Isolation source matters: sclerotia and ectomycorrhizal roots provide different views of genetic diversity in Cenococcum geophilum

ABSTRACT

Cenococcum geophilum forms sclerotia and ectomycorrhizas with host plants in forest soils. We demonstrated the differences in genetic diversity of C. geophilum between cultured isolates from sclerotia and those from ectomycorrhizal roots in the same 73 soil samples based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences and newly developed microsatellite markers. Based on GAPDH sequences, 759 cultured isolates (553 from sclerotia and 206 from ectomycorrhizas) were classified into 107 “genotypes” with sequence variation of up to 8.6%. The total number of GAPDH genotypes per soil sample ranged from 1 to 9, but genotypes that were shared between sclerotia and ectomycorrhizas were uncommon (0–3 per soil sample). More than 50% of GAPDH genotypes were unique to one source in most soil samples. Unique GAPDH genotypes were detected from either scleotia or ectomycorrhizal roots in most of the soil samples. Multilocus analysis using nine microsatellite markers provided additional resolution to differentiate fungal individuals and supported the results of GAPDH genotyping. The results indicated that sampling both sclerotia and ectomycorrhizal roots maximizes the detection of diversity at the soil core scale. On the other hand, when all isolates were viewed together, 82 GAPDH genotypes were unique to sclerotia whereas only 6 GAPDH genotypes were unique to ectomycorrhizas. Rarefaction analysis indicated that GAPDH genotypic diversity is significantly higher in sclerotia than ectomycorrhizal roots and the diversity within sclerotia is nearly the same as that of both sclerotia and ectomycorrhizas together. These findings suggest that sampling sclerotia alone is likely to detect the majority of GAPDH genotypes in Cenococcum at the regional scale. When deciding whether to sample sclerotia, ectomycorrhizas, or both types of tissues from Cenococcum, it is critical to consider the spatial scale and also the main questions and hypotheses of the study.

KEYWORDS:

• Anamorph
• ectomycorrhizal root
• genotype
• glyceraldehyde-3-phosphate dehydrogenase gene
• microsatellite
• sclerotium

ACKNOWLEDGMENTS

We thank the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for performing DNA sequencing, Austin Cary Memorial Forest and Ordway Swisher Biological Station for access to study sites, Dr. Martina Peter (Swiss Federal Research Institute WSL, Birmensdorf, Switzerland) for giving information about extraction of pure genomic DNA from mycelia of Cenococcum, and collaborators at the Smith mycology laboratory at the University of Florida for their assistance in collecting samples.

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Funding

This study was supported by Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for Research Abroad (to K. Obase), with additional funding from the University of Florida Institute for Food and Agricultural Sciences (IFAS) (to M. E. Smith).