Characterising the pathogenicity of Cylindrocarpon pauciseptatum associated with black foot disease on grapevines

Black-foot disease caused by Cylindrocarpon/Ilyonectria species is a widespread disease of grapevines. In a 2005 survey of symptomatic plants in New Zealand, 95% of the pathogenic species recovered were “Cylindrocarpon” destructans, I. liriodendri and ”C”. macrodidymum. However, 11 C. pauciseptatum isolates were also recovered. In this study, the 11 isolates of C. pauciseptatum were identified by morphology and DNA sequences, their pathogenicity investigated and the activity of secreted laccase measured. Phylogenetic analyses of individual ITS, β – tubulin, EF1-α and histone datasets showed all 11 isolates grouped into well-supported clades along with voucher specimens. The 11 isolates formed a monophyletic unit in the rRNA gene and histone gene trees, while they were paraphyletic based on β – tubulin, EF1-α phylogenies. For the NJ tree generated from β-tubulin sequences eight isolates, including the avocado isolate were identical to a New Zealand isolate recovered from Erica melanthera (CBS100819) and grapevine (CBS113550), but did not group with voucher isolates recovered from other countries. There were 9 bp differences between these seven isolates and the other four isolates which had grouped with international voucher specimens in clade 1. The genetic diversity analysis of the 11 C. pauciseptatum isolates showed that they were genetically diverse. A total of 65 bands (loci) were produced by five primers, with 46% polymorphism. Two major groups within the dendrogram were generated by the UP-PCR data for the 11 C. pauciseptatum isolates. Group 1 contained five isolates, whereas the other group contained 6 isolates. No clonal isolates were detected with all 11 isolates having unique genotypes. Each genetic group was composed of isolates from different geographic regions and vineyards. Vegetative compatibility groups showed that 64% of the paired reactions were compatible despite the distinct genotypes of the isolates. Two main VCGs were identified between the pairings of C. pauciseptatum isolates. In addition, 3 sub-groups which overlapped in a complicated manner with each other were observed. No correlation was found between VCGs and genotypic diversity based on NJ tree. Microscopic analysis of interactions between C. pauciseptatum isolates showed that there were anastomoses and hyphal fusions within the actively growing hyphae of isolates in compatible reactions. Pathogenicity tests with eight isolates on detached root assay of three rootstock cultivars showed that isolates Mar6a, Mar14b, Ack2b Ack2e CO6g and Hb3b were present in lesions and that isolates Mar6a, Mar14b, CO6g and Hb3b could move endophytically within the root. No isolates were recovered from inoculated potted vines. There was no relationship between the genetic group determined by UP-PCR/ β–tubulin and pathogenicity of the C. pauciseptatum isolates. Unfortunately, a high degree of contamination by botryosphaeriaceous fungi interfered with the recovery of the inoculated pathogen from infected plant tissue in both assays. The preliminary study on in vitro production of laccase by C. pauciseptatum isolates showed both type of laccase activity (PPO-I and PPO-II) were measurable for all isolates with varied levels. There was no relationship between pathogenicity and laccase activity, but a trend could be observed for some isolates. Some of the isolates produced laccase levels similar to the positive controls. Using degenerate PCR the gene encoding lcc1 laccase was isolated. Only 40% of the putative laccase gene was isolated and sequenced. The active sites and the specific Cu-oxidase domain were not amplified. There were no polymorphic amino acid residues observed between the C. pauciseptatum isolates. There were 13 amino acid differences observed between I. liriodendra/I.novozelandicai and C. pauciseptatum, in which twelve were non-conservative changes and only one was a conservative substitution (Δ73I→V). Overall this study identified the 11 isolates as C. pauciseptaum, confirmed that they were present in lesions formed on detached roots and showed that they produced laccase in vitro. This study has improved understanding of this species in New Zealand and its ability to infect grapevines