In planta pathobiota of grapevines

In both managed and natural ecosystems, plants live together with their microbiome including wide spectrum of endophytic fungi, which colonize plant tissues during some period of their life cycle yet cause no visible symptoms to their hosts. Varieties of grapevines (Vitis vinifera) also represent reservoirs of these endophytes, which may have effect on its growing, health status and grape production. These microorganisms might also serve as protective agents against various pathogens. Although, there is an increasing number of studies focusing on grapevine microbiome, our information is still limited on the communities of some cultivars and their potential role. The visualization of the fungal endophytes within the plant tissues is also not a routine task, hence we have limited information on their structures and nature of their presence in the host plant. Due to their potential effect on plant survival, production and on features of vine, investigation of endophytes will serve major findings and general results with agronomical importance.

Our aims are to identify the fungal microbiome using culture-based and culture-independent methods, found the common and dominant members of the community and visualize fungal endophytes in above ground tissues of V. vinifera cv. Furmint, one of the most cultivated white grapevine variety in distinct Hungarian vineyards.



Different aboveground organs of the plants were sampled from four and two sites from two wine regions of Tokaj and Eger in different seasons for three years. For culture-based and culture-independent methods and visualization of fungal endophytes both young and matured leaves, flowers, and bunch of grapes were collected in different phenological stages and set aside for investigation. Isolates gained from surface sterilized different plant tissues were identified based on their nrDNA ITS (Internal Transcribed Spacer) region. For the culture-independent (Next-Generation Sequencing, NGS) investigation, after the total DNA extraction from the plant material, also the ITS region was used to gain information on the endophytic fungal community. We also studied the soil fungal communities using this method. For specific staining and visualization of fungal endophytes, the fluorescence dye, a WGA (Wheat Germ Agglutinin) conjugate was applied after clearing the plant tissues.

In our study, we have collected until now more than 1200 plant samples from Furmint varieties in different wine regions, and approximately 500 fungal isolates were gained, preserved and identified. Using culture-based and culture-independent methods to characterize the endophytic fungal communities in different above ground tissues of the plant in different seasons and phenological states, we found the common and dominant taxa and various lineages. We successfully applied the WGA-based dying method, based on which extensive fungal colonization was found in majority of plant organs/parts. We also found structures characteristic to certain dominant taxa strengthening their dominant presence and precise location within the plant tissues. In this study, we gain substantial information that might reveal and bring us closer to understand the function, structure and general features of the fungal endophytic community of the agronomically important grapevine.