Ophiocordyceps zhenxingensis sp. nov. and its microbiota during sexual and asexual stages in nature

Abstract

Ophiocordyceps holds significant importance in forest arthropod population regulation, biological control of agricultural pests, and the development of bioactive substances. Investigating the microbial community composition of wild Ophiocordyceps is significant for understanding its individual development process, understory cultivation, and taxonomy. In this study, a novel insect pathogenic fungus, Ophiocordyceps zhenxingensis, parasitizing Hymenoptera larvae, originating from Liaoning Province, China, was delineated based on morphology and phylogenetics. The microbial communities in the sclerotium and host surface complex of its different reproductive stages (sexual and asexual) were investigated. O. zhenxingensis was detected within the Hirsutella citriformis subclade, being closely related to Hirsutella gigantea and Ophiocordyceps elongata. It was morphologically characterized by solitary or multiple stromata, semi-submerged perithecia, and viscous basidiospores. Proteobacteria, Actinobacteriota, and Bacteroidota were the major bacterial taxa, and Basidiomycota and Ascomycota were the major fungal taxa. Tomentella, Sebacina, and Russula were abundant in the host surface complex that belongs to mycorrhizal fungi. The microbial co-occurrence network suggested that microbial relationships were predominantly positive correlations, and positive correlations were more pronounced in the asexual stage than the sexual stage. The microbial co-occurrence network of O. zhenxingensis was more complex during the asexual stage; it exhibited greater metabolic activity during the asexual stage. These results have enhanced our understanding of the composition, diversity, and functions of the endophytic microbial community of Ophiocordyceps, furnishing additional evidence for classifying its sexual and asexual stages and establishing a theoretical basis for subsequent research on its individual development.

Importance: Ophiocordyceps exhibits two distinct life stages (asexual and sexual) in its natural environment. The symbiotic microorganisms associated with the fungus play a crucial role in its growth and development. We have identified a new species, Ophiocordyceps zhenxingensis, which belongs to the Hirsutella citriformis subclade. A large number of mycorrhizal fungi were found in the insect appendages of O. zhenxingensis, whereas the fungal community within the sclerotium was predominantly composed of Ascomycota. During the asexual stage, O. zhenxingensis exhibited greater microbial diversity and stronger positive correlations among microorganisms. Additionally, it possesses a richer array of metabolic pathways. These results have deepened our knowledge of the composition, diversity, and roles of the microbial community in Ophiocordyceps, providing further evidence for distinguishing its sexual and asexual stages and laying a theoretical foundation for future research on its developmental processes.

Keywords: Ophiocordyceps zhenxingensis; community composition; function predictions; new species; phylogenetic analyses; sexual and asexual stages.

Fig 1

Phylogenetic tree based on the combined data set of nrSSU, nrLSU, tef-1α, rpb1, and ITS showing the relationship of Ophiocordyceps zhenxingensis with other Ophiocordyceps species. Values at the nodes before and after the backslash are BI posterior probabilities (BI-PP greater than 0.60) and ML bootstrap proportions (ML-BP greater than 50%), respectively. New species described in this paper are shown in bold.

Fig 2

Ophiocordyceps zhenxingensis (A) sexual stromata of fungus arising from lepidopterous larvae, (B) asexual stromata of fungus arising from lepidopterous larvae, (C) fertile part with infertile tip, (D through F) perithecia arrangement, (G) host, (H) ascospores, (I and J) spore germination, (K and L) asci, (M through P) conidiogenous cells and conidia, (Q and R) colony on PDA. Scale bars: 1 cm (A, B, Q, R) ,0.1 mm (C), 0.2 mm (D, F), 1 mm (E, G), 20 µm (H, J, K, L, O, P), and 10 µm (I, M, N).

Fig 3

The microbial composition of O. zhenxingensis. (A) The composition of the bacterial community at the phylum and genus levels. (B) The composition of the fungal community at the phylum and genus levels. (C) Venn diagrams of fungi at the genus level. (D) Venn diagrams of bacteria at the genus level. “Uncultured” denotes that the taxonomic classification of the microorganism is based solely on sequence similarity, in the absence of an associated pure culture isolate. A-N-P-R: Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium.

Fig 4

Species diversity. (A) The alpha diversity of fungi. (B) The beta diversity of fungi. (C) The alpha diversity of bacteria. (D) The beta diversity of bacteria. Different lowercase letters represent statistically significant differences (P < 0.05).

Fig 5

Microbial network structure characterization (OTUs greater than 1% of sequence sum): (A) asexual host surface complex, (B) sexual host surface complex, (C) asexual sclerotium, (D) sexual sclerotium, (E) host surface complex, and (F) sclerotium.

Fig 6

KEGG pathway composition of bacteria: (A) pathway clustering at the secondary classification level, (B) pathway abundance composition at the secondary classification level. Different symbols in the figure represent different KEGG pathways at the primary classification level.

Fig 7

Schematic diagram of the structure of O. zhenxingensis. The left portion of the figure illustrates the phenotypic characteristics associated with the asexual stage, whereas the right portion depicts the characteristics of the sexual stage. The perithecia serve as the critical criterion for differentiating between these two stages.