Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/1997
Publication Date: N/A
Citation: Interpretive Summary: Phomopsis seed decay of soybean is a major cause of poor quality soybean seeds in the United States and other soybean-growing areas of the world. The disease is caused primarily by a fungus known as Phomopsis longicolla, but other related fungi also cause the disease. Infected seeds are one means of introducing the pathogen into new areas. The fungi can cause off-color in soybean seeds, which is one of the primary quality-rating factors that negatively influences the market grade of seeds. Most seed-testing laboratories in the United States do not routinely test soybean seed lots for the causal fungi, although seeds for export to Europe need a phytosanitary certificate that states no more than 15% Phomopsis seed infection can occur (EC Directive 9.92). Specific conserved DNA sequences of fungal DNA were used to detect the fungus in symptomless soybean seeds and plants. These results show that molecular-based methods can be used to detect the pathogenic fungi in soybeans seeds and this technology may eventually be routinely used by seed-testing laboratories worldwide.
Technical Abstract: Polymerase chain reaction (PCR) based on restriction fragment length polymorphisms was used to distinguish Phomopsis longicolla and Diaporthe phaseolorum isolates from other soybean fungal pathogens. Primers made to the conserved sequences of nuclear ribosomal DNA were used to amplify the internal transcribed spacer (ITS) regions of P. longicolla and D. phaseolorum var. meridionalis. The PCR products were cloned and then sequenced. Diaporthe/Phomopsis specific-primers, Phom.I and Phom.II, were designed from the polymorphic regions of Diaporthe/Phomopsis isolates to distinguish them from other soybean fungal pathogens. These ITS-derived primers amplified a 337 bp-specific DNA fragment from P. longicolla, D. phaseolorum var. meridionalis, D. phaseolorum var. caulivora, D. phaseolorum var. sojae and Phomopsis spp. from 20 different hosts. No amplified product was observed from DNA of seven other soybean fungal pathogens or soybean DNA. The sensitivities of these primers were tested by amplifying the Diaporthe/Phomopsis specific bands at 5 x 10-5 dilution of DNA extracted from 10 soybean seeds derived from a seed lot with 25% infection of Phompsis and 1:15 dilution (Phomopsis:soybean) for pure P. longicolla DNA at 10 ng/#l. Using PCR with the specific primers, Diaporthe/Phomopsis were detected in symptomless soybean seeds and plants.