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ARS Home » Midwest Area » Urbana, Illinois » Soybean/maize Germplasm, Pathology, and Genetics Research » Research » Publications at this Location » Publication #71974
Title: MOLECULAR DIFFERENTIATION OF FUNGI ASSOCIATED WITH BROWN STEM ROT AND DETECTION PHIALOPHORA GREGATA IN RESISTANT AND SUSCEPTIBLE SOYBEAN CULTIVARS

Author
item CHEN, WEIDONG - UNIV OF ILLINOIS
item Gray, Lynn
item GRAU, CRAIG - UNIV OF WISCONSIN

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/5/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Soybean brown stem rot is an important vascular disease of soybean that is caused by a fungus called Phialophora gregata. This fungus is very slow growing and extremely difficult to identify. Presently it takes a minimum of six weeks in pure culture before this fungus can be identified microscopically. In addition, there are other fungi that are associated with the soybean vascular system that microscopically look like individuals (called isolates) of Phialophora. The present work was undertaken to determine if certain molecular biology techniques (Polymerase Chain Reaction) could be used to rapidly identify Phialophora isolates in culture as well as in infected plant tissue. DNA was isolated from a large number of individuals of Phialophora collected from different soybean fields and from other plants. The Polymerase Chain Reaction with specific primers that allow amplification of certain DNA pieces was done on all individuals of Phialophora. Sequence data from certain individuals was obtained and from this data specific primers were designed that allow only amplification of DNA from the Phialophora individuals. Using PCR with the specific primers it was possible to positively identify each Phialophora individual in culture and also to detect the fungus in infected soybean stem and leaf tissue. This technology should be useful to soybean pathologists to rapidly identify individuals of Phialophora that will be used in disease screening tests as well as detecting the fungus in diseased soybean plants.

Technical Abstract: A collection of 79 isolates of Phialophora gregata obtained from several midwestern states, Brazil, and Japan was studied for intraspecific genetic variation in the nuclear rDNA. All the isolates of P. gregata shared one unique banding pattern after restriction enzyme digestion of 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 completely sequenced on both strands. The isolates of P. gregata isolated from soybean from the U.S. and Brazil had identical ITS sequences. The ITS sequence of P. gregata isolated from adzuki bean from Japan was 98% similar to that of the P. gregata from soybean. At least two groups of Acremonium spp. were found to be 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. The specific primers were used in PCR to amplify a 483-bp DNA fragment in isolates of P. gregata from soybean and mung bean, but not from P. gregata from adzuki bean at specified annealing temperature. PCR with the specific primers did not detect the DNA fragment in Acremonium spp. and in any other fungi tested, nor in soybean DNA. PCR with the specific primers and traditional isolation technique were used to detect P. gregata in artificially inoculated soybean cultivars BSR101 and Century. The specific DNA fragment was also detected in naturally infected stems of soybean.