Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/10/1999
Publication Date: N/A
Citation: Interpretive Summary: Diaporthe and Phomopsis are fungi that occur on a variety of plant hosts. Some of them cause severe diseases in economically important crops. The identification of Phomopsis and Diaporthe species using traditional characteristics often is inadequate for clearly delimiting species. Keeping in mind the worldwide importance and implications of successful soybean pathogen diagnosis, this study developed a fast and economical DNA extraction method for a single soybean seed. With the use of a specialized detection system (TaqMan'/SDS), a quick method was developed to identify and detect D. phaseolorum and P. longicolla based on their DNA extracted from soybean seed. This advanced research leads the way into using new technologies to detect seedborne pathogens. This research is important to other scientists interested in detecting seedborne pathogens and to seed testing laboratories. The impact of this research could affect the procedures used to detect seedborne organisms in seeds.
Technical Abstract: Species specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and TaqMan' chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break cells. DNA fragments lengths ranged from 200 to 1200 bp with the majority of fragments, 500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) of ribosomal DNA, three TaqMan' primer/probe sets were designed. Primer/probe set PL-5 amplified a 96 bp fragment within the ITS I region of P.longicolla, D. p. var. caulivora, D. p. var. meridionalis, and D. p. var. sojae. Set PL-3 amplified a 86 bp DNA fragment within the ITS II region of P. longicolla. Set DPC-3 amplified a 151 bp DNA fragment within the ITS II region of D.j p. var. caulivora. TaqMan' primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA Among 13 soybean seed lots from Italy and the U.S., the total Diaporthe and Phomopsis detected using a traditional seed plating technique ranged from 0 to 32%. P. longicolla was most prevalent followed by D. p. var. sojae. D. p. var caulivora only occurred in 0.5% of the Italian seedlots but was not detected in the U.S. seed lots. D. p. var. merodionalis was not detected from either the U.S. or Italian seed lots. Using TaqMan' primer/ probe PL-3, the frequency of P. longicolla was 18% in seed lot 13 which was similar to the frequency obtained from PCR-RFLP and PDA plating detection. The frequency of D. phaseolorum and P. longicolla in each seed lot obtained from the different detection methods were comparable with respect to total infection and individual species detection.