Characterization of Two Mitochondrial Genomes and Gene Expression Analysis Reveal Clues for Variations, Evolution, and Large-Sclerotium Formation in Medical Fungus Wolfiporia cocos.

Wolfiporia cocos, a precious mushroom with a long history as an edible food and Asian traditional medicine, remains unclear in the genetic mechanism underlying the formation of large sclerotia. Here, two complete circular mitogenomes (BL16, 135,686 bp and MD-104 SS10, 124,842 bp, respectively) were presented in detail first. The salient features in the mitogenomes of W. cocos include an intron in the tRNA (trnQ-UUG2), and an obvious gene rearrangement identified between the two mitogenomes from the widely geographically separated W. cocos strains. Genome comparison and phylogenetic analyses reveal some variations and evolutional characteristics in W. cocos. Whether the mitochondrion is functional in W. cocos sclerotium development was investigated by analyzing the mitogenome synteny of 10 sclerotium-forming fungi and mitochondrial gene expression patterns in different W. cocos sclerotium-developmental stages. Three common homologous genes identified across ten sclerotium-forming fungi were also found to exhibit significant differential expression levels during W. cocos sclerotium development. Most of the mitogenomic genes are not expressed in the mycelial stage but highly expressed in the sclerotium initial or developmental stage. These results indicate that some of mitochondrial genes may play a role in the development of sclerotium in W. cocos, which needs to be further elucidated in future studies. This study will stimulate new ideas on cytoplasmic inheritance of W. cocos and facilitate the research on the role of mitochondria in large sclerotium formation.

Development of crossbreeding high-yield-potential strains for commercial cultivation in the medicinal mushroom Wolfiporia cocos (Higher Basidiomycetes).

Wolfiporia cocos is a well-known medicinal mushroom, and its dried sclerotia has been widely used as a traditional medicine in China, Japan, and other Asian countries for centuries. However, long-term asexual reproduction of the breeding system in W. cocos results in a current universal degeneration of cultivated strains. To develop a W. cocos breeding program that will benefit commercial cultivation, we previously developed an optimum method for indoor induction of W. cocos fruiting bodies and clarified the nature of preponderant binuclear sexual basidiospores. In this paper, we first show that the majority of W. cocos single-spore isolates cannot form sclerotium in field cultivation. We then investigated the possibility of breeding new strains by crossbreeding. Three types of mating reactions were observed in both intra-strain pairings and inter-strain pairings, and a total of fifty-five hybrids were selected by antagonistic testing and allele-specific polymerase chain reaction (PCR). Field cultivation of hybrids demonstrated that some hybrids can form sclerotium via two cultivated methods. Two new high-yield strains were identified. This report will stimulate new thinking on W. cocos and promote further extensive studies on crossbreeding in W. cocos, a new topic related to the development of more efficient protocols for the discrimination of hybrids in W. cocos.

Biological characteristics of teleomorph and optimized in vitro fruiting conditions of the Hoelen medicinal mushroom, Wolfiporia extensa (Higher Basidiomycetes).

Wolfiporia extensa is a basidiomycetous brown rot fungus and is of well-known medicinal import in China, Japan, and other Asiatic countries. Fruiting body induction is of major relevance for basic biological research and for their use in industrial applications. Based on the evaluation of the effects of temperature, time in the dark before induction and culture, and wounding treatment on fruiting, this report describes the most efficient protocol for inducing fruiting of W. extensa growing on agar plates. Furthermore, several biological characteristics of teleomorph, such as the locations of hymenium, the configuration of basidiospores and primary mycelia, and events involved in basidiosporogenesis in W. extensa, were analyzed for the first time using fluorescence microscopy. The results showed that the hymenium born on both sides of the wall of the honeycomb-like structure on the surface of fruiting bodies and the hymenophoral trama situated in the middle. Each basidia has 4 binuclear basidiospores, and the primary mycelia are multinucleate without clamp connections. These results broaden our knowledge about this brown rot fungus and promote further studies of the sexual reproduction, fruiting body development, and advancement of breeding program, new topics related to the contents of pharmacologically active substances in W. extensa fruiting bodies.

Effect of revulsive cultivation on the yield and quality of newly formed sclerotia in medicinal Wolfiporia cocos.

Wolfiporia cocos is a well-known medicinal mushroom widely used in China, Japan and other Asiatic countries for its various therapeutic effects. 'Revulsive cultivation' is a newly developed method for promoting sclerotia growth in W. cocos field cultivation in China. In this report, we have systematically examined the effects of 'revulsive cultivation' on the yield and quality of newly formed sclerotia. The results showed that the genetic differences between the cultivated strain and the revulsive strain of T1 used in this study did not affect the formation process of new, large sclerotia in which the mycelia of the cultivated strain grew on pine logs directionally assembled on the revulsive strain. Additionally, 'revulsive cultivation', in which the cultivated strain and the revulsive strain used had the same or different genotypes, could remarkably increase the yield, lower the water content, and increase the water-soluble polysaccharide content of the newly formed sclerotia. Moreover, we observed that the changes in the values of the tested economic traits obtained from different genotype combinations through 'revulsive cultivation' were dissimilar. The correlations of these changes with the original sclerotium-forming ability of the cultivated strains and the genetic differences between the cultivated strain and the revulsive strain were not significant. These results will broaden our knowledge regarding the field cultivation of this medical fungus, stimulate new thinking on the study of sclerotium formation in some sclerotium-forming fungi, and promote further studies on the mechanism of sclerotium formation in W. cocos.