engleski

PCR-RFLP identification of the Phomopsis/Diaporthe species on soybean seeds

Testing for and monitoring of pathogens is a component of seed quality control. The European soybean seeds are certified for Diaporthe phaseolorum infection (max infection allowed 15%), without specifying the relative incidence of Phomopsis longicolla, Diaporthe phaseolorum var. caulivora and Diaporthe phaseolorum var. sojae. The variable and overlapping cultural characteristics among them make the morphological identification very confusing, and time consuming. As they cause different economical damages in soybean fields, we set up a molecular method based upon ITS PCR-RFLP, to identify each Diaporthe/Phomopsis species infecting soybean seeds, Diaporthe phaseolorum var. meridionalis including. Soybean seeds were incubated on PDA plates and after 3-4 days mycelium of Phomopsis/Diaporthe species was scraped off for DNA extraction using a rapid method. The DNA of isolates was amplified with universal primers ITS4 and ITS5, and the amplification product was digested with AluI, RseI, HhaI and MseI restriction enzymes. The banding patterns obtained with each enzyme allowed the identification of all isolates, as they matched the specific banding patterns of each species. More than five hundred isolates obtained from Italy, U.S.A. Croatia and Argentina seed samples, were identified in the last five years, with a 100% of reliability for Phomopsis longicolla, Diaporthe phaseolorum var. caulivora and Diaporthe phaseolorum var. meridionalis. As part of the validation of this PCR-based approach, we compared molecular identification with classical methods of detection and identification. We propose these PCR-based assays to interested seed health testing laboratory for a comparative test with the ultimate aim to propose the method as a standardized ISTA method to detect these soybean seedborne pathogens.

soybean; PCR-RFLP; Phomopsis/Diaporthe species; seed

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