Diversity and bioactivity of endophytic actinobacteria from the grapevine: potential for inhibition of grapevine trunk disease pathogens : A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Science at Lincoln University

Abstract

Grapevine trunk diseases (GTDs) are one of the most problematic diseases in grapevines production caused by multiple groups of fungal pathogens that usually invade the vine through pruning wounds. Infection by these pathogens result in a wide range of symptoms negatively affecting vine longevity and productivity in grape-growing regions worldwide including New Zealand. Since chemical options to control GTDs are limited, biological control strategies using plant beneficial microorganisms are of increasing interest. Grapevine tissues harbour a variety of microbes, with endophytic actinobacteria being of particular interest due to their capacity to produce a range of bioactive compounds which ability to inhibit phytopathogens. There have, however, been no studies investigating the diversity of actinobacteria associated with grapevines and their bioactivity against GTD pathogens in New Zealand vineyards. The first aim of this research was to identify the diversity of culturable endophytic actinobacteria associated with different grapevine tissues in New Zealand vineyards and determine the most effective method and media for isolation of endophytic actinobacteria from different grapevine tissues. Grapevine tissues (leaf, stem, and roots) were sampled from Sauvignon blanc vines in both a conventionally and an organically managed vineyard site at Lincoln University. Two isolation methods were tested on surface sterilised tissue: (i) direct plating of tissue pieces, and (ii) tissue maceration, with the tissue homogenized prior plating to dilutions. The tissue samples were plated on four media, International Streptomyces Project 2 agar (ISP2), Starch Casein agar (SC), Tap Water Yeast Extract agar (TWYE) and Actinomycete Isolation agar (AIA). The recovered actinobacteria isolates were identified based on morphology and sequencing of the 16S rRNA gene. A higher frequency of isolates were iii recovered from roots (20 isolates), than leaves (1 isolate) and stems (0 isolates), with more actinobacteria isolated from the organically managed vineyard and using the tissue maceration technique. AIA (8 isolates), ISP2 (5 isolates), SC (5 isolates), and TWYE (3 isolates) were effective at isolating actinobacteria from grapevine tissues. Isolates identified as Streptomyces spp. (20 isolates) and Mycolicibacterium sp. (1 isolate) were recovered. The culturable endophytic actinobacteria species/genera associated with tissues sampled from grapevines of two different ages, young newly planted (2-year-old) and mature (25-year old) Sauvignon blanc vines established in the same vineyard site were identified using the methods developed. Endophytic actinobacteria isolates were more frequently isolated from the mature vines (n=20) than the newly planted vines (n=2) and all isolates were recovered from the root tissues. Sequencing of the 16S rRNA gene revealed that all 22 isolates belonged to the genus Streptomyces. The second aim was to investigate the potential biocontrol activity of actinobacteria isolates to inhibit key pathogens responsible for grapevines trunk diseases (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum and N. luteum) and whether these endophytes produce bioactive compounds related to plant growth promotion traits using a range of in vitro bioactivity assays. The inhibitory activity of 40 endophytic actinobacterial isolates was further determined. Thirteen endophytic actinobacterial isolates showed either strong (+++) or moderate (++) antagonistic activity against all five phytopathogenic fungi. In addition to biocontrol activity, four isolates exhibited high siderophore activity on CAS-LB plates, 6 isolates showed moderate or low ability to solubilize phosphate on TCP agar, and 3 isolates were indicated to have high indole acetic acid (IAA) production. Overall, this study revealed the diversity and bioactive potential of culturable endophytic actinobacteria associated with grapevine tissues in New Zealand vineyards. This study identified several isolates belonging to the genus Streptomyces with potential as biocontrol agents against grapevine trunk pathogens. Further study is required to determine their biocontrol activity in planta.