Caracterización del micoviroma de aislados de campo de dos especies del hongo botrytis

Fungi of the genus Botrytis are necrotrophic plant pathogens, which develop and infect damaged or senescent tissue. This genus comprises 22 species, with most of them having a worldwide distribution. Botrytis cinerea is the only generalist species in the genus, being able to infect more than 1000 plant species, including more than 200 crops worldwide. It is a highly widespread and destructive pathogen in economically important crops such as grapes, tomatoes, peppers, etc. B. cinerea causes grey mold, leading to significant economic losses worldwide in both field and post-harvest stages. Controlling this fungus is challenging, with chemical fungicides being the primary control measure. However, the emergence of resistant strains and environmental concerns have made it necessary to search for less polluting alternatives. Biological control could be a more environmentally friendly alternative. The discovery of mycoviruses and their potential application in biological control strategies against plant pathogenic fungi, has stimulated their study in B. cinerea with the aim of developing strategies for controlling grey mold disease. The symptoms caused by mycoviruses infecting plant pathogenic fungi are diverse, some may induce hypovirulence, reducing the virulence of the fungus in the plant. This makes them promising candidates for the development of biocontrol strategies against fungal diseases in plants. Therefore, the search of mycoviruses with potential applications for biocontrol and its biological and molecular characterization have been encouraged. Furthermore, mycoviruses can serve as important biotechnological tools for elucidating the molecular mechanisms of fungal infection. The methods used for mycovirus identification have evolved over the years in parallel with the analysis techniques. Currently, the most widespread method for mycovirus identification that have allowed an important advance in the knowledge mycoviruses is RNA-Seq. Additionally, it is known that fungi use the machinery of gene silencing as defence mechanism against mycovirus. This study aims to expand the knowledge of the mycovirome of B. cinerea by analysing three field isolates of two Botrytis species. RNA-Seq data were used to identify mycoviruses in the isolates, and sRNA data to describe fungal gene silencing on each of the mycoviruses. These data were previously analysed in 2017, but due to the improvement of bioinformatics tools and the discovery of new viruses, mycoviruses that had not been detected in the initial analysis should be identified in this new analysis. The method employed in this work for the search of mycovirus consisted of a bioinformatic pipeline composed of four steps: reads cleaning, assembly of the clean reads, viral sequence identification using an updated viral database, and mapping of the viral sequences with the clean reads. Afterwards, molecular characterization of all the identified mycoviral sequences and their mapping with the sRNA library were carry out. Concluding the analysis, it was possible to identify all the mycoviruses that had already been described infecting these field isolates, confirming the correct implementation of the bioinformatic pipeline. Additionally, five new viral sequences were identified in the samples: Botrytis cinerea alpha-like virus 1 (BcALV1) isolate Pi258.8, Botrytis cinerea alpha-like virus 1 (BcALV1) isolate V448, Plasmopara viticola lesion-associated ourmia-like virus 80 (PvaOLV80) isolate V446, Botrytis narnavirus 1 (BNV1) isolate V446, and Botrytis cinerea narnavirus 1 (BcNV1) isolate V448. The first two are mycoviruses from the same species as BcALV1, which is taxonomically unclassified. The third is a mycovirus of the Deltascleroulivirus betaplasmoparae species, as PvaOLV80. And the last two are new mycoviral species that had not been described until now, BcNV1 and BNV1. Finally, it has been verified that all the mycoviruses described are targets of the gene silencing machinery of the fungal host, and more remarkable, that for the first time has been showed that this machinery is also active against narnaviruses.