primers were necessary to detect P. gregata in its nat ural habitats. PCR with the specific primers and the traditional isola tion technique were used to detect P. gregata in artificially inoculat ed soybean cvs. BSR101 and Century, which are resistant and susceptibl e to brown stem rot, respectively. No differences were found in the in fection and movement of the pathogen between the two soybean cultivars . The specific DNA fragment also was detected in naturally infected st ems of soybean cvs. Bell, BSR101, Newton, and Sturdy collected from fi elds, and sequence analyses verified that these amplified fragments we re from P. gregata. Results of PCR with specific primers confirmed fie ld observations that cv. BSR101 may not be resistant to brown stem rot under certain conditions.