Botryosphaeriaceous infection in New Zealand vineyards: Identification, population structure and genetic diversity

A survey of 43 vineyards from six wine growing regions in New Zealand collected 238 grapevine wood samples displaying characteristic symptoms, including cankers, trunk necrosis, dieback and decline. Isolation from the symptomatic material showed that botryosphaeriaceous species infection was present in 88% of the vineyards and 68% of the 238 samples, which yielded 336 isolates of botryosphaeriaceous species. The incidence of infection varied between regions (P<0.001), being highest in Gisborne (96%) and lowest in Otago (23%), and between age groups (P<0.001), being highest in grapevines 6-10 years old. Infection incidence differed between scion varieties (P<0.001), being highest in Sauvignon blanc and higher in grafted than non-grafted grapevines (P<0.001). Analysis of all isolates using morphological characteristics and the molecular tool ARDRA identified nine species as Neofusicoccum parvum, N. luteum, N. australe, N. ribis, Diplodia mutila, D. seriata, Dothiorella sarmentorum, Botryosphaeria dothidea and also Do. iberica, which was a first record in New Zealand. The relative frequencies of these species showed that N. parvum was predominant (34%) followed by D. mutila (18%), D. seriata (16%), N. luteum (14%), N. australe (11%), N. ribis (3%), Do. iberica (2%), Do. sarmentorum (1%) and B. dothidea (1%). Neofusicoccum parvum and N. luteum were isolated most frequently from the North Island, and N. australe most frequently from the South Island. The Diplodia species were mostly found in South Island vineyards. The species, N. parvum, N. luteum, N. australe, D. mutila and D. seriata, varied significantly (P<0.05) in their optimum temperatures for maximum growth rate. High genetic diversity intra- and inter-vineyard and between regions was demonstrated for populations of N. parvum, N. luteum, N. australe and D. mutila using UP-PCR and neighbour joining analysis. Nei’s genetic diversity using data from 8 UP-PCR primers for N. parvum was H=0.2581 and with 5 UP-PCR primers for N. luteum, N. australe and D. mutila populations was 0.1791, 0.2417 and 0.2347, respectively. Vegetative compatibility tests with 11 N. parvum isolates selected from different branches of the neighbour joining tree showed incompatible, partially compatible and compatible interactions, with overlap between three of the four VCGs identified. For D. mutila the 14 isolates within three VCGs largely overlapped between groups. Microscopic analysis of different compatibility reactions for N. parvum and D. mutila revealed many anastomoses within and between the isolates’ colonies. Pathogenicity varied between isolates of N. parvum, N. luteum, N. australe and D. mutila in assays with excised grapevine green shoots and 1 year old potted grapevines. However, the isolates were ranked in a similar order in both assays with respect to their mean lesion lengths. On potted grapevines, the greatest ranges in lesion lengths were caused by isolates of N. parvum (0-56 mm) and N. australe (47-139 mm). On potted vines, N. luteum isolates caused the largest lesions (100-178 mm) with the greatest endophytic movement (153-268 mm) beyond lesions. Isolates from the same genetic groups in N. parvum and N. luteum neighbour joining tree generally showed similar pathogenicity levels. Co-inoculation with N. parvum and N. luteum isolates on potted grapevine showed that the infections were not synergistic or competitive, although co-inoculation caused greater downward movement from the inoculation point than single inoculations. Preliminary assays showed that N. parvum isolates could produce laccase in vitro but the variable levels from different isolates showed no relationship with their pathogenicity. From the UP-PCR fingerprints, endogenous markers were identified in N. parvum isolate B2141 and N. luteum isolate G51a2, so PCR assays were developed to identify them in environmental samples. For N. parvum B2141, a nested PCR-RFLP based on sequence polymorphism allowed the TaqI restriction endonuclease to differentiate it from other N. parvum isolates and to detect as little as 0.5 pg genomic DNA. For N. luteum G51a2, a standard PCR assay at an annealing temperature of 63ºC amplified a unique 510 bp product for isolate G51a2 but not for other N. luteum isolates, and had a sensitivity of 5 pg of genomic DNA. Neither assay amplified bands in the other botryosphaeriaceous species tested. In a field experiment with conidia of these endogenous markers of N. parvum and N. luteum, the method could detect rainwater splashed propagules up to 2 m from the conidium source.