RESUMO
AIM: 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. METHODS AND RESULTS: 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. CONCLUSION: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.
Assuntos
Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Pythium/isolamento & purificação , Sequência de Bases , Colocasia/microbiologia , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Polimorfismo de Nucleotídeo Único , Pythium/genética , Análise de Sequência de DNARESUMO
Ceratocystis fimbriata is a complex of many species that cause wilt and cankers on woody plants and rot of storage roots or corms of many economically important crops worldwide. In Brazil, C. fimbriata infects different cultivated crop plants that are not native to Brazil, including Gmelina arborea, Eucalyptus spp., Mangifera indica (mango), Ficus carica (fig), and Colocasia esculenta (inhame). Phylogenetic analyses and inoculation studies were performed to test the hypothesis that there are host-specialized lineages of C. fimbriata in Brazil. The internal transcribed spacer region ribosomal DNA sequences varied greatly but there was little resolution of lineages based on these sequences. A portion of the MAT1-2 mating type gene showed less variation, and this variation corresponded more closely with host of origin. However, mango isolates were found scattered throughout the tree. Inoculation experiments on the five exotic hosts showed substantial variation in aggressiveness within and among pathogen populations. Native hosts from the same families as the exotic hosts tended to be less susceptible than the cultivated hosts, but there was little correlation between aggressiveness to the cultivated and native hosts of the same family. Cultivation and vegetative propagation of exotic crops may select for strains that are particularly aggressive on those crops.