Five years investigation of female and male genotypes in périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction strategies.

The Périgord black truffle (Tuber melanosporum Vittad.) is a heterothallic ascomycete that establishes ectomycorrhizal symbiosis with trees and shrubs. Small-scale genetic structures of female genotypes in truffle orchards are known, but it has not yet been studied in male genotypes. In this study, our aim was to characterize the small-scale genetic structure of both male and female genotypes over five years in an orchard to better understand the T. melanosporum sexual reproduction strategy, male genotype dynamics, and origins. Two-hundred forty-one ascocarps, 475 ectomycorrhizas, and 20 soil cores were harvested and genotyped using microsatellites and mating type genes. Isolation by distance analysis revealed pronounced small-scale genetic structures for both female and male genotypes. The genotypic diversity was higher for male than female genotypes with numerous small size genotypes suggesting an important turnover due to ascospore recruitment. Larger and perennial female and male genotypes were also detected. Only three genotypes (1.5%) were found as both female and male genotypes (hermaphrodites) while most were detected only as female or male genotype (dioecy). Our results suggest that germinating ascospores act as male genotypes, but we also proposed that soil mycelium could be a reservoir of male genotypes.

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells. While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.