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

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

Distribution of Botryosphaeriaceae species and genotypes within a rootstock mother vine indicates multiple inoculum sources

Infected rootstock and scion cuttings by Botryosphaeriaceae fungi have been reported as major sources of infection for young grapevines. To investigate the potential infection pathways of Botryosphaeriaceae species within a rootstock mother vine, a universally-primed polymerase chain reaction (UP-PCR) was conducted. This method differentiated the genotypes within two Neofusicoccum species, namely N. luteum and N. parvum. The first study showed that multiple Botryosphaeriaceae species and genotypes can infect a single mother vine. It further showed that the trunk and shoot isolates of the same species from the same vine were of the same or different genotypes, suggesting multiple infections from different inoculum sources. The second study that investigated the spatial distribution of Botryosphaeriaceae fungi within an entire dormant cane also showed that multiple species and genotypes were distributed along the cane, but most isolates were sited within the bark and less frequently in the wood, suggesting they were latent in surface tissues. Some adjacent wood and bark infections were caused by the same genotypes also suggesting that wood infection may have originated from the bark. The third study further showed that the Neofusicoccum isolates recovered by washing the cuttings were of the same or different genotypes from those isolated from adjacent internal tissues, again suggesting multiple sources of external inoculum. These fungi appear to cause latent infections in the bark of dormant cuttings which are used in plant propagation, thus providing an additional infection pathway for a disease that is known to show obvious symptoms only in older vineyards

Spatial distribution of Neofusicoccum species within a rootstock mother vine indicates potential infection pathways

Past studies have shown that the major source of infection of young grapevine plants by Botryosphaeriaceae fungi were through the use of infected rootstock and scion cuttings. To investigate the potential infection pathways of Botryosphaeriaceae species within a rootstock mother vine, three genotyping studies using universally-primed polymerase chain reaction (UP-PCR) were conducted with two Neofusicoccum species, namely N. luteum and N. parvum. The investigations identified genotypes of the fungal isolates in trunk and shoot infections of the same mother vine. Results showed that the trunk and shoot isolates from the same vine were of the same or different genotypes, suggesting multiple infections from different inoculum sources. This study further showed that the Neofusicoccum isolates recovered from the surfaces of the cuttings were of the same or different genotypes from those isolated from adjacent internal tissues, again suggesting multiple sources of external inoculum. Investigations into the spatial distribution of Botryosphaeriaceae fungi within an entire dormant cane also showed that multiple species and genotypes were distributed along the cane but most isolates were sited within the bark, being less frequently in the wood, which suggested that they were latent on surface tissues. Since some adjacent wood and bark infections were caused by the same genotypes, this indicated that wood infection may have originated from the bark. These fungi appear to cause latent infections in the bark of dormant cuttings which are used in plant propagation, thus providing an additional infection pathway for a disease that is known to show obvious symptoms only in older vineyards

Virulence affected by assay parameters during grapevine patholgenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1-year-old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP-PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method

Inoculum sources of Botryosphaeriaceae species in New Zealand grapevine nurseries

This study investigated the sources of inoculum of Botyosphaeriaceae species in three commercial grapevine nurseries in New Zealand. Botryosphaeriaceae propagules were detected on the surfaces of 33 to 100 % of canes and 33 % of dead grapevine materials using microscopy, plating assays and PCR with Botryosphaeriaceae multi-species primers (Bot100F/Bot472R). Isolations also showed that 15 to 68 % of the canes were internally infected by Botryosphaeriaceae species, with Neofusicoccum luteum and N. parvum being the most predominant species. Incidence varied between nurseries, with Nursery 5 having highest N. luteum incidence (53 %) and Nursery 3 having highest N. parvum incidence (88 %). Botryosphaeriaceae DNA was detected in rain-water run-off from the mother-vine canopy, but not in soil samples collected around infected mother-vines. However, samples from the nursery propagation stages had no visible or viable Botryosphaeriaceae propagules although PCR using multi-species primers detected Botryosphaeriaceae DNA from wash and hydration tanks, on grafting tools and in callusing media from the different nurseries. The single-stranded conformational polymorphism (SSCP) was able to identify N. parvum, N. luteum and Diplodia mutila as the most common species present in the nursery system. Overall results indicated that the canes grown in mother-vine blocks were the most likely source of inoculum for infection of young grafted plants by Botryosphaeriaceae species

Management of Botryosphaeriaceae species infection in grapevine propagation material

In New Zealand grapevine propagation nurseries, Botryosphaeriaceae species have been reported to infect the source blocks of the nursery propagators leading to infection of the propagation materials. This research investigated the efficacy of different control methods which could prevent infection or eradicate the pathogen from harvested canes prior to plant propagation. In the source blocks, attempts to reduce infection of shoots by protecting trimming wounds were partially successful (P=0.036), with 19.5% incidence in fungicide-treated shoots and 24.3% infection in the control shoots. Further sampling showed that overall 19.9% of these infections were in the bark and 9.6% in the wood. Hot water treatment (HWT) of dormant rootstock 5C canes, previously infected with Neofusicoccum luteum and N. parvum , at 50°C for 30 min resulted in internal infection incidences of 55 and 100%, respectively. HWT at 53°C reduced infection incidence to 0 and 8.5%, respectively, but killed the buds. In naturally infected canes, HWT of 50°C for 30 min reduced infection incidence from 35% in controls, to 0–15% over all Botryosphaeriaceae species. Shorter periods of HWT, at 55°C for 10 min, designed to kill bark infections, were effective in Sauvignon blanc but killed the buds of Pinot noir. Sauvignon blanc canes superficially infected with N. luteum were soaked for 30 min in the fungicides carbendazim, tebuconazole, thiophanate methyl and flusilazole, with and without a polyether-modified trisiloxane adjuvant. Results showed that carbendazim with no adjuvant and tebuconazole with 0.5 mL L-1 adjuvant eliminated 100% of bark infections. A further experiment that soaked 2,000 canes (Sauvignon blanc and Pinot noir) in a carbendazim solution prior to rooting found that all canes were free of Botryosphaeriaceae species infection, compared to 17% natural incidence. These results have indicated the potential efficacy of several methods for preventing or reducing infection Botryosphaeriaceae species in grapevine propagating materials

Prevalence and distribution of Botryosphaeriaceae species in New Zealand grapevine nurseries

To determine presence of infection by botryosphaeriaceous fungi in plant materials from different grapevine nurseries, different types of grafted plants and cuttings were requested from nine grapevine nurseries around New Zealand. Of the 311 propagation materials and plants collected, 23 % were infected by botryosphaeriaceous fungi. 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, most isolates (80 %) were from near the graft unions while infections on rootstocks and scion cuttings were mostly from the middle (39 %) and basal parts (46 %). Identification of isolates by morphological and molecular methods showed that seven species were present 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. These results show that Botryosphaeriaceae species were present in propagation materials and in the grafted plants supplied by nurseries, indicating that some of the current vineyard infections may have originated in the propagation nurseries