Decoding the RNA virome of the tree parasite Armillaria provides new insights into the viral community of soil-borne fungi.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

Composition and diversity of soil microbial communities change by introducing Phallus impudicus into a Gastrodia elata Bl.-based soil.

BACKGROUND: The Gastrodia elata Bl. is an orchid, and its growth demands the presence of Armillaria species. The strong competitiveness of Armillaria species has always been a concern of major threat to other soil organisms, thus disrupting the equilibrium of soil biodiversity. Introducing other species to where G. elata was cultivated, could possibly alleviate the problems associated with the disequilibrium of soil microenvironment; however, their impacts on the soil microbial communities and the underlying mechanisms remain unclear. To reveal the changes of microbial groups associated with soil chemical properties responding to different cultivation species, the chemical property measurements coupled with the next-generation pyrosequencing analyses were applied with soil samples collected from fallow land, cultivation of G. elata and Phallus impudicus, respectively. RESULTS: The cultivation of G. elata induced significant increases (p < 0.05) in soil pH and NO3-N content compared with fallow land, whereas subsequent cultivation of P. impudicus reversed these G. elata-induced increases and was also found to significantly increase (p < 0.05) the content of soil NH4+-N and AP. The alpha diversities of soil microbial communities were significantly increased (p < 0.01) by cultivation of G. elata and P. impudicus as indicated with Chao1 estimator and Shannon index. The structure and composition of soil microbial communities differed responding to different cultivation species. In particular, the relative abundances of Bacillus, norank_o_Gaiellales, Mortierella and unclassified_k_Fungi were significantly increased (p < 0.05), while the abundances of potentially beneficial genera such as Acidibacter, Acidothermus, Cryptococcus, and Penicillium etc., were significantly decreased (p < 0.05) by cultivation of G. elata. It's interesting to find that cultivation of P. impudicus increased the abundances of these genera that G. elata decreased before, which contributed to the difference of composition and structure. The results of CCA and heatmap indicated that the changes of soil microbial communities had strong correlations with soil nutrients. Specifically, among 28 genera presented, 50% and 42.9% demonstrated significant correlations with soil pH and NO3-N in response to cultivation of G. elata and P. impudicus. CONCLUSIONS: Our findings suggested that the cultivation of P. impudicus might have potential benefits as result of affecting soil microorganisms coupled with changes in soil nutrient profile.

Full-length genome characterization of a novel mitovirus isolated from the root rot fungus Armillaria mellea.

Members of the genus Armillaria belong to the group of pathogenic and facultative saprotrophic fungi that are generally known as one of the causative agents of white root rot in infected plants including deciduous and evergreen trees and shrubs. Although several single-stranded RNA mycoviruses were previously described in different Armillaria species, there is no report on mitoviruses (one of the simplest RNA viruses of fungal hosts) known to infect Armillaria taxa. In this study, a new mitovirus denominated "Armillaria mellea mitovirus 1" (AmMV1) was identified in the sporophore samples of Armillaria mellea, commonly known as honey mushroom. AmMV1 has a genome length of 4440 nucleotides and a G + C content of 48%. It encompasses a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). Comparison through BLASTp analysis revealed that the RdRp domain of AmMV1 shares a sequence identity ranging from 33.43% to 43.27% with RdRp domains of Duamitovirus genus members, having the highest similarity (43.27%) to Rhizoctonia solani mitovirus 94. According to phylogenetic analysis, AmMV1 is classified as a member of the genus Duamitovirus belonging to the Mitoviridae family. This marks the initial instance of a mitovirus identified in Armillaria spp..

Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

Comparison of the Secondary Metabolism of the Basidiomycetes Armillaria mellea and Desarmillaria ectypa.

During the course of our ongoing studies on the secondary metabolism of cultures of Basidiomycota, a new meroterpenoid named 10, 15-dihydroxydihydromelleolide (1) was isolated along with the known armillaridin (2) and arnamiol (3) from cultures of the rare saprotrophic species, Desarmillaria ectypa. These are the first secondary metabolites that were ever isolated from the latter species. A concurrently studied strain of the common pathogenic A. mellea yielded other melleolides, with 5'-O-methylmelledonal (4), melledonal C (5), 10 α-hydroxydihydromelleolide (6) and melledonal (7). The chemical structures were elucidated using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). All compounds were studied for their antimicrobial and cytotoxic effects against a panel of microbes and mammalian cell lines, and the results are also reported.

Network Pharmacology Analysis of Liquid-Cultured Armillaria ostoyae Mycelial Metabolites and Their Molecular Mechanism of Action against Gastric Cancer.

Armillaria sp. are traditional edible medicinal mushrooms with various health functions; however, the relationship between their composition and efficacy has not yet been determined. Here, the ethanol extract of liquid-cultured Armillaria ostoyae mycelia (AOME), a pure wild Armillaria sp. strain, was analyzed using UHPLC-QTOF/MS, network pharmacology, and molecular docking techniques. The obtained extract affects various metabolic pathways, such as JAK/STAT and PI3K/AKT. The extract also contains important compounds such as 4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide, isoliquiritigenin, and 7-hydroxycoumarin. Moreover, the extract targets key proteins, including EGFR, SCR, and IL6, to suppress the progression of gastric cancer, thereby synergistically inhibiting cancer development. The molecular docking analyses indicated that the main compounds stably bind to the target proteins. The final cell culture experimental data showed that the ethanol extract inhibited MGC-803 gastric cancer cells. In summary, our research revealed the beneficial components of AOME for treating gastric cancer and its associated molecular pathways. However, further research is needed to confirm its effectiveness and safety in gastric cancer patients.

[Effects of soil relative water content on growth characteristics of Armillaria spp. and seedling production of Gastrodia elata f. glauca].

The seedling survival rate, yield, and individual weight of Gastrodia elata is closely related to the soil relative water content(RWC) and the growth characteristics of the associated fungi Armillaria spp. This study explored the effects of the soil RWC on the growth characteristics of Armillaria spp. and the seedling production of G. elata f. glauca, aiming to provide guidance for breeding G. elata f. glauca and selecting elite strains of Armillaria. According to the growth characteristics on the medium for activation, thirty strains of Armillaria were classified into 4 clusters. Two strains with good growth indicators were selected from each cluster and cultiva-ted with immature tuber(Mima) and the branches of the broad-leaved trees in a water-controlled box. The results showed that the Armillaria clusters with uniaxial branches of rhizoid cords, such as clusters Ⅲ and Ⅳ, were excellent clusters in symbiosis with G. elata f. glauca. The soil RWC had significant effects on the growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca. The growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca in the case of the soil RWC being 75% were significantly better than those in the case of other soil RWC. Cultivating Mima with elite strains of Armillaria, together with branches of broad-leaved trees, in the greenhouses with the artificial control of the soil RWC, can achieve efficient seedling production and Mima utilization of G. elata f. glauca.

Whole genome sequencing and analysis of Armillaria gallica Jzi34 symbiotic with Gastrodia elata.

BACKGROUND: Armillaria species are plant pathogens, but a few Armillaria species can establish a symbiotic relationship with Gastrodia elata, a rootless and leafless orchid, that is used as a Chinese herbal medicine. Armillaria is a nutrient source for the growth of G. elata. However, there are few reports on the molecular mechanism of symbiosis between Armillaria species and G. elata. The genome sequencing and analysis of Armillaria symbiotic with G. elata would provide genomic information for further studying the molecular mechanism of symbiosis. RESULTS: The de novo genome assembly was performed with the PacBio Sequel platform and Illumina NovaSeq PE150 for the A. gallica Jzi34 strain, which was symbiotic with G. elata. Its genome assembly contained ~ 79.9 Mbp and consisted of 60 contigs with an N50 of 2,535,910 bp. There were only 4.1% repetitive sequences in the genome assembly. Functional annotation analysis revealed a total of 16,280 protein coding genes. Compared with the other five genomes of Armillaria, the carbohydrate enzyme gene family of the genome was significantly contracted, while it had the largest set of glycosyl transferase (GT) genes. It also had an expansion of auxiliary activity enzymes AA3-2 gene subfamily and cytochrome P450 genes. The synteny analysis result of P450 genes reveals that the evolutionary relationship of P450 proteins between A. gallica Jzi34 and other four Armillaria was complex. CONCLUSIONS: These characteristics may be beneficial for establishing a symbiotic relationship with G. elata. These results explore the characteristics of A. gallica Jzi34 from a genomic perspective and provide an important genomic resource for further detailed study of Armillaria. This will help to further study the symbiotic mechanism between A. gallica and G. elata.

Nonribosomal peptide synthetase gene clusters and characteristics of predicted NRPS-dependent siderophore synthetases in Armillaria and other species in the Physalacriaceae.

Fungal secondary metabolites are often pathogenicity or virulence factors synthesized by genes contained in secondary metabolite gene clusters (SMGCs). Nonribosomal polypeptide synthetase (NRPS) clusters are SMGCs which produce peptides such as siderophores, the high affinity ferric iron chelating compounds required for iron uptake under aerobic conditions. Armillaria spp. are mostly facultative necrotrophs of woody plants. NRPS-dependent siderophore synthetase (NDSS) clusters of Armillaria spp. and selected Physalacriaceae were investigated using a comparative genomics approach. Siderophore biosynthesis by strains of selected Armillaria spp. was evaluated using CAS and split-CAS assays. At least one NRPS cluster and other clusters were detected in the genomes studied. No correlation was observed between the number and types of SMGCs and reported pathogenicity of the species studied. The genomes contained one NDSS cluster each. All NDSSs were multi-modular with the domain architecture (ATC)3(TC)2. NDSS clusters of the Armillaria spp. showed a high degree of microsynteny. In the genomes of Desarmillaria spp. and Guyanagaster necrorhizus, NDSS clusters were more syntenic with NDSS clusters of Armillaria spp. than to those of the other Physalacriaceae species studied. Three A-domain orthologous groups were identified in the NDSSs, and atypical Stachelhaus codes were predicted for the A3 orthologous group. In vitro biosynthesis of mainly hydroxamate and some catecholate siderophores was observed. Hence, Armillaria spp. generally contain one highly conserved, NDSS cluster although some interspecific variations in the products of these clusters is expected. Results from this study lays the groundwork for future studies to elucidate the molecular biology of fungal phyto-pathogenicity.

Characterization of the transcriptional responses of Armillaria gallica 012m to GA3.

Gastrodia elata needs to establish a symbiotic relationship with Armillaria strains to obtain nutrients and energy. However, the signaling cross talk between G. elata and Armillaria strains is still unclear. During our experiment, we found that the vegetative mycelium of Armillaria gallica 012m grew significantly better in the media containing gibberellic acid (GA3) than the blank control group (BK). To explore the response mechanism, we performed an RNA-sequencing experiment to profile the transcriptome changes of A. gallica 012m cultured in the medium with exogenous GA3. The transcriptome-guided differential expression genes (DEGs) analysis of GA3 and BK showed that a total of 1309 genes were differentially expressed, including 361 upregulated genes and 948 downregulated genes. Some of those DEGs correlated with the biological process, including positive regulation of chromosome segregation, mitotic metaphase/anaphase transition, attachment of mitotic spindle microtubules to kinetochore, mitotic cytokinesis, and nuclear division. These analyses explained that GA3 actively promoted the growth of A. gallica to some extent. Further analysis of protein domain features showed that the deduced polypeptide contained 41 candidate genes of GA receptor, and 27 of them were expressed in our samples. We speculate that GA receptors exist in A. gallica 012m. Comparative studies of proteins showed that the postulated GA receptor domains of A. gallica 012m have a higher homologous correlation with fungi than others based on cluster analysis.

Genomic Comparisons of Two Armillaria Species with Different Ecological Behaviors and Their Associated Soil Microbial Communities.

Armillaria species show considerable variation in ecological roles and virulence, from mycorrhizae and saprophytes to important root pathogens of trees and horticultural crops. We studied two Armillaria species that can be found in coniferous forests of northwestern USA and southwestern Canada. Armillaria altimontana not only is considered as a weak, opportunistic pathogen of coniferous trees, but it also appears to exhibit in situ biological control against A. solidipes, formerly North American A. ostoyae, which is considered a virulent pathogen of coniferous trees. Here, we describe their genome assemblies and present a functional annotation of the predicted genes and proteins for the two Armillaria species that exhibit contrasting ecological roles. In addition, the soil microbial communities were examined in association with the two Armillaria species within a 45-year-old plantation of western white pine (Pinus monticola) in northern Idaho, USA, where A. altimontana was associated with improved tree growth and survival, while A. solidipes was associated with reduced growth and survival. The results from this study reveal a high similarity between the genomes of the beneficial/non-pathogenic A. altimontana and pathogenic A. solidipes; however, many relatively small differences in gene content were identified that could contribute to differences in ecological lifestyles and interactions with woody hosts and soil microbial communities.

Transcriptome analysis of Armillaria gallica 012 m in response to auxin.

Gastrodia elata is an achlorophyllous and fully mycoheterotrophic orchid which obtains carbon and other nutrients from Armillaria species in its life cycle. Many researchers suggested that plant hormones, as signing molecules, play a central role in the plant-fungi interaction. In the process of Armillaria gallica 012 m cultivation, both exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) distinctly stimulated the growth of mycelia in solid media. The differential expression genes (DEGs) of A. gallica 012 m with IAA versus blank control (BK) and IBA versus BK were investigated. The results showed that more than 80% of DEGs of the IAA group were coincident with the DEGs of the IBA group, and more than half of upregulated DEGs and most of the downregulated DEGs of the IAA group coincided with those DEGs of the IBA group. Above research implied that A. gallica 012 m could perceive IAA and IBA, and possess similar responses and signaling pathways to IAA and IBA. The overlapping differential genes of the IAA group and IBA group were analyzed by GO term, and the results showed that several DEGs identified were related to biological processes including positive regulation of the biological process and biological process. The downregulated NmrA-like and FKBP_C genes might be benefit to the growth of mycelia. Those results can explain that exiguous IAA and IBA improved the growth of A. gallica to some extent. We speculate that IAA and IBA are signaling molecules, and regulate the expression of growth-related genes of A. gallica 012 m by the same signaling pathway.

UHPLC-MS-Based Metabolomics Reveal the Potential Mechanism of Armillaria mellea Acid Polysaccharide in and Its Effects on Cyclophosphamide-Induced Immunosuppressed Mice.

Armillaria mellea (Vahl) P. Kumm is commonly used for food and pharmaceutical supplements due to its immune regulatory function, and polysaccharides are one of its main components. The aim of this research is to study the immunological activity of the purified acidic polysaccharide fraction, namely, AMPA, isolated from Armillaria mellea crude polysaccharide (AMP). In this study, a combination of the immune activity of mouse macrophages in vitro and serum metabonomics in vivo was used to comprehensively explore the cell viability and metabolic changes in immune-deficient mice in the AMPA intervention, with the aim of elucidating the potential mechanisms of AMPA in the treatment of immunodeficiency. The in vitro experiments revealed that, compared with LPS-induced RAW264.7, the AMPA treatment elevated the levels of the cellular immune factors IL-2, IL-6, IgM, IgA, TNF-α, and IFN-γ; promoted the expression of immune proteins; and activated the TLR4/MyD88/NF-κB signaling pathway to produce immunological responses. The protein expression was also demonstrated in the spleen of the cyclophosphamide immunosuppressive model in vivo. The UHPLC-MS-based metabolomic analysis revealed that AMPA significantly modulated six endogenous metabolites in mice, with the associated metabolic pathways of AMPA for treating immunodeficiency selected as potential therapeutic biomarkers. The results demonstrate that phosphorylated acetyl CoA, glycolysis, and the TCA cycle were mainly activated to enhance immune factor expression and provide immune protection to the body. These experimental results are important for the development and application of AMPA as a valuable health food or drug that enhances immunity.

[Screening of an Armillaria gallica strain for Gastrodia elata cultivation].

This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.

An exploration of mechanism of high quality and yield of Gastrodia elata Bl. f. glauca by the isolation, identification and evaluation of Armillaria.

BACKGROUND: Gastrodia elata Bl. f. glauca, a perennial herb of G.elata Bl. in Orchidaceae, is one of the most valuable traditional Chinese medicines. G. elata Bl. is a chlorophyll-free myco-heterotrophic plant, which must rely on the symbiotic growth of Armillaria, but not all Armillaria strains can play the symbiotic role. Additionally, Armillaria is easy to degenerate after multiple generations, and the compatibility between the strains from other areas and G. elata Bl. f. glauca in Changbai Mountain is unstable. Therefore, it is incredibly significant to isolate, identify and screen the symbiotic Armillaria suitable for the growth of G. elata Bl. f. glauca in Changbai Mountain, and to explore the mechanism by which Armillaria improves the production performance of G. elata Bl. f. glauca. RESULTS: Firstly, G. elata Bl. f. glauca tubers, and rhizomorphs and fruiting bodies of Armillaria were used for the isolation and identification of Armillaria. Five Armillaria isolates were obtained in our laboratory and named: JMG, JMA, JMB, JMC and JMD. Secondly, Armillaria was selected based on the yield and the effective component content of G. elata Bl. f. glauca. It was concluded that the yield and quality of G. elata Bl. f. glauca co-planted with JMG is the highest. Finally, the mechanism of its high quality and yield was explored by investigating the effects of different Armillaria strains on the soil, its nutrition element contents and the soil microbial diversity around G. elata Bl. f. glauca in Changbai Mountain. CONCLUSIONS: Compared with commercial strains, JMG significantly increased the content of Na, Al, Si, Mn, Fe, Zn, Rb and the absorption of C, Na, Mg, Ca, Cr, Cu, Zn and Rb in G. elata Bl. f. glauca; it improved the composition, diversity and metabolic functions of soil microbial communities around G. elata Bl. f. glauca at phylum, class and genus levels; it markedly increased the relative abundance of bacteria such as Chthoniobacter and Armillaria in the dominant populations, and enhanced such functions as Cell motility, amino acid metabolism and Lipid metabolism; it dramatically decreased the relative abundance of Bryobacter and other fungi in the dominant populations, and reduced such functions as microbial energy metabolism, translation and carbohydrate metabolism. This is the main reason why excellent Armillaria strains promote the high quality and yield of G. elata Bl. f. glauca in Changbai Mountain.

Two new sesquiterpene aryl esters from Armillaria gallica 012m.

Two new sesquiterpene aryl esters, armimelleolides A and B (1 and 2), and four known ones, were isolated from the EtOAc extract of Armillaria gallica 012 m by column chromatography on silica gel, reversed-phase C18 silica gel and semi-preparative HPLC. Their structures were elucidated on the basis of spectroscopic methods, including extensive 1 D NMR, 2 D NMR and MS. All these compounds showed potential antitumor activities against at least one of the human cancer cell lines (A549, HCT-116, M231 and W256), with IC50 ranging from 2.57 to 19.94 µM.

Development of RT-PCR for rapid detection of ssRNA ambi-like mycovirus in a root rot fungi (Armillaria spp.).

Letter to the editor (No abstract) Keywords.

Molecular characterization of a ssRNA mycovirus isolated from the forest pathogenic fungus Armillaria ostoyae.

Letter to the editor (no abstract) Keywords.

Detrimental and essential elements in fruiting bodies of wild-growing fungi Coprinus comatus, Flammulina velutipes, and Armillaria ostoyae.

Edible medicinal wild-growing fungi Coprinus comatus, Flammulina velutipes, and Armillaria ostoyae were analyzed with regard to the contents of 21 elements in their fruiting bodies. The samples were collected from selected sites in South Bohemia, the Czech Republic. C. comatus concentrated Ag, Cd, Cu, Se, and Rb with bioconcentration factors of 12, 2.5, 2.3, 1.8, and 1.1, respectively. High contents (all values expressed in mg kg-1 dry matter) of Al (260), Ca (480), Cu (61), Fe (340), Mg (1400), and Zn (86) were determined for this species. F. velutipes was characterized with markedly high contents of Ca (360), Fe (110), Mg (1200), Mn (26), and Zn (98), respectively. A considerably high content of Ag (5.6) was revealed for A. ostoyae. High contents of Ca (150), Cu (28), Fe (190), Mg (1100), Mn (30), and Zn (40) were determined in fruiting bodies of this species as well. The data concerning the detrimental elements in fruiting bodies of studied fungi indicate no considerably negative effect on human health if they are consumed as a delicacy or used in alternative medicine.

[Analysis and evaluation of paddy-upland rotation of Gastrodia elata- Oryza sativa in Dabie Mountains].

Gastrodia elata, as the valuable Chinese medicinal material, has been used for more than 2 000 years in China. With the increasing market demand for G. elata, the traditional wild resources have been unable to transform into commodities. At present, local authorities give full play to the advantages of natural resources and vigorously cultivate G. elata to form the cultivation mode and technical system with local characteristics. Huanggang Comprehensive Experimental Station of National Technical System of Chinese Medicinal Materials Industry has optimized and summarized the paddy-upland rotation of G. elata-Oryza sativa in Dabie Mountains of Hubei province through field visits and guidance for four consecutive years. Based on the ecological adaptability and planting characteristics of G. elata and O. sativa, and the actual production experience of farmers, analyzed the principle of paddy-upland rotation from production environment selection and fungus treatment, and evaluated the paddy-upland rotation of G. elata-O. sativa from production status, ecological benefits, and economic benefits. The paddy-upland rotation of G. elata-O. sativa has achieved efficient cultivation of G. elata and produced considerable economic benefits. Through the summary, analysis, and evaluation of the paddy-upland rotation mode of G. elata-O. sativa in Dabie Mountains, the present study put forward the optimization strategy of cultivation technology for G. elata in low-altitude areas, i.e., to use artificial Armillaria sticks instead of traditional cut-log for substitute cultivation of G. elata, which can effectively alleviate the "bacteria-forest contradiction" arising from the cultivation of G. elata. It can also improve the ecological environment and production status of the Dabie Mountains and even the G. elata producing areas at the same altitude.