A Molecular Marker Identifying Subspecific Populations of the Soybean Brown Stem Rot Pathogen, Phialophora gregata

ABSTRACT A molecular marker was developed to separate and identify subspecific populations of Phialophora gregata, the causal agent of soybean brown stem rot. A variable DNA region in the intergenic spacer of the nuclear rDNA was identified. Two specific primers flanking the variable region were developed for easy identification of the genotypes using polymerase chain reaction (PCR). These two specific primers amplified three DNA products. The three PCR products were used to separate isolates of P. gregata into distinct genotypes: A (1,020 bp), B (830 bp), and C (660 bp). Genotype C was found in isolates obtained from Adzuki beans from Japan, whereas all 292 isolates obtained from soybean and the 8 isolates from mung bean belonged to either genotype A or B. The original nondefoliating (type II) strain ATCC 11073 (type culture of P. gregata) belonged to genotype B. The difference between genotypes A and B was due only to an 188-bp insertion or deletion; genotype C, however, differs from genotypes A and B at 58 point mutations, in addition to the length difference. Isolates of both genotypes A and B were widespread in seven Midwestern states. Genotype A was found mostly in certain susceptible soybean cultivars like Sturdy and Pioneer 9305, whereas genotype B was found predominately in brown stem rot-resistant soybean cvs. Bell, IA 3003, and Seiben SS282N. The specific primers were also used to directly detect cultivar-preferential infection by the two genotypes in infected soybean stems growing in the same field. Data from direct detection in soybean stems showed that cultivar-preferential infection by the two genotypes of P. gregata was significant.