USING PCR TO DISTINGUISH DIAPORTHE-PHASEOLORUM AND PHOMOPSIS-LONGICOLLA FROM OTHER SOYBEAN FUNGAL PATHOGENS AND TO DETECT THEM IN SOYBEAN TISSUES

Restriction fragment length polymorphism analyses of polymerase chain reaction (PCR) amplified DNA were used to distinguish Diaporthe phaseo lorum and Phomopsis longicolla isolates from other soybean fungal path ogens. Primers made to the conserved sequences of nuclear ribosomal DN A amplified the internal transcribed spacer (ITS) regions of D. phaseo lorum var. meridionalis and P. longicolla. The PCR products were clone d and then sequenced. Specific-primers, Phom.I and Phom.II, were desig ned from the polymorphic regions of D. phaseolorum and P. longicolla i solates from soybean to distinguish them from other soybean fungal pat hogens. These ITS-derived primers amplified a 337-bp-specific DNA frag ment from P. longicolla, D. phaseolorum var. meridionalis, D. phaseolo rum var. caulivora, D. phaseolorum var. sojae, and Phomopsis spp. from 20 different hosts. No amplified product was observed using DNA of se ven other soybean fungal pathogens or soybean DNA. The detection limit of PCR using primers Phom.I and Phom.II was 2.5 x 10(-7) dilution of fungal DNA extracted from samples of 10 pooled seeds and as low as a 1 :15 (Phomopsis:soybean) ratio when using 10 ng of DNA per pi from each P. longicolla and soybean. PCR did not produce products using primers Phom.I and Phom.II with DNA extracted from noninfected seeds, but spe cific bands were observed from samples of 10 pooled seeds and from ind ividually infected seeds. A specific band was observed as well from DN A extracts of tissue samples from symptomless plants inoculated with P . longicolla and D. phaseolorum var. sojae.