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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.

Black foot disease of grapevines is a significant problem in New Zealand and throughout the world. Recent taxonomic revision of one of the main causal agents of this disease, "C." macrodidymum, resulted in the delimination of seven monophyletic species, namely I. macrodidyma, I.estremocensis, I. novozelandica, I. torresensis, llyonectria sp. 1 and llyonectria sp. 2. This has resulted in the need to carry out new studies to determine the incidence of the individual species. The aim of this study was to use multi-gene sequence analysis to identify the prevalence of these species in New Zealand from a collection of 40 isolates previously identified as "C". macrodidymum, the development of a rapid identification system for these species and to determine variability in virulence of the different species. Multi-gene sequence analysis based on part of the histone H3, β-tubulin gene, translation elongation factor 1-α and the internal transcribed spacers on both sides of the 5.85 nuclear ribosomal RNA genes was able to identify all isolates to species level. The most informative gene was histone H3 ,and the least informative was internal transcribed spacers. Three species were identified as beingpresent in New Zealand, the most predominant species identified was I. macrodidyma (58%, n=23), followed by similar occurrence of I. novozelandica (22%, n=9) and I. torresensis (20%, n=8). There was no significant difference in species distribution between the North and South Island (0.623), although the sample size was considered too small to be definitive. This study developed and optimised two rapid molecular identification methods, PCR-RFLP and SSCP. In both techniques the initial step was to amplify the histone H3 gene using PCR. Restriction analysis of the amplicons using the restriction endonucleases, MnII and HinFI, allowed identification of all seven species in silica, and all three of the species present in New Zealand experimentally. lntraspecific polymorphism was low and detected only in I. torresensis. For the resolution of the banding patterns, acrylamide gels were found to have a higher resolving power in comparison to molecular grade agarose gels. SSCP analysis of the amplicons could resolve isolates of I. macrodidyma, I. novozelandica and I. torresensis and, on the basis of sequence differences, was predicted to be able to resolve between the other species in this complex. This method could also detect all three species in a mixed sample. Virulence did not vary between genetically distinct isolates of I. macrodidyma, I. novozelandica and I.torresensis or between the three species on detached root assay using two rootstock varieties, 3309 and 5C (P=0.255). All isolates except two in one replication produced visible lesions of varying degrees on the roots indicating all isolates were pathogenic. Rootstock variety 5C was significantly more resistant to lesion development than 3309 (P=0.008). Using degenerate PCR the genes encoding the laccase 1 (lcc1l) of I. macrodidyma, I. novozelandica and I. torresensis were isolated. For this gene approximately 1100 bp was isolated, and translated into a putative 364 residue polypeptide which was 65% of the predicted 558 residue protein. There was no variation between the predicted amino acid sequences of the three species or between the examined isolates.

Black foot disease, caused by species of Cylindrocarpon, is a significant problem in New Zealand and throughout the world. From a 2005 study of symptomatic grapevine material contributed by 49 grape growers encompassing eight grape growing areas, 174 cultures of Cylindrocarpon-like isolates were recovered. The isolates were identified using a combination of morphological grouping, species specific PCR and sequencing of taxonomically informative genes. The collection contained five species of Cylindrocarpon, namely, C. liriodendri (57 isolates), C. destructans (53 isolates), C. macrodidymum (41 isolates),C. pauciseptatum (11 isolates) and Cylindrocarpon sp. (4 isolates). All three of the main Cylindrocarpon species reported worldwide were found throughout New Zealand, however, they differed in their distribution. A high proportion of the isolates recovered in the South Island were C. destructans (39%, n=46/118) as compared to the North Island (17%, n=8/48). A high proportion of the isolates recovered in the North Island were C. macrodidymum (45%, n=22/48) as compared to South Island (17%, n=20/118). The proportion of C. liriodendri isolates recovered from the North Island (27%, n=13/48) and from South Island (37%, n=44/118) were similar. Similar numbers of C. pauciseptatum and Cylindrocarpon sp. were isolated from both islands. The distribution of the three main species correlated with optimal temperature for growth where C. destructans had the lowest optima of 17.7°C and C. macrodidymum had the highest optima of 19.3°C (P