A PCR-RFLP assay for identification and detection of Pythium myriotylum, causal agent of the cocoyam root rot disease

Abstract

Development of a PCR-RFLP assay that could reliably distinguish strains of Pythium myriotylum that are pathogenic to cocoyam from nonpathogens, as well as in planta detection of the pathogen. Sequences of the internal transcribed spacer regions of nuclear ribosomal DNA (rDNA-ITS) containing ITS1 and ITS2 of P. myriotylum isolates from cocoyam and other hosts were aligned and a restriction map was generated. rDNA-ITS alignment report revealed a new single nucleotide polymorphism (SNP; thymine/cytosine) downstream to previously published SNP (guanine/adenine) between isolates of P. myriotylum that are pathogenic to cocoyam and nonpathogenic strains. This new SNP is within the restriction site of the endonuclease AarI. Based on this SNP, a PCR-RFLP assay was developed for specific detection of P. myriotylum. The PCR amplicons of all isolates of P. myriotylum that infect cocoyam were cleaved by AarI, resulting to two bands (600/400 bp); but those from other hosts showed a single band (1000 bp), confirming the presence and specificity of the AarI restriction site. Also, the assay was effective in in planta detection of the pathogen on infected cocoyam roots without prior isolation of a pure culture. A PCR-RFLP method was developed that differentiates isolates of P. myriotylum that are pathogenic to cocoyam from nonpathogens as well as from other fungi commonly found in the cocoyam rhizosphere. Early and rapid detection of the pathogen could be of great importance in certifying planting materials as disease-free, enhancing sustainable management practices and limiting economic losses.