Utilization of used fungus-growing materials of Gastrodia elata / 中国中药杂志

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

Association Study Between Rhizoma Gastrodia elata Brown-rot and Soil Microbial Communities / 中国实验方剂学杂志

Objective:To study the microbial community composition and diversity of brown-rot Gastrodia elata and its surface soil，in order to explain the relationship between brown-rot G. elata and soil microflora in G. elata planting process and provide theoretical basis for revealing the reasons of G. elata brown-rot disease. Method:Used internal transcribed spacer region（ITS） and 16S rDNA high-throughput sequencing technologies to detect the microbial diversity，community structure composition and community structure similarity of fungi and bacteria in healthy tuber，Brown-rot tuber，healthy soil and Brown-rot soil. Result:Compared with health groups，the number of fungi and bacteria operational taxonomic units（OTUs） was increased in brown-rot G. elata and its soil， and the abundance and diversity of fungi and bacteria in brown-rot G. elata soil were significantly decreased. The diversity of fungi in the tubers of brown-rot G. elata was significantly reduced，while the diversity of bacteria was significantly decreased. At the genus level， Mortierella was dominant fungi genus in healthy tuber and healthy soil，which was reduced 7.62% and 15.75% respectively in brown-rot tuber and brown-rot soil. And the dominant bacteria genus was Bradyrhizobium and Burkholderia-Paraburkholderia respectively. Ilyonectria was dominant fungi genus in brown-rot tuber and brown-rot soil，the dominant bacteria genus was Serratia and Bradyrhizobium respectively. Conclusion:The fungal flora in the tuber of brown-rot G. elata had a very high degree of similarity to that in the surrounding soil. These results indicated that the change of soil microbial fungal community caused the occurrence of G. elata brown-rot disease to a certain extent. And the pathogenic fungal Ilyonectria was dominant genus in fungi community of brown-rot tuber and brown-rot soil. Ilyonectria may have the main G. elata brown-rot disease pathogen.

Effect of Gastrodia elata Planting on Soil Microbial Community Structure / 中国实验方剂学杂志

Objective:To analyze the changes of soil microbial community structure before and after planting Gastrodia elata in different producing areas，and to investigate the response of soil microorganisms to the planting of G. elata. Method:ITS and 16S rDNA high-throughput sequencing technologies were used to detect fungal and bacterial community compositions in the soil，including the soil without planting G. elata（CK1，CK2），the soil around G. elata（GE1，GE2）before harvesting, and the soil around the rhizomorph of Armillaria（AGE1，AGE2） in Dafang, Guizhou and Jinzhai, Anhui respectively. Result:Principal component analysis (PCA) showed that the soil microorganisms changed significantly after G. elata planting as compared with the control soil. The sequencing results showed that the planting of G. elata increased the OTUs number of fungi and bacteria. As compared with the control soil，the diversity and abundance of fungal and bacterial communities showed an increase trend after the cultivation of G. elata in soil of Dafang, Guizhou， such changes of fungal communities were not significant, but the abundance of soil bacteria communities increased in Jinzhai, Anhui as compared with the control soil. The abundance of genera Ilyonectria and Nitrospira increased，while genera Russula decreased significantly both in the soil of Guizhou and Anhui. Furthermore，the abundance of Fusarium and Mortierella increased significantly in the soil of Dafang, Guizhou. Conclusion:The soil microorganisms were out of balance after planting of G. elata, and the abundance of pathogenic microorganisms such as Ilyonectria and Fusarium increased，which may be related to the plant diseases and insect pests of G. elata.

Effects of High Temperature Stress on Growth Characteristics of Armillaria / 中国实验方剂学杂志

Objective:To explore the effects of high temperature stress on the growth characteristics of different Armillaria strains，and to provide guidance for screening excellent Armillaria strains with high-temperature resistance. Method:14 strains of Armillaria from different G. elata producing areas were used as experimental materials to observe the growth characteristics and conduct phenotypic classification for the strains. rDNA-IGS sequence analysis was used for molecular identification to further determine the genetic relationship of the tested strains.The strain growth rate, biomass，mycelial length and other indicators under the condition of 23 ℃ （CK） and 30 ℃ high temperature stress were recorded. Result:All the 14 strains of Armillaria had the highest similarity and the closest relationship with Armillaria gallica，but there were significant differences in growth characteristics among different G. elata producing areas. The 14 strains of Armillaria were classified into Ⅳ groups，and the growth status was groupⅠ>group Ⅱ>group Ⅲ>group Ⅳ. After treatment with high temperature stress，the tolerance of each strain to high temperature also showed obvious differences，as shown in the average growth rate of the mycelial was GZ16>SX1>GZ1. The rank of relative mycelial length was GZ16>SX1>GZ3 and the relative biomass was GZ16>SX4>GZ1>HB1>AH2. Conclusion:Under high temperature stress，GZ16 was best in growth rate，relative length of mycelial，relative biomass and growth state，followed by SX1 and GZ1 strains. The results indicate that strains GZ16，SX1 and GZ1 have the strong resistance to high temperature and excellent growth characteristics at normal temperature，so these three strains are suitable to be produced in main G. elata producing areas in China.

Isolation and Identification of Mycena of Pleurotus in Wumeng Mountain Area and Optimization of Its Production Conditions / 中国实验方剂学杂志

Objective:Isolate and identify Mycena, expand the resources of geminating fungus of Gastrodia elata and optimize the culture conditions of Mycena，in order to provide information and guidance for the production of geminating fungus of G. elata. Method:Juvenile tuber tissue mass transfer separation and purification technology was used for the separation and purification of strains，traditional morphology microscopy was used to isolate the colony mycelia spores morphological characteristics, such as identification，polymerase chain reaction（PCR） amplification rDNA （Ribosomal DNA） internal transcribed spacer（ITS） was used for sequencing analysis and further homology with NCBI database retrieval，MEGA6 software was used to establish Phylogenetic tree by the Maximum likelihood method （MaximumLikelihood，M-L）, so as to classify and identify isolated strains. At the same time，orthogonal test was used to optimize the optimal growth conditions of Mycena. Result:A total of 86 strains were isolated, which belong to 21 species in 12 genera. WMMFJ，SHXG，WMM-21 and MFJ8103 were identified as M. purpureofusca, and ZT01-6 and ZT01-8 were identified as M. cf. purpureofusca. The growth rate of Mycena in wheat bran medium was significantly higher than in PDA medium. The optimal medium composition for the growth of germinating bacteria was 100 g potato，150 g wheat bran and 20 g corn flour，100 g glucose. And 1，3，5-Trihydroxybenzene significantly promoted the growth of WMMFJ，and played a role in promoting the growth of WMM-21 and ZT01-6，and 2-Methoxyphenol promoted the growth of WMMFJ. Conclusion:Six strains of Mycena were isolated and identified，four of them are M. purpureofusca，and two of them are M. cf. Purpureofusca. The separation method improved the separation effect of germinating bacteria.

Analysis of ecological recycling modes for Gastrodia elata cultivation / 中国中药杂志

Gastrodia elata is a kind of precious traditional Chinese medicine. In artificial cultivation, it has not got rid of its dependence on forest resources. In order to maintain the balance of the ecological system and reduce the waste of resources as much as possible, based on the information from field investigation at many places, this paper introduced the new ecological circulation planting patterns of G. elata, such as "forest-G. elata" supporting planting, G. elata-edible mushroom rotation, forest-G. elata-edible mushroom three-dimensional planting, fungus material classification planting technology, and so on. In this paper, we expounded the ecological problems solved by several planting patterns in G. elata production and analyzed their shortcomings. Finally, based on the exis-ting models, a complete ecological planting system of G. elata was summarized. This planting system emphasizes: ① The follow-up forests should be started before the planting of G. elata. And the economic forests were used to cultivation of G. elata. ② The classified utilization of fungus-growing materials. The leaves were used to cultivate germination bacteria of G. elata, the small branches were used to cultivate protocorm and juvenile tuber, the large branches were used to cultivate immature tuber, and the tree trunk was used to cultivate mature tuber. ③ Recycle utilization G. elata fungus material. The old fungus materials were used to produce strains or cultivate edible fungus. This design project not only solves the problems of the source of G. elata fungus material, the efficient utilization of fungus material and land resources, but also enriches the industrial structure. Using limited time and land resources to obtain greater economic benefits. It has certain guiding significance for poverty alleviation and ecological improvement.

Ecological benefits of Gastrodia elata-Phallus impudicus sequential planting pattern / 中国中药杂志

Gastrodia elata is a kind of precious traditional Chinese medicine. In the process of cultivation of G. elata, due to the influence of continuous cropping obstacles and other factors, the fungus materials and land that have been planted with G. elata are often abandoned, resulting in a great waste of resources. Based on the planting characteristics of G. elata and Phallus impudicus and the previous research experience in ecological agriculture, this paper analyzed the ecological adaptability characteristics of G. elata and P. impudicus, and summarized the key techniques of the G. elata-P. impudicus sequential planting pattern. Keeping track of the planting area, fungus-growing materials consumption and market sales of G. elata-P. impudicus sequential planting pattern, the ecological benefits of G. elata-P. impudicus sequential planting pattern from the aspects of utilization rate of fungus-growing materials were analyzed, the value of land resources per unit area, ecological environmental protection, labor cost and economic benefits were consi-dered. The technical principle of G. elata-P. impudicus sequential planting pattern was expounded according to their ecological habit, the season of harvest and planting, the difference of composition of fungus-growing materials, and the microbial ecology. The sequential planting pattern of G. elata-P. impudicus not only realized the double production of medicinal materials and edible fungi, reduced the waste of old fungus-growing materials, but also transformed the energy from nutrition-supplied fungi to edible and medicinal fungi, which guaranteed the ecological recycling and utilization of G. elata in the process of cultivation.

Effect of Gastrodiae elata-Phallus impudicus sequential planting pattern on soil microbial community structure / 中国中药杂志

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.

Comparative study on infection and degradation of Armillaria gallica and Phallus impudicus to fungus-growing materials / 中国中药杂志

The phenomenon that waste of fungus-growing materials in the planting process of Gastrodia elata is very common. It has been proved by practice that the used fungus-growing materials planted with G. elata can be used to plant Phallus impudicus. But the mechanism is unclear. In this study, we compared the different infested-capacity of Armillaria gallica and Phallus impudicus by morphological anatomy of the used fungus-growing materials. We also compared the differences on the two fungi consumed the main contents of fungus-growing materials, cellulose, lignin and hemicellulose, by using nitric acid-95% ethanol method, sulfuric acid method and tetrabromide method respectively, so that to explore the mechanism of A. gallica and P. impudicus recycle the fungus-growing materials, and to provide scientific basis for recycling the used fungus-growing materials of G. elata. The results showed that A. gallica had a strong ability to invade some parts outside the vascular cambium, but it had a weak ability to invade some parts inside the vascular cambium, while P. impudicus had a strong ability to invade the same parts. The contents of lignin and cellulose, which from inside and outside the vascular cambium of fungus-growing materials were significantly different. In the parts of outside the vascular cambium of fungus-growing materials, A. gallica degraded more lignin and cellulose, while P. impudicus degraded more hemicellulose. In the parts of inside the vascular cambium of fungus-growing materials, A. gallica degraded more cellulose, while P. impudicus degraded more hemicellulose. The present results suggested that A. gallica and P. impudicus made differential utilization of the carbon source in the fungus-growing materials to realize that P. impudicus recycle the used fungus-growing materials of G. elata. A. gallica used lignin and cellulose as the main carbon source, while P. impudicus used hemicellulose as the main carbon source.

Investigation, analysis and identification of disease of Gastrodia elata f. glauca / 中国中药杂志

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.

Separation Identification of Chemical Constituents of Lysimachia capillipes / 中国实验方剂学杂志

Objective:To systemically study the chemical constituents of n-butanol fraction from Lysimachia capillipes. Method:The whole plant of L. capillipes was crushed into power,extracted by 70% methanol,concentrated under reduced pressure,and then its n-butanol extract was obtained by fractional extraction. The compounds from n-butanol fraction were isolated and purified by macroporous resin column chromatography,medium pressure ODS,silica gel,Sephadex LH-20 and preparative HPLC. Their structures were identified on the basis of spectral analysis and comparison with literature data. Result:Fifteen compounds including 6 saponins and 9 flavonoid glycosides were isolated from L. capillipes,and were identified as ascapilliposide B(1) and capilliposide C(2),kaempferol-3-O-β-D-xylopyranosyl(1→3)-[4-O-E-p-coumaroyl-α-L-rhamnopyranosyl(1→2)] [β-D-glucopyranosyl(1→6)]-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside(3),kaempferol-3-O-{[β-D-xylopyranosyl(1→3)-α-L-rhamnopyranosyl(1→6)] [α-L-rhamnopyranosyl(1→2)]}-β-D-3-trans-p-coumaroylgalactopyranoside (4),capilliposide K (5),3β-O-{α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→4)-[O-β-D-glucopyranosyl-(1→2)]-α-L-arabinopyranosyl)}-16α-hydroxyolean-28,13β-olide (6),capilliposide I(7),quercetin-3-O-(2″,6″-di-O-α-rhamnopyranosyl)-β-galactopyranoside(8),kaempferol-3-O-{[β-D-xylopyranosyl(1→3)-α-L-rhamnopyranosyl(1→6)] [α-L-rhamnopyranosyl-(1→2)]}-β-D-galactopyranoside(9),kaempferol-3-O-[2-glucopyranosyl(1→3)rhamnopyranosyl-6-rhamnopyranosyl]-β-D-galactopyranoside(10),kaempferol-3-O-α-L-rhamnopyranosy-(1→2)-[α-L-rhamnopyranosy-(1→6)]-β-D-galactopyranoside(11),capilliposide I(12),kaempferol-3-O-{(β-D-glucopyranosyl-(1→3)-[4-O-(E-p-coumaroyl)]-α-L-rhamnopyranosyl-(1→6)-(β-D-galactopyranoside)}-7-O-α-L-rhamnopyranoside (13),kaempferol-3-O-{[β-D-glucopyranosyl(1→3)]-4-O-(E-p-coumaroyl)}-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside-7-O(4-O-acetyl)-α-L-rhamnopyranoside (14),and (3β,20S,23S,24R)-3,20,23,24,25,29-hexahydroxydammaran-21-oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→6)-β-D-gluco-pyranoside(15). Conclusion:The compounds 3,4,6,9,10,13-15 were isolated from this plant for the first time.

Characteristics of CDR3 of TCR beta on CD8+ T cells in chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the characteristics of CDR3 of TCRbeta on CD8+ T cells in chronic hepatitis B patients.</p><p><b>METHODS</b>Eight patients with chronic hepatitis B (ALT more than 2 ULN) were enrolled in this study. CD8+ T cells were isolated from peripheral blood. RT-PCR was proformed to amplify the CDR3 of TCRbeta, and the PCR products were sequenced and analyzed.</p><p><b>RESULTS</b>The chronic hepatitis B patients showed obvious clonal expansion of T cell, and three perturbation patterns of T cell expansion were showed in the CDR3 of TCRbeta, including monoclonicity, oligoclonicity and skewed peak patterns. The number of perturbation families of CD8+ subpopulation was significantly higher than that of CD8- subpopulation (10.6+/-4.7 vs. 4.1+/-3.1, t = 6.619, P less than 0.01). In 3 out of 8 patients, the number of perturbation families of CD8+ subpopulation was also higher than that of PBMCs without depleting CD8+ subpopulation.</p><p><b>CONCLUSIONS</b>The characteristics of CDR3 of TCRbeta may help to understand the inflammatory response in CHB patients.</p>

Imperatorin is responsible for the vasodilatation activity of Angelica Dahurica var. Formosana regulated by nitric oxide in an endothelium-dependent manner / 中国结合医学杂志

<p><b>OBJECTIVE</b>To demonstrate the vasodilatation activity of the coumarin-containing Angelica dahurica var. formosana and to further analyze active components in the herb extracts.</p><p><b>METHODS</b>(1) The vasodilatation effects induced by different extracts (cyclohexane, ethyl acetate, acetone, methanol, 95 % ethanol and water) of Angelica dahurica var. formosana on mouse thoracic aorta pre-contracted with phenylephrine were investigated. (2) The amount of imperatorin and isoimperatorin in each extract was measured by high-performance liquid chromatography. (3) The vasodilatation effects of imperatorin and isoimperatorin on mouse thoracic aorta were compared using the same in vitro method. (4) The vasodilatation mechanism of imperatorin in the mouse thoracic aorta pre-contracted with phenylephrine was studied using the methods of denuded endothelium, NG-nitro-L-arginine methylester (L-NAME, a nitric oxide synthase inhibitor), and propranolol.</p><p><b>RESULTS</b>(1) The cyclohexane and ethyl acetate extracts of Angelica dahurica var. formosana decreased the maximal response of phenylephrine-induced mouse thoracic aorta contraction dose-dependently, with 50% inhibiting concentration (IC(50)) values of 35.3+/-12.4 mg/L and 40.5+/-12.0 mg/L, respectively. The vasodilatation effect of imperatorin and isoimperatorin was dose-dependent. (2) The cyclohexane extract, showing the strongest vasodilatation effect, possessed the highest contents of imperatorin (4.09%) and isoimperatorin (0.27%, w/w). There was a correlation between the vasodilatation activity and the contents of imperatorin and isoimperatorin in the extracts. (3) The vasodilatation effect of imperatorin was about 4-fold stronger than that of isoimperatorin. (4) The vasodilatation effect of imperatorin was signifificantly attenuated to 24.88%+/-4.06% in the denuded endothelium group compared with the intact endothelium group. And 1 mmol/L L-NAME reduced the imperatorin-induced vasorelaxation by 32.18 %+/-11.29 %.</p><p><b>CONCLUSIONS</b>The principal effective component of Angelica dahurica var. Formosana was found to be imperatorin. Imperatorin-induced vasodilatation is at least partially regulated by nitric oxide, and has no correlation to beta-receptor.</p>

Analysis of T cell receptor BV dominant usage and CDR3 sequences during acute exacerbation in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To understand the role cellular immunology plays in the pathogenesis of chronic hepatitis B (CHB) through analysis of T cell receptor (TCR) beta chain variable region gene (BV) family dominant usage and beta chain complementarity determining region3 (CDR3) sequences of peripheral blood mononuclear cells of the patients.</p><p><b>METHODS</b>TCR BV families were amplified by inverse polymerase chain reaction (RT-PCR), and the dominant usage of BV families and CDR3 repertoire were analyzed by immunoscope technology for 8 CHB patients during their acute exacerbations and for 4 healthy blood donors who served as controls. The clonality of the T cells suspected by immunoscope was further confirmed by CDR3 sequencing.</p><p><b>RESULTS</b>The TCR BV CDR3 repertoire of the 4 healthy blood donors showed a Gaussian distribution. In the 8 CHB patients, however, the clonal expansion of T cells showed different TCR BV families with each patient. The T cells of the clonal expansion shared different CDR3 sequences.</p><p><b>CONCLUSION</b>The peripheral blood T cells of CHB patients during their acute exacerbation showed significantly a clonal expansion and their T cell clonal expansion may be stimulated by several HBV epitopes. These results indicate that cellular immunology is involved in the pathogenesis of the liver inflammation process of CHB.</p>