Development of biotools for vineyard-associated pest and disease control based on entomopathogenic nematode symbiotic bacteria stars

Abstract

Grapevine is one of the most important socio-economic crops worldwide. However, the vine sector faces numerous challenges today, ranging from climate change and various forms of environmental degradation to severe pest and disease outbreaks. The development of new sustainable tools to address these challenges and protect vineyards is necessary. In this context, symbiotic bacteria of entomopathogenic nematodes, specifically Xenorhabdus spp. and Photorhabdus spp., emerge as potential biocontrol agents due to their ability to produce bioactive compounds with insecticidal and antifungal properties. We hypothesized that these symbiotic bacteria and their by-products might effectively manage selected pests and diseases that pose significant threats to grapevines. Therefore, the specific objectives to accomplish during this Thesis were as follows: (i) to evaluate the insecticidal and antifungal effect of the soluble toxins produced by Xenorhabdus and Photorhabdus for controlling vineyard pests and diseases; (ii) to assess the deterrent and the antifungal activity of the volatile organic compounds (VOCs) emitted by Xenorhabdus and Photorhabdus fermentations; and (iii) to evaluate the effect that bacterial secondary metabolites may have on the defence of the harvested fruit. We conducted in vitro experiments to assess the insecticidal and antifungal properties of various bioproducts derived from Xenorhabdus spp. and Photorhabdus spp. To obtain these bacterial bioproducts, we inoculated individual colonies of each pure culture in Triptone Soya Broth (TSB) media, maintaining the liquid cultures for 72 hours under continuous orbital shaking (150 rpm) at a temperature of 22 ºC in a light-protected environment. The resulting bacterial by-products included the soluble secondary metabolites (bacterial cell-free supernatants and the unfiltered ferments), the volatile organic compounds (VOCs) emitted from the ferments and the crude bacteria isolates. For our bioassays, we employed two insect species: Philaenus spumarius, the primary vector responsible for transmitting Xylella fastidiosa in Europe, the causative agent of Pierce’s disease in grapevines; and Lobesia botrana, one of the most economically significant pests affecting European vineyards. Our disease studies focused on Botrytis cinerea, the pathogen responsible for grape rot. Each set of experiments was conducted under controlled conditions. We employed a randomized complete block design with multiple replications to conduct our experiments. All the experiments were performed 2-3 times to confirm the observations. The results demonstrated that the soluble secondary metabolites produced by symbiotic bacteria associated with entomopathogenic nematodes possess insecticidal properties against L. botrana larvae and P. spumarius nymphs and antifungal activity against B. cinerea grape rot. Unfiltered bacterial ferments exhibited the highest insecticidal and antifungal capacity compared to bacterial cell-free supernatants studied against L. botrana larvae and B. cinerea, respectively. Moreover, the crude isolate of the specie Photorhabdus laumondii subsp. laumondii showed an equivalent antifungal degree to the commercial Bacillus amyloliquefaciens (Serenade® ASO fungicide). On the other hand, the so far little studied VOCs produced by the bacteria had a potential control effect on grapevine pests. The VOCs emitted by Xenorhabdus nematophila and P. laumondii subsp. laumondii ferments demonstrated feeding and anti-oviposition deterrent effects against L. botrana larvae and adult instars, respectively. Furthermore, the VOCs emitted by X. nematophila and P. laumondii subsp. laumondii TSB ferments inhibited > 60 % of B. cinerea mycelial growth in in vitro tests and limited the lesion area of B. cinerea to 0.5 % and 2.2 % on the grapes, respectively. Finally, we tested whether the VOCs may have a preventive effect on the grapes. The results showed that the volatile natural products emitted by X. nematophila and P. laumondii subsp. laumondii showed potential for inducing preventive effects on both damaged and intact post-harvest grapes, protecting against potential Botrytis rot infection. The mechanisms are still undescribed. In conclusion, the findings suggest that Xenorhabdus and Photorhabdus hold significant potential as effective biocontrol agents for managing pests and diseases associated with vineyards. These symbiotic bacteria offer a promising source of new biotools for sustainable viticulture.