[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.

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 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.

Peniterester, a carotane-type antibacterial sesquiterpene from an artificial mutant Penicillium sp. T2-M20.

Peniterester (1), a new tricyclic sesquiterpene, together with 6 known compounds (2-7) were isolated from the secondary metabolites of an artificial mutant Penicillium sp. T2-M20 which was obtained from the parental strain Penicillium sp. T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. Peniterester was only produced by the mutant T2-M20 on the basis of LC-MS analysis. Meanwhile, the results of in vitro bioactivities screening indicated that peniterester owned obvious antibacterial activities against Bacillus subtilis, Escherichia coli and Staphylococcus aureus with MICs of 8.0, 8.0 and 4.0 µg/mL, respectively.

[Molecular identification of Armillaria gallica by PCR-RFLP analysis].

Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.

[Identification of four Armillaria strains and their effects on quality and yield of Gastrodia elata f. glauca].

In order to improve the quality and yield of Gastrodia elata f. glauca,determine the suitable Armillaria strains for the accompanying experiment in Xiaocaoba,Yiliang,four Armillaria strains were selected. They were used for G. elata cultivation,and the gene sequence,r DNA-ITS,ß-tubulin and EF1-α of four Armillaria strains,were compared and analyzed. The yield was mesured in November which was based on previous laboratory research. The tubers were washed and steamed,then dried and powdered. The content of gastrodin and p-hydroxybenzyl alcohol was determined by UPLC,the polysaccharide was determined by phenol-concentrated sulfuric acid method. The results showed that the strains M1,M2,M3 and M4 were Armillaria gallica group but there were differences in the yield and active ingredient content when they were cultivated with the same G. elata. The yield of G. elata( Jian Ma) was the lowest when cultivated with Armillaria strain M3,but it was not the same when used M1,0. 981 kg·m-2,the highest yield in the four stains.The content of gastrodin was 0. 581%,the total content of gastrodin and p-hydroxybenzyl alcohol was 0. 595%,when accompanied with M1 strains. It was higher than other strains. The content of G. elata polysaccharide was 2. 132%,which was similar to the content of M3 strain,higher than that of M2 and M4 strain. Selecting phylogenesis of Armillaria strians,the content of active ingredient,and the yield as indicators,it was concluded concerned that the M1 strain was the best of four strains. The results will provide a theoretical basis and guidance for higher yield and quality in cultivation of G. elata in Yiliang.

Phylogenetic Analyses of Armillaria Reveal at Least 15 Phylogenetic Lineages in China, Seven of Which Are Associated with Cultivated Gastrodia elata.

Fungal species of Armillaria, which can act as plant pathogens and/or symbionts of the Chinese traditional medicinal herb Gastrodia elata ("Tianma"), are ecologically and economically important and have consequently attracted the attention of mycologists. However, their taxonomy has been highly dependent on morphological characterization and mating tests. In this study, we phylogenetically analyzed Chinese Armillaria samples using the sequences of the internal transcribed spacer region, translation elongation factor-1 alpha gene and beta-tubulin gene. Our data revealed at least 15 phylogenetic lineages of Armillaria from China, of which seven were newly discovered and two were recorded from China for the first time. Fourteen Chinese biological species of Armillaria, which were previously defined based on mating tests, could be assigned to the 15 phylogenetic lineages identified herein. Seven of the 15 phylogenetic lineages were found to be disjunctively distributed in different continents of the Northern Hemisphere, while eight were revealed to be endemic to certain continents. In addition, we found that seven phylogenetic lineages of Armillaria were used for the cultivation of Tianma, only two of which had been recorded to be associated with Tianma previously. We also illustrated that G. elata f. glauca ("Brown Tianma") and G. elata f. elata ("Red Tianma"), two cultivars of Tianma grown in different regions of China, form symbiotic relationships with different phylogenetic lineages of Armillaria. These findings should aid the development of Tianma cultivation in China.

Chemical, pharmacological, and biological characterization of the culinary-medicinal honey mushroom, Armillaria mellea (Vahl) P. Kumm. (Agaricomycetideae): a review.

Recently, studies have been conducted on the chemical composition of fruiting bodies of the culinary-medicinal Honey mushroom, Armillaria mellea (Vahl.) P. Kumm. (higher Basidiomycetes). It is considered in Europe and Asia as edible and medicinal, when appropriately prepared, and has demonstrated the presence of different groups of organic compounds, including carbohydrates, sterols, sphingolipids, fatty acids, sesquiterpenes, non-hallucinogenic indole compounds, peptides, enzymes, adenosine derivatives, and many other components. Most of these metabolite groups possess potential therapeutic and dietary values. The results of quantitative analyses of indole compounds and heavy metals signal potential health hazards for humans. Some of the studies reviewed herein describe in detail the mechanism of symbiosis between A. mellea and the orchid species Gastrodia elata. This orchid is native to Asia, Australia, and New Zealand, and is used in therapeutics in official Chinese medicine.

Tian ma, an ancient Chinese herb, offers new options for the treatment of epilepsy and other conditions.

Our purpose is to bring attention to the antiepileptic properties of the Chinese herb tian ma and its constituents, as well as to suggest the potential for the development of new antiepileptic drugs (AEDs) related to this herb. All available literature regarding the chemistry, pharmacology, animal data, and clinical use of tian ma and its constituents are reviewed, showing that tian ma, its constituents, and its symbiotic fungus Armillaria mellea have antiepileptic properties in in vitro and in vivo models. One clinical study reportedly demonstrated the AED effects of a component of tian ma, vanillin. Thus, tian ma, its constituent vanillin, and its symbiotic fungus armillaria hold promise as cost-effective and less toxic alternatives to standard AEDs. In addition, similar chemical compounds may be developed as AEDs.

[The changes of cell structure in the courses of Armillaria mellea penetrating the nutritional stems of Gastrodia elata].

OBJECTIVE: To study the cell structure changes of Gastrodia elata after Armillaria mellea infection as well as the nutritional resource of this important medicinal plant. METHOD: Observation of the serial sections on the nutritional stems of G. elata, and measure new corms of G. elata when cut off the old corms connection with rhizomorph of A. mellea. RESULT: After G. elata nutritional stems infected by A. mellea, the rhizomorph of A. mellea separate into several hyphal layers and penetrate hyphal stream which infect into the cells of cortex layer in the direction of outside and infect directly into layer cells in the direction of inside, these hypha can be used the nutrition of G. elata the new corms will be stop growth if cut off the rhizomorph of A. mellea connected with G. elata. CONCLUSION: After the rhizomorph of A. mellea infected G. elata nutritional stems, hyphal coils and the hyphal stream will breakthrough passage cells as well as large cells surround the whole nutritional stems, so the passage cells is the key nutritional resource in the whole growth period of G. elata.