Characterization of chestnut bark fungal communities in healthy trees and blight recovered through natural or introduced hypovirulence

Various fungal species together with Cryphonectria parasitica (Murr.) Barr. have been isolated from chestnut tissues with blight symptoms. Microfungi remain in cankers during tissue healing, which occurs by transmitting hypovirulence by Cryphonectria hypovirus 1 (CHV1) compatible strains. However, studies focused on the diversity and ecology of the non-C. parasitica taxa on chestnut bark are lacking. This work evaluated the composition and richness of microfungi species associated with healthy chestnut trees, those with cankers healed by natural hypovirulence and those treated with hypovirulent strains (artificially introduced hypovirulence). Microfungi from diseased trees were isolated from six randomly selected points in the inner and external areas of the healed canker. In healthy trees, tissue samples were collected from 12 random locations on each tree’s trunk. Fungal species were identified based on morphological characteristics and ITS region sequencing using the universal primers ITS1 and ITS4. Four hundred thirty-one fungal isolates were obtained from which 38 operational taxonomic units (OTUs) were identified. The fungal communities varied from tree to tree and did not display similar patterns. The endophyte Biscogniauxia mediterranea and epiphyte Cytospora eucalypticola fungi were detected in all study locations and tree health conditions. Notably, C. parasitica (virulent and hypovirulent strains) was dominant in the inner area of healed cankers, accounting for 64.3% of the isolates, and the saprobe fungi Penicillium glabrum was dominant among non-C. parasitica microfungi species. Dissimilarity analyses showed low similarity between the microfungi communities found in the inner and external areas of the healed cankers. The study reveals the long-life span of C. parasitica in healed cankers and the therapeutic effect of natural and introduced hypovirulence.This study was funded by the Project BioChestnut PDR2020-101-030959