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Conventional and real-time PCR assays for specific detection and quantification of Fusarium oxysporum f. sp. ciceris in plants using intergenic spacer region-based marker

Priyanka, Kumari, Dubey, Sunil C., Singh, Arun K.
Biologia 2015 v.70 no.3 pp. 314-319
Fusarium oxysporum, Fusarium wilt, chickpeas, detection limit, fungi, host plants, intergenic DNA, melting, microbial detection, pathogens, quantitative polymerase chain reaction
Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris (Foc) is one of the most important fungal diseases worldwide. The detection of the pathogen at reasonable time period is of great importance, which requires rapid and sensitive detection methods. The intraspecific divergence sequences found in the intergenic spacer region (IGS) were selected and utilized with the aim to develop a molecular marker specifically to identify the Foc. A marker set, ISR52 F1 and R1 developed, was tested for their specificity as well as sensitivity using conventional as well as real-time polymerase chain reaction (PCR). The specificity of the marker was tested against Foc, other Fusarium species which are closely related to Foc as well as with artificially infected host plant samples. The detection limits of conventional PCR assay was up to 100 pg of infected plant DNA. It proved possible to amplify the IGS region in different portion of a Foc infected host plant by this PCR method. Furthermore, the real-time assay showed more sensitivity and was able to detect the pathogen in infected chickpea plant samples at the DNA concentration of 5 pg. A single melting peak obtained at 87.5°C showed the specificity of the marker towards Foc. Thus, real-time PCR assay proved their potentiality for same-day diagnosis of fungal infection and can be used as a rapid and effective procedure for routine detection and identification of Foc in chickpea samples.