[Inhibitory effect of three strains of biocontrol microbes on pathogens causing rhizome rot of Polygonatum cyrtonema].

Rhizome rot is one of the main disease in the cultivation of Polygonatum cyrtonema, and it is also a global disease which seriously occurs on the perennial medicinal plants such as Panax notoginseng and P. ginseng. There is no effective control method at present. To identify the effects of three biocontrol microbes(Penicillium oxalicum QZ8, Trichoderma asperellum QZ2, and Brevibacillus amyloliquefaciens WK1) on the pathogens causing rhizome rot of P. cyrtonema, this study verified six suspected pathogens for their pathogenicity on P. cyrtonema. The result showed that Fusarium sp. HJ4, Colletotrichum sp. HJ4-1, and Phomopsis sp. HJ15 were the pathogens of rhizome rot of P. cyrtonema, and it was found for the first time that Phomopsis sp. could cause rhizome rot P. cyrtonema. Furthermore, the inhibitory effects of biocontrol microbes and their secondary metabolites on three pathogens were determined by confrontation culture. The results showed that the three tested biocontrol microbes significantly inhibited the growth of three pathogens. Moreover, the secondary metabolites of T. asperellum QZ2 and B. amyloliquefaciens WK1 showed significant inhibition against the three pathogens(P<0.05), and the effect of B. amyloliquefaciens WK1 sterile filtrate was significantly higher than that of high tempe-rature sterilized filtrate(P<0.05). B. amyloliquefaciens WK1 produced antibacterial metabolites to inhibit the growth of pathogens, and the growth inhibition rate of its sterile filtrate against three pathogens ranged from 87.84% to 93.14%. T. asperellum QZ2 inhibited the growth of pathogens through competition and antagonism, and P. oxalicum QZ8 exerted the inhibitory effect through competition. The research provides new ideas for the prevention and treatment of rhizome rot of P. cyrtonema and provides a basis for the di-sease control in other crops.

Transcriptomic and Metabolomic Approaches Deepen Our Knowledge of Plant-Endophyte Interactions.

In natural systems, plant-symbiont-pathogen interactions play important roles in mitigating abiotic and biotic stresses in plants. Symbionts have their own special recognition ways, but they may share some similar characteristics with pathogens based on studies of model microbes and plants. Multi-omics technologies could be applied to study plant-microbe interactions, especially plant-endophyte interactions. Endophytes are naturally occurring microbes that inhabit plants, but do not cause apparent symptoms in them, and arise as an advantageous source of novel metabolites, agriculturally important promoters, and stress resisters in their host plants. Although biochemical, physiological, and molecular investigations have demonstrated that endophytes confer benefits to their hosts, especially in terms of promoting plant growth, increasing metabolic capabilities, and enhancing stress resistance, plant-endophyte interactions consist of complex mechanisms between the two symbionts. Further knowledge of these mechanisms may be gained by adopting a multi-omics approach. The involved interaction, which can range from colonization to protection against adverse conditions, has been investigated by transcriptomics and metabolomics. This review aims to provide effective means and ways of applying multi-omics studies to solve the current problems in the characterization of plant-microbe interactions, involving recognition and colonization. The obtained results should be useful for identifying the key determinants in such interactions and would also provide a timely theoretical and material basis for the study of interaction mechanisms and their applications.

Endophytic fungi, host genotype, and their interaction influence the growth and production of key chemical components of Dendrobium catenatum.

To examine how host plant genotype, endophytic fungal species, and their interaction may affect growth and key chemical content and composition in an important orchid species, we assessed four Dendrobium catenatum cultivars co-cultured with three fungi previously isolated from D. catenatum. Fungal endophytes (Tulasnella sp., Leptosphaeria microscopica, and Guignardia sp.) specifically affected the growth and chemical composition of the four cultivars. Fungal infection significantly increased certain growth traits, especially mid-stem thickness, stem biomass, stem polysaccharide and ethanol-soluble extractive content, and leaf flavonoid and phenol content. Presence or abundance of some key chemical components was also altered by fungal treatment. These increases and alterations were highly dependent on the host genotype. The findings of this study contribute to our understanding of Dendrobium and endophytic fungi interactions, and provide vital information for improving the development and use of endophytic fungi in D. catenatum breeding.

[Occurrence regularity of Dendrobium catenatum southern blight disease].

In order to scientifically prevent and control Dendrobium catenatum southern blight disease,the main factors related to this disease occurrence,the pathogen( Sclerotium delphinii),environmental factors( temperature and humidity) and D. catenatum germplasms,were investigated. The results showed that reaching 25-30 ℃ temperature and over 95% humidity simultaneously should be the main conditions for the occurrence and prevalence of D. catenatum southern blight disease. Moreover,the S. delphinii-infected plants and their contaminated substrates were the disease spreading sources. Therefore,removing the infected plants,dealing with the contaminated substrates,keeping air ventilation,and reducing air humidity are the effective ways to prevent and control the occurrence and prevalence of D. catenatum southern blight disease. The research also indicated that D. catenatum has different resistances to the southern blight disease depending on germplasm. The present study lays important foundations for the breeding of D. catenatum diseaseresistant varieties and the further analysis of the infection and resistance mechanisms underlying southern blight disease.

[Effects of germplasm and host tree trunk on endophytic fungal communities in epiphytic Dendrobium catenatum].

In order to investigate the effects of germplasm and host tree trunk on endophytic fungal communities in epiphytic Dendrobium catenatum, a total of 3 835 isolates were recovered from roots, stems and leaves of four D. catenatum germplasms attached to one kind of host tree trunk and one germplasm attached to four kinds of epiphyte-host tree trunks. A total of 152 taxa were identified and classified based on the fungal cultural characteristics and phylogenetic analyses of ITS sequences. The taxa were assigned to 60 genera, 35 families, 21 orders and 5 classes of 2 phyla. The results indicated that D. catenatum cultivated in stereo cultivation harbor variety of fungi. The dominant fungal groups were different between Lin'an and Yiwu. Moreover, several groups showed geographical specificity, such as Arthrinium, Coniochaeta, Fusarium, Neofusicoccum and Zopfiella only dominating in Panshan of Lin'an, while Alternaria, Bjerkandera, Cercophora, Nigrospora and Trichoderma only dominating in Shangxi of Yiwu. There was no significant difference in diversity or species richness of endophytic fungi neither among germplasm nor host tree trunk. However, the richness and diversity indices exhibited a strong dependence on tissue type (P<0.05). The germplasm and host tree trunk impact the distribution patterns of endophytic fungi less than tissue type. Nevertheless, the relative frequencies of the dominant fungal groups were different among germplasms or host tree trunk types. Furthermore, there were some fungal species specific to certain germplasm or host tree trunk. This might be due to the distinctions in growth traits and chemical compositions of D. catenatum owning to the differences in D. catenatumgenetic background and microenvironment of host tree. Most of fungal taxa exhibit tissue specificity or preference. These results provide the basis for the study on the relationship between endophytic fungi and D. catenatum in stereo cultivation mode.

[Effects of cultivation environments on Dendrobium catenatum].

The study was aimed to clarify the effect of three cultivation environments on the growth and metabolism of Dendrobium catenatum C13 group. There were three different cultivation conditions including rock epiphytic cultivation, pear epiphytic cultivation and pot cultivation. Morphological characteristics and agronomic characters of D. catenatum were observed and measured. Microstructure, contents of polysaccharide and alcohol-soluble extracts were measured by paraffin section method, phenol-sulfuric acid method and hot-dip method, respectively. The result showed that the cultivation environment significantly affected the growth of D. catenatum, the leaves of D. catenatum that cultivated on the rock and pear were sparse and small, the stems were short and purple and the root system was developed. Compare with potted cultivation, D. catenatum from rock epiphytic cultivation and pear epiphytic cultivation showed the following characteristics in the microstructure: the upper epidermis became thicker, the epidermal hair in the epidermis became denser, stomatal showed smaller and denser, the cell wall of exodermis, endoderm and medulla became thicker, the cell of velamen, exodermis, endoderm and medulla were smaller and arranged more closely, but the cultivation environment did not produce specific tissue structure, mainly changed in the structural parameters of size and quantity. The growth environments also influenced contents of polysaccharides and alcohol-soluble extracts. The dontents of polysaccharides and alcohol-soluble extracts in D. catenatum from rock epiphytic were the highest, reached 37.34% and 11.66%, the second was pear epiphytic, both higher than pot cultivation, alcohol-soluble extracts contents in D. catenatum from rock epiphytic are more complex, which shows that rock epiphytic is conducive to the accumulation of secondary metabolites in D. catenatum.

[Ecological basis of epiphytic Dendrobium officinale growth on cliff].

In order to make Dendrobium officinale return to the nature, the temperature and humidity in whole days of the built rock model with different slopes and aspects in the natural distribution of wild D. officinale in Tianmu Mountain were recorded by MH-WS01 automatic recorder. The results showed that the slope has a significant impact on the extreme temperature on the surface of the rocks. In summer, the extreme temperature on the surface of horizontal or soft rock can reach to 69.4 ℃, while the temperatures were lower than 50 ℃ on the vertical rock. In winter, the temperatures on the surface of vertical rock were higher and the low temperature duration was shorter than those on the horizontal or soft rock. Also, the humidity of the rocks was significantly influenced by the slope. The monthly average humidity on the surface of vertical rock was above 80%RH. Furthermore, the aspect had a significant impact on the temperature and humidity on the surface of the rocks, but had no significant effect on the daily mean temperature and extreme temperature on the surface of vertical rock. Therefore, the slope affects the survival of D. officinale by affecting the extreme temperature of rocks and affects the growth of D. officinale by affecting the humidity. The choice of slope is the key to the success of cliff epiphytic cultivation for D. officinale.

[Effects of endophytic fungi from Dendrobium officinale on host growth and components metabolism of tissue culture seedlings].

The paper aims to study the effects of endophytic fungi from D. officinale cultivated on living trees on growth and components metabolism of tissue culture seedlings. Morphological characteristics and agronomic characters of tissue culture seedlings infected and uninfected by endophytic fungus were observed and measured. Polysaccharides and alcohol-soluble extracts contents were determined by phenol-sulfuric acid method and hot-dipmethod, respectively. Monosacchride composition of polysaccharides and alcohol-soluble extracts components were analyzed by pre-column derivatives HPLC and HPLC method, respectively. It showed that effects of turning to purple of stem nodes could be changed by endophytic fungus. Besides, the endophytic fungus could affect the contents and constitutions of polysaccharides and alcohol-soluble extracts. The strains tested, expect DO34, could promote growth and polysaccharides content of tissue culture seedlings. The strains tested, expect DO12, could promote the accumulation of mannose. Furthermore, DO18, DO19 and DO120 could increase alcohol-soluble extracts. On the basis, four superior strains were selected for mechanism research between endophytic fungus and their hosts and microbiology engineering.

[Screening of meaningful endophytic fungi in Dendrobium officinale based on polysaccharides and flavonoids].

To reveal the relationship between endophytic fungi and the functional components, saccharides and flavonoids in the mycelia or fermented liquor of 21 endophytic fungi in D.officinale were detected using HPLC and UV spectrophotometer.The results showed that the ethyl acetate extracts from 21 fungal strains all contain flavonoids.According to the chromatographic retention time of HPLC and UV spectra characteristics of flavonoids, strain DO49 was found produce naringenin, strains DO23, DO81 and DO83 were found produce rutin.The water-soluble extracts from 21 strains all had polysaccharides.However, there was difference in the composition of monosaccharides derived from polysaccharides among different strains.According to the composition of monosaccharides and the peak area ratio of mannose and glucose, the fungal strains including DO23, DO26, DO81, DO54, DO55, DO83 product polysaccharides associated with D.officinale were selected.In conclusion, based on the saccharides and flavonoids, the excellent endophytic fungal strains DO23, DO81 and DO83 were selected, which could produce the same flavonoids and similar polysaccharides in D.officinale.

Cytotoxic and Antifungal Constituents Isolated from the Metabolites of Endophytic Fungus DO14 from Dendrobium officinale.

Two novel cytotoxic and antifungal constituents, (4S,6S)-6-[(1S,2R)-1, 2-dihydroxybutyl]-4-hydroxy-4-methoxytetrahydro-2H-pyran-2-one (1), (6S,2E)-6-hydroxy-3-methoxy-5-oxodec-2-enoic acid (2), together with three known compounds, LL-P880γ (3), LL-P880α (4), and Ergosta-5,7,22-trien-3b-ol (5) were isolated from the metabolites of endophytic fungi from Dendrobium officinale. The chemical structures were determined based on spectroscopic methods. All the isolated compounds 1-5 were evaluated by cytotoxicity and antifungal effects. Our present results indicated that compounds 1-4 showed notable anti-fungal activities (minimal inhibitory concentration (MIC) ≤ 50 µg/mL) for all the tested pathogens including Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, Aspergillus fumigatus. In addition, compounds 1-4 possessed notable cytotoxcities against human cancer cell lines of HL-60 cells with the IC50 values of below 100 µM. Besides, compounds 1, 2, 4 and 5 showed strong cytotoxities on the LOVO cell line with the IC50 values were lower than 100 µM. In conclusion, our study suggested that endophytic fungi of D. officinale are great potential resources to discover novel agents for preventing or treating pathogens and tumors.

[Study on accumulation of polysaccharides and alcohol-soluble extracts contents of Dendrobium officinale leaves].

This paper revealed the accumulation regularity of polysaccharides and alcohol-soluble extracts contents of Dendrobium officinale leaves, which have provided basis for the development and utilization of the leaves. The polysaccharides and alcohol-soluble extracts contents of three D. officinale strains leaves collected in different growing periods were determined by phenol-sulfric acid method and hot-dip method respectively. The results showed that the content of polysaccharides in leaves was 4.45% -12.17%, and was about a quarter in stems. The alcohol-soluble extracts content in leaves was 7.45% - 29.34%, and was 1.5 times that of stems. The quality variation of polysaccharides in leaves was closely related to the phenophase. The leaves with lower level of metabolism in three stages: winter, early germination stage and deciduous period, which led to lower content of polysaccharides. The leaves at the vigorous growth stage with higher content of polysaccharides. The alcohol-soluble extracts were closely associated to the formation and germination of buds. The content of alcohol-soluble extracts peaked before sprout, and promoted the growth of new shoots.

[Effect of strains and parts on amino acids of Dendrobium officinale].

The aim of the paper is to reveals the variations of Dendrobium officinale amino acids in different strains and parts for breeding excellent varieties, and providing scientific basis for the expanding of medicinal or edible parts. The contents of 17 amino acids in 11 strains of D. officinale were determined by hydrochloric acid hydrolysis method. The total amino acids content of leaves was from 6.76 to 7.97 g per 100 g, and the stems was from 1.61 to 2.44 g per 100 g. As the content of amino acids in leaves was significantly higher than that of stems, and the composition was close to the ideal protein standard proposed by FAO/WHO. The leaves of D. officinale had the good prospect for the development of functional foods. The 9 x 66 strain which with high yield and polysaccharide content had the highest amino acids content both in stems and leaves, indicated crossbreeding could improve the quality of varieties. Compared the amino acids content of D. officinale in two main harvest periods, the harvest time has a significant impact on amino acids content of D. officinale. This study demonstrates that the harvesting time of D. officinale stems is suitable for leaves as well, which is the period before bolssom.

[Variation of polysaccharides and alcohol-soluble extracts content of Dendrobium officinale].

To reveal the variation of polysaccharides and alcohol-soluble extract contents of Dendrobium officinale, the polysaccharides and alcohol-soluble extracts contents of three D. officinale strains were determined by phenol-sulfuric acid method and hot-dip method, respectively. The results showed that the contents of polysaccharides and alcohol-soluble extracts and their total content were significantly different among D. officinale samples collected in different periods, and the variations were closely related to the phenology of D. officinale. Additionally, the quality variation of polysaccharides was closely related to the flowering of D. officinale, while the alcohol-soluble extracts was closely associated to the formation and germination of buds. According to the dynamic variation of these two compounds, it is more reasonable to harvest D. officinale at biennials pre-bloom than at specific harvesting month considering polysaccharides content. It is better to harvest before the germination of buds considering alcohol-soluble extracts. While with regards to both polysaccharides and alcohol-soluble extract, it is better to harvest this plant at the period from the sprouting to pre-bloom next year.

[Effects of post-harvest processing and extraction methods on polysaccharides content of Dendrobium officinale].

OBJECTIVE: To reveal the quality variation of polysaccharide in Dendrobium officinale by post-harvest processing and extraction methods, and provide a basis for post-harvest processing and clinical and hygienical applications of Tiepifengdou (Dendrobii Officinalis Caulis). METHOD: The content of polysaccharides were studied by 4 post-harvest processing methods, i. e. drying by drying closet, drying after scalding by boiling water, drying while twisting, and drying while twisting after scalding by boiling water. And a series of temperatures were set in each processing procedure. An orthogonal test L9 (3(4)) with crushed degrees, solid-liquid ratio, extraction time and extraction times as factors were designed to analyze the dissolution rate of polysaccharides in Tiepifengdou processed by drying while twisting at 80 degrees C. RESULT: The content of polysaccharides was ranged from 26.59% to 32.70% in different samples processed by different processing methods, among which drying while twisting at 80 degrees C and 100 degrees C respectively were the best. Crushed degree was the most important influence on the dissolution rate of polysaccharides. The dissolution rate of polysaccharides was extremely low when the sample was boiled directly without crushing and sieving. CONCLUSION: Drying while twisting at 80 degrees C was the best post-harvest processing method, which can help to dry the fresh herbs and improve the accumulation of polysaccharides. Boiling the uncrushed Tiepifengdou for a long time as traditional method could not fully extract polysaccharides, while boiling the crushed Tiepifengdou can efficiently extract polysaccharides.

Cytotoxic metabolites from Perenniporia tephropora, an endophytic fungus from Taxus chinensis var. mairei.

Based on bioactivity-oriented isolation, the EtOAc extract of a culture broth of the endophytic fungus Perenniporia tephropora Z41 from Taxus chinensis var. mairei, with strong anti-Pyricularia oryzae activity, afforded a new sesquiterpenoid, perenniporin A (1), together with three known compounds, ergosterol (2), rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]genfuran-5-ol (3), and albicanol (4). Their structures were elucidated by means of spectroscopic methods. All the isolated compounds and the EtOAc extract of P. tephropora Z41 (EPT) were evaluated for their cytotoxic activity against three human cancer cell lines (HeLa, SMMC-7721, and PANC-1). EPT demonstrated significant cytotoxicity with IC(50) values ranging from 2 to 15 µg/mL. Compound 2 was the most cytotoxic constituent against the tested cell lines with IC(50) values of 1.16, 11.63, and 11.80 µg/mL, respectively, while compounds 1, 3, and 4 exhibited moderate cytotoxicity with IC(50) values ranging from 6 to 58 µg/mL. We conclude that the endophytic fungus P. tephropora is a promising source of novel and cytotoxic metabolites.

[Field experiment of F1 generation and superior families selection of Dendrobium officinale].

Based on randomized block design of experiment, agronomic traits and yields of 14 F1 generations of Dendrobium officinale were determined. The results showed that the differences in agronomic traits and yields among families were significant, and the hybrid vigor was obvious. Families of 6b x 2a, 9 x 66 and 78 x 69 were selected with the remarkable superiority of yields, agronomic traits and product customization. Correlation analysis between agronomic traits and yields showed that plant height, stem diameter, leaf number, blade length and blade width were all significantly correlated with biological yields and economic yields. Among which, stem diameter, leaf number and blade length were the most significant, and an optimal linear regression model could be established. When the number of shoots was fewer than 4.5, both biological yields and economic yields increased with the increasing number of shoots, but it could not much affect yields when the number of shoots was larger than 4.5. Shoots number, stem diameter and leaf index were basic stability when compared biennial traits to annual, which could be used for early selection.

[Quantitive variation of polysaccharides and alcohol-soluble extracts in F1 generation of Dendrobium officinale].

Using phenol-sulfuric acid method and hot-dip method of alcohol-soluble extracts, the contents of polysaccharides and alcohol-soluble extracts in 11 F1 generations of Dendrobium officinale were determined. The results showed that the polysaccharides contents in samples collected in May and February were 32.89%-43.07% and 25.77%-35.25%, respectively, while the extracts contents were 2.81%-4.85% and 7.90%-17.40%, respectively. They were significantly different among families. The content of polysaccharides in offspring could be significantly improved by hybridization between parents with low and high polysaccharides contents, and the hybrid vigor was obvious. Cross breeding was an effective way for breeding new varieties with higher polysaccharides contents. Harvest time would significantly affect the contents of polysaccharides and alcohol-soluble extracts. The contents of polysaccharides in families collected in May were higher than those of polysaccharides in families collected in February, but the extracts content had the opposite variation. The extents of quantitative variation of polysaccharides and alcohol-soluble extracts were different among families, and each family had its own rules. It would be significant in giving full play to their role as the excellent varieties and increasing effectiveness by studying on the quantitative accumulation regularity of polysaccharides and alcohol-soluble extracts in superior families (varieties) of D. officinale to determine the best harvesting time.