Fungal endophytes of grapevine trunks: community structure and implication for grapevine trunk diseases : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

The grapevine trunk is colonized by a diverse community of fungi, collectively referred to as the endophytic mycota. These communities are shaped by different factors, both biotic and abiotic, including diseases status. Pathogens and beneficial fungi play important roles in grapevines, and these endophytes are considered key players that enhance plant growth, reduce plant disease and can improve the ability to adapt to stress. Some pathogenic fungi are known to be the causal agents of grapevine trunk disease (GTD) and are considered latent pathogens. Studying variation in their disease expression in response to the endomycobiome can guide the identification of disease suppressive microbial antagonists against (or that interact with) GTDs pathogens. Understanding of the processes by which these endophytic fungi initiate, maintain, or modulate trunk diseases is lacking. This thesis focused on characterising the taxonomic diversity and the composition of the trunk endophytic mycota (endomycobiome) colonizing symptomatic and asymptomatic trunk tissues of old and young vines from different management systems, by using both culture- dependent and high-resolution culture-independent approaches. Samples were taken from nine vineyards in the Marlborough region. The samples encompassed 60 mature vines (>10 years old) and 30 young vines (<9 years old), with each age group consisting of equal numbers of apparently healthy and symptomatic vines. A culture collection of 2116 fungal isolates from 90 vines (270 tissue samples) were created. As some endophytic fungi are uncultured, this work was complemented by amplicon sequencing to characterise both the unculturable and culturable endophytes. This approach generated a total of 10 M reads. These were divided into 1892 distinct operational taxonomic units (OTUs). The results from both approaches demonstrated a complex community that was dominated by Ascomycetes species. The community analysis revealed that health, tissue age, and type of management were major drivers of the trunk endophytic community structure. The same fungal genera from culturome were identified as the most abundant using metabarcoding. Consequently, the key indicator fungal isolates recovered from trunk tissues were evaluated for their co-occurrence and interaction with GTD pathogens. Isolates of Botrytis cinerea recovered from symptomatic and asymptomatic woody tissues were pathogenic on grapevine stems. The interaction between Botrytis cinerea and two common botryosphaeriaceous species (N. parvum and D. seriata) on the expression of symptoms in young grapevine plants were explored using detached and attached assays. The co-inoculation on detached material did not show any evidence of synergism. However, co inoculation onto attached shoots on potted plants produced contrasting results. Inhibition of N. parvum lesion development by B. cinerea was observed. Next, in detached and attached grapevine assays the interaction between A. pullulans and the grapevine trunk pathogens and their effect on symptom expression demonstrated that A. pullulans could reduce the disease expression/colonisation by both N. parvum and D. seriata indicating possible antagonistic activity against these pathogens by this fungal species. The interactions between Seimatosporium spp. and the grapevine trunk pathogens N. parvum and D. seriata in detached and attached grapevine shoots showed that Seimatosporium spp. were effective in reducing the colonisation of shoot tissues by D. seriata in detached experiments, but had no significant effect in attached shoot tissues. Additionally, both S. vitis and S. sp. nov were effective in reducing lesion length caused by N. parvum in detached shoots, while S. vitis also showed a reduction in lesion length in attached shoots of potted vines. The overall results of this thesis showed that culturome and NGS metabarcoding complement each other and can be applied to gain knowledge about microbial dynamics inside the grapevine trunk. This is the first study investigating the combined effect of three factors on the endomycobiome. The findings of this study provide valuable insights into the complex interactions between pathogens associated with GTD and other fungal species isolated as endophytes from the same grapevine woody tissues.