Real‐time quantitative PCR assays for the rapid detection and quantification of Fusarium oxysporum f. sp. phaseoli in Phaseolus vulgaris (common bean) seeds

Abstract

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long‐distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real‐time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop‐infected seeds with Fop‐free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.