Trophic relationships between protists and bacteria and fungi drive the biogeography of rhizosphere soil microbial community and impact plant physiological and ecological functions.

Rhizosphere microorganisms play a vital role in enhancing plant health, productivity, and the accumulation of secondary metabolites. Currently, there is a limited understanding of the ecological processes that control the assembly of community. To address the role of microbial interactions in assembly and for functioning of the rhizosphere soil microbiota, we collected rhizosphere soil samples from Anisodus tanguticus on the Tibetan Plateau spanning 1500 kilometers, and sequenced the bacteria, fungi, archaea, and protist communities. We observed a significant but weak distance-decay relationship in the microbial communities of rhizosphere soil. Our comprehensive analysis of spatial, abiotic, and biotic factors showed that trophic relationships between protists and bacteria and fungi predominantly influenced the alpha and beta diversity of bacterial, fungal, and protistan communities, while abiotic factors had a greater impact on archaeal communities, including soil pH, available phosphorus, total phosphorus and mean annual temperature. Importantly, microbial interactions had a more significant influence on Anisodus tanguticus physiological and ecological functions compared to individual microorganisms. Network analyses revealed that bacteria occupy a central position of the co-occurrence network and play a crucial role of connector within this community. The addition of protists increased the stability of bacterial, fungal, and archaeal networks. Overall, our findings indicate that trophic relationships play an important role in assembly and for functioning of the rhizosphere soil microbiota. Bacterial communities serve as a crucial link between different kingdoms of microorganisms in the rhizosphere community. These findings help us to fully harness the beneficial functions of rhizosphere microorganisms for plants and achieve sustainable use of biological resources.

Mycena subpiligera sp. nov., a Symbiotic Species from China Associated with the Seed Germination of Gastrodia elata.

Mycena subpiligera, a new taxon in sect. Fragilipedes that can strongly enhance the germination efficiency of Gastrodia elata seeds, was discovered in subtropical areas of China. As revealed by a morphological comparison with related Mycena species as well as maximum likelihood (ML) and Bayesian phylogenetic analyses based on sequences of the internal transcribed spacer (ITS) and the large subunit (LSU) regions of nuclear ribosomal RNA, the new taxon can be distinguished from phenotypically similar and phylogenetically related species. Optimal cultural conditions for M. subpiligera basidiomata are reported, and the germination rate of the new species is compared with that of M. citrinomarginata.

First Report of Colletotrichum fructicola Causing Anthracnose on Camellia yuhsienensis Hu in China.

Camellia yuhsienensis Hu is an endemic species from China, where is the predominant oilseed crop due to its anthracnose resistance (Kuang 2015; J. Li et al. 2020; Nie et al. 2020). In April 2019, anthracnose symptoms were observed on C. yuhsienensis in a plantation in Youxian, Zhuzhou, Hunan Province, China (113.32°E, 26.79°N). It was detected approximately 10% anthracnose incidence in 500 two-year-old plants in a 5000 m2 cultivated area. Diseased leaves showed irregular grayish brown spots with dark brown edges and dark brown undersides. Symptomatic tissues (4 to 5 mm2) were surface-disinfected for 90 s in 75% ethanol, then rinsed twice with sterile water, and finally incubated on PDA (potato dextrose agar) at 28℃ (Jiang et al. 2018). Pure cultures were obtained by the single-spore isolation method. A total of 100 fungal isolates were obtained from 85 symptomatic leaves, from which 81 had similar colony morphology. Colonies on PDA were white, fluffy and cottony, and becoming dark gray after 5 days. The character of the reverse of the colony were similar to that of the upper of the colony, but the color was darker at the same time. The isolates produced a large number of single-celled, hyaline, straight and cylindrical conidia, with 10.35 to 17.58 length × 3.46 to 5.69 µm width (x=13.61 × 4.63 µm, n = 30). The isolates were preliminarily identified as Colletotrichum spp. according to morphological features (Weir et al. 2012). Representative isolate YX2-5-2 was used for molecular identification: internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic DNA regions were amplified by PCR (Weir et al. 2012). Gene sequences were deposited in GenBank (GenBank accession no. MW398863 for ACT, MW886232 for CHS-1, MW398864 for GAPDH, MW398865 for ITS). BLAST analysis revealed that DNA sequences of YX2-5-2 at the ITS, GAPDH, ACT, and CHS-1 loci showed 100%, 99.25%, 100%, and 99.33% sequence identity, respectively to their corresponding loci in strains ZH6 (GenBank accession no. MT476840.1), ICKP18B4 (LC494274.1), YN17 (MN525804.1), and ICKG4 (LC469131.1) of C. fructicola. A Maximum Likelihood phylogenetic tree based on the combined ACT, CHS-1, ITS and GAPDH sequences revealed that the representative isolate YX2-5-2 clustered with C. fructicola. In addition, the morphological features of YX2-5-2 were similar to C. fructicola which has been reported (Weir et al. 2012). Pathogenicity was tested using isolate YX2-5-2 by inoculating leaves of 2-year-old C. yuhsienensis. Four leaves of each healthy C. yuhsienensis were sprayed with a conidial suspension (105 conidial/mL) of isolate YX2-5-2, and the above steps were repeated three times. Two additional mock-inoculated control plants were sprayed with sterilized liquid potato dextrose medium. The plants were incubated in a greenhouse at 28℃ and 90% humidity with a 12 h photoperiod. Anthracnose-like symptoms were observed 5 days post-inoculation. The control plant tissues remained healthy. C. fructicola was re-isolated on PDA from lesions, and the morphological features were consistent with YX2-5-2, confirming Koch's postulates. To our knowledge, this is the first report of anthracnose of C. yuhsienensis caused by C. fructicola in China. Anthracnose of Camellia. oleifera has been reported for a long time (H. Li et al. 2016). C. yuhsienensis, as a wild relative of C. oleifera (commonly known as tea-oil tree), has been concerned about its resistance to anthracnose. Therefore, the occurrence of C. yuhsienensis anthracnose hindered the control of anthracnose tea-oil tree. This finding will lay the foundation for studying the pathogenesis of anthracnose of tea-oil tree and developing effective prevention methods. References: Jiang, S. Q., et al. 2018. Plant Dis. 102: 674. Kuang, R. 2015. Forest Pest and Disease. Li, H., et al. 2016. PLoS One 11: e0156841. Li, J., et al. 2020. Microorganisms 8: 1385. Nie, Z., et al. 2020. Mitochondrial. DNA. B. 5: 3016. Weir, B. S., et al. 2012. Stud. Mycol. 73: 115.

First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China.

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies' undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5-20.6×3.5-6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

First Report of Colletotrichum brevisporum Causing Anthracnose of Dalbergia odorifera in China.

Dalbergia odorifera T. Chen (family Fabaceae) is one of four prized species of mahogany plant in China. In June 2017, an investigation of the condition of anthracnose was carried out on apporximately 333 hectares of D. odorifera plantations in Haikou City, Hainan Province (110.19°E, 20.03°N). Approximately 40% of D. odorifera plants had disease symptoms. Lesions on leaves were brown to grayish-white containing black dots and dark-brown borders, occasionally surrounded by a yellowish-green halo. Leaf spots generally occurred along the edge of the leaf. Severely infected leaves became withered and died. Hyphal growth was recovered from symptomatic leaf tissue, surface-sterilized with a 75% ethanol solution for 30s, rinsed with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 26°C in the dark. The representative isolate JXHTC19 was recovered by transferring a hyphal tip to a fresh PDA plate to obtain a pure culture. Fungal colonies had white aerial mycelium initially, turning pale gray after 3 days. At 7 days, colonies had a cottony appearance ranging from white to dark gray with orange masses of conidia. The colony surface was slimy and aerial mycelium was sparse. Isolates displayed single-celled, cylindrical, hyaline conidia that were rounded at both ends, and were 9.7 - 16.4 µm long (avg. 13.5 µm) × 3.6 - 6.2 µm wide (vg. 4.8 µm) (n = 100). To further identify the fungus, genomic DNA was extracted from single conidial cultures of JXHTC19. The rDNA internal transcribed spacer (ITS) region, glutamine synthetase (GS) gene, partial sequence of glyceraldeyde-3-phosphate dehydrogenase-like (GAPDH) gene, actin (ACT) gene, and beta-tubulin (TUB2) gene were amplified using the primer pairs ITS4/ITS5, GS-F/GS-R, GDF1/GDR1, ACT-512F/ACT-783R, and TUB2-T1/Bt-2b (Weir et al 2012), respectively. The results showed that the ITS, GS, GAPDH, ACT and TUB2 genes of the target strain (JXHTC19) have 100%, 95%, 100%, 97% and 98% sequence homology with C. brevisporum, respectively. The sequences obtained were deposited in GenBank (MF993572, MN737615, MN737614, MG515612, and MG515615[LJ1]). All five sequences were analyzed together with representative sequences from type or ex-type specimens of the Colletotrichum genus (Yang et al. 2011, Weir et al. 2012) and a phylogenetic tree was generated via the neighbor-joining method using MEGA6. The tree placed the isolate in the same group as C. brevisporum. Thus, both morphological and molecular characteristics identified the pathogen as C. brevisporum. To verify Koch's postulates, two-year-old leaves of healthy potted D. odorifera plants (n = 6) were inoculated with a spore suspensions of JXHTC19 that contained 105 conidia/ml. Plants were sprayed with water to serve as mock-inoculated controls [LJ2](Garibaldi et al, 2020). Six plants per treatment were used in each test. The test was repeated once.Plants were incubated in moist chambers at 26°C and monitored daily for symptom development. After five days, eleven of twelve isolates [LJ3]caused lesions on all inoculated plants, whereas no symptoms developed on the mock-inoculated controls. Koch's postulates were fulfilled by reisolating the same fungus and verifying its colony and morphological characters as C. brevisporum. To our knowledge, this is the first report of this species causing anthracnose of D. odorifera in China. Corresponding measures must be adopted to manage this disease such as reducing the planting density of D. odorifera and increasing the species diversity of undergrowth vegetation. These results could help develop better monitoring and management practices for this disease.

First Report of Fusarium fujikuroi Causing Leaf spot of Dalbergia odorifera T.Chen in China.

Dalbergia odorifera T. Chen is a national second-grade protected and one of the four famous trees in China, with high medicinal and economic value. Leaf spot disease in this plant can cause the leaves to dry up, perforate or even fall off, which affects the growth and development, and also has a great influence on its products. In May 2019, the leaf spot of Dalbergia odorifera T. Chen was found and observed in Chengmai County (N19°40', E110°0'), Hainan Province, China, and the symptomatic leaves were brought back to the laboratory for research; According to our survey at that time, the incidence of the disease was between 10% and 15%. A sterile stainless-steel scalpel was used to cut the tissues at the junction of the leaf lesions and placed on a clean bench, soaked in alcohol (75 %) for 30 s, and rinsed thrice with sterile water. Then it was inserted obliquely onto lactic acid-containing potato dextrose agar (PDA) and incubated at 28 °C for 5 days. The growing prominent colonies were singled out and re-inoculated on PDA and SNA plates. Preliminary identification was based on morphological characteristics, followed by molecular identification of strains by evaluating genes for translation elongation factor-1α(TEF-1α), beta-tubulin, mitochondrial small subunit (mtSSU)( Duan et al. 2019; Cao et al.2019; Stenglein et al.2010), and histone H3 (Jacobs, et al. 2010) . Through morphological observation, the isolate was identified as Fusarium fujikuroi. At the initial stage of growth on PDA, the strain produced a large number of white hyphae, followed by pink and purple-brown hyphae in the center of the colony which spread to the surrounding area. The microspores were abundant, colorless, elliptic or clavate, without septum or at 1-2 septate, and the size was about 3.3 to 13.5 × 1.2 to 3.2 µm. After nine days of culturing on SNA medium, few, large conidia were observed, typically sickle-like, with 3-4 septa with a size of about 20 to 40.2 × 2.3 to 4.4 µm. The identity of the strains was determined by comparing the gene sequences of TEF-1α, mtSSU, beta-tubulin and histone H3 by NCBI BLAST. The results showed that TEF-1 α (MN958396), mtSSU (MN958394), ß - tubulin (MN958395), and histone H3 (MN958397) from the target strain (jxht0302) had 100% sequence homology with F. fujikuroi (GenBank, accession numbers KF604040.1, MF984420.1, XM023575231.1, and MF356523.1 respectively). Next, the infection of D. odorifera T. Chen seedlings with and without injury was studied using a fungus block, with PDA as a control. Two days after inoculation with injury, obvious lesions were observed on the leaves, which appeared at least 5 days post- inoculation without injury, with no lesions in the control group. F. fujikuroi could be re-isolated from the leaves with lesions, but not from the control group. F. fujikuroi causes Black Rot of Macleaya cordata and maize ear rot (Yull et al.2019; Duan et al. 2019). As far as we know, this is the first report of F. fujikuroi causing leaf spot disease of D. odorifera T. Chen. Given the importance of D. odorifera T. Chen products, this disease needs more attention to tackle it.

Isolation and characterization of Bacillus subtilis strain 1-L-29, an endophytic bacteria from Camellia oleifera with antimicrobial activity and efficient plant-root colonization.

Endophytic bacteria, which are common in plant tissues, may help to control plant pathogens and enhance plant growth. Camellia oleifera, an oil-producing plant, is widely grown in warm, subtropical, hilly regions in China. However, C. oleifera is strongly negatively affected by C. oleifera anthracnose, which is caused by Colletetrichum fructicola. To find a suitable biocontrol agent for C. oleifera anthracnose, 41 endophytes were isolated from the stems, leaves, and roots of C. oleifera. Bacterial cultures were identified based on analyses of 16S rDNA sequences; most strains belonged to the genus Bacillus. The antagonistic effects of these strains on C. fructicola were tested in vitro. In total, 16 strains inhibited C. fructicola growth, with B. subtilis strain 1-L-29 being the most efficient. Strain 1-L-29 demonstrated antagonistic activity against C. siamense, C. asianum, Fusarium proliferatum, Agaricodochium camellia, and Pseudomonas syringae. In addition, this strain produced indole acetic acid, solubilized phosphate, grew on N-free media, and produced siderophores. To facilitate further microecological studies of this strain, a rifampicin-resistant, green fluorescent protein (GFP)-labeled strain, 1-L-29gfpr, was created using protoplast transformation. This plasmid had good segregational stability. Strain 1-L-29gfpr was re-introduced into C. oleifera and successfully colonized root, stem, and leaf tissues. This strain remained at a stable concentration in the root more than 20 d after inoculation. Fluorescence microscopic analysis showed that strain 1-L-29gfpr thoroughly colonized the root surfaces of C. fructicola as well as the root vascular tissues of Arabidopsis thaliana.

Traceability of Geographical Origin in Gentiana straminea by UPLC-Q Exactive Mass and Multivariate Analyses.

The root of Gentiana straminea Maxim. (Gentianaceae), is officially listed as "Qin-Jiao" in the Chinese Pharmacopoeia for the treatment of rheumatic arthritis, icteric hepatitis, constipation, pain, and hypertension. To establish the geographical origin traceability in G. straminea, its chemical profiles were determined by a UPLC-Q exactive mass spectrometer, from which 43 compounds were identified by comparing retention times and mass spectrometry. Meanwhile, a pair of isomers (loganin and secologanol) was identified by mass spectrometry based on their fragmentation pathway. A total of 42 samples from difference habitats were determined by an UPLC-Q exactive mass spectrometer and the data were assayed with multivariate statistical analysis. Eight characteristic compounds were identified to determine the geographical origin of the herb. To estimate the key characteristic markers associated with pharmacological function, the inhibiting activities of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages were examined. This finding is crucial in realizing the determination of botanical origin and evaluating the quality of G. straminea.

Efficient Accumulation and In Vitro Antitumor Activities of Triterpene Acids from Submerged Batch--Cultured Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes).

Triterpene acids are among the major bioactive constituents of lucidum. However, submerged fermentation techniques for isolating triterpene acids from G. lucidum have not been optimized for commercial use, and the antitumor activity of the mycelial triterpene acids needs to be further proven. The aim of this work was to optimize the conditions for G. lucidum culture with respect to triterpene acid production, scaling up the process, and examining the in vitro antitumor activity of mycelial triterpene acids. The key conditions (i.e., initial pH, fermentation temperature, and rotation speed) were optimized using response surface methodology, and the in vitro antitumor activity was evaluated using the MTT method. The optimum key fermentation conditions for triterpene acid production were pH 6.0; rotation speed, 161.9 rpm; and temperature, 30.1°C, resulting in a triterpene acid yield of 291.0 mg/L in the validation experiment in a 5-L stirred bioreactor; this yield represented a 70.8% increase in titer compared with the nonoptimized conditions. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and a triterpene productivity of 47.9 mg/L/day was achieved, which is, to our knowledge, the highest reported in the large-scale fermentation of G. lucidum. In addition, the mycelial triterpene acids were found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro. Chemical analysis showed that the key active triterpene acid compounds, ganoderic acids T and Me, predominated in the extract, at 69.2 and 41.6 mg/g, respectively. Thus, this work develops a simple and feasible batch fermentation technique for the large-scale production of antitumor triterpene acids from G. lucidum.

Improved Production and Antitumor Properties of Triterpene Acids from Submerged Culture of Ganoderma lingzhi.

Triterpene acids (TAs) are the major bioactive constituents in the medicinal fungus Ganoderma lingzhi. However, fermentative production of TAs has not been optimized for commercial use, and whether the TAs isolated from G. lingzhi submerged culture mycelia possess antitumor activity needs to be further proven. In this study, enhanced TA yield and productivity were attained with G. lingzhi using response surface methodology. The interactions of three variables were studied using a Box-Benhnken design, namely initial pH, dissolved oxygen (DO) and fermentation temperature. The optimum conditions were an initial pH of 5.9, 20.0% DO and 28.6 °C. These conditions resulted in a TA yield of 308.1 mg/L in a 5-L stirred bioreactor. Furthermore, the optimized conditions were then successfully scaled up to a production scale of 200 L, and maximum TA production and productivity of 295.3 mg/L and 49.2 mg/L/day were achieved, which represented 80.9% and 111.5% increases, respectively, compared with the non-optimized conditions. Additionally, the triterpene acid extract (TAE) from G. lingzhi mycelia was found to be cytotoxic to the SMMC-7721 and SW620 cell lines in vitro, and the TAE exhibited dose-dependent antitumor activity against the solid tumor sarcoma 180 in vivo. Chemical analysis revealed that the key active triterpene compounds, ganoderic acid T and ganoderic acid Me, predominated in the extract.

Population Genetic Analyses of the Fungal Pathogen Colletotrichum fructicola on Tea-Oil Trees in China.

The filamentous fungus Colletotrichum fructicola is found in all five continents and is capable of causing severe diseases in a number of economically important plants such as avocado, fig, cocoa, pear, and tea-oil trees. However, almost nothing is known about its patterns of genetic variation and epidemiology on any of its host plant species. Here we analyzed 167 isolates of C. fructicola obtained from the leaves of tea-oil tree Camellia oleifera at 15 plantations in seven Chinese provinces. Multilocus sequence typing was conducted for all isolates based on DNA sequences at fragments of four genes: the internal transcribed spacers of the nuclear ribosomal RNA gene cluster (539 bp), calmodulin (633 bp), glutamine synthetase (711 bp), and glyceraldehyde-3-phosphate dehydrogenase (190 bp), yielding 3.52%, 0.63%, 8.44%, and 7.89% of single nucleotide polymorphic sites and resulting in 15, 5, 12 and 11 alleles respectively at the four gene fragments in the total sample. The combined allelic information from all four loci identified 53 multilocus genotypes with the most frequent represented by 21 isolates distributed in eight tea-oil plantations in three provinces, consistent with long-distance clonal dispersal. However, despite evidence for clonal dispersal, statistically significant genetic differentiation among geographic populations was detected. In addition, while no evidence of recombination was found within any of the four gene fragments, signatures of recombination were found among the four gene fragments in most geographic populations, consistent with sexual mating of this species in nature. Our study provides the first insights into the population genetics and epidemiology of the important plant fungal pathogen C. fructicola.

[Community composition and diversity of endophytic fungi from roots of Sinopodophyllum hexandrum in forest of Upper-north mountain of Qinghai province].

High throughput sequencing technology is also called Next Generation Sequencing (NGS), which can sequence hundreds and thousands sequences in different samples at the same time. In the present study, the culture-independent high throughput sequencing technology was applied to sequence the fungi metagenomic DNA of the fungal internal transcribed spacer 1(ITS 1) in the root of Sinopodophyllum hexandrum. Sequencing data suggested that after the quality control, 22 565 reads were remained. Cluster similarity analysis was done based on 97% sequence similarity, which obtained 517 OTUs for the three samples (LD1, LD2 and LD3). All the fungi which identified from all the reads of OTUs based on 0.8 classification thresholds using the software of RDP classifier were classified as 13 classes, 35 orders, 44 family, 55 genera. Among these genera, the genus of Tetracladium was the dominant genera in all samples(35.49%, 68.55% and 12.96%).The Shannon's diversity indices and the Simpson indices of the endophytic fungi in the samples ranged from 1.75-2.92, 0.11-0.32, respectively.This is the first time for applying high through put sequencing technol-ogyto analyze the community composition and diversity of endophytic fungi in the medicinal plant, and the results showed that there were hyper diver sity and high community composition complexity of endophytic fungi in the root of S. hexandrum. It is also proved that the high through put sequencing technology has great advantage for analyzing ecommunity composition and diversity of endophtye in the plant.

[Dynamic study on two lignans contents in different parts of Sinopodophyllum hexandrum].

The contents of two lignans, namely 4'-demethylpodophyllotoxin and podophyllotoxin in cultivated and wild Sinopodophyllum hexandrum plants were extracted by ultrasonicaction and determined by HPLC. According to the result showed, the order of parts of cultivated plants containing 4'-demethylpodophyllotoxin from high to low is as follows: stem > root, no 4'-demethypodophyllotoxin was detected in leaves of cultivated plants; The order of parts of wild plants 4'-demethylpodophyllotoxin from high to low is as follows: lateral root > petiole > rhizome > leaf, no 4'-demethypodophyllotoxin was detected in fruit. The order of parts of cultivated and wild S. hexandrum containing podophyllotoxin from high to low is as follows: root > petiole > leaf ( > fruit). Both of the lignan contents in different parts of cultivated plant varied in a " W" curve with the changes in seasons, with the highest content in July.

[ITS sequences variation and phylogenetic analysis on 31 geographical populations of Notopterygium incisum].

In this study, 31 Notopterygium incisum populations were analyzed using ITS sequences to investigate the genetic structure. The results showed that: the ITS region ranged in size from 634 to 635 bp and base composition was with high G + C content of 57.8%. Thirty-one polymorphic sites were detected from 402 sequences of 31 populations of N. incisum, and the proportion of polymorphic sites was 4.88%, in which parsimony informative sites were up to 12. And 31 haplotypes were identified based on these polymorphic sites. Molecular variance analysis (AMOVA) indicated that high genetic differentiation (57%) existed among population, and gene flow was low (N(m) = 0.38) among populations. Phylogenetic relationships of 31 haplotypes were analyzed using NJ method with N. forbesiias an out-group. Phylogenetic analysis showed that 31 haplotypes from different populations mixed together and did not form distinct geographically separated clades.

[Soil microfauna diversity among Cunninghamia lanceolata plantations based on pyrosequencing].

In order to study the function of soil microfauna and its responses to environmental changes, we used metagenome analyses of the 18S rDNA gene region to identify differences in microfauna diversity and community structure among fifteen soil samples belonging to five different Cunninghamia lanceolate plantations. The plantations were located in Youxian County, Hunan Province in central China. The trees in these plantations were of different ages (3, 13, and 26 years) and belonged to different ecological successions (first, second, and third successions). The total dataset comprised 94922 high quality sequences with an average length of 436 bp. The dominant taxonomic groups across all samples were Chordata, Annelida, Arthropoda, Nematoda, Rotifera and Platyhelminthes with each accounting for 60.8%, 24.0%, 7.4%, 3.6%, 1.5% and 1.2% of the sequences, respectively. There were significant differences in ACE index and Shannon index among the five plantations. The lowest diversity of soil microfauna was in the 13-year old plantation of the first ecological succession. The correlation analysis showed that both ACE and available potassium concentration were negatively correlated to the Chaol index. However, there were no significant correlations between the Shannon, Simpson indices and the physical-chemical properties of soil. Overall, the Jaccard's similarity coefficient was less than 0.4 among samples at each site, and significant differences were found among plantations.

[Prediction of chlorophyll content of leaves of oil camelliae after being infected with anthracnose based on Vis/NIR spectroscopy].

The prediction model of chlorophyll content of leaves in canopies of oil camelliae under disease was explored and built by analyzing the Vis/NIR spectroscopy characteristics of oil camelliae canopies after being injected with anthracnose. Through field survey of disease index (DI), chlorophyll content and spectral data of leaves in canopies surviving different severity of disease were acquired. The first order differential of spectral data combined with moving average filter was pretreated. The prediction model of BP neural network of chlorophyll content was built by extracting sensitive wave band from spectral resample data. The results showed that with the disease being aggravated, reflection peaks and valleys of spectra of oil camelliae canopies in visible-light region vanished gradually, steep red edges from red light to near infrared leveled little by little, and reflectivity of healthy oil camelliae was far larger than that of ill ones. The sensitive wave band of absorption and reflection of chlorophyll lay in the region of 84-512, 533-565, 586-606 and 672-724 nm. The correlation coefficient r and RMSE between predictive values calculated from BP neural network using sensitive wave band as input variables and observed values was 0.9921 and 0.0458 respectively. It was therefore feasible to utilize Vis/NIR spectroscopy technology to forecast the chlorophyll content of oil camelliae after being infected with anthracnose.

[Moderate and severe sudden deafness treated with low-energy laser irradiation combined with auricular acupoint sticking].

OBJECTIVE: To test the therapeutic effect on moderate and severe sudden deafness treated with low-energy laser irradiation on acupoint and external auditory canal combined with auricular point sticking (APS) and as compared with electroacupuncture. METHODS: Two hundred and fifty-eight cases of moderate and severe sudden hearing loss were randomly divided into an observation group 1, an observation group 2 and a control group, 86 cases in each group. In three groups, 10% low molecular Dextran 500 mL were used for intravenous infusion. Based on the above treatment, the observation group 1 was treated with low-energy laser irradiation on acupoint and external auditory canal (such as Ermen (TE 21), Tinggong (SI 19) and Tinghui (GB 2)), combined with APS at Gan (liver), Shen (kidney) and Neifenmi (endorine), etc. The observation group 2 was treated with electroacupuncture at the same acupoints as those point irradiation in observation group 1. Fifteen days made one session. The therapeutic effects were evaluated after one and two sessions. RESULTS: After two sessions, The cured rate was 40.7% (35/86) in observation group 1 and 38.4% (33/86) in observation group 2,which were superior to 25.6% (22/86) in control group (both P < 0.05). Compared with one session, the therapeutic effects after two sessions were better in two observation groups (both P < 0.05), but there was no significant difference between two groups (both P > 0.05). In comparison of the improvements of frequency audiometry and auditory function, the two observation groups were better than those in control group (P < 0.05, P < 0.01), and the improvements after two sessions were better in two observation groups (both P < 0.01). CONCLUSION: Both of low-energy laser irradiation on acupoint and external auditory canal combined with APS and electroacupuncture are able to decrease frequency audiometry, improve auditory function, and the therapeutic effects are better with prolongation of treatment time. The clinical efficacy of above two the rapies on moderate and severe sudden deafness is superior remarkably to that of conventional treatment. The therapy of low-energy laser irradiation on acupoint and external auditory canal combined with APS can replace the electroacupuncture therapy in treating moderate and severe sudden deafness.

[Determination of trace determination of elements in medicinal materials of cultivated and wild Rhizoma et Radix Notopterygii vegetated in different months by flame atomic absorption spectrometry].

The contents of six trace elements Ca, Mg, Fe, Mn, Cu and Zn in the medicinal materials of cultivated and wild Rhizoma et Radix Notopterygii vegetated in different months were determined by flame atomic absorption spectrometry (FAAS) with air-acetylene flame, and the feature of trace elements contents and their seasonal dynamics were analyzed using SPSS16.0 analysis software. The recovery rate obtained by standard addition method ranged between 96.33% and 105.25%, and the relative standard deviation was 0.84%-2.98%. This showed that the method had good precision and definition. The study indicated that there are abundant microelements in medicinal materials of cultivated and wild Rhizoma et Radix Notopterygii. The contents of Ca are both highest in the medicinal materials of cultivated and wild Rhizoma et Radix Notopterygii, and the contents of Cu element are lowest; the contents order is Ca > Mg > Fe > Mn > Zn > Cu; the seasonal dynamics of Ca, Mg, Fe, Mn, Cu and Zn had obvious regularity in the medicinal materials of cultivated and wild Rhizoma et Radix Notopterygii. It is concluded that the amount of six elements in wild Rhizoma et Radix Notopterygii are more than the cultivated; the data can provide reference for artificial cultivation of wild herbs.

[Season features of mineral elements in the root of cultivated and wild Anisodus tanguticus].

The Anisodus tanguticus (Maxim.) Pascher is a Chinese traditional medicinal material and Tibet herb of local Qing-hai. The authors collected, tanguticus and wild Anisodus tanguticus (Maxim.) Pascher, and analyzed the contents of mineral elements such as K, Ca, Fe, Na and Mg by atomic absorption spetrometer, and P by 721 spectrophotometer. The results show that: samples in different places are all rich in Mg and Na while poor in P. At the same time, Ca has a negative correlation with other elements. Compared to the others, the amount of 4 elements is higher in wild Anisodus tanguticus is higher.

[Transcutaneous electrical acupoint stimulation combined with auricular acupoint sticking for treatment of primary nocturnal enuresis].

OBJECTIVE: To observe the therapeutic effect of transcutaneous electrical acupoint stimulation (TEAS) combined with auricular acupoint sticking (AAS) for treatment of primary nocturnal enuresis (PNE). METHODS: Two hundred and fifty cases of PNE patients were randomly divided into an observation group and a control group, 125 cases in each group. Two groups were treated on basis of the routine treatment, the observation group was treated with TEAS at Zhongji (CV 3), Qihai (CV 6) and Sanyinjiao (SP 6) etc. combined with AAS at Xin (heart), Shen (kidney), and Jiaogan (sympathytic) etc., once each day, 15 days constituting one course, an interval of 5 days between two courses, the therapeutic effect was evaluated after 3 courses of continuous treatment. The control group was treated with oral administration of Desmopressin Acetate (DA) and Meclofenoxate, the therapeutic effect was evaluated after 2 months of continuous treatment. RESULTS: The total effective rate of 96.8% (121/125) in observation group was superior to that of 90.4% (113/125) in control group (P < 0.05), the observation group was superior to control group in reducing the times of nocturia and enuresis, onset time and duration of effect (all P < 0.05). CONCLUSION: TEAS combined with AAS can reduce the times of nocturia and enuresis of PNE children, with significant therapeutic effect, taking quick effect and keeping long effect duration.