Molecular strategies to assess Botrytis cinerea in the context of viticulture and oenology

One of the most destructive pathogens in agriculture is the fungus Botrytis cinerea. It infects a wide range of crops, including grapes and vines. Due to its multiple and complex modes of attack, controlling the disease is a challenge in itself, but this is aggravated by growing concerns about traditional treatment strategies, climate change and the problematic predictability of infection sites. This work provides fundamental information on the laccase enzyme, a key component in overcoming plant defences, at the genomic level to understand potential differences in Botrytis cinerea strains based on regionality, vintage and laccase activity. To this end, eight strains were selected based on their origin, vintage and laccase activity and next generation sequencing was performed. The strains were then analysed using bioinformatic tools to assess differences in laccase genome regions. The study also investigated a classification method of Botrytis cinerea by molecular fingerprinting. Based on available microsatellite markers, 59 strains were analysed by SSR-PCR and the effectiveness of two methods, agarose gel electrophoresis and capillary sequencing, was compared. The aim of this aspect was to develop a simple method for strain differentiation and to evaluate potential markers for regionality, vintage and laccase activity. It also aimed to provide information on the general strain diversity in vineyards. The established strain differentiation method was used for a study on growth- and laccasemodulating properties of polyphenols. The last major part of this study was to optimise a qPCR method for the early detection of Botrytis cinerea on grapes, before it is visible to the eye. The detection method was adapted to reliably quantify biomass without cross-amplification of other pathogens relevant in vineyards, and the influence of different Botrytis cinerea strains on the quantification result was investigated. In addition to refining the method, the early detection limit was evaluated under laboratory conditions. The results of the bioinformatic evaluation of the genome indicate that different laccase gene regions are highly variable among each other and among different strains. While regional and evolutionary aspects seem to influence the genomic relationship, there was no clear correlation for laccase activity. There was also no clear correlation in the promoter and terminator regions of the genes. The results highlight the importance of gene expression studies, transcriptomics and proteomics, but also provide new targets for further research by analysing either highly conserved or evolutionarily changing laccase gene regions. This is particularly true for laccase gene regions that have not been the focus of previous experiments. The results for the molecular biology methods underline the diversity of Botrytis cinerea in vineyards. Strains from locally distant regions showed a similar level of differences as strains from locally closer regions. Although the complexity of Botrytis cinerea is high, it seems that general assumptions based on research on specific strains can be adapted to different regions, but it has to be considered that some strain characteristics can influence experimental results. Almost all strains could be assigned individual fingerprints with both methods implemented, although the capillary sequencer gave slightly better differentiation than agarose gel electrophoresis. However, the difference was marginal and agarose gel electrophoresis is much cheaper and can be performed by untrained personnel. The effectiveness for strain differentiation also became evident in the study of growth- and laccase- modulating properties, where a classification of strains for the research question was possible to discuss and interpret the obtained data. For the qPCR quantification and early detection method, the cross amplification of Cladosporium sp., Trichothecium roseum and especially Penicillium expansum could be excluded. There were some effects of the choice of strain on the quantification result. The detection of Botrytis cinerea in grapes under laboratory conditions was possible four days before it was visible to the naked eye. This component of the research signifies a substantial advancement in the implementation of this methodology in viticulture, enabling precises targeted treatments, thereby enhancing predictability while reducing the necessity for treatments. The findings offer invaluable insights into the Botrytis cinerea fungus, providing a foundation for research and agriculture concurrently. The results of the study have the potential to provide an important basis for the monitoring of the evolving strains of Botrytis cinerea in the context of climate change.