Molecular differentiation of fungi associated wtih brown stem rot and detection of Phialophora gregata in resistant and susceptible soybean cultivars

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 nuclear ribosomal DNA (rDNA). The Phialophora isolates also were compared with 16 isolates of Acremonium spp. isolated from soybean. All the isolates of P. gregata shared one unique banding pattern after restriction enzyme digestion of the polymerase chain reaction (PCR)-amplified internal transcribed spacer (ITS) and the 5' end of the large subunit rDNA. Isolates of Acremonium spp. from soybean were clearly differentiated from P. gregata isolates. The ITS region of isolates representing various DNA groups, based on restriction digestion, were sequenced completely on both strands. The isolates of P. gregata from soybean from the United States and Brazil had identical ITS sequences. The ITS sequence 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 groups could be a Plectosporium sp., based on ITS sequence comparisons. Two PCR primers, BSR1 and BSR2, based on the ITS sequence, were designed specifically for P. gregata from soybean to detect the pathogen in infected plants. The specific primers were used in PCR to amplify a 483-hp DNA fragment in isolates of P. gregata from soybean and mung bean but not from P. gregata from adzuki bean at a specified annealing temperature. 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 that the specific primers were necessary to detect P. gregata in its natural habitats. PCR with the specific primers and the traditional isolation technique were used to detect P. gregata in artificially inoculated soybean cvs. BSR101 and Century, which are resistant and susceptible to brown stem rot, respectively. No differences were found in the infection and movement of the pathogen between the two soybean cultivars. The specific DNA fragment also was detected in naturally infected stems of soybean cvs. Bell, BSR101, Newton, and Sturdy collected from fields, and sequence analyses verified that these amplified fragments were from P. gregata. Results of PCR with specific primers confirmed field observations that cv. BSR101 may not be resistant to brown stem rot under certain conditions.