Botryosphaeria infections in New Zealand grapevine nurseries: Sources of inoculum and infection pathways

Abstract

The botryosphaeriaceous fungi can cause decline, dieback and death of grapevines. Anecdotal evidence has indicated that these pathogens might be present in the young vines sold by propagation nurseries, so this study investigated their role in spread of this disease. Sampling of grapevine nurseries across New Zealand showed that botryosphaeriaceous infections were present in eight out of nine nurseries with infection incidence ranging from 5 to 63%. Of the 311 propagation materials and plants received, 23% were positive for botryosphaeriaceous infection, with a total of 120 isolates recovered. The highest incidence was in failed grafted plants (33%), followed by Grade 1 plants (28%), rootstock cuttings (19%), scion cuttings (17%) and Grade 2 plants (7%). For grafted plants, the majority of botryosphaeriaceous species (49%) were isolated near the graft unions while infections on rootstocks and scion cuttings were mostly from the middle and basal parts. Identification of isolates by morphological and molecular methods showed that the six species commonly found in vineyards also occurred in nurseries, being Neofusicoccum luteum (57%), N. parvum (18%), N. australe (8%), Diplodia mutila (8%), Botryosphaeria dothidea (5%) and D. seriata (3%), with one novel isolate of N. macroclavatum and two unidentified botryosphaeriaceous isolates. Pathogenicity tests using one-year-old Sauvignon blanc rooted canes and green shoots showed that the seven identified botryosphaeriaceous species from the nurseries were pathogenic but pathogenicity differed significantly between species and isolates within a species, with N. parvum being the most pathogenic in both assays (P≤0.001). Genetic variability analysis using UP-PCR showed that N. luteum isolates of different pathotypes were genetically diverse with intra- and inter-plant and nursery variability but no association between genotype and pathogenicity was observed. Susceptibility tests using three isolates each of N. luteum, N. parvum and N. australe against the most commonly used scion and rootstock varieties (six of each) found that all varieties were susceptible to the three species with 5C and SO4 being the most susceptible of the rootstock varieties, and Merlot and Pinot noir being the most susceptible of the scions. Investigations into the sources of inoculum conducted in three nurseries in 2009 using conventional and molecular methods showed that the mothervines used to provide cuttings for propagation were the most likely source of botryosphaeriaceous infections. Propagules were detected on the surfaces of cuttings and dead grapevine materials, as well as in rain-water run-off, but not in soil samples collected from the mothervine blocks. The different botryosphaeriaceous isolates recovered from canes were mostly sited within the bark suggesting presence of latent infections. Since the isolates from mothervine trunk and canes were distributed in isolated patches and the UP-PCR assessment showed that they belonged to multiple genotypes and species, this indicated that they were spread from external sources. Microscopy and plating assays were not able to detect botryosphaeriaceous contamination from any of the nursery propagation stages but molecular methods using multi-species primers detected botryosphaeriaceous DNA in samples from a wash pit, pre-cold storage hydration tanks, post-grafting hydration tanks, grafting tools and callusing medium from the different nurseries. An investigation into the probable survival of N. luteum conidia during the processing of cuttings showed that they adhered rapidly and could not be totally washed from the surfaces of cuttings after 0 h, with minimal recovery of conidia after 1, 2 and 4 h incubation at room temperature. During this time they germinated and colonised the periderm, phloem and xylem but not the pith of the cuttings. At storage temperatures of 2 and 8°C some conidia were able to germinate. At 2°C, conidial germination was 4% after 48 h, the number gradually increasing to 17% after 3 weeks. At 8°C, conidial germination was 23% after 24 h, the number gradually increasing to 34% after 72 h with no further increase observed up to 2 weeks. Cold storage did not kill all conidia, since conidia from 72 h and 3 weeks storage at 2°C had 84% and 33% viability, respectively, while those from 8°C storage had 72% and 39% viability after 72 h and 2 weeks storage, respectively. The N. luteum conidia stored at different temperatures were similarly pathogenic compared to freshly-harvested conidia but higher pathogenicity was observed on conidia stored at 2°C for 48 to 72 h. Overall results suggest that botryosphaeriaceous infection in new vineyards may originate from latent infection that began in the nurseries, thus management of this disease should start at the nursery level to prevent this pathogen from being carried over into new vineyards.