Genome-Wide Identification and Characterization of the PHT1 Gene Family and Its Response to Mycorrhizal Symbiosis in Salvia miltiorrhiza under Phosphate Stress.

Phosphorus (P) is a vital nutrient element that is essential for plant growth and development, and arbuscular mycorrhizal fungi (AMF) can significantly enhance P absorption. The phosphate transporter protein 1 (PHT1) family mediates the uptake of P in plants. However, the PHT1 gene has not yet been characterized in Salvia miltiorrhiza. In this study, to gain insight into the functional divergence of PHT1 genes, nine SmPHT1 genes were identified in the S. miltiorrhiza genome database via bioinformatics tools. Phylogenetic analysis revealed that the PHT1 proteins of S. miltiorrhiza, Arabidopsis thaliana, and Oryza sativa could be divided into three groups. PHT1 in the same clade has a similar gene structure and motif, suggesting that the features of each clade are relatively conserved. Further tissue expression analysis revealed that SmPHT1 was expressed mainly in the roots and stems. In addition, phenotypic changes, P content, and PHT1 gene expression were analyzed in S. miltiorrhiza plants inoculated with AMF under different P conditions (0 mM, 0.1 mM, and 10 mM). P stress and AMF significantly affected the growth and P accumulation of S. miltiorrhiza. SmPHT1;6 was strongly expressed in the roots colonized by AMF, implying that SmPHT1;6 was a specific AMF-inducible PHT1. Taken together, these results provide new insights into the functional divergence and genetic redundancy of the PHT1 genes in response to P stress and AMF symbiosis in S. miltiorrhiza.

Crosstalk between H2O2 and Ca2+ signaling is involved in root endophyte-enhanced tanshinone biosynthesis of Salvia miltiorrhiza.

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.

Different techniques reveal the difference of community structure and function of fungi from root and rhizosphere of Salvia miltiorrhiza Bunge.

Fungi have essential functions in plant health and performance. However, the plant-associated functions of many cultured fungi have not been established in detail. Here, the fungal species diversity in Salvia miltiorrhiza roots and rhizosphere was assessed for the first time using culturomics and high-throughput sequencing. We present a comprehensive functional metagenomic analysis of these fungi and verified activity of cellulase and chitinase predicted in the metagenomic analysis. We first collected and cultured fungi from the root and rhizosphere of S. miltiorrhiza. We found 92 species across 37 families and five phyla, with Ascomycota being dominant. Many rDNA internal transcribed spacer sequences could not be assigned to lower taxonomic levels. There were 19 genera of endophytic fungi and 37 genera of rhizosphere fungi. The culturomics approach had lower taxonomic diversity than high-throughput sequencing, but some fungi were only found in cultures. Structural analyses indicated that the dominant species differed in cultured and non-cultured samples at other levels, apart from the phylum level. Functional analysis mapped 223 carbohydrate enzyme families and 393 pathways in the CAZy and KEGG databases, respectively. The most abundant families were glycoside hydrolases and those involved in carbohydrate metabolism. As predicted by metagenomics, we experimentally verified cellulase and chitinase activity for 29 and 74 fungi, respectively. We provide the first evidence of biomass recycling by fungi that are associated with plants. Culturing is essential to reveal the hidden microbial community and critical functions in plant-microbe interactions.

CuO, ZnO, and γ-Fe2O3 nanoparticles modified the underground biomass and rhizosphere microbial community of Salvia miltiorrhiza (Bge.) after 165-day exposure.

To investigate whether metal oxide nanoparticles exhibit toxicity or positive effects on medicinal plants, CuO, ZnO, and γ-Fe2O3 nanoparticles (NPs), at concentrations of 100 and 700 mg kg-1, were introduced into the cultivation of Salvia miltiorrhiza (Bge.). Metal elemental contents, chemical constituents, biomass and the structure of the rhizosphere microbial community was used to estimate this effect. The results indicated CuO NPs increased the Cu content and ZnO NPs increased the Zn content significantly as exposure increased, γ-Fe2O3 NPs had no significant effect on Fe content in S. miltiorrhiza roots, while 100 mg kg-1 ZnO and CuO NPs significantly decreased the Fe content in roots. Additionally, ZnO and γ-Fe2O3 NPs increased the underground biomass, and diameter of S. miltiorrhiza roots. However, these three metal oxide nanoparticles had no significant effect on total tanshinones, while the 700 mg kg-1 γ-Fe2O3 NPs treatment increased salvianolic acid B content by 36.46%. High-throughput sequencing indicated at 700 mg kg-1 ZnO NPs, the relative abundance of Humicola (Zn superoxide dismutase producer), was notably increased by 97.46%, and that of Arenimonas, Thiobacillus and Methylobacillus (taxa related to heavy metal tolerance) was significantly increased by 297.14%, 220.26% and 107.00%. The 700 mg kg-1 CuO NPs exposure caused a significant increase in the relative abundances of Sphingomonas (a copper-resistant and N2-fixing genus) and Flavisolibacter (stripe rust biocontrol bacteria) by 127.32% and 118.33%. To our best knowledge, this is the first study to examine the potential impact of NPs on the growth and rhizosphere microorganisms of S. miltiorrhiza.

Endophytic fungus Mucor circinelloides DF20 promote tanshinone biosynthesis and accumulation in Salvia miltiorrhiza root.

As a traditional Chinese medicine, Salvia miltiorrhiza rhizome is mainly used to treat cardiovascular diseases. Symbiosis of endophytic fungi with their host plants, is an effectively regulatory means to promote the growth and secondary metabolism of medicinal plants. Here, an endophytic fungus Mucor circinelloides DF20 was co-cultivated with the sterile seedlings of S. miltiorrhiza, to clarify the promoting mechanism on tanshinone biosynthesis and accumulation in S. miltiorrhiza root. The assay of promoting-growth activities in vitro showed that DF20 have the ability to produce IAA and siderophores. DF20 could significantly promote the biosynthesis and accumulation of tanshinones in the root of S. miltiorrhiza, especially the content of tanshinone ⅡA, reaching 4.630 ± 0.342 mg/g after 56 days of DF20 treatment, which is 22-fold of the control group. The result also showed that the hyphae of M. circunelloides DF20 mainly colonized in the root tissue interspace of S. miltiorrhiza, and a small amount of hyphae were located inside the cells. The results of florescent real-time quantitative RT-PCR showed that DF20 colonization significantly increase the expression level of some key enzyme genes (DXS, DXR, HMGR, GGPPS) in tanshinone biosynthesis pathway, but the regulatory effect mainly occurred in the early stage of co-culture, while the expression level decreased in different degrees in the later stage. In conclusion, the endophytic fungus M. circunelloides DF20 can form an interaction relationship with its host, then to promote the biosynthesis and accumulation of tanshinones in root by upregulating the key enzyme genes expression levels of the biosynthesis pathway.

Salchaetoglobosins A and B: Cytochalasan alkaloids from Chaetomium globosum D38, a fungus derived from Salvia miltiorrhiza.

Chemical investigation on the solid rice culture of Chaetomium globosum D38, an endophytic fungus derived from Salvia miltiorrhiza, has afforded two new 19,20-seco-chaetoglobosins, salchaetoglobosins A (1) and B (2), along with three known analogues, chaetoglobosins E (3), Fex (4), and Vb (5). Their structures and absolute configurations were elucidated by a set of spectroscopy and single-crystal X-ray crystallography. Compounds 1-5 were evaluated for their cytotoxic activities against HCT-116 (colorectal carcinoma) and PC3 (prostate cancer) cells, as well as the NO production inhibitory effects in LPS-stimulated RAW264.7 cells.

Foetidibacter luteolus gen. nov., sp. nov., isolated from the rhizosphere of Salvia miltiorrhiza.

A polyphosphate-producing bacterium, YG09T, was isolated from the rhizosphere of Salvia miltiorrhiza. Its colonies were 2.0-3.0 mm in diameter, smooth, circular, convex and yellow after growth on R2A at 28 °C for 72 h, with aerobic, Gram-stain-negative, non-motile and rod-shaped bacteria. The strain was found to grow at 10-40 °C (optimum 37 °C), pH 5.5-8.0 (optimum 6.0), with 0-0.6% (w/v) NaCl (optimum 0). Chemotaxonomic analysis showed menaquinone-7 as the only quinone present; C15: 1 iso G, C15: 1 iso, C16: 0, C16: 0 3OH, C17: 0 iso 3OH, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) as the major fatty acids (> 5%), and phosphatidylethanolamine, three unidentified phospholipids, four unidentified polar lipids, three unidentified aminolipids, and one unidentified amino phospholipid as the polar lipids. The DNA G + C content was 44.6 mol%. The 16S rRNA gene sequences of the isolate showed highest similarities to Panacibacter ginsenosidivorans Gsoil 1550T (93.6%), Filimonas endophytica SR2-06T (93.4%), Parasegetibacter terrae SGM2-10T (92.8%), and Arvibacter flaviflagrans C-1-16T (92.7%), within the family Chitinophagaceae of the phylum Bacteroidetes. The ANI values between strain YG09T and Panacibacter ginsenosidivoran Gsoil 1550T, Filimonas endophytica SR2-06T and Filimonas lacunae YT21T were 69.4, 68.3 and 68.7%, respectively. Based on phenotypic, genotypic and phylogenetic analyses, strain YG09T represents a novel genus in the family Chitinophagaceae, for which the name Foetidibacter luteolus gen. sp. nov. is proposed. The type strain is Foetidibacter luteolus YG09T (= MCCC 1K04042T = KCTC 72595T).

Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

A new fusicoccane diterpene and a new polyene from the plant endophytic fungus Talaromyces pinophilus and their antimicrobial activities.

A new fusicoccane diterpene, pinophicin A (1), and a new polyene, pinophol A (2), were isolated from the plant endophytic fungus Talaromyces pinophilus obtained from the aerial parts of Salvia miltiorrhiza. The structures and relative configurations of 1-2 were determined by the analysis of extensive spectroscopic data, chemical method, and comparison with known compounds. Compound 2 exhibited weak antibacterial activity against Bacterium paratyphosum B with an MIC value of 50 µg/mL.

[Promoting tanshinone synthesis of Salvia miltiorrhiza root by a seed endophytic fungus, Phoma herbarum D603].

The interaction of endophytes and host plant is an effective mean to regulate the growth and secondary metabolism of medicinal plants. Here we want to elucidate the effects and mechanism of Phoma herbarum D603 on the root development and tanshinone synthesis in root of Salvia miltiorrhiza by endophyte-plant coculture system. The mycelium of P. herbarum D603 was colonized in the root tissue space, and formed a stable symbiotic relationship with host plant. The in vitro activities analysis showed that the concentration of IAA produced by D603 can reach(6.45±0.23) µg·mL~(-1), and this strain had some abilities of phosphorus solubilization and siderophore production activities. The coculture experiment showed that strain D603 can significantly promote the synthesis and accumulation of tanshinones in the root of S. miltiorrhiza, in which after 8 weeks of treatment with D603, the content of tanshinone Ⅱ_A in the roots reached up to(1.42±0.59) mg·g~(-1). By the qRT-PCR analysis results, we found that D603 could improve the expression levels of some key genes(DXR, DXS, GGPP, HMGR, CPS) of tanshinone biosynthesis pathway in host plant S. miltiorrhiza, but the promoting effect mainly occurred in the early stage of the interaction, and the enzyme activity level decreased in varying degrees of the later stage. In summary, seed-associated endophyte P. herbarum D603 can promote the growth and root development of S. miltiorrhiza by producing hormones, promoting nutrient absorption and siderophore production, and promote the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway in S. miltiorrhiza.

Two novel eremophylane acetophenone conjugates from Colletotrichum gloeosporioides, an endophytic fungus in Salvia miltiorrhiza.

Two novel eremophylane acetophenone conjugates, colletotricholides A (1) and B (2), were isolated from the solid fermentation cultures of an endophytic fungus Colletotrichum gloeosporioides XL1200 isolated from the aerial parts of Salvia miltiorrhiza. The chemical structures of 1-2 were characterized by extensive spectroscopic methods and single-crystal X-ray crystallography. Structurally, compounds 1-2 are two unusual eremophylane acetophenone conjugates originating from the hybrid pathways of polyketide synthase and sesquiterpene synthase. In addition, compounds 1-2 were inactive against tested pathogens.

Transcriptome sequencing of Salvia miltiorrhiza after infection by its endophytic fungi and identification of genes related to tanshinone biosynthesis.

Context: Salvia miltiorrhiza Bunge (Labiatae) is a traditional Chinese herb. Endophytic fungi, which are biotic elicitors, can induce accumulation of secondary metabolites in their host plants.Objective: To analyze the interaction mechanism between S. miltiorrhiza and endophytic fungi.Materials and methods: Endophytic fungi U104 producing tanshinone IIA were isolated from the healthy disease-free tissue of root of S. miltiorrhiza by conventional methods. The endophytic fungus U104 of S. miltiorrhiza was co-cultured with the sterile seedlings of S. miltiorrhiza for 20 d (temp:day/night = 26 °C/18 °C, photoperiod:12/12 h, illuminance:2000 Lx). Transcriptome sequencing of S. miltiorrhiza seedlings after 20 d of co-cultivation was performed using the Illumina platform.Results: A total of 3713 differentially expressed genes (DEGs) were obtained. These different expression genes, such as STPII, LTP2, MYB transcription factors, CNGC, CDPK, Rboh, CaM, MAP2K1/MEK1, WRKY33, SGT1/SGT and Hsp90/htpG, showed that host S. miltiorrhiza had biological defence response in the initial stage of interaction. Under the induction of endophytic fungi, 14 key enzyme genes were up-regulated in the tanshinone biosynthesis pathway: DXS, DXS2, DXR, HMGR3, AACT, MK, PMK, GGPPS2, GPPS, KSL, IDI, IPII, FDPS and CPS.Discussion and conclusions: A total of 14 key genes were obtained from the tanshinone component synthesis and metabolic pathways, providing a reasonable explanation for the accumulation of tanshinone components, an accumulation induced by endophytic fungi, in the host plants. The large amounts of data generated in this study provide a strong and powerful platform for future functional and molecular studies of interactions between host plants and their endophytic fungi.

A label-free immunoassay protocol for aflatoxin B1 based on UV-induced fluorescence enhancement.

As one of the most toxic chemical carcinogens, aflatoxin B1 (AFB1) has attracted extensive attention due to its severe impairment to human health. There exists urgent demand to develop facile and sensitive method for rapid screening of AFB1. Here magnetic beads modified with mouse monoclonal antibody (McAb) were adopted for capture and enrichment of the mycotoxin in sample matrix. Then UV radiation at 365 nm was utilized to induce the enhancement of fluorescent (FL) emission of the captured AFB1 with an addition reaction. The FL signal of the derivative at 435 nm was collected to quantify AFB1. The immunoassay method for AFB1 showed a wide detection range of 1.0-1000 ng mL-1, with a low detection limit of 0.21 ng mL-1 (3σ). It was applied to detect AFB1 in herbal medicines including Astragalus membranaceus and Salvia Miltiorrhiza, with acceptable recovery values of 95.4-107.7%. It shows many merits including facile manipulation, low cost, high sensitivity and ideal selectivity. Due to its simple detection mechanism, the UV-induced FL derivatization-based label-free immunoassay can be furtherly extended to detection of other mycotoxins with similar chemical structures.

[Community structure and diversity of soil bacteria in rhizosphere of Salvia miltiorrhiza and Salvia miltiorrhiza f. alba based on pyrosequencing].

Rhizosphere bacteria play a vital role in plant nutrition absorption,growth and disease resistance. In this study,high-throughput sequencing technology was used to analyze the rhizosphere bacterial communities of Salvia miltiorrhiza and S. miltiorrhiza f. alba. Moreover,the function of dominant rhizosphere bacterial communities was analyzed. We found that Sphingobacteriales,Sphingomonadales and Nitrosomonadaceae were both dominant and specific bacteria in the rhizosphere of S. miltiorrhiza. The main functions of dominant rhizosphere bacteria communities in both species include promoting transformation of soil nutrients,improving plant immunity and ability of stress tolerance. This study was the first to compare rhizobacterial communities structure and function of S. miltiorrhiza and S. miltiorrhiza f. alba,which provided a new theoretical reference for studing the rhizosphere mechanism of healthy S. miltiorrhiza planting in the future.

[Effect of soil pH on efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza].

By comparing the effects of soil pH on the efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza， the study is aimed to provide guidance for the use of mycorrhiza in the cultivation of S. miltiorrhiza. In this experiment, the inoculant treated and the non-inoculant treated control were grown in different soil pH. The data was collected after 60 days of cultivation including rate of mycorrhizal infection, biomass, and three chemical constituents with known medicinal action. The results showed that Glomus versiforme was more apt to infect S. miltiorrhiza (F>94.00%; M>69.45%; m>73.66%) and promote the growth of S. miltiorrhiza under pH 5-9 soil. The mycorrhizal contribution to the growth of S. miltiorrhiza was the highest when grown in pH 8 soil. Plants grown with mycorrhiza in pH 8 soil had above-ground biomass more than 2 times and root biomass more than 5 times. The uninoculated plants grew better under acidic and neutral conditions, but the inoculated plants grew better under alkaline (pH 8) conditions. This result showed mycorrhiza can play a role in the adaptability of S. miltiorrhiza to the environment. Inoculation of mycorrhiza significantly increased the accumulation of rosmarinic acid, salvianolic acid B, and dihydrotanshinone by 6.59,5.03 and 2.20-folds. Based on our results alkaline soil (pH 8) is most suitable for the cultivation of S. miltiorrhiza by inoculation with the mycorrhiza G. versiforme.

Core Microbiome of Medicinal Plant Salvia miltiorrhiza Seed: A Rich Reservoir of Beneficial Microbes for Secondary Metabolism?

Seed microbiome includes special endophytic or epiphytic microbial taxa associated with seeds, which affects seed germination, plant growth, and health. Here, we analyzed the core microbiome of 21 Salvia miltiorrhiza seeds from seven different geographic origins using 16S rDNA and ITS amplicon sequencing, followed by bioinformatics analysis. The whole bacterial microbiome was classified into 17 microbial phyla and 39 classes. Gammaproteobacteria (67.6%), Alphaproteobacteria (15.6%), Betaproteobacteria (2.6%), Sphingobacteria (5.0%), Bacilli (4.6%), and Actinobacteria (2.9%) belonged to the core bacterial microbiome. Dothideomycetes comprised 94% of core fungal microbiome in S. miltiorrhiza seeds, and another two dominant classes were Leotiomycetes (3.0%) and Tremellomycetes (2.0%). We found that terpenoid backbone biosynthesis, degradation of limonene, pinene, and geraniol, and prenyltransferases, were overrepresented in the core bacterial microbiome using phylogenetic examination of communities by reconstruction of unobserved states (PICRUSt) software. We also found that the bacterial genera Pantoea, Pseudomonas, and Sphingomonas were enriched core taxa and overlapped among S. miltiorrhiza, maize, bean, and rice, while a fungal genus, Alternaria, was shared within S. miltiorrhiza, bean, and Brassicaceae families. These findings highlight that seed-associated microbiomeis an important component of plant microbiomes, which may be a gene reservoir for secondary metabolism in medicinal plants.

The Plant Growth-Promoting Fungus (PGPF) Alternaria sp. A13 Markedly Enhances Salvia miltiorrhiza Root Growth and Active Ingredient Accumulation under Greenhouse and Field Conditions.

Plant growth-promoting fungi (PGPF) have attracted considerable interest as bio-fertilisers due to their multiple beneficial effects on plant quantity and quality and their positive relationship with the ecological environment. Advancements in the development of PGPF for crops and economic plant cultivation applications have been achieved, but such improvements for the use of PGPF with popular medicinal herbs, such as Salvia miltiorrhiza, are rare. In this study, we collected S. miltiorrhiza specimens inhabiting wild, semi-wild, farmland and pot-cultured areas in the Henan province of China and isolated endophytes from the roots, shoots and leaves of these samples. Twenty-eight strains of the dominant genus Alternaria were identified and selected as candidate PGPF. Under greenhouse conditions, Alternaria sp. A13 simultaneously enhanced the dry root biomass and secondary metabolite accumulation of S. miltiorrhiza as the optimal PGPF of the 28 candidate isolates. To further assess the interaction between S. miltiorrhiza and Alternaria sp. A13, the effects on seedlings growth, active ingredient accumulation, and the activity of key enzymes for effective biosynthetic pathways were investigated over a period of six months under field conditions. Compared to uninoculated seedlings, S. miltiorrhiza seedlings colonised by Alternaria sp. A13 showed significant increment of 140% in fresh weight, 138% in dry weight, and enhancement in the contents of total phenolic acid, lithospermic acids A and B (LAA and LAB, respectively) of 210%, 128% and 213%, respectively. Examination of the related enzyme activities showed that the elicitation effect of A13 on LAB accumulation correlated with cinnamic acid 4-hydroxylase (C4H) activity in the phenylpropanoid pathway under field conditions. Our results confirmed that Alternaria sp. A13 not only contributes to the stimulation of S. miltiorrhiza root growth, but also boosts the secondary metabolism, thus demonstrating its application potential as a bio-fertiliser for S. miltiorrhiza cultivation, especially in areas outside of its native growth regions.

Phoma glomerata D14: An Endophytic Fungus from Salvia miltiorrhiza That Produces Salvianolic Acid C.

In recent years, more and more researches focus on endophytic fungi derived from important medicinal plants, which can produce the same bioactive metabolites as their host plants. Salvia miltiorrhiza Bunge is a traditional medicinal plant with versatile pharmacological effects. But the wild plant resource has been in short supply due to the overcollection for bioactive metabolites. Our study was therefore conducted to isolate endophytic fungi from S. miltiorrhiza and get candidate strains that produce the same bioactive compounds as the plant. As a result, an endophyte that produces salvianolic acid C was obtained and identified as Phoma glomerata D14 based on its morphology and internal transcribed spacer analysis. Salvianolic acid C was found present in both the mycelia and fermentation broth. Our study indicates that the endophytic fungus has significant industrial potential to meet the pharmaceutical demands for salvianolic acid C in a cost-effective, easily accessible, and reproducible way.

[Effects of arbuscular mycorrhizal fungi on endogenous hormones in Salvia miltiorrhiza].

To investigate the dynamic changes of endogenous hormones contents in Salvia miltiorrhiza after the inoculation of Glomus versiforme(GV).The contents of endogenous hormones of ABA, ZR, GA, IAA and MeJA by ELISA were measured. Infection rata of GV reached plateau of 90% at the 90th d of inoculation; fresh weight of overground part and leaf number were significantly higher in GV group, with 2.7 and 1.96 fold than that of control; contents of all endogenous hormones apart from ABA in over- and under-ground part were markedly lower (P<0.05) in GV group at the 75th and 90th d, respectively, with 63% to 75% and 45% to 81% of that in control, and were significantly higher (P<0.05) in both over- and under-ground part in GV group at the 105th d, with 1.4 to 1.7 fold higher than that of control; content of ABA in underground part increased significantly at 60th, 75th and 105th d. Arbuscular mycorrhizal fungi could promote the growth of S. miltiorrhiza, and affect the dynamic changes of endogenous hormones contents in different infection periods.

[Correlation analysis between foliar endophytic fungi of Salvia miltiorrhiza and effective components].

The aim of this study was to comprehensively investigate the correlations between foliar fungal endophyte communities and effective components accumulations in Salvia miltiorrhiza. Foliar samples of S. miltiorrhiza were collected in 5 different areas. Their fungal endophyte communities and effective component contents were determined by denaturing gradient gel electrophoresis (DGGE) and high performance liquid chromatography (HPLC), respectively. The results showed that, for characteristics of foliar fungal endophyte communities and effective component contents, there were both similarities and differences among the five samples. Correlation analysis of DGGEs' band and 24 effective components revealed a significant correlations (P < 0.01). For examples, 4 bands (15, 18, 23 and 26) were all significantly correlated with the accumulations of caffeic acid, salvianolic acid B, salvianolic acid C and dihydrotanshinone I.