Development of primers and method for identification and detection of diaporthe phaseolorum var. meridionalis in soybean seeds

Abstract

In spite of the great variability of the Diaporthe/Phomopsis complex, the routine seed health testing, used for detection of the Diaporthe, is not reliable, taking consideration into that for its identification morphological characteristics of the colonies are used. By considering, this work had as objective to develop an accurate, fast and reliable method for Diaporthe phaseolorum var. meridionalis (Dphm) detection and identification in soybean seeds. A sample of seeds from cultivar IAS5, analyzed previously for sanity, without Diaporthe (SS) was inoculated with Dphm (S.I.). Samples were collected with the following SI:SS rations: 1:10, 1:50, 1:100, 1:200 and 1:400. DNA mycelium extraction was necessary first to incubate, the seeds, during 3 to7 days, using blotter test modified with 2,4 D and washing them with buffer extraction (1% of SDS, 10 mM of TRIS/HCl and 25 mM of EDTA) for 20 min, on an orbital shaker (100 rpm). An aliquot of 350 μL of the supernatants was transferred to microcentrifuge tubes with 350 μL of silica resin gel of the Wizard kit (Promega) remaining in contact during one minute. DNA extracted was used as template in the reaction of PCR by using the following primers: DphLe (TCG GCC TTG GAA GTA GAA GAC) and DphRi (ACT GAA TGC GTT GCG ATT CT). Using these primers, it was possible to detect the presence of D.phaseolorum var meridionalis in soybean seeds, in the ratio of one infected seed with Dphm in samples of 400 seeds (0.25% of incidence), using blotter test modified with 2,4 D associated with the PCR technique.