Preliminary identification of endophytic actinobacteria in different grapevine tissues

Abstract

Grapevine trunk diseases are an economically important issue for the New Zealand viticulture industry. The limited control strategies effective against these pathogens and the increasing awareness of the potential environmental impacts of fungicide applications has led to an increased interest in identifying alternative control strategies. Endophytic actinobacteria are of particular interest due to their capacity to produce bioactive compounds that inhibit phytopathogens. However, there have been no studies in New Zealand to investigate the diversity and bioactivity of endophytic actinobacteria against grapevine trunk pathogens. This study aimed to 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 at Lincoln University. Two isolation techniques were tested on surface sterilised tissue: 1) direct plating of tissue pieces (2 mm²); and 2) tissue maceration, with tissue (0.1 g) homogenized in 1 mL water and 1520 metal beads (2 mm diam.). 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. More isolates were recovered from roots (22 isolates), than leaves (1 isolates) or stems (0 isolates), with more actinobacteria isolated from the conventionally managed vineyard and using the tissue maceration technique. AIA (8 isolates), ISP2 (7 isolates), SC (5 isolates), and TWYE (3 isolates) were effective at isolating actinobacteria from grapevine tissues. Isolates identified as Streptomyces spp. (22 isolates) and Mycolicibacterium sp. (1 isolate) were recovered. Future work will use these methods to determine endophytic actinobacteria diversity associated with grapevines, and their ability to inhibit key grapevine trunk pathogens.