Characterization of Rhizosphere and Endophytic Microbial Communities Associated with Stipa purpurea and Their Correlation with Soil Environmental Factors.

This study was to explore the diversity of rhizosphere and endophytic microbial communities and the correlation with soil environmental factors of Stipa purpurea on the Qinghai-Tibetan Plateau. The bacterial phylum of Proteobacteria, Firmicutes and Bacteroidota, and the fungal phylum of Ascomycota, Basidiomycota and Zygomycota were dominant in microbial communities of S. purpurea in all three sampling sites. Multiple comparison analysis showed that there were significant differences in the composition of microbial communities in the roots, leaves and rhizosphere soil. Whether it is fungi or bacteria, the OTU abundance of rhizosphere soils was higher than that of leaves and roots at the same location, while the difference among locations was not obvious. Moreover, RDA analysis showed that Zygomycota, Cercozoa, Glomeromycota, Chytridiomycota and Rozellomycota possessed strongly positive associations with altitude, dehydrogenase, alkaline phosphatase, neutral phosphatase, available kalium and available phosphate, while Ascomycota was strongly negatively associated. Changes in ammonium nitrate, alkaline phosphatase, polyphenol oxidase, total phosphorus, and altitude had a significant impact on the bacterial communities in different habitats and altitudes. Taken together, we provide evidence that S. purpurea has abundant microbial communities in the alpine grassland of the Qinghai-Tibetan Plateau, whose composition and diversity are affected by various soil environmental factors.

Effects of Allelochemicals, Soil Enzyme Activities, and Environmental Factors on Rhizosphere Soil Microbial Community of Stellera chamaejasme L. along a Growth-Coverage Gradient.

Allelochemicals released from the root of Stellera chamaejasme L. into rhizosphere soil are an important factor for its invasion of natural grasslands. The aim of this study is to explore the interactions among allelochemicals, soil physicochemical properties, soil enzyme activities, and the rhizosphere soil microbial communities of S. chamaejasme along a growth-coverage gradient. High-throughput sequencing was used to determine the microbial composition of the rhizosphere soil sample, and high-performance liquid chromatography was used to detect allelopathic substances. The main fungal phyla in the rhizosphere soil with a growth coverage of 0% was Basidiomycetes, and the other sample plots were Ascomycetes. Proteobacteria and Acidobacteria were the dominant bacterial phyla in all sites. RDA analysis showed that neochamaejasmin B, chamaechromone, and dihydrodaphnetin B were positively correlated with Ascomycota and Glomeromycota and negatively correlated with Basidiomycota. Neochamaejasmin B and chamaechromone were positively correlated with Proteobacteria and Actinobacteria and negatively correlated with Acidobacteria and Planctomycetes. Allelochemicals, soil physicochemical properties, and enzyme activity affected the composition and diversity of the rhizosphere soil microbial community to some extent. When the growth coverage of S. chamaejasme reached the primary stage, it had the greatest impact on soil physicochemical properties and enzyme activities.

Genome Assembly and Pathway Analysis of Edible Mushroom Agrocybe cylindracea.

Agrocybe cylindracea, an edible mushroom, is widely cultivated for its abundance of nutrients and flavor, and many of its metabolites are reported to have beneficial roles, such as medicinal effects on tumors and chronical illnesses. However, the lack of genomic information has hindered further molecular studies on this fungus. Here, we present a genome assembly of A. cylindracea together with comparative genomics and pathway analyses of Agaricales species. The draft, generated from both next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing platforms to overcome high genetic heterozygosity, is composed of a 56.5 Mb sequence and 15,384 predicted genes. This mushroom possesses a complex reproductive system, including tetrapolar heterothallic and secondary homothallic mechanisms, and harbors several hydrolases and peptidases for gradual and effective degradation of various carbon sources. Our pathway analysis reveals complex processes involved in the biosynthesis of polysaccharides and other active substances, including B vitamins, unsaturated fatty acids, and N-acetylglucosamine. RNA-seq data show that A. cylindracea stipes tend to synthesize carbohydrate for carbon sequestration and energy storage, whereas pilei are more active in carbon utilization and unsaturated fatty acid biosynthesis. These results reflect diverse functions of the two anatomical structures of the fruiting body. Our comprehensive genomic and transcriptomic data, as well as preliminary comparative analyses, provide insights into the molecular details of the medicinal effects in terms of active compounds and nutrient components.

Neutral Lipid Content in Lipid Droplets: Potential Biomarker of Cordycepin Accumulation in Cordycepin-Producing Fungi.

To clarify the relationship between neutral lipid content and cordycepin accumulation in Cordyceps militaris, mutants were generated from mixed spores of two C. militaris strains with varying cordycepin-producing capacities. Fifteen stable mutants producing from 0.001 to 2.363 mg/mL cordycepin were finally selected. The relative fluorescence intensities of the 15 mutants, two C. militaris strains and an Aspergillus nidulans strain at different concentrations of lyophilized mycelium powder were then investigated using the Nile red method. The mutant CM1-1-1 with the highest relative fluorescence intensity among the eighteen strains was selected for optimizing the Nile red method. Relative fluorescence intensity was linearly correlated with cordycepin concentration in liquid broth (R2 = 0.9514) and in lyophilized mycelium powder (R2 = 0.9378) for the 18 cordycepin-producing strains under identical culture conditions and with cordycepin concentration in liquid broth (R2 = 0.9727) and in lyophilized mycelium powder (R2 = 0.9613) for CM1-1-1 under eight different sets of conditions. In addition, the cordycepin content in lyophilized mycelium powder measured by the Nile red method was linearly correlated with that determined by an HPLC method (R2 = 0.9627). In conclusion, neutral lipids in lipid droplets are required during cordycepin accumulation; these neutral lipids are potential biomarkers of cordycepin biosynthesis.

Therapeutic Potential and Biological Applications of Cordycepin and Metabolic Mechanisms in Cordycepin-Producing Fungi.

Cordycepin(3'-deoxyadenosine), a cytotoxic nucleoside analogue found in Cordyceps militaris, has attracted much attention due to its therapeutic potential and biological value. Cordycepin interacts with multiple medicinal targets associated with cancer, tumor, inflammation, oxidant, polyadenylation of mRNA, etc. The investigation of the medicinal drug actions supports the discovery of novel targets and the development of new drugs to enhance the therapeutic potency and reduce toxicity. Cordycepin may be of great value owing to its medicinal potential as an external drug, such as in cosmeceutical, traumatic, antalgic and muscle strain applications. In addition, the biological application of cordycepin, for example, as a ligand, has been used to uncover molecular structures. Notably, studies that investigated the metabolic mechanisms of cordycepin-producing fungi have yielded significant information related to the biosynthesis of high levels of cordycepin. Here, we summarized the medicinal targets, biological applications, cytotoxicity, delivery carriers, stability, and pros/cons of cordycepin in clinical applications, as well as described the metabolic mechanisms of cordycepin in cordycepin-producing fungi. We posit that new approaches, including single-cell analysis, have the potential to enhance medicinal potency and unravel all facets of metabolic mechanisms of cordycepin in Cordyceps militaris.

Cycloartane-type triterpenoids from Astragalus hoantchy French.

A new cycloartane-type triterpenoid, 3ß-acetoxy-9ß,19-cyclolanost-24E-ene-1α,16ß-diol-27-O-ß-d-glucopyranoside (1), together with three known ones (2-4) were isolated from the aerial parts of Astragalus hoantchy. The structure of compound 1 was elucidated by detailed spectroscopic analyses including 1D and 2D NMR, HRESIMS, and IR experiments. All of the four compounds were isolated from A. hoantchy for the first time. The antifungal activities of compounds 1-4 against a phytopathogenic fungus (Alternaria solani) were evaluated with the result that compound 3 exhibited a moderate activity with an IC50 value of 284 µg/mL.

Characterization of rhizosphere and endophytic fungal communities from roots of Stipa purpurea in alpine steppe around Qinghai Lake.

Stipa purpurea is among constructive endemic species in the alpine steppe on the Qinghai-Xizang Plateau. To reveal the fungal community structure and diversity in the rhizosphere and roots of this important grass and to analyze the potential influence of different habitats on the structure of fungal communities, we explored the root endophyte and the directly associated rhizosphere communities of S. purpurea by using internal transcribed spacer rRNA cloning and sequencing methods. We found that the roots of S. purpurea are associated with a diverse consortium of Basidiomycota (59.8%) and Ascomycota (38.5%). Most fungi obtained from rhizosphere soil in S. purpurea have been identified as Ascomycetes, while the high proportion detected in roots were basidiomycetous endophytes. The species richness, diversity, and evenness of fungal assemblages were higher in roots than in the rhizosphere soil. Fungi inhabiting the rhizosphere and roots of S. purpurea are significantly different, and the rhizosphere and endophyte communities are largely independent with little overlap in the dominant phyla or operational taxonomic units. Taken together, these results suggested that a wide variety of fungal communities are associated with the roots and rhizosphere soil of S. purpurea and that the fungal assemblages are strongly influenced by different habitats.

The complete mitochondrial genome of Hequ Tibetan Mastiff Canis lupus familiaris (Carnivora: Canidae).

The Hequ Tibetan Mastiff Canis lupus familiaris (Carnivora: Canidae) is a primitive breed of large dogs native to the northeastern Qinghai-Tibetan Plateau of China. In this study, its complete mitochondrial genome sequence has been assembled and characterized using high-throughput Illumina sequencing technology. The circular genome is 16 730 bp in length, and possesses all genomic components as typically found in most other metazoan mitogenomes. The gene arrangement is identical to those of most other vertebrates. Except for ND4L with GTG as its start codon, all the other PCGs are initiated with an ATR (ATA/ATG) codon. Three distinct stop codons are employed, i.e. AGA for CYTB, TAA for ATP6, ATP8, COX1, COX2, ND1, ND4L, ND5 and ND6, and an incomplete stop codon T for COX3, ND2, ND3 and ND4. The nucleotide composition is asymmetric (31.6% A, 25.5% C, 14.2% G and 28.7% T) with an overall A + T content of 60.3%. These data would contribute to our better understanding its evolutionary history.

Phylogenic diversity and tissue specificity of fungal endophytes associated with the pharmaceutical plant, Stellera chamaejasme L. revealed by a cultivation-independent approach.

The fungal endophytes associated with medicinal plants have been demonstrated as a reservoir with novel natural products useful in medicine and agriculture. It is desirable to explore the species composition, diversity and tissue specificity of endophytic fungi that inhabit in different tissues of medicinal plants. In this study, a culture-independent survey of fungal diversity in the rhizosphere, leaves, stems and roots of a toxic medicinal plant, Stellera chamaejasme L., was conducted by sequence analysis of clone libraries of the partial internal transcribed spacer region. Altogether, 145 fungal OTUs (operational taxonomic units), represented by 464 sequences, were found in four samples, of these 109 OTUs (75.2 %) belonging to Ascomycota, 20 (13.8 %) to Basidiomycota, 14 (9.7 %) to Zygomycota, 1 (0.7 %) to Chytridiomycota, and 1 (0.7 %) to Glomeromycota. The richness and diversity of fungal communities were strongly influenced by plant tissue environments, and the roots are associated with a surprisingly rich endophyte community. The endophyte assemblages associated with S. chamaejasme were strongly shaped by plant tissue environments, and exhibited a certain degree of tissue specificity. Our results suggested that a wide variety of fungal assemblages inhabit in S. chamaejasme, and plant tissue environments conspicuously influence endophyte community structure.

Isolation and identification of chemical constituents from the bacterium Bacillus sp. and their nematicidal activities.

A strain SMrs28 was isolated from the rhizosphere soil of a toxic plant Stellera chamaejasme and identified as Bacillus sp. on the basis of morphological and partial 16S rRNA gene sequence analysis. The crude extract of SMrs28 fermentation broth showed strong nematocidal activities in preliminary test. To define the active nematocidal metabolites of SMrs28, a novel compound (1), 4-oxabicyclo[3.2.2]nona-1(7), 5,8-triene, along with five known compounds (2-6), were isolated from the strain by various column chromatographic techniques and characterized on the basis of spectroscopic analysis. Results of the in vitro nematicidal tests showed that the metabolites presented different levels of activity at certain exposure conditions. Compounds (1-3) displayed LC50 values of 904.12, 451.26, 232.98 µg/ml and 1594.0, 366.62, 206.38 µg/ml against Bursaphelenchus xylophilus and Ditylenchus destructor at 72 h, respectively. This is the first report of the nematicidal activity of the compounds as constituents of Bacillus sp.. Our findings help to find potential chemical structures to develop nematicides from microbial source for the management of nematode-infected plant diseases.

Isolation and characterization of bacteria from the rhizosphere and bulk soil of Stellera chamaejasme L.

This study is the first to describe the composition and characteristics of culturable bacterial isolates from the rhizosphere and bulk soil of the medicinal plant Stellera chamaejasme L. at different growth stages. Using a cultivation-dependent approach, a total of 148 isolates showing different phenotypic properties were obtained from the rhizosphere and bulk soil. Firmicutes and Actinobacteria were the major bacterial groups in both the rhizosphere and bulk soil at all 4 growth stages of S. chamaejasme. The diversity of the bacterial community in the rhizosphere was higher than that in bulk soil in flowering and fruiting stages. The abundance of bacterial communities in the rhizosphere changed with the growth stages and had a major shift at the fruiting stage. Dynamic changes of bacterial abundance and many bacterial groups in the rhizosphere were similar to those in bulk soil. Furthermore, most bacterial isolates exhibited single or multiple biochemical activities associated with S. chamaejasme growth, which revealed that bacteria with multiple physiological functions were abundant and widespread in the rhizosphere and bulk soil. These results are essential (i) for understanding the ecological roles of bacteria in the rhizosphere and bulk soil and (ii) as a foundation for further evaluating their efficacy as effective S. chamaejasme growth-promoting rhizobacteria.

Allelochemicals in the rhizosphere soil of Euphorbia himalayensis.

Weed infestation has been known to cause considerable reductions in crop yields, thereby hindering sustainable agriculture. Many plants in genus Euphorbia affect neighboring plants and other organisms by releasing chemicals into the environment. In view of the serious threat of weeds to agriculture, the allelochemicals of Euphorbia himalayensis and their allelopathic effects were investigated. The extract of root exudates from rhizosphere soil exhibited allelopathic activities against crops (wheat, rape, and lettuce) and grasses (Poa annua, Festuca rubra, and red clover). Bioassay-guided fractionation and isolation from the root extract of E. himalayensis led to the characterization of two ellagic acid derivatives and a jatrophane diterpene, which observably showed phytotoxic activities against lettuce, Festuca arundinacea, and F. rubra. They were further confirmed by ultra-performance liquid chromatography-tandem mass spectrometry to have concentrations of 3.6, 3.8, and 8.99 nmol/g in the rhizospere soil, respectively. Bioassay indicated that the combination of the allelochemicals could be selective plant growth regulator in agriculture.