Development of a PCRRFLP method to distinguish species within the <i>Ilyonectria macrodidyma</i> complex

Species within the Ilyonectria macrodidyma complex are known plant pathogens and several are implicated as the causal agents of black foot disease of grapevines. The seven species within the complex can be identified by DNA sequencing of the histone H3 gene. In this study, a PCR-RFLP method to identify the species was developed. In silico digestion of the 500 bp histone H3 amplimer using MnlI showed that it could identify Ilyonectria sp. 1, Ilyonectria sp. 2, I. alcacerensis and I. macrodidyma. Subsequent in silico digestion with Hinf1 identified I. estremocensis, I. novozelandica and I. torresensis. The PCR-RFLP was validated using a collection of 40 I. macrodidyma complex isolates that had been recovered from symptomatic grapevines. Ilyonectria macrodidyma, I. novozelandica and I. torresensis were detected in that collection. Intraspecific polymorphism was only detected in I. torresensis. This method provides a rapid procedure for identifying individual species of the I. macrodidyma species complex.