Multiplex PCR to detect Curtobacterium flaccumfaciens pv. flaccumfaciens and the common bean root pathogens Fusarium oxysporum f. sp. phaseoli and Fusarium solani f. sp. phaseoli

Abstract

Root diseases that affect common beans (Phaseolus vulgaris L.) may share similar symptoms, such as Fusarium wilt (Fusarium oxysporum f. sp. phaseoli), dry root-rot (Fusarium solani f. sp. phaseoli) and Curtobacterium wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens). The difficulties for their accurate diagnosis include multiple infections in the field or a single plant, but molecular tools can overcome such drawbacks and identify causal agents accurately. The objective of this work was to develop a multiplex PCR (m-PCR) method to simultaneously identify three common bean pathogens, F. oxysporum f. sp. phaseoli (Fop), F. solani f. sp. phaseoli (Fs) and C. flaccumfaciens pv. flaccumfaciens (Cff). The approach included the design and validation of the new primers IGS1FOR and IGS1REV for F. solani f. sp. phaseoli and adjustments on primer concentration and annealing temperature. Multiplex PCR using IGS1FOR/ IGS1REV, CffFOR2/CffREV4 and A280/B310 primers did not change the pre-established procedures for detecting F. solani f. sp. phaseoli, C. flaccumfaciens pv. flaccumfaciens and F. oxysporum f. sp. phaseoli, and reactions with 0.4 to 0.8 μM of primers and annealing at 57 ºC resulted in clear, spaced amplicons in agarose gel, respectively with 143 (Fs), 306 (Cff)and 609 base pairs (Fop). The multiplex PCR technique detected pathogens in bean seeds infected by Curtobacterium and Fusarium spp. similarly to the standard ISTA method, even with a shorter seed immersion time; however, only Fusarium oxysporum f. sp. phaseoli was identified, with Fusarium solani f. sp. phaseoli not being detected in the tested cultivars. This is the first report of an m-PCR method to detect common bean phytopathogens.