Differential coexistence of multiple genotypes of Ophiocordyceps sinensis in the stromata, ascocarps and ascospores of natural Cordyceps sinensis

Abstract

Objective: To examine the differential occurrence of Ophiocordyceps sinensis genotypes in the stroma, stromal fertile portion (SFP), and ascospores of natural Cordyceps sinensis. Methods: Immature and mature C. sinensis were harvested. Mature C. sinensis were continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and ascospores of C. sinensis were collected for microscopic and molecular analyses using species-/genotype-specific primers. Results: Fully and semi-ejected ascospores were collected from the same specimens. The semi-ejected ascospores tightly adhered to the surface of the asci. The multicellular heterokaryotic ascospores showed uneven staining of nuclei. The ascospores were found to contain several genotypes of O. sinensis, Samsoniella hepiali, and an AB067719-type fungus. The genotypes within AT-biased Cluster-A occurred in all compartments of C. sinensis, but those within AT-biased Cluster-B were absent in the ascospores. Genotypes #13?14 of O. sinensis occurred differentially in fully and semi-ejected ascospores, which featured DNA segment substitutions and genetic material recombination between the genomes of the parental fungi, Hirsutella sinensis and the AB067719-type fungus. These offspring genotypes combined with varying abundances of S. hepiali in the 2 types of ascospores participated in the control of the development, maturation and ejection of the ascospores. Conclusion: Multiple genotypes of O. sinensis coexist differentially in the SFP and 2 types of C. sinensis ascospores, along with S. hepiali and the AB067719-type fungus. The fungal components in different combinations and their dynamic alterations in the compartments of C. sinensis during maturation play symbiotic roles in the lifecycle of natural C. sinensis .

Altered GC- and AT-biased genotypes ofOphiocordyceps sinensisin the stromal fertile portions and ascospores of naturalCordyceps sinensis

“…All reagents and biochips required for MassARRAY SNP MALDI-TOF MS genotyping were from Sequenom, USA, and experiments were performed at CapitalBio Co., Beijing. The humidity of our laboratory was maintained by spraying water using an atomizer twice a day in the morning and evening [61]. [61] shows a collection of the fully ejected ascospores of C. sinensis using double layers of autoclaved weighing paper.…”

“…The humidity of our laboratory was maintained by spraying water using an atomizer twice a day in the morning and evening [61]. [61] shows a collection of the fully ejected ascospores of C. sinensis using double layers of autoclaved weighing paper. It also showed numerous ascospores adhered to the outer surface of asci during massive ascospore ejection, which failed to be brushed off for collection using an autoclaved brush; hence, these ascospores were instead gently scratched off using a disinfected inoculation shovel or ring and referred to as semi-ejected ascospores.…”

“…The fully and semi-ejected ascospores were cleaned separately by 2 washes with 10% and 20% sucrose solutions and 10-min centrifugation at 1,000 rpm (desktop centrifuge, Eppendorf, Germany); the supernatant was discarded after each centrifugation. The pellets (ascospores) were subsequently washed with 50% sucrose solution and centrifuged for 30 min, and the ascospores that floated on the liquid were collected [61][62]. The 2 types of ascospores were stored in a -80°C freezer prior to further processing.…”

“…These 2 offspring genotypes of O. sinensis were characterized by large DNA segment reciprocal substitutions and genetic material recombination between 2 parental fungi (Group A H. sinensis and an AB067719-type Group E fungus [8][9][10]61]). The ITS1-5.8S-ITS2 sequences of Genotypes #13-14 were alternatively segmentally inherited from their parental fungi [61]. The segment sequences of Genotypes #13-14 are either completely identical to or far different from those of Genotype #1, and the primer pairs for the first-step PCR listed in Table 1 are unfavorable for amplifying the ITS sequences of Genotypes #13-14.…”

“…Li et al [61] reported the absence of genotypes of AT-biased Cluster-B (Genotypes #4 and #15; cf. 9) in semi-ejected ascospores was due to the considerably increased intensity of Peak T, the genotypes of AT-biased Cluster-A.…”