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.

Diverse Mycena Fungi and Their Potential for Gastrodia elata Germination.

It remains to be determined whether there is a geographical distribution pattern and phylogenetic signals for the Mycena strains with seed germination of the orchid plant Gastrodia elata. This study analyzed the community composition and phylogenetics of 72 Mycena strains associated with G. elata varieties (G. elata. f. glauca and G. elata. f. viridis) using multiple gene fragments (ITS+nLSU+SSU). We found that (1) these diverse Mycena phylogenetically belong to the Basidiospore amyloid group. (2) There is a phylogenetic signal of Mycena for germination of G. elata. Those strains phylogenetically close to M. abramsii, M. polygramma, and an unclassified Mycena had significantly higher germination rates than those to M. citrinomarginata. (3) The Mycena distribution depends on geographic site and G. elata variety. Both unclassified Mycena group 1 and the M. abramsii group were dominant for the two varieties of G. elata; in contrast, the M. citrinomarginata group was dominant in G. elata f. glauca but absent in G. elata f. viridis. Our results indicate that the community composition of numerous Mycena resources in the Zhaotong area varies by geographical location and G. elata variety. Importantly, our results also indicate that Mycena's phylogenetic status is correlated with its germination rate.

Discovery of seed germinating fungi (Mycetinis scorodonius) from Gastrodia elata Bl. f. glauca S. chow in Changbai Mountain and examination of their germination ability.

Multi-generational asexual reproduction of Gastrodia elata Bl. will cause seedling species degeneration. Sexual reproduction of Gastrodia elata Bl. seed is an effective method to solve the problem of degeneration. The development of Gastrodia elata Bl. seeds cannot be separated from the germination fungus. However, there are few strains of germination fungus in production, and there is also the problem of species degradation in application for many years. It is very important for the sexual reproduction of Gastrodia elata Bl. to isolate more new strains of excellent germination fungus from the origin. This study used the Gastrodia elata Bl. f. glauca S. chow seeds germination vegetative propagation corms capture method to isolate its symbiotic germination fungus, and comprehensively identified the species of germination fungus by colony morphology, ITS, sporocarps regeneration and germination function, and compared the growth characteristics and germination ability with other germination fungus (Mycena purpureofusca, Mycena dendrobii and Mycena osmundicola). The germination fungus was isolated from the vegetative propagation corms of Gastrodia elata Bl. f. glauca S. chow seeds and named GYGL-1. After comprehensive identification, GYGL-1 was Mycetinis scorodonius. Compared with other germination fungus, GYGL-1 has fast germination speed, vigorous growth, and high germination ability for Gastrodia elata Bl. f. glauca S. chow seeds. Innovated the isolation method of Gastrodia elata Bl. seeds germination fungus, obtained the regenerated sporocarps of the germination fungus, and discovered that Mycetinis scorodonius has a new function of germinating Gastrodia elata Bl. f. glauca S. chow seeds, enriching the resource library of Gastrodia elata Bl. germination fungus.

Research Status and Application Prospects of the Medicinal Mushroom Armillaria mellea.

Armillaria is one of the most common diseases underlying chronic root rot in woody plants. Although there is no particularly effective way to prevent it, soil disinfection is a common effective protective measure. However, Armillaria itself has important medicinal value and is a symbiotic fungus in the cultivation of Gastrodia elata and Polyporus umbellatus. Therefore, researching Armillaria is of great practical significance. In this review, the biological characteristics, cultivation methods, chemical components, food and medicinal value and efficacy of Armillaria were all reviewed, and its development and utilization direction were analyzed and discussed.

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.

Phialocetones A-J, C12 lactones from the rhizospheric soil-derived fungus Phialocephala sp. YUD18001 associated with Gastrodia elata.

Ten undescribed C12 polyketide phialocetones A-J, featuring twelve-, six- and five-membered lactone moieties, were isolated from a rhizospheric soil-derived Phialocephala sp. YUD18001 associated with Gastrodia elata. Their structures were established by NMR spectroscopic analysis and HRMS, while their absolute configurations were determined by computational methods and chemical reactions. All isolated compounds were evaluated for their anti-inflammatory and cytotoxic activities. As a result, phialocetone D exhibited moderate effects against NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells with an IC50 value of 14.77 µM, while phialocetone E showed cytotoxicity against HL-60 and SW480 cell lines with IC50 values of 19.04 and 10.22 µM, respectively.

[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.

[Resistance of different ecotypes of Gastrodia elata to tuber rot].

Tuber rot has become a serious problem in the large-scale cultivation of Gastrodia elata. In this study, we compared the resistance of different ecotypes of G. elata to tuber rot by field experiments on the basis of the investigation of G. elata diseases. The histological observation and transcriptome analysis were conducted to reveal the resistance differences and the underlying mechanisms among different ecotypes. In the field, G. elata f. glauca had the highest incidence of tuber rot, followed by G. elata f. viridis, and G. elata f. elata and G. elata f. glauca×G. elata f. elata showed the lowest incidence. Tuber rot showcased obvious plant source specificity and mainly occurred in the buds and bottom of G. elata plants. After infection, the pathogen spread hyphae in host cortex cells, which can change the endophytic fungal community structure in the cortex and parenchyma of G. elata. G. elata f. glauca had thinner lytic layer and more sugar lumps in the parenchyma than G. elata f. elata. The transcription of genes involved in immune defense, enzyme synthesis, polysaccharide synthesis, carbohydrate transport and metabolism, hydroxylase activity, and aromatic compound synthesis had significant differences between G. elata f. glauca and G. elata f. elata. These findings suggested that the differences in resis-tance to tuber rot among different ecotypes of G. elata may be related to the varied gene expression patterns and secondary metabolites. This study provides basic data for the prevention and control of tuber rot and the improvement of planting technology for G. elata.

[Correlation analysis between continuous cropping obstacle of Gastrodia elata and Ilyonectria fungi and relieving strategy].

The continuous cropping obstacle of Gastrodia elata is outstanding, but its mechanism is still unclear. In this study, microbial changes in soils after G. elata planting were investigated to explore the mechanism correlated with continuous cropping obstacle. The changes of species and abundance of fungi and bacteria in soils planted with G. elata after 1, 2, and 3 years were compared. The pathogenic fungi that might cause continuous cropping diseases of G. elata were isolated. Finally, the prevention and control measures of soil-borne fungal diseases of G. elata were investigated with the rotation planting pattern of "G. elata-Phallus impudicus". The results showed that G. elata planting resulted in the decrease in bacterial and fungal community stability and the increase in harmful fungus species and abundance in soils. This change was most obvious in the second year after G. elata planting, and the soil microbial community structure could not return to the normal level even if it was left idle for another two years. After G. elata planting in soils, the most significant change was observed in Ilyonectria cyclaminicola. The richness of the Ilyonectria fungus in soils was significantly positively correlated with the incidence of G. elata diseases. When I. cyclaminicola was inoculated in the sterile soil, the rot rate of G. elata was also significantly increased. After planting one crop of G. elata and one to three crops of P. impudicus, the fungus community structure in soils gradually recovered, and the abundance of I. cyclaminicola decreased year by year. Furthermore, the disease rate of G. elata decreased. The results showed that the cultivation of G. elata made the Ilyonectria fungi the dominant flora in soils, and I. cyclaminicola served as the main pathogen of continuous cropping diseases of G. elata, which could be reduced by rotation planting with P. impudicus.

Correlation analysis between continuous cropping obstacle of Gastrodia elata and Ilyonectria fungi and relieving strategy / 中国中药杂志

The continuous cropping obstacle of Gastrodia elata is outstanding, but its mechanism is still unclear. In this study, microbial changes in soils after G. elata planting were investigated to explore the mechanism correlated with continuous cropping obstacle. The changes of species and abundance of fungi and bacteria in soils planted with G. elata after 1, 2, and 3 years were compared. The pathogenic fungi that might cause continuous cropping diseases of G. elata were isolated. Finally, the prevention and control measures of soil-borne fungal diseases of G. elata were investigated with the rotation planting pattern of "G. elata-Phallus impudicus". The results showed that G. elata planting resulted in the decrease in bacterial and fungal community stability and the increase in harmful fungus species and abundance in soils. This change was most obvious in the second year after G. elata planting, and the soil microbial community structure could not return to the normal level even if it was left idle for another two years. After G. elata planting in soils, the most significant change was observed in Ilyonectria cyclaminicola. The richness of the Ilyonectria fungus in soils was significantly positively correlated with the incidence of G. elata diseases. When I. cyclaminicola was inoculated in the sterile soil, the rot rate of G. elata was also significantly increased. After planting one crop of G. elata and one to three crops of P. impudicus, the fungus community structure in soils gradually recovered, and the abundance of I. cyclaminicola decreased year by year. Furthermore, the disease rate of G. elata decreased. The results showed that the cultivation of G. elata made the Ilyonectria fungi the dominant flora in soils, and I. cyclaminicola served as the main pathogen of continuous cropping diseases of G. elata, which could be reduced by rotation planting with P. impudicus.

Resistance of different ecotypes of Gastrodia elata to tuber rot / 中国中药杂志

Tuber rot has become a serious problem in the large-scale cultivation of Gastrodia elata. In this study, we compared the resistance of different ecotypes of G. elata to tuber rot by field experiments on the basis of the investigation of G. elata diseases. The histological observation and transcriptome analysis were conducted to reveal the resistance differences and the underlying mechanisms among different ecotypes. In the field, G. elata f. glauca had the highest incidence of tuber rot, followed by G. elata f. viridis, and G. elata f. elata and G. elata f. glauca×G. elata f. elata showed the lowest incidence. Tuber rot showcased obvious plant source specificity and mainly occurred in the buds and bottom of G. elata plants. After infection, the pathogen spread hyphae in host cortex cells, which can change the endophytic fungal community structure in the cortex and parenchyma of G. elata. G. elata f. glauca had thinner lytic layer and more sugar lumps in the parenchyma than G. elata f. elata. The transcription of genes involved in immune defense, enzyme synthesis, polysaccharide synthesis, carbohydrate transport and metabolism, hydroxylase activity, and aromatic compound synthesis had significant differences between G. elata f. glauca and G. elata f. elata. These findings suggested that the differences in resis-tance to tuber rot among different ecotypes of G. elata may be related to the varied gene expression patterns and secondary metabolites. This study provides basic data for the prevention and control of tuber rot and the improvement of planting technology for G. elata.

The selective anti-fungal metabolites from Irpex lacteus and applications in the chemical interaction of Gastrodia elata, Armillaria sp., and endophytes.

The investigation of the metabolites from endophyte Irpex lacteus cultured in host "tian ma" (Gastrodia elata) revealed five new tremulane sesquiterpenes (1-5), and a new tetrahydrofuran derivative (6). Compound 1 was the first tremulane glucoside, and 6 possessed a rare tetrahydropyran-tetrahydrofuran scaffold. Main metabolite (2,3-dihydroxydodacane-4,7-dione, 14) from I. lacteus showed significant selectivity for antifungal activity against phytopathogen and endophytes associated with G. elata rather than against Armillaria sp. providing nutrition for the host G. elata. 14 accounted for 27.4% of isolated compounds from G. elata medium, and 69.3% by co-culturing with Armillaria sp. So the I. lacteus tended to promote the growth of Armillaria sp. in co-culture by producing 2,3-dihydroxydodacane-4,7-dione (14) to selective inhibit the phytopathogen and endophyte existed in host G. elata for the benefit of G. elata-Armillaria symbiosis. And the results were in accord with the real environment of G. elata depending on the nutrition of Armillaria. Some metabolites had anti-fungal activities against phytopathogens of G. elata with MICs ≤8 µg/mL.

Transcriptome Reveals Roles of Lignin-Modifying Enzymes and Abscisic Acid in the Symbiosis of Mycena and Gastrodia elata.

Gastrodia elata is a well-known medicinal and heterotrophic orchid. Its germination, limited by the impermeability of seed coat lignin and inhibition by abscisic acid (ABA), is triggered by symbiosis with fungi such as Mycena spp. However, the molecular mechanisms of lignin degradation by Mycena and ABA biosynthesis and signaling in G. elata remain unclear. In order to gain insights into these two processes, this study analyzed the transcriptomes of these organisms during their dynamic symbiosis. Among the 25 lignin-modifying enzyme genes in Mycena, two ligninolytic class II peroxidases and two laccases were significantly upregulated, most likely enabling Mycena hyphae to break through the lignin seed coats of G. elata. Genes related to reduced virulence and loss of pathogenicity in Mycena accounted for more than half of annotated genes, presumably contributing to symbiosis. After coculture, upregulated genes outnumbered downregulated genes in G. elata seeds, suggesting slightly increased biological activity, while Mycena hyphae had fewer upregulated than downregulated genes, indicating decreased biological activity. ABA biosynthesis in G. elata was reduced by the downregulated expression of 9-cis-epoxycarotenoid dioxygenase (NCED-2), and ABA signaling was blocked by the downregulated expression of a receptor protein (PYL12-like). This is the first report to describe the role of NCED-2 and PYL12-like in breaking G. elata seed dormancy by reducing the synthesis and blocking the signaling of the germination inhibitor ABA. This study provides a theoretical basis for screening germination fungi to identify effective symbionts and for reducing ABA inhibition of G. elata seed germination.

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm-isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)-like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria-colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high-affinity sucrose-specific proton-dependent importer. Plasma-membrane/tonoplast localization of GeSUT4-GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant-microbe interactions.

Transcriptome analysis reveals underlying immune response mechanism of fungal (Penicillium oxalicum) disease in Gastrodia elata Bl. f. glauca S. chow (Orchidaceae).

BACKGROUND: Gastrodia elata Bl. f. glauca S. Chow is a medicinal plant. G. elata f. glauca is unavoidably infected by pathogens in their growth process. In previous work, we have successfully isolated and identified Penicillium oxalicum from fungal diseased tubers of G. elata f. glauca. As a widespread epidemic, this fungal disease seriously affected the yield and quality of G. elata f. glauca. We speculate that the healthy G. elata F. glauca might carry resistance genes, which can resist against fungal disease. In this study, healthy and fungal diseased mature tubers of G. elata f. glauca from Changbai Mountain area were used as experimental materials to help us find potential resistance genes against the fungal disease. RESULTS: A total of 7540 differentially expressed Unigenes (DEGs) were identified (FDR < 0.01, log2FC > 2). The current study screened 10 potential resistance genes. They were attached to transcription factors (TFs) in plant hormone signal transduction pathway and plant pathogen interaction pathway, including WRKY22, GH3, TIFY/JAZ, ERF1, WRKY33, TGA. In addition, four of these genes were closely related to jasmonic acid signaling pathway. CONCLUSIONS: The immune response mechanism of fungal disease in G. elata f. glauca is a complex biological process, involving plant hormones such as ethylene, jasmonic acid, salicylic acid and disease-resistant transcription factors such as WRKY, TGA.

Penctrimertone, a bioactive citrinin dimer from the endophytic fungus Penicillium sp. T2-11.

Penctrimertone (1), a novel citrinin dimer bearing a 6/6/6/6 tetracyclic ring scaffold, along with two known compounds xerucitrinic acid A (2) and citrinin (3) were isolated from the endophytic fungus Penicillium sp. T2-11. Their structures were unequivocally established by a comprehensive interpretation of the spectroscopic data, with the stereochemistry for 1 was defined by a combination of TDDFT-ECD calculations and the DP4+ probability analysis based on NMR chemical shift calculations. Bioassays revealed that compound 1 exhibited noticeable antimicrobial activities and moderate cytotoxicity. A plausible biosynthetic pathway of 1 was also proposed.

Draft genomic sequence of Armillaria gallica 012m: insights into its symbiotic relationship with Gastrodia elata.

Armillaria species (Basidiomycota, Physalacriaceae) are well known as plant pathogens related to serious root rot disease on various trees in forests and plantations. Interestingly, some Armillaria species are essential symbionts of the rare Chinese medicinal herb Gastrodia elata, a rootless and leafless orchid used for over 2000 years. In this work, an 87.3-M draft genome of Armillaria gallica 012m strain, which was symbiotic with G. elata, was assembled. The genome includes approximately 23.6% repetitive sequences and encodes 26,261 predicted genes. In comparison with other four genomes of Armillaria, the following gene families related to pathogenicity/saprophytic phase, including cytochrome P450 monooxygenases, carbohydrate-active enzyme AA3, and hydrophobins, were significantly contracted in A. gallica 012m. These characteristics may be beneficial for G. elata to get less injuries. The genome-guided analysis of differential expression between rhizomorph (RH) and vegetative mycelium (VM) showed that a total of 2549 genes were differentially expressed, including 632 downregulated genes and 1917 upregulated genes. In the RH, most differentially expressed genes (DEGs) related to pathogenicity were significantly upregulated. To further elucidate gene function, Gene Ontology enrichment analysis showed that the upregulated DEGs significantly grouped into monooxygenase activity, hydrolase activity, glucosidase activity, extracellular region, fungal cell wall, response to xenobiotic stimulus, response to toxic substance, etc. These phenomena indicate that RH had better infection ability than VM. The infection ability of RH may be beneficial for G. elata to obtain nutrition, because the rhizomorph constantly infected the nutritional stems of G. elata and formed the hyphae that can be digested by G. elata. These results clarified the characteristics of A. gallica 012m and the reason why the strain 012m can establish a symbiotic relationship with G. elata in some extent from the perspective of genomics.

[Effect of Gastrodiae elata-Phallus impudicus sequential planting pattern on soil microbial community structure].

Gastrodia elata is a heterotrophic plant that needed to be symbiotic with Armillaria. The obstacle of continuous cropping in G. elata is serious during the G. elata cultivation, and the mechanism of obstacle in G. elata continuous cropping had not been solved. The planting of G. elata-Phallus impudicus is a new sequential planting pattern adopted in Guizhou province, but the effect of the cultivation on soil microbial community structure is still unclear. In this study, we collected four soil samples for the research including the soil without planted G. elata as control(CK), rhizosphere soil samples tightly adhering to the G. elata surface(GE), rhizosphere soil samples tightly adhering to Armillaria which was symbiotic with G. elata(AGE), the rhizosphere soil of P. impudicus planting after G. elata cultivation(PI). In order to explore the mechanism, the research study on the soil of G. elata-P. impudicus by using ITS and 16 S rDNA high-throughput sequencing technologies to detect soil microbial community structure including fungi and bacteria in the soil of CK, AGE, GE and PI. OTU clustering and PCA analysis of soil samples showed that the soil microbial diversity was relatively similar in AGE and GE. And the soil microbial in PI and CK clustered together. The results showed that AGE and GE had similar soil microbial diversity, as well as PI and CK. Compared with CK, the soil microbial diversity and abundance in AGE and GE were significantly increased. But the microbial diversity and abundance decreased in PI compared with AGE and GE. The annotation indicated that the abundance of Basidiomycota, Acidobacteria and Chloroflexi decreased, and that of Ascomycota, Zygomycota and Proteobacteria increased in AGE and GE compared with CK. In contrast to AGE and GE, PI was the opposite. The abundance of Basidiomycota, Acidobacteria and Chloroflexi increased in PI compared with AGE and GE. The abundance of microorganisms in the soil of PI and CK was similar. In addition, the co-culture of Armillaria and P. impudicus indicated that P. impudicus had obvious antagonistic effects on the growth of Armillaria. Therefore, it is speculated that the mechanism of G. elata-P. impudicus planting pattern related to the change of soil microbial. And we supposed that P. impudicus might inhibit the growth of Armillaria and change the soil microbial community structure and the abundance of soil microbial. And the soil microbial community structure was restored to a state close to that of uncultivated G. elata. Thus, the structure of soil microbial community planting G. elata could be restored by P. impudicus planting.

[Investigation, analysis and identification of disease of Gastrodia elata f. glauca].

Fungal disease is an important factor restricting the healthy development of Gastrodia elata industry. The control of fungal disease in G. elata is an important issue in production. This paper makes a detailed investigation on the current situation of G. elata disease in China through statistics on the failure rate, rotten pit rate and occurrence rate of G. elata disease in the main producing areas of China. It was found that G. elata disease was mainly infected from the top bud and junction, causing the occurrence rate of disease was 6%-17%, and the yield decreased by 10%-30%. The 23 dominant fungi were isolated from 18 typical G. elata disease samples. Through identification of colony morphology, mycelium morphology, spore morphology and genetic characteristics, they were finally identified as 13 species, belonging to 7 families and 7 genera. Trichoderma harzianum, Ilyonectria sp. and Ilyonectria destructans are the most frequently separated. Their isolation frequency were 22.22%,16.67%,16.67% respectively. Ilyonectria sp. and I. destructans were the first time isolated from G. elata disease samples. They may be the main pathogens causing soil-borne diseases of G. elata. T. harzianum has certain potential as Gastrodia biocontrol bacteria. This study can provide a theoretical basis for the research and development of control technology of Gastrodia fungi disease.

Cellulomonas endophytica sp. nov., isolated from Gastrodia elata Blume.

A Gram-stain-positive, facultatively anaerobic and non-motile strain, designated SYSUP0004T, was isolated from the tubers of Gastrodia elata Blume collected from Yunnan Province, PR China. The 16S rRNA gene sequence result showed that the strain SYSUP0004T shared low similarity (97.7 %) with the type strain of Cellulomonas marina. SYSUP0004T grew at pH 6.0-9.0 (optimum, pH 8.0), temperature 4-30 °C (optimum, 28 °C) and could tolerate NaCl up to 4 % w/v (optimum in the absence of NaCl). The cell-wall peptidoglycan type was A4ß with an interpeptide bridge l-ornithine-d-glutamic acid. Cell-wall sugars were mannose, ribose, glucose, galactose and fucose. The menaquinone was MK-9(H4). The major fatty acids were anteiso-C15:0, anteiso-C15 : 1 A, C16 : 0 and anteiso-C17 : 0. The polar lipids of SYSUP0004T were diphosphatidylglycerol, unidentified phosphoglycolipid, phosphatidylinositol mannosides and unidentified glycolipid. The genomic DNA G+C content was 76.5 %. The average nucleotide identity values between SYSUP0004T and members of the genus Cellulomonas were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Thus, based on the above results strain SYSUP0004T represents a novel species of the genus Cellulomonas, for which the name Cellulomonas endophytica sp. nov. is proposed. The type strain, SYSUP0004T (=KCTC 49025T=CGMCC 1.16405T).