In planta-polymerase-chain-reaction detection of the wilt-inducing pathotype of Fusarium oxysporum f.sp. ciceris in chickpea (Cicer arietinum L.)

A 1.6 kb fragment of random amplified polymorphic DNA (RAPD-PCR, polymerase chain reaction), which was specific for race 5, a wilt-inducing isolate of Fusarium oxysporum f.sp. ciceris (FOG)I was cloned and sequenced. This fragment was not detected in RAPD-PCR reactions with DNA from yellowing-inducing pathotypes of Foc, or from other fungi tested. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless chickpea plants, 16 days after inoculation. A single, I 5 kb PCR product was only observed in PCR reactions with DNA from plants infected with a wilt-inducing isolate. No products were observed in reactions with DNA from plants infected with yellowing-inducing pathotypes, or from DNA isolated from uninfected chickpea cultivar controls. Southern hybridization demonstrated homology between the second PCR product and the original specific wilt-associated RAPD fragment. PCR products were detected with DNA extracted from roots and stem tissue, but no fungal DNA was detected in leaf tissue of the same infected plants. In a blind trial, the specific primers correctly identified the fungal pathotype in four different, wilt-infected chickpea cultivars. (C) 1998 Academic Press.