Identification of Ilyonectria species associated with and determining their role in avocado decline

Abstract

Black root rot is a significant problem for grapevine growers worldwide. In recent years, black root rot pathogens Ilyonectria and Calonectria species have been isolated from avocado roots of trees showing signs of decline. In 2013, avocado roots from an orchard in Northland showed signs of decline and Dr Eirian Jones isolated thirteen Ilyonectria and Calonectria type isolates from these roots. The aim of this study was to identify these Ilyonectria and Calonectria type isolates using molecular and morphological tools and to determine the pathogenicity of these species on avocado roots. Multi-gene sequence analysis using the histone H3 gene and β-tubulin gene was able to identify all thirteen isolates down to species level. These thirteen isolates were identified into six species; I. macrodidyma, I. liriodendri, I. liligena, I. robusta, I. europaea and C. pacifica. Of these species I. macrodidyma belongs to the I. macrodidyma complex and I. liligena, I. robusta and I. europaea belong to the I. radicicola complex. In this study the most informative gene was histone H3 that had high bootstrap values for the phylogenetic tree. The least informative gene was β-tubulin, which was unable to resolve between I. robusta and I. europaea. Morphological observations identified different colony colours, textures and macroconidia size between the different isolates. The colony diameter measured over 14 days allowed for analysis to differentiate between the growth patterns of the different species x identified through sequencing. The results of the analysis found significant results (P<0.01) for the majority of assessment days. Colony diameter data also helped to confirm the differences within the I. liligena isolates seen from the sequence data, where isolate LUPP2503 was clustered in a different group to the other four New Zealand I. liligena isolates and showed significantly smaller colony diameter. Colony characteristics were however different to that of published descriptions for many of the species. The pathogenicity of one isolate representing each of the four Ilyonectria species and one Calonectria pacifica isolate could not be determined in this study using a detached root assay as lesions also developed on uninoculated control roots. Isolates characteristic of C. pacifica was only recovered from lesions which developed on C. pacifica inoculated Bounty® rootstocks indicating that it maybe pathogenic to avocado roots. Unfortunately cross-contamination by Ilyonectria likely occurred in the glasshouse resulting in Ilyonectria type colonies being isolated from the majority of replicates for not only the Ilyonectria inoculated roots, but also the uninoculated control for all three avocado rootstocks used. To determine the pathogenicity of Ilyonectria and Calonectria species on avocado roots, another detached root assay should be done using rootstocks that were not clean of Ilyonectria and Calonectria contamination.