Biological control of grapevine trunk diseases using bacterial endophytes from grapevines

Jennifer Niem

Research output: ThesisDoctoral Thesis

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Abstract

Grapevine trunk diseases (GTDs) such as the Esca complex, Eutypa dieback and Botryosphaeria dieback are considered major constraints to grapevine production. These diseases can cause dieback, cankers and wood decay affecting the long-term sustainability and productivity of vineyards. Current management of these diseases is through protection of grapevine propagation materials in nurseries, remedial surgery and fungicide treatment of pruning wounds. However, registered fungicides for GTDs are limited and some only offer short term protection. Thus, there is a need to find alternate solutions to combat these diseases for the wine grape industry.

Endophytes are plant-associated microorganisms that reside within plant tissues. These may be beneficial, commensal or pathogenic to their host plant. In recent years, the use of beneficial endophytes in plant disease control has gained popularity as the application of fungicides raises concerns for human and environmental safety. This research explored the diversity of the grapevine endomicrobiome with the aim of identifying endophytes that could potentially be used as biocontrol agents (BCAs) for GTDs.

The diversity of microbial endophytes associated with grapevine wood was investigated using next-generation sequencing. Characterisation of the grapevine endophytic community was undertaken using DNA extracted from both asymptomatic and GTD symptomatic vines collected from the Hilltops and Hunter Valley regions of New South Wales (NSW), Australia. Both the bacterial 16S rRNA gene and ITS region of fungal rRNA genes were sequenced. The analysis determined that Pseudomonas spp. predominated the bacterial community in asymptomatic grapevine wood from both regions, comprising 56-75% of the total population. In contrast, the Pseudomonas population in GTD symptomatic tissues were significantly lower representing 29% and 2% of the bacterial community in Hilltops and Hunter Valley, respectively. The fungal community was dominated by the genus Phaeomoniella in both asymptomatic and symptomatic grapevines, representing 74-89% and 59-78% of the total fungi, respectively. The other two abundant genera were Phaeoacremonium spp. and Inonotus spp. Phaeomoniella chlamydospora and Ph. minimum(syn. Ph. aleophilum) and some Inonotus spp. have been implicated in Petri and Esca disease of grapevines.

The abundance of Pseudomonas spp. in asymptomatic grapevines led to further investigation of its potential antagonistic activity against GTD pathogens. Isolation of fluorescent Pseudomonas from grapevine wood resulted in 47 isolates for examination. In dual-culture assays, 10 of these were antagonistic towards GTD pathogens (a testing panel consisting of nine species of Botryosphaeria dieback, three species of Eutypa dieback, and two Esca/Petri disease pathogens). Identification through 16S rRNA gene sequencing determined that nine Pseudomonas isolates represented strains of P. poae and one isolate, a strain of P. moraviensis.

The ability of the antagonistic strains of Pseudomonas to suppress GTD infection in grapevine plants were investigated using Neofusicoccum luteum as a model pathogen. The 10 antagonistic strains of Pseudomonas were screened for their ability to protect grapevine pruning wounds from infection by N. luteum using detached canes. Re-isolations from canes treated with P. poae strains BCA13, 14, 16, 17, 18, and 19 showed 67-78% reduction in the recovery of N. luteum while all inoculated control canes were 100% positive. Wounds created by pruning glasshouse potted vines were treated with P. poae strains BCA13, BCA14 and BCA17 prior to inoculation with N. luteum. BCA17 significantly reduced the recovery of N. luteum by 80% relative to the control, while BCA13 and BCA14 were not effective in reducing pathogen recovery. A quantitative assay (qPCR) developed for N. luteum further elucidated the efficacy of BCA17. The qPCR analysis indicated a 40-fold reduction in the amount of pathogen DNA in vines that were treated with BCA17 compared to the positive control. Pathogenicity tests using BCA17 showed this strain was not pathogenic to detached grapevine leaves, berries or single node canes.

A rifampicin-resistant mutant (RifMut) strain of BCA17 was generated to assess its establishment in grapevine tissue. Re-isolations of RifMut from inoculated vines showed that this strain can persist in the host tissue for up to 6 months. The RifMut strain could also be re-isolated from dormant canes dipped in RifMut bacterial suspension, indicating this strain was potentially transmissible in propagation material.

Investigation of the underlying mechanisms involved in the inhibition of GTD pathogens and suppression of GTD infection in grapevine plants by BCA17 was conducted. All 10 antagonistic Pseudomonas spp. produced siderophores in chrome azurol S (CAS) media. The cell-free filtrate of BCA17 inhibited mycelial growth and spore germination of all four GTD pathogens tested (Diplodia seriata, Neofusicoccum parvum, N. luteum, Eutypa lata). The lipopeptide profile of the antagonistic (BCA17) and the non-antagonistic (JMN1) strains were compared using MALDI-TOF-MS analysis. A dominant lipopeptide was detected in a crude filtrate of BCA17 but not in that of JMN1. However, due to the lack of a comprehensive lipopeptide database with specific mass spectra, this lipopeptide remains to be identified. Draft genomes of the four strains (BCA13, BCA14, BCA17, and JMN1) were generated and analysis revealed putative gene clusters that may impart biocontrol activity against plant pathogens including: lipopeptide antibiotics, siderophores, proteases, detoxification, lipopolysaccharide, multidrug resistance, microbe-associated molecular proteins (MAMPS), biofilms, and a number of nonribosomal proteins.

This research identified Pseudomonas spp. as abundant in healthy grapevine wood and further showed that they may provide some protection against GTDs. From this, the research characterised an antagonistic strain of Pseudomonas (BCA17) that could potentially be used as a BCA to manage GTD pathogens.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Charles Sturt University
Supervisors/Advisors
  • Savocchia, Sandra, Principal Supervisor
  • Baaijens, Reggie, Principal Supervisor
  • Stodart, Ben, Principal Supervisor
Place of PublicationAustralia
Publisher
Publication statusPublished - 31 Jan 2021

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