Strain breeding is much less advanced in the edible and medicinal species Agaricus subrufescens than in Agaricus bisporus, the button mushroom. Both species have a unifactorial system of sexual incompatibility, a mating type locus tightly linked to a centromere, and basidia producing both homokaryotic (n) and heterokaryotic (n + n) spores. In A. bisporus, breeding is mainly based on direct selection among the heterokaryotic offspring and on hybridization between homokaryotic offspring. The parental heterozygosity is highly maintained in the heterokaryotic offspring due to suppression of recombination and preferential pairing in the spores of nuclei, each one per second meiotic divisions; such "non-sister nuclei" heterokaryons are fertile. In A. subrufescens, recent studies revealed that recombination is not suppressed and that nuclei from the same second meiotic division can also be paired in a spore that give rise to a "sister nuclei" heterokaryon in which the nuclei bear the same mating type allele. The objective of the present work was to investigate the potential function of the different categories of spores in A. subrufescens and their possible use in a genetic breeding program. Using eight co-dominant molecular markers, we found that half of the offspring of the A. subrufescens strain WC837 were heterokaryotic, one quarter of them being sister nuclei heterokaryons. These heterokaryons were infertile and behaved like homokaryons, being even able to cross between each other. In contrast, non-sister nuclei heterokaryons could fruit but inconsistently due to inbreeding depression. Potential roles of these two categories of heterokaryons in nature and consequences for strain breeding are discussed.

Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the Agaricus genus comprises the most cultivable or potentially cultivable species. Agaricus bisporus, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. subrufescens, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus. The 13 main LG of A. subrufescens were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. subrufescens LG. Electronic mapping of a collection of A. subrufescens expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of Agaricus species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. subrufescens.

Scanning electron microscopy enabled the process of colonization and reproduction to be observed within the formation of phialides, conidiophores and verticils of L. fungicola. The formation of calcium oxalate crystals by the pathogen was also visible using the X-ray microanalysis, both at the hyphae in the Petri plate and at basidiocarp infection site.