The effect of hot water treatment (HWT) of grapevine propagation material on endophytic bacterial communities and susceptibility to Botryosphaeria dieback : A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Horticultural Science at Lincoln University

Grapevine trunk diseases (GTDs) are a major economic issue for grapevine production worldwide. Within this group is Botryosphaeria dieback, currently known to be caused by 10 different fungal genera, is an important disease in New Zealand vineyards. Neofusicoccum luteum and N. parvum have been identified as the most common and virulent species both in vineyards and nurseries in New Zealand. Botryosphaeriaceae species can produce spore inoculum throughout the year and can infect grapevines at all crop stages. In addition, these pathogens can be introduced into vineyards through infected propagation material. Hot water treatments (HWT) are carried out by nurseries to reduce the incidence and infection of the planting material by pathogenic bacteria, fungi and nematodes. The overall hypothesis of this study was that HWT can also affect the communities of microbial endophytes colonising the grapevine propagation material as well as causing physiological effects on the grapevine propagation material. Both factors may affect the susceptibility of the grapevine canes to subsequent challenge by the GTD pathogens N. luteum and N. parvum. The first aim of this research was to determine the effect of HWT on the susceptibility of grapevine canes to Botryosphaeriaceae infection. For this, Neofusicoccum luteum isolate CC445 and N. parvum isolate G69a1, both highly pathogenic isolates, were inoculated onto hot water treated Sauvignon blanc and Pinot noir dormant and rooted canes. The susceptibility of the planting material to Neofusicoccum spp. infection was determined by measuring infection progression. Results for dormant canes demonstrated a higher (p=0.025) colonisation by Neofusicoccum spp. in Pinot noir than in Sauvignon blanc. The HWT’s at 48⁰C x 30 min, 50⁰C x 15 min and 50⁰C x 30 min resulted in greater (p<0.001) length of tissue colonised by the pathogens compared to the non-HWT control. For rooted canes, the interaction between cultivar and HWT was significant for cane growth variables. Across all treatments, the number of shoots per cane and E-L number were greater (p<0.001) for Sauvignon blanc when compared to Pinot noir. A significant decrease (p<0.05) in the number of shoots per cane was found for Pinot noir canes treated at 50⁰C x 30 min compared to the other HWT’s, but no differences were found for Sauvignon blanc. Further, for each HWT the percentage of canes which developed shoots and roots were also higher in Sauvignon blanc. The assessment of pathogenicity was completed only for Sauvignon blanc as the Pinot noir plants did not re-sprout after winter dormancy. The length of tissue colonised by the pathogens was higher (p<0.05) for Sauvignon blanc rooted canes treated at 50⁰C compared to non-HWT canes. For both dormant (p=0.001) and rooted (p= 0.034) canes, N. parvum G69a1 was showed to be more pathogenic than N. luteum CC445. The second aim was to estimate the effect of HWT on the endophytic bacteria colonising dormant canes. This was determined by culture-dependent and culture-independent (PCR-DGGE) methods. The ability of culturable bacterial isolates to inhibit the growth of N. luteum and N. parvum was determined using dual plate assays. Twelve isolates showed medium to high antagonistic (++, +++) activity against Neofusicoccum spp. No isolates showing high inhibition (+++) were recovered from canes treated at 50⁰C. 16S sequencing and phylogenetic analysis revealed that 10 bacterial isolates were identified as Bacillus sp. and 1 as Streptomyces sp. PCR-DGGE analysis demonstrated a higher total bacterial richness (p<0.001) for Pinot noir in each HWT excluding the non-HWT control. For both total and gamma (γ)-proteobacteria communities’ similarity, the interaction between cultivar and HWT was significant (p=0.001), both factors significantly affecting (p<0.005) community similarity when analysed independently as well. For Pinot noir, the communities from the HWT canes were more homogenous, tending to cluster more tightly, compared with the community from the non-HWT canes. The findings of this study indicate that HWT increased the susceptibility of dormant and rooted canes to Neofusicoccum spp. infection. Despite HWT causing changes to the endophytic bacterial richness and community similarity, the increase in the susceptibility of the propagation material to pathogen infection after HWT cannot be completely ascribed to these changes in the endophytic bacterial communities. The effect of HWT on canes growth observed in the study indicates that changes in the physiology of the canes could be associated with the increased susceptibility to Botryosphaeria dieback and further study is required in this area. This study provided valuable information to the viticulture industry that HWT at 50⁰C not only negatively affects the growth and establishment of grapevine propagation material but also increased the susceptibility of the material to infection by Botryosphaeriaceae. Based on the results of the study it is recommended that if HWT of propagation material is required then for cool climate viticulture regions a lower temperature protocol should be implemented.