Based on archaeological evidences, there is a history of over 5 000 years for mankind’s knowledge of cordycipitoid fungi, with culture, art, history, philosophy, religion, convention, medicine, as well as life science involved. The history can be divided into worship, herbal, descriptive, and molecular eras, based on the characters of various periods. (1) Worship era from Hongshan culture to coming-out of Shennong’s Herbal Classics. Archaeological excavation revealed jade silkworm pendants and jade cicada laid in the mouth of the deads, which showed early barbaric knowledge characteristic of worship on reincarnation. Currently, there retain a cicada stele in China and scarab antiques in Egypt, while Maori of New Zealand reserve tattoo tradition by use of Ophiocordyceps robertsii to make tattoo dye. (2) Herbal era (coming-out of Shennong’s Herbal Classics-establishment of the genus Cordyceps in 1818. Cuneiform medical texts of ancient Sumerian revealed that people might use a cordycipitoid fungus against skin diseases. Chinese have been using Beauveria bassiana-infected silkworms since 2 000 years ago, and then Cordyceps chanhua-infected cicadas and Ophiocordyceps sinensis-infected caterpillars for medicine up to the present. In this period, the notion of materialization influenced eastern and western people, resulting in the materializational cognition of cordycipitoid fungi as “animalized plant”, “cicada turned from bamboo root”, “interconversion between insect and grass”. (3) Descriptive era (from 1818 to the end of the 1900’s): In 1818, Fries opened a 300 year chapter of cordycipitoid science development by establishing the genus Cordyceps. The notion of materialization was replaced gradually with modern science, and a vast Cordyceps family was formed gradually. Popular illustration of codycitipoid fungi in Europe and Japan accelarated the popularization of Cordyceps knowledge. Industries of fungal insecticides and foods and medicines by use of Cordyceps in China rose at the mid 1900’s and late 1900’s, respectively. (4) Molecular era (since the beginning of the 2000’s). Driven and supported by contemporary biological science and technology, studies of cordycipitoid fungi developed into a molecular era with uneven advances in different areas. Meanwhile, internal and international exchange became active, promoting the development of the culture of cordycipitoid fungi.
Cordyceps militaris is an important edible and medicinal fungus, and the stroma can be artificially cultivated on a large scale. However, the cultivated strains are easily degenerate, hindering industrialized cultivation. Germplasms for cultivation mainly depend on investigation and collection of wild resources. In this study, the resources of C. militaris in partial areas of Northeast China and Shandong Province were investigated, and the ecological distribution and morphology of the fungus were systematically surveyed. A total of 414 wild C. militaris specimens was collected from five locations. Most of the hosts were pupae of Lepidoptera, and a few of them were larvae and cocoons. The wild C. militaris stromata are solitary or 2-25-clustered, 1-17 cm long, rod-, flat-shaped, or irregularly deformed, branched and come out from various parts of the host, mainly from the head. The ground part of stroma is 1-6 cm long and shows deep orange-yellow. The fertile part of stroma is 0.5-4 cm long, 1.2-6 mm wide, and most stromata have a sharp boundary between the fertile and sterile parts. The underground part is 0.5-11 cm, and the length is correlated with the thickness of humus. The rhizomorph was clearly observed for the first time in some fruiting bodies of C. militaris, which connected with the pupa and the stroma, or grown from the pupa and stroma alone. The rhizomorph was morphologically different from the mycelia of prosenchyma and the middle of hyphae of the rhizomorph is enlarged. The macroscopic morphological differences between artificially cultivated and wild stromata were obvious, but no difference was found in the morphology of asci and ascospores. Enormous wild C. militaris germplasm resources have been observed in this survey, being helpful for the development of C. militaris industry in China.
Chinese cordyceps (Ophiocordyceps sinensis) is a rare species in the Qinghai-Tibet Plateau with high medicinal and economic values, and the quantitative characteristics, spatial distribution pattern and suitability of its resources are still unclear. In this study, the suitability and spatial distribution of Chinese cordyceps in Qinghai Province were analyzed by geographic information system (GIS) spatial analysis using altitude, vegetation, soil, temperature and precipitation as the main indicators, and verified by field sampling. The study showed that Yushu City, Zadoi County, Chindu County and Qumarleb County in Yushu Tibetan Autonomous Prefecture and Maqen County, Darlag County and Gade County in Tibetan Autonomous Prefecture of Golog were the most suitable areas for the growth of Chinese cordyceps. Qilian County and Gangca County in Tibetan Autonomous Prefecture of Haibei, Zekog County, Henan County and Tongren County in Tibetan Autonomous Prefecture of Huangnan, Zhidoi County and Nangqen County in Yushu Tibetan Autonomous Prefecture, Xinghai County, Tongde County and Guinan County in Tibetan Autonomous Prefecture of Hainan and some other areas were more suitable areas, while Datong County of Xining City, Huzhu County in Haidong City and the low altitude areas in Haixi Mongolian and Tibetan Autonomous Prefecture are unsuitable areas. The habitat of Chinese cordyceps covers approximately 76.2% of the regional area in whole Qinghai-Tibet Plateau. The habitat of Chinese cordyceps in Yushu Tibetan Autonomous Prefecture and Golog Tibetan Autonomous Prefecture covers 88.3%-99.6% of the regional area of the two Prefectures, while the habitat in Tibetan Autonomous Prefecture of Haibei, Haidong City, Autonomous Prefecture of Hainan, Haixi Mongolian and Tibetan Autonomous Prefecture, Tibetan Autonomous Prefecture of Huangnan and Xining covers 30.2%-96.5% of the regional area of these regions. The comprehensive zoning map can reflect the spatial distribution pattern of Chinese cordyceps resources in Qinghai Province, and the zoning results are consistent with the field survey results, indicating that it is feasible to use the spatial analysis method to delineate the habitable areas of Chinese cordyceps.
DnaJ, namely heat shock protein 40, plays an important role in regulating growth and development and resisting adversity of organisms. CgDnaJ05 cloned from Cordyceps guangdongensis strain GDGM30035 has coding sequence of 2 361 bp, encoding a protein of 786 amino acids. Bio-informatics analysis showed that CgDnaJ05 protein contained J domain and was basic, unstable and hydrophilic. 240 bp antisense fragment from the conserved domain of CgDnaJ05 protein was promoted dually, and used for constructing the vector of gene silence. The vector was transferred to C. guangdongesis mycelia via Agrobacterium tumefaciens-mediated transformation method, and 9 relatively stable CgDnaJ05-silenced transformants were obtained. The results of qRT-PCR showed that relative expression levels of CgDnaJ05 in transfortmants decreased to 20%-82% of that in wild-type strain. Compared with the wild-type strain, the mycelial growth rate of CgDnaJ05-silenced strains significantly decreased, and the more significantly CgDnaJ05 expression was down-regulated, the more obviously mycelial growth rate slowed down. Furthermore, gene silence of CgDnaJ05 markedly inhibited the primordial formation. The results indicate that CgDnaJ05 obviously affects mycelial growth and primordial formation of C. guangdongensis, and shows an important role in regulating growth and development of the cordyceps.
Cordyceps herbal samples were extracted by ultrasonic extraction with 85% ethanol for obtaining free sterols, and extracted by reflux extraction with 0.5 mol/L potassium hydroxide-ethanol solution for total sterols. The sample solution was purified by solid phase extraction with green solvent (ethanol and water). Determination of three kinds of sterols (ergosterol, cholesterol and sitosterol) separated from the samples was carried out by HPLC-ELSD, and performed on a Poroshell 120 EC-C18 column (100 mm×4.6 mm, 2.7 μm) with green mobile phase (ethanol:water=89:11) at flow rate of 0.5 mL/min. The results showed that the developed analytical method had a good linear relation, precision and accuracy. All the 20 batches of cordyceps samples contain free and conjugated sterols. All the samples of cultivated and wild Ophiocordyceps sinensis contain three kinds of sterols of similar content. Cordyceps liangshanensis (wild), C. militaris (cultivated) and C. chanhua (wild) contain ergosterol. This method can be used to determine sterol content of cordyceps samples for improving the quality evaluation of cordyceps products.
Cordyceps fungi are widely distributed throughout the world, with a variety of biological activities and great potential for use as food and medicine. In order to enrich and develop natural resources of cordyceps fungi, a wild Cordyceps fruiting body was sampled from Danxia Mountain, Shaoguan, Guangdong, China. The fungal isolate ZYJ0835 obtained from the natural fruiting body was employed to evaluate its biological activities and probability of artificial cultivation. Based on morphology and multi-gene phylogenetic analyses, ZYJ0835 was identified as Cordyceps blackwelliae. Five different media were used for liquid fermentation, and it was found that fermentation supernatant and mycelia of the strain showed both antioxidant and fibrinolytic activities. In addition, the strain also showed antimicrobial activities to some extent. Fruiting bodies were successfully induced on solid media consisted of rice, wheat, or tussah pupae, and the artificially-cultivated fruiting bodies also displayed antioxidant and fibrinolytic activities. This study reports, for the first time, the distribution of C. blackwelliae in China, and development and utilization of this cordyceps resource can be expected.
Strain degeneration occurs often in Cordyceps militaris during subculture and long-term storage at low temperature, resulting in a decrease in yield and a great loss in income. In this study, normal strains and degenerated strains due to long-term storage at 4 °C PDA slant and continuous subculture of C. militaris were observed to survey the morphological variation of fruiting bodies, colonies and mycelia and sporulation capacity of conidia. The nuclei, mitochondria, reactive oxygen species accumulation in cell, and lipid droplets in blastospore were observed by staining method, and the content of cordycepin, adenosine and ergosterol in mycelia was analyzed. The results showed that most of the phenotypical variation of degenerated strains caused by long-term storage at 4 °C PDA slant and continuous subculture were consistently displayed as: the decrease of fruiting body yield, the presence of adhesive and knotted hyphae, significant decrease of conidia, increased level of reactive oxygen species, reduction of mitochondria in the mycelia, mergence of dispersed and small lipid droplets in the blastospores into large droplets. There is no color change after being exposed to light for the colony of degenerated strains caused by long-term storage at 4 °C, whereas color change is unstable during the continuous subculture. The number of nuclei in the mycelia of degenerate strains undergoing continuous subculture is reduced whereas no change in that undergoing long-term storage at 4 °C. The content of cordycepin, adenosine and ergosterol in the mycelia of degenerated strains of long-term preservation was reduced by 58%, 41% and 70% as compared with that of the normal strains, but the ergosterol content of the degenerated strains of continuous subculture was unchanging. Microscopic observation of adhesive and knotted mycelia, and staining the mycelia by nitrogen blue tetrazolium (NBT) to detect the reactive oxygen species are easy to operate and not time-consuming. This method can be used to detect the degeneration of C. militaris strains on a large scale. Mitochondrial and lipid droplet staining can also be used to identify the degenerated strains. The commonly used method of judging the quality of spawn by color conversion after light irradiation needs to be cautious. Obvious degeneration of the strains usually appears in fourth generation during subculture, so the strains used for cultivation should be selected within three generations.
Illumina MiSeq high-throughput sequencing techniques were used to analyze the soil fungal community of four vegetation restoration types (plantations of Robinia pseudoacacia, Pinus tabulaeformis, Hippophae rhamnoides and Caragana korshinskii) in the ecological reclamation area of the Antaibao Open-pit Coal Mine, and correlation between fungal communities and soil environmental factors was explored. In total, 821 508 valid sequences and 1 841 OTUs were obtained by high-throughput sequencing. Ascomycota and Basidiomycota were the dominant phyla, accounting for 88.44% of total numbers of fungal sequences, and 3.72% of the sequences were unidentified. The results of α diversity analysis showed that the Sobs index, Chao1 index, Ace index and Shannon index of soil fungi in R. pseudoacacia, H. rhamnoides and C. korshinskii plantations were significantly higher than those in P. tabulaeformis plantation (P<0.01), while the Simpson index of soil fungi in P. tabulaeformis plantation was significantly higher as compared with that in the other three vegetation restoration types (P<0.05). The results of β diversity analysis showed that the composition of the soil fungal communities of the four plantation types was significantly different (R=0.709 9, P=0.005). The soil fungal community in the P. tabulaeformis plantation type was far different from that in the other three types of plantations, with significant difference of P<0.01, while that in plantations of the other three vegetation restoration types were similar to each other and not significantly different (P<0.05). Determination of soil physical and chemical properties, canonical correspondence analysis (CCA), redundancy analysis (RDA), and Spearman correlation analysis showed that soil organic matter, total nitrogen and available phosphorus were the main environmental factors that significantly affected the composition of the soil fungal community in the plantations of four vegetation restoration types. In summary, R. pseudoacacia vegetation restoration type displayed the highest species abundance of soil fungi in the process of ecological reclamation in the mining area, and P. tabulaeformis vegetation restoration type showed the richest dominant communities. The research results provide a reference for the development and utilization of soil fungal resources in the coal mine reclamation area for vegetation restoration.
From 2018 to 2021, 66 samples of radish (Raphanus sativus) with visible symptoms of root internal discoloration were collected from Xining, Qinghai Province and its surrounding areas. Eighty strains of Verticillium were isolated and purified by conventional plate separation method, numbered VL1-VL80. The strains grew slowly on potato dextrose agar, and could produce microsclerotia and verticillate-arranged conidiophores bearing conidia. Microsclerotia are elongated and irregularly-shaped, 53.91-164.42×8.91-29.81 μm in size and the average ratio of length to width is greater than 2. Conidia are hyaline and elongate, 5.35-9.31×2.12-4.78 μm. Chlamydospores and dormant hyphae were not observed. Direct sequencing of PCR products using the primers ITS1/ITS4, ActFa1/VactR, and ActF2d1/VactR were conducted to identify these isolates, then four isolates were chosen for direct sequencing of four protein coding genes EF, GPD, OX and TS. Phylogenetic analyses based on the ITS region indicated that these strains were located in the same branch of Verticillium longisporum clade. Phylogenetic analyses using five protein coding genes showed the four isolates belonged to the V. longisporum lineage A1/D1. The pathogenicity test showed that the strains could infect radish and cause the symptoms of root internal discoloration, indicating the causal agent of radish root internal discoloration in Qinghai, China was V. longisporum lineage A1/D1. To our knowledge, this is the first report of V. longisporum lineage A1/D1 in China.
Beauveria bassiana is a broad spectrum insecticidal fungus. In order to determine the ability of transcription factor BbMSN2 to recognize the promoter binding sites, the BbMSN2 protein was expressed and purified. Three nucleic acid probes with different number of binding site sequences (AGGGG/CCCCT) and six nucleic acid probes with point mutations were synthesized. The protein of BbMSN2 was used to combined with the nucleic acid probes in electrophoretic mobility shift assay. The result showed that BbMSN2 protein could interact with the nucleic acid probes containing the binding sites, but the interaction with point mutation probe was significantly weakened. Moreover, the synergistic effect of the binding sites was not significant. These data indicate that the transcription factor BbMSN2 specifically interact with the nucleic acid probe containing the binding sites. Our study provides foundation for further exploring the transcriptional regulation mechanism of BbMSN2.
The witches’ broom disease widespread in Yucheng District of Ya’an city in Sichuan Province has caused serious harm to Phyllostachys violascens. To clarify the occurring rules of this disease and biological character of the pathogen, an investigation was conducted in six bamboo forests in Ya’an city, and diseased samples were collected. The pathogen was identified as Aciculosporium take on the basis of combining morphology observation and multi-gene phylogenetic analyses of a combined seven-gene dataset (ITS, LSU, SSU, tef1-α, rpb2, mcm7 and tub2). The culture experiments under different conditions revealed that the optimum medium for growth and sporulation of the pathogen was PDA at pH value of 7.5. Carbon source was glucose, and nitrogen source was beef extract and yeast extract, and 24-hour light was optimal for growth. Fungicide plate tests showed that 10% difenoconazole (WG) gained the best control effect (EC50=0.019 μg/mL). In the field test, the control efficiency based on disease index was 96.57%, adopting difenoconazole (WG) with a concentration of 400 μg/mL.
Based on the genome sequence of Lentinula edodes strain L808, 300 insertion/deletion (InDel) markers were developed. Through the preliminary screening of five strains, 82 polymorphic markers evenly distributed on the genome were selected to analyze the genetic background of 42 L. edodes germplasm resources. Under the condition of facility cultivation, the phenotypes of germplasm resources such as growth period, colouring of log, hardness of log, budding stage and yield were investigated. The results showed that the genetic diversity level of L. edodes germplasm resources was high. The genetic distance between cultivated strains and wild strains was far, and the average value of genetic similarity coefficient was 0.51. Population structure analysis divided the germplasm resources into six subpopulations, which was consistent with the result of systematic clustering based on genetic distance. The average genetic differentiation index was 0.290, indicating the selected germplasm resources were suitable for cross breeding. The phenotypic results showed that the agronomic characters varied among strains under facility cultivation, and growth period, budding stage and yield of strains among subpopulations were significantly different. The results of diversity analysis provide a basis for the cross breeding of L. edodes.
