Microbiome plays an important role during the tobacco aging process which was an indispensable link in the production and processing of cigarettes. However, the structure and functions of microbiome have not been clarified during the tobacco aging process. In this study, 16S rDNA and ITS amplicon sequencing techniques were used to analyze the core microbiome of 15 tobacco samples from five different aging stages. The whole bacterial microbiome was classified into 29 microbial phyla and 132 orders. Enterobacteriales (63%), Pseudomonadales (16%), Sphingomonadales (8%), Xanthomonadales (4%), Burkholderiales (4%), Rhizobiales (3%), and Bacillales (2%) comprised the core bacterial microbiome. The whole fungal microbiome was classified into five microbial phyla and 52 orders. Incertae_sedis_Eurotiomycetes (27%), Wallemiales (25%), Sporidiobolales (17%), Capnodiales (5%), Eurotiales (2%), an unclassified Ascomycota (12%), and an unidentified Eurotiomycetes (4%) comprised the core fungal microbiome. FAPROTAX function prediction suggested that the core microbiome has a substantial potential for the carbon cycle, nitrate metabolism, aromatic compound degradation, chitinolysis, cellulolysis, and xylanolysis, but simultaneously, the core microbiome is also a source of human pathogens. The dynamics of the bacterial community were primarily determined by the total nitrogen in tobacco leaves during the aging process, while those of the fungal microbiome were primarily determined by total organic carbon. This study indicated that the core microbiome activities may play an important role in regulating the loss of carbon organic compounds and enhancing the secondary metabolites during tobacco leaves aging process.

Hirsutella liboensis was isolated from the larva of Cossidae (Lepidoptera) in Libo Natural Reserves, Guizhou Province. The fungus produces fasciculate synnemata and mono- and polyphialidic conidiogenous cells with necks twisted in two or three helical turns. Conidia are one-celled or (rarely) one-septate, fusiform or like orange segments that are enveloped in a mucous sheath. Morphological characters and phylogenetic analyses of ITS1-5.8S-ITS2 sequences support this fungus as a new species.

Purpose: Microorganisms are important in tobacco aging. These are used to improve the quality of tobacco leaves after threshing and redrying. However, the response of microbial community to the storage environment and time during the tobacco aging process has been less explored. This study aimed to characterize the dynamic changes in microbial community composition and diversity in tobacco leaf samples. Methods: In this study, 16S and ITS rRNA gene amplicon sequencing techniques were used to characterize the composition, diversity, and co-occurrence of the microbial community in tobacco leaves stored in two different cities during the 24-month aging. Furthermore, the activities of several enzymes were measured spectrophotometrically, and the correlation between the microbiota and enzyme activity was analyzed by network analysis. Results: Shannon diversity and Chao richness of bacterial communities gradually increased during the first 18 months, whereas those of the fungal community decreased. The relative abundance of Proteobacteria decreased, whereas that of Actinobacteria and Bacteroidetes increased. The proportion of Ascomycota gradually increased during the first 18 months and then rapidly decreased, whereas the proportion of Basidiomycota exhibited a completely opposite pattern. The change in the composition of bacterial community and dominant genera in leaves was not significant between Guiyang city and Maotai city storerooms, but that in the fungal community was significant. The network analysis revealed that fungal networks were more complex and compact than bacterial networks, and a strong negative correlation existed between bacteria and fungi. Moreover, the bacterial microbiome showed a strong positive association with amylase activity, while the fungal microbiome positively correlated with cellulase activity. Conclusions: This study demonstrated a significant spatiotemporal heterogeneity in the composition of the microbial community during tobacco aging and highlighted the possible influence of the interactions and enzyme activity on microbial diversity and composition. The findings provided a scientific basis for using microorganisms to regulate and control tobacco aging.

Background: The genus Lecanicillium W.Gams & Zare is a recognized insect pathogen but members of the genus have been found parasitizing various hosts including arthropods, nematodes, plants, and fungi. The new classification system for fungi proposed to reject Lecanicillium and transfer some of the species to the genus Akanthomyces. However, the attribution problem of most species in the original genus Lecanicillium remains unsolved. The current study aimed to improve understanding of the pivotal internal phylogeny in Lecanicillium by estimating the divergence times of Lecanicillium to provide additional insights into the status of this genus within the family Cordycipitaceae.Results: Dating analyses support the supposition that the ancestor of Lecanicillium was in the Cretaceous period (84.36 Mya, 95% HPD: 72.12–94.74 Mya). After originating from a common ancestor, eight clades of Lecanicillium were derived and evolved independently in parallel with other genera of Cordycipitaceae. Based on the clear divergence age estimates, Lecanicillium clade 8 originated earlier as an independent group in the Cretaceous period (75.61 Mya, 95% HPD: 63.31–87.54 Mya), while Lecanicillium clades 1–7 originated later as an independent group in the boundary of the Cretaceous and Paleogene periods (64.66 Mya, 95% HPD: 52.75–76.74 Mya). Lecanicillium huhutii formed an independent branch in a polytomy together with a clade containing Lecanicillium tenuipes (BI posterior probabilities 1, ML bootstrap 100%).Conclusion: The pivotal internal phylogeny, origin, and evolutionary history of Lecanicillium in the family Cordycipitaceae were investigated. Phylogenetic and morphological analyses indicated that there are eight representative clades (four representative branches of evolutionary history), including clade 1 (members have a relatively uniform sporulation structure comprising globose heads with a higher number of conidia), clade 8 (including all members of Gamszarea), clades 2–5 (the differences of the divergence time estimations were smaller compared with other clades), and clade 6–7 (members are close to Gibellula, Hevansia, and Ascopolyporus). Based on the above findings, a novel spider-pathogenic fungus, Lecanicillium huhutii, is described. All other species in Lecanicillium clade 1 (Lecanicillium araneogemum, L. nodulosum, L. pissodis, and L. uredinophilum) should be transferred to the genus Akanthomyces. Furthermore, the monotypic genus Parengyodontium should be merged with the genus Gamszarea. More novel species need to be discovered to thoroughly resolve the attribution problem of Lecanicillium. Finally, no major lineages of Lecanicillium were correlated with the nearby Cretaceous-Tertiary extinction event, indicating that the diversity of Lecanicillium is more likely to be caused by long-term environmental adaptation and coevolution with insects rather than by dramatic extinction events.

Karst habitats are hotspots of diversity and endemism. Their naturally fragmented distributions across broad geographic landscapes have led to a complex array of smaller evolutionary ecosystems. Comprehensive biodiversity assessments of karst habitats have revealed that these ecosystems contain a high level of endemism. During a survey of cordycipitoid fungi in the karst region of southwest Guizhou, China, we identified and proposed two new species, based on morphology and multi-locus (nrSSU, nrLSU, tef, rpb1, and rpb2) phylogenetic analyses. O. cystidiatasp. nov. is characterized by gray-white to yellow fertile part, verrucose phialides, and conidia enveloped in a cystic thickened mucus sheath, distinguishing it from other species. O. sinocampessp. nov. is distinguished by long tapering phialides with inflated bases. Phylogenetic analyses using five loci reveal that O. cystidiata shares a close relationship with O. fenggangensis, O. musicaudata, O. alboperitheciata, and Hirsutella kuankuoshuiensis, while O. sinocampes is closely related to O. multiperitheciata and H. strigosa. Herein, we completed the descriptions, illustrations and molecular phylogeny of these two new species. The host diversity of O. sinocampes has also been documented within the orders Lepidoptera and Coccoidea. Our research further enriches the diversity of cordycipitoid species in the typical karst landform areas of Guizhou, China.

Cordyceps cicadae is an entomogenous fungus with important uses in traditional Chinese medicine. However, its wild resources have not met consumers’ demand due to excessive harvesting practices. Artificial cultivation is therefore an important alternative, but research on cultivating C. cicadae in natural habitats has not been reported. In this study, we aimed to explore the viability of cultivating C. cicadae in a natural habitat, in the soil of Pinus massoniana forest. We assessed and compared the yield, metabolite contents and bacterial community composition of C. cicadae grown in the Antheraea pernyi pupae at different growth stages, and under different cultivation conditions, in the soil of a natural habitat and in sterile glass bottles. Our results showed that cultivating C. cicadae in a natural habitat is feasible, with up to 95% of pupae producing C. cicadae fruiting bodies. The content of nitrogen compounds (amino acids) in C. cicadae cultivated in a natural habitat was significantly higher than in glass bottles, while the yield and carbon compound (mannitol and polysaccharide) and nucleoside (cordycepin and adenosine) contents were lower. Different bacterial genera were enriched in C. cicadae at different growth stages and cultivation environments, and these bacterial genera were closely related to metabolites contents during growth. This study demonstrated the viability of a novel cultivation method of C. cicadae, which could be used as an alternative to wild stocks of this fungus. These findings provided new insights into the growth mechanism of C. cicadae and its interaction with soil microorganisms.