Gut microbiome promotes mice recovery from stress-induced depression by rescuing hippocampal neurogenesis.

Studies from rodents to primates and humans indicate that individuals vary in how resilient they are to stress, and understanding the basis of these variations may help improve treatments for depression. Here we explored the potential contribution of the gut microbiome to such variation. Mice were exposed to chronic unpredictable mild stress (CUMS) for 4 weeks then allowed to recover for 3 weeks, after which they were subjected to behavioral tests and categorized as showing low or high stress resilience. The two types of mouse were compared in terms of hippocampal gene expression using RNA sequencing, fecal microbiomes using 16S RNA sequencing, and extent of neurogenesis in the hippocampus using immunostaining of brain sections. Fecal microbiota were transplanted from either type of mouse into previously stress-exposed and stress-naïve animals, and the effects of the transplantation on stress-induced behaviors and neurogenesis in the hippocampus were examined. Finally, we blocked neurogenesis using temozolomide to explore the role of neurogenesis promoted by fecal microbiota transplantation in enhancing resilience to stress. Results showed that highly stress-resilient mice, but not those with low resilience, improved significantly on measures of anhedonia, behavioral despair, and anxiety after 3-week recovery from CUMS. Their feces showed greater abundance of Lactobacillus, Bifidobacterium and Romboutsia than feces from mice with low stress resilience, as well as lower abundance of Staphylococcus, Psychrobacter and Corynebacterium. Similarly, highly stress-resilient mice showed greater neurogenesis in hippocampus than animals with low stress resilience. Transplanting fecal microbiota from mice with high stress resilience into previously CUMS-exposed recipients rescued neurogenesis in hippocampus, facilitating recovery from stress-induced depression and cognitive decline. Blockade of neurogenesis with temozolomide abolished recovery of recipients from CUMS-induced depression and cognitive decline in mice transplanted with fecal microbiota from mice with high stress resilience. In conclusion, our results suggested that remodeling of the gut microbiome after stress may reverse stress-induced impairment of hippocampal neurogenesis and thereby promote recovery from stress-induced depression.

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.

First report of Botrytis cinerea causing grey mold on Corydalis yanhusuo in Panan, China.

Yanhusuo (Corydalis yanhusuo (Y. H. Chou & Chun C. Hsu) W. T. Wang ex Z.Y. Su & C.Y. Wu), a perennial herbaceous plant of the Papaveraceae family and genus Corydalis, is also known as Yuanhu and used as medicine by its tuberous roots. It is mainly planted in Zhejiang, Jiangsu and Anhui provinces of China, with the best quality produced in Panan County of Zhejiang province. Yanhusuo has the effects of promoting blood circulation, invigorating the flow of qi and relieving pain, and is widely used in Chinese traditional medicines. In surveys carried out in summer of 2020-2023, grey mold disease was found occurred on C. yanhusuo in Panan County. This disease begins at April, and lasts to July, with incidence of 20% to 70%. The diseased plants showed a large number of gray mold layers adhere to the leaves. When the disease infects from the leaf tips, it form V-shaped lesions; when the leaves are severely infected, the entire leaves die, shrink, curl, and have a large number of gray mold layers on the surface. To identify the causal agent of this leaf disease, diseased leaves were collected from Yanhusuo field at Panan County of Zhejiang province in China since 2020, and tissues at the junction of the healthy and diseased areas were cut off, disinfected with 75% ethanol for 30 seconds, rinsed with sterile water for 3 times for 1 minute each time, air-dried under sterile conditions, and then were inoculated in PDA medium and cultured in a 25℃ incubator. After 2-3 days, picked the edge hyphae of the fungi that grew on the PDA plate and cultured them on a new PDA plate. After 5 days, picked the single spore and inoculated on a new PDA plate for continuous cultivation until pure culture strains were obtained. Thirty strains were isolated from 30 samples that collected from 3 Yanhusuo fields in Panan County. One of the thirty purified strains was named "YH8" for further identification. When cultured on PDA medium, mycelia were initially whitish and turned gray with age. The hyphae accumulate into clusters, and no sclerotia are produced during the cultivation. The conidiophores are slender, septate. The base of the conidiophore is enlarged or slightly enlarged. The conidiophore often has branches and produces a large number of conidia, which are similar to grape clusters. The conidia are monosporous, ovoid, and colorless, 6.08 µm-12.76 µm×8.42 µm-19.34 µm, with an average size of 9.55 µm×14.50 µm. To further identify the species, YH8 genomic DNA was extracted, and the internal transcribed spacer (ITS), heat shock protein (HSP60), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes were amplified with the primers ITS1/4 (White et al. 1990), HSP60-F/HSP60-R, and G3PDH-F/G3PDH-R (Staats et al. 2005), respectively. A multilocus phylogenetic tree was constructed with the ITS, HSP60, and G3PDH reference sequences, and the sequences of PCR amplicons (Genbank number: PP388281, PP376066 and PP376067) were 100% (518 bp out of 518 bp), 99% (994 bp out of 995 bp) and 100% (880 bp out of 880 bp) identical to the Botrytis cinerea strain 5-3, respectively, and the grouping of strain YH8 was supported by 99% bootstrap value. To fulfill the Koch's postulates, spore suspension (approximately 103 CFU/mL) of YH8 was sprayed onto leaves of 3-week Yanhusuo seedlings, and sterile water was sprayed as negative control, 15 seedlings for each treatment, and the experiments was repeated for times. The seedlings were incubated in a growth chamber under 28℃ and 80% humidity. Seven days after inoculation, leaves of noninoculated controls were green and healthy, while the seedlings inoculated with spore suspension of YH8 showed lesions and molds, which were same with field symptoms. The causal pathogen was then reisolated from the lesions, and the gained pathogen showed same colony and spore morphology with YH8, which suggested the confirmation of Koch's postulates. Based on the morphological characteristics and molecular identification, the strain YH8 was identified as B. cinerea. To our knowledge, this is the first report of B. cinerea causing gray mold on the Corydalis yanhusuo in China. This report will provide guide to growers and local technicians for diagnostic and controlling grey mold disease of Yanhusuo.

Chronic exposure to aflatoxin B1 increases hippocampal microglial pyroptosis and vulnerability to stress in mice.

BACKGROUND: Chronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear. METHODS: Mice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice. RESULTS: Mice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1ß) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU+, BrdU-DCX+, and BrdU-NeuN+ cells in the hippocampal dentate gyrus, as well as the percentages of BrdU+ cells that were NeuN+ in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX+ cells correlated negatively with the percentage of ameboid microglia, TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro. CONCLUSION: Chronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.

First Report of Pectobacterium aroidearum Causing Soft Rot of Pinellia ternata in China.

Pinelliae rhizoma is the dried tuber of Pinellia ternata (Thunb.) Breit., and has been used for thousand of years in traditional Chinese medicine as an antivomit, anticough, and analgesic (Ying et al. 2007). In September 2022, P. ternata planted in Bijie, Guizhou Province, showed severe soft rot symptoms with incidence of about 50%. The diseased plants showed water-soaked symptoms and produced a foul soft rot smell, and finally the whole plant collapsed. Lesions were first observed at the tip of a leaf or wound, and symptoms of the disease spread rapidly, with the entire plant collapsing and dying within a week. The tissue sections of six plants with typical symptoms from the diseased field were disinfected with 75% ethanol for 30 seconds and 0.3% NaClO for 3 minutes. The tissue sections were then washed with sterile water for three times. A small piece of tissue (5x5mm) was removed from the edge of the lesion and mashed in a 1.5 ml centrifuge tube containing 20 µl of sterile water. The tissue liquid was then diluted 100 times with prepared sterile water. The bacteria were streaked on LB (tryptone/yeast extract/NaCl) AGAR medium and cultured at 37°C for 48 h (Kravitz, 1962). Isolated colonies were streaked on Luria-Bertani (LB) AGAR medium to obtain single colonies for further identification. A total of 13 representative isolates were selected for PCR amplification using primers targeting the conserved region of the 16S rDNA gene, which were in turn analyzed via the BLASTn search engine on the NCBI website. The results of the analysis revealed that seven of the isolates were similar to P. aroidearum strain SCRI 109 (GenBank accession no. NR_159926), with strain BX13 exhibiting the highest similarity to P. aroidearum (99.93% similarity), and therefore, this strain was selected for further investigation. The strain BX13 was incubated on LB solid medium for 24 h at 37 °C, and the single colonies were creamy white, translucent and round, slightly elevated in the center, with smooth surfaces and neat edges (Figure S1 B1). Then,the Scanning Electron Microscope revealed that the thalli of strain BX13 were short rod-shaped and somewhat blunt round at both ends (Figure S1 B2). The steward genes (icdA, gapA, proA) of BX13 were amplified and sequenced for further identification. The sequences of the amplified fragments were all deposited in GenBank 16S rDNA (OQ874505,) icdA (OQ954122)，gapA (OQ954123), proA (OQ954124). Sequence analysis using the BLASTn program at the NCBI revealed gene icdA, gapA, and proA had 100% identity to P. aroidearum strain QJ002 (GenBank accession no. CP090597).. Meanwhile, a maximum likelihood phylogenetic tree was constructed based on multigene sequence analysis of BX13 16S rDNA and steward genes (gapA, icdA, proA) by MEGA X (Liang et al. 2022). Phylogenetic results also showed that BX13 and P. aroidearum strain QJ002 gathered in the same clade(Figure S2). Accordingly, the morphological and molecular characteristics of strain BX13 indicate that it is P. aroidearum. (Nabhan S., et al.2013,Xu et al. 2020). In order to confirm the pathogenicity of strain BX13, a bacterial suspension containing 107 CFU/ml (10 ml/ inoculation point) was injected into the base of a one-week-old P. ternata stems, control seedlings were inoculated with sterile water, inoculated and control seedlings (each of six plants) were kept in a growth chamber maintained at 26°C with a relative humidity range of 70% to 80%. Plants were watered as needed. After 3 days, the stem base of the plants inoculated with bacteria solution showed water-soaked necrosis and stems began to rot, while the plants inoculated with water did not show this symptom. The strains were then successfully re-isolated from the symptomatic P. ternata. Then the strain re-isolated was identified using the BLASTn program at the NCBI and found that it has the same 16S rDNA, icdA, gapA, and proA sequences as strain BX13, thus completing the Koch's postulates. To our knowledge, this is the first report of P. aroidearum causing P. ternata soft rot in China, which expands its known host range. Accordingly, this study provides essential information for the breeding of P. ternata resistant to bacterial soft rot and the development of control measures in China.

Asperosaponin VI ameliorates the CMS-induced depressive-like behaviors by inducing a neuroprotective microglial phenotype in hippocampus via PPAR-γ pathway.

BACKGROUND: The natural compound asperosaponin VI has shown potential as an antidepressant, but how it works is unclear. Here, we explored its effects on mice exposed to chronic mild stress (CMS) and the underlying molecular pathways. METHODS: Mice were exposed to CMS for 3 weeks followed by asperosaponin VI (40 mg/kg) or imipramine (20 mg/kg) for another 3 weeks. Depression-like behaviors were assessed in the forced swimming test (FST), sucrose preference test (SPT), tail suspension test (TST). Microglial phenotypes were evaluated using immunofluorescence staining, real-time quantitative PCR and enzyme-linked immunosorbent assays in hippocampus of mice. In some experiments, stressed animals were treated with the PPAR-γ antagonist GW9662 to examine its involvement in the effects of asperosaponin VI. Blockade of PPAR-γ in asperosaponin VI-treated primary microglia in the presence of lipopolysaccharide (LPS) was executed synchronously. The nuclear transfer of PPAR-γ in microglia was detected by immunofluorescence staining in vitro and in vivo. A co-cultured model of neuron and microglia was used for evaluating the regulation of ASA VI on the microglia-neuron crosstalk molecules. RESULTS: Asperosaponin VI ameliorated depression-like behaviors of CMS mice based on SPT, TST and FST, and this was associated with a switch of hippocampal microglia from a pro-inflammatory (iNOS+-Iba1+) to neuroprotective (Arg-1+-Iba1+) phenotype. CMS reduced the expression levels of PPAR-γ and phosphorylated PPAR-γ in hippocampus, which asperosaponin VI partially reversed. GW9662 treatment prevented the nuclear transfer of PPAR-γ in asperosaponin VI-treated microglia and inhibited the induction of Arg-1+ microglia. Blockade of PPAR-γ signaling also abolished the ability of asperosaponin VI to suppress pro-inflammatory cytokines while elevating anti-inflammatory cytokines in the hippocampus of CMS mice. The asperosaponin VI also promoted interactions between hippocampal microglia and neurons by enhancing CX3CL1/CX3CR1 and CD200/CD200R, and preserved synaptic function based on PSD95, CamKII ß and GluA levels, but not in the presence of GW9662. Blockade of PPAR-γ signaling also abolished the antidepressant effects of asperosaponin VI in the SPT, TST and FST. CONCLUSION: CMS in mice induces a pro-inflammatory microglial phenotype that causes reduced crosstalk between microglia and neuron, inflammation and synaptic dysfunction in the hippocampus, ultimately leading to depression-like behaviors. Asperosaponin VI may ameliorate the effects of CMS by inducing microglia to adopt a PPAR-γ-dependent neuroprotective phenotype.

[Thoughts and suggestions on ecological cultivation of Gastrodia elata].

Due to the special biological characteristics, Gastrodia elata suffers from high resource consumption and low utilization rate in modern agricultural production, which significantly block the green and healthy development of this industry. Based on the theory and technology in ecological cultivation of Chinese medicinal materials, this study analyzed the challenges in ecological cultivation of G. elata, such as waste of fungus material, a few cultivation modes available, continuous cropping obstacles, frequent occurrence of diseases, and poor stability of ecological structure. According to the production practice, the following suggestions were proposed for ecological cultivation of G. elata: following the principle of environmental protection and no pollution, selecting suitable habitats to yield high-quality medicinal materials, committing to green control of diseases and pests, upgrading industrial structure to maximize the benefits, establishing a sound mechanism for protecting the genetic diversity of wild G. elata, carrying out simulative habitat cultivation to improve medicinal material quality, adopting science-based planning of fungus resources to relieve forestry pressure, enhancing the recycling and utilization of fungus materials, and applying diversified cultivation modes to improve the stability of ecological structure. The result is expected to provide a reference for the quality development of G. elata industry.

[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.

[Utilization of used fungus-growing materials of Gastrodia elata].

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.

[Content changes of triterpene saponins in crude and sweated Dipsacus asper:an iTRAQ-based analysis].

The present study aimed to explore the mechanism of the sweating of Dipsacus asper on content changes of triterpene sa-ponins by detecting the total triterpene saponins and the index component asperosaponin Ⅵ in the crude and sweated D. asper, and analyzing the differentially expressed proteins by isobaric tags for relative and absolute quantification(iTRAQ) combined with LC-MS/MS. After sweating, the content of total triterpene saponins decreased manifestly, while that of asperosaponin Ⅵ increased significantly. As revealed by the iTRAQ-LC-MS/MS analysis, 140 proteins with significant differential expression were figured out, with 50 up-regulated and 90 down-regulated. GO analysis indicated a variety of hydrolases, oxido-reductases, and transferases in the differential proteins. The results of activity test on two differentially expressed oxido-reductases were consistent with those of the iTRAQ-LC-MS/MS analysis. As demonstrated by the analysis of enzymes related to the triterpene saponin biosynthesis pathway, two enzymes(from CYP450 and UGT families, respectively, which are involved in the structural modification of triterpene saponins) were significantly down-regulated after sweating. The findings suggested that sweating of D. asper presumedly regulated triterpene saponins by affecting the expression of downstream CYP450 s and UGTs in the biosynthesis pathway of triterpene saponins of D. asper.

Insight to shape of soil microbiome during the ternary cropping system of Gastradia elata.

BACKGROUND: The ternary cropping system of Gastradia elata depends on a symbiotic relationship with the mycorrhizal fungi Armillaria mellea, which decays wood to assimilate nutrition for the growth of G. elata. The composition of microbe flora as key determinants of rhizoshere and mycorrhizoshere soil fertility and health was investigated to understand how G. elata and A. mellea impacted on its composition. The next generation pyrosequencing analysis was applied to assess the shift of structure of microbial community in rhizoshere of G. elata and mycorrhizoshere of A. mellea compared to the control sample under agriculture process. RESULTS: The root-associated microbe floras were significantly impacted by rhizocompartments (including rhizoshere and mycorrhizoshere) and agriculture process. Cropping process of G. elata enhanced the richness and diversity of the microbial community in rhizoshere and mycorrhizoshere soil. Furthermore, planting process of G. elata significantly reduced the abundance of phyla Basidiomycota, Firmicutes and Actinobacteria, while increased the abundance of phyla Ascomycota, Chloroflexi, Proteobacteria, Planctomycetes, and Gemmatimonadetes in rhizoshere and mycorrhizoshere. Besides, A. mellea and G. elata significantly enriched several members of saprophytoic and pathogenic fungus (i.e., Exophiala, Leptodontidium, Cosmospora, Cercophora, Metarhizium, Ilyonectria, and Sporothrix), which will enhance the possibility of G. elata disease incidence. At the same time, the ternary cropping system significantly deterred several members of beneficial ectomycorrhizal fungus (i.e., Russula, Sebacina, and Amanita), which will reduce the ability to protect G. elata from diseases. CONCLUSIONS: In the ternary cropping system of G. elata, A. mellea and G. elata lead to imbalance of microbial community in rhizoshere and mycorrhizoshere soil, suggested that further studies on maintaining the balance of microbial community in A. mellea mycorrhizosphere and G. elata rhizosphere soil under field conditions may provide a promising avenue for high yield and high quality G. elata.

Comprehensive separation of iridoid glycosides and triterpenoid saponins from Dipsacus asper with salt-containing solvent by high-speed countercurrent chromatography coupled with recycling mode.

The roots of Dipsacus asper Wall as a commonly used traditional Chinese medicine are used for tonifying liver and kidney and strengthening bones and muscles. However, an effective separation strategy for comprehensive and rapid separation of the main active compounds from the roots of D. asper is nonexistent. This investigation provided an effective separation method based on AB-8 macroporous resin column chromatography using different ratios of ethanol in water and two different modes of high-speed countercurrent chromatography with salt-containing solvent system for rapid enrichment and separation from the roots of D. asper. The macroporous resin column chromatography was performed on AB-8 resin using ethanol in water ratios of 10, 30, 40, 50, and 80% as the optimized enrichment conditions for iridoid glycosides and triterpenoid saponins with different polarities. For high-speed countercurrent chromatography separation, the conventional and recycling modes were combined together to develop a strategy for 12 compounds (1-12) from the enriched parts of 30, 40, and 80% ethanol, including six high-polarity iridoid glycosides (1-6) using inorganic salt-containing solvent system and six triterpenoid saponins (7-12). Recycling high-speed countercurrent chromatography separation was successfully applied to separate two isomers (9 and 10) after 11 cycles.

[Ecological benefits of Gastrodia elata-Phallus impudicus sequential planting pattern].

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.

[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.

[Analysis of ecological recycling modes for Gastrodia elata cultivation].

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.

[Technical evaluation and principle analysis of simulative habitat cultivation of Dendrobium nobile].

The technique of "simulative habitat cultivation" is to preserve the quality of traditional Chinese medicine by simulating the original habitat and site environment of wild Chinese medicine resources. Dendrobium nobile is the most representative variety of traditional Chinese medicine which reflects the coordinated development of medicinal material production and ecological environment. In this paper, the main technical points of the simulated cultivation model of D. nobile were summarized as follows: rapid propagation of seedling tissue technology to ensure the genetic stability of provenance; line card+fermented cow manure+live moss method to improve the survival rate; epiphytic stone cultivation to improve the quality of medicinal materials; and the integration of mycorrhizal fungi to improve the quality stability of medicinal materials. On the basis of summarizing the ecological benefits, economical and social benefits generated by the application of the technology, the paper systematically analyzes the principle of the technology for the cultivation of D. nobile to promote the excellent quality, the light, gas, heat and fertilizer resources of the undergrowth niche are in line with the wild site environment of D. nobile. The rich and complex soil microbial community in the forest laid the foundation for the species diversity needed for the growth of D. nobile.The stress effect on the growth of D. nobile resulted in the accumulation of secondary metabolites. The symbiotic relationship between the symbiotic fungi such as bryophytes and D. nobile promotes the synthesis of plant secondary metabolites. The high quality D. nobile was produced efficiently by improving and optimizing the cultivation techniques.

Association between dipsacus saponin VI level and diversity of endophytic fungi in roots of Dipsacus asperoides.

Dipsacus asperoides contains multiple pharmacologically active compounds. The principal are saponins. The plant can be cultivated, but it contains lower levels of bioactive compounds than the plant in the wild. It may be the reason to exploit the endophytic fungi that colonize the plant roots in order to produce bioactive compounds. However, the endophytic fungi of D. asperoides have not been analyzed in detail. In this study, we isolated and identified 46 endophytic fungal strains from the taproots, lateral roots and leaves, and we used morphological and molecular biological methods to assign them into 15 genera: Fusarium sp., Ceratobasidium sp., Chaetomium sp., Penicillium sp., Aspergillus sp., Talaromyces sp., Cladosporium sp., Bionectria sp., Mucor sp., Trichoderma sp., Myrothecium sp., Clonostachys sp., Ijuhya sp., Leptosphaeria sp. and Phoma sp. Taproots contained abundant endophytic fungi, the numbers of which correlated positively with level of dipsacus saponin VI. Primary fermentation of several endophytic fungal strains from taproots showed that Fusarium, Leptosphaeria, Ceratobasidium sp. and Phoma sp. can produce the triterpenoid saponin. These results may guide efforts to sustainably produce bioactive compounds from D. asperoides.

[Analysis of heavy metals in Guizhou soil planting Eucommia ulmoides].

In order to understand the pollution of heavy metals in Guizhou soil planting Eucommia ulmoides,the paper has determined the contents of five heavy metals respectively( Cu,Cd,Pb,Hg and As) by atomic absorption spectrophotometry. The soil environmental quality by single factor pollution index was evaluated,nemerow integrated pollution index and potential ecological risk index,the correlation of 5 heavy metals was analyzed and the relationship between heavy medals content and p H value was discussed. The results showed that: ①The soils of LP,SB and ZA-2 had low heavy metals content,LP soil met the first national standard,and other soils met the second national standard; ②The soils of LP,ZA-1 and ZA-2 were all in a clean state,while HX and BZ-1 were in a light pollution state,and the pollution degree was Cu>Pb>As>Cd>Hg; ③The soil of BZ-2 was light warning( medium ecological risk),the soils of HX,BZ-1 and MT were early warning( low ecological risk),the main warning source was Cu and Pb,and the rest were no warning( no ecological risk); ④Cd and As were positively correlated with Pb and Hg,and Cd,Pb and As were positively correlated with p H. The research showed that the soils of LP,SB,ZA-1 and ZA-2 were clean and could be used for E. ulmoides to continue planting,the soils of HX,MT,BZ-1 and BZ-2 were polluted and should be treated in time by effective measures.

[Study on commodity specification and grade standard and quality evaluation of Dipsaci Radix].

Through the records of herbal and market investigations,the samples collected from different market and origin were analyzed,and the appearance character indexes were determined in order to revise the commodity specification and grade standard of Dipsaci Radix combined with production practice. This is also to analyze association of the appearance with quality different of intrinsic components. The investigation results indicated the root's long and thick was better,and atrovirens of fracture surface,there are most of the market is dominated by the ungraded goods. Through principal component analysis,variance analysis and cluster analysis,combining with the feasibility of actual operation and herbal record,the length,middle diameter and phloem color were filtrated and the specification was divided into 2 ranks: the selection and ungraded goods,and the grade was divided into 2 ranks for the selection goods: big,small selected goods. Moreover,there were no significant correlation between the appearance and the intrinsic quality index of Dipsaci Radix,the content of extractum were significantly positive correlation with the moisture,but had extremely negative correlation with the total ash. Multiple comparisons indicated that the content of the moisture,extractum and asperosaponin VI of the selected goods were higher than the ungraded goods,while the total ash content were lower,and they hasn't significant difference. The commodity specification and grade standard of Dipsaci Radix as a basis provide commodity specification and grade standard of communities and standardizing market trade order.

[Analysis of medicinal resources diversity of 33 pilots (districts) in Guizhou province].

This study is based on the data analysis of medicinal plant resources and diversity collected from the fourth Chinese traditional medicine resource survey( pilot). Through the analysis of relevant data from 33 census pioneer plots in Guizhou province( area),a total of 265 families,1 432 genera and 5 296 species of medicinal resources were reported,including algae,fungi,lichens,mosses,a total of 43 genera and 35 families,57,48 families,120 genera and 453 species of ferns,gymnosperms 11 families,22 genera and 61 species,167 families,1 243 genera and 4 721 species of angiosperms,4 genera and 4 families four medicinal animals.Compared with the data related to the third survey of traditional Chinese medicine resources,the number of ferns,gymnosperms and angiosperms in the fourth survey has increased far more than that of the third survey. From the regional distribution of medicinal resources,the composition of the genus,the type of life,and the location of the medicine,the richness of the medicinal plant resources in Guizhou province is not only reflected in many types,but also in the variety of medicinal resources. These studies provide a scientific basis for vigorously developing the Chinese herbal medicine industry and the sustainably using medicinal plant resources in Guizhou province.