Identification of the individual species within the llyonectria macrodidyma complex that cause black foot disease of grapevines in New Zealand : a dissertation submitted in partial fulfilment of the requirements for the Degree of Bachelor of Science (Honours)

Abstract

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.