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

2014; Wiley; Volume: 64; Issue: 2 Linguagem: Inglês

10.1111/ppa.12257

1365-3059

Marcella Viana de Sousa, José da Cruz Machado, H. E. Simmons, Gary P. Munkvold,

Plant pathogens and resistance mechanisms

F usarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested P haseolus 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 (q PCR ) protocol was developed for detection of F op 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 F op‐infected seeds with F op‐free seeds. Direct comparisons between SYBR G reen and T aq M an q PCR methods were performed using primers based on the F op virulence factor ftf1 . The primers developed in this study produced a 63 bp product for highly virulent strains of F op 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 q PCR assays detected F op infection at low levels (0·25%); however, the results suggest the T aq M an assay was more reliable at quantification than the SYBR G reen assay. Linear regression models were fitted to the relationships between results of q PCR 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 T aq M an assay developed in this study is a useful tool for the detection and quantification of F op in bean seeds.