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.

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.

[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.

[RNA interference and its effect of CYP76AH1 in biosynthesis of tanshinone].

Tanshinones, the main bioactive compounds of Salvia miltiorrhiza, are the diterpenoid pigments, multiple genes were proved to be involved in their biosynthesis in plants. CYP76AH1 is the initial P450 gene in the tanshinones biosynthetic pathway, its function has been validated by yeast expression and in vitroenzymatic reaction. In order to clarify the function of CYP76AH1 in vivo, in this study, we constructedthe RNA interference of CYP7AH1 in S. miltiorrhiza hairy root. The RNA interference vector with a hairpin structure was constructed using the Gateway technology, and then the interference fragment was integrated into the genome of S. miltiorrhiza mediated by Agrobacterium rhizogenes. Several highly CYP76AH1 interference S. miltiorrhiza hairy roots were obtained for further analysis.

[Optimization of induction and culture conditions for hairy roots of Salvia miltiorrhiza].

To establish induction and liquid culture system for hairy roots of Danshen (Salvia miltiorrhiza), Agrobacterium rhizogenes A4, LBA9402, 15834 as test bacterium were used to infect aseptic leaves of Danshen. The hairy roots were induced and positive transgenic hairy roots were selected with PCR using rolB and rolC as the target gene. Then hairy roots of S. miltiorrhiza were harvested and salvianolic acids were extracted with 70% methanol containing 1% formic acid. The content of salvianolic acid B (SalB) and rosmarinic acid (RA) were determined by HPLC. According to the above research results, the Danshen hairy roots induced by A. rhizogenes LBA9402 were inoculated into the following group of culture media: MSOH, MS, B5, and 6,7-V liquid media. Then the same methods of extraction and determination for the content of Danshen hairy roots were adopted. Last, the hairy roots of S. miltiorrhiza induced by A. rhizogenes LBA9402 were inoculated into the MSOH liquid media with different pH values. The content of salvianolic acid were extracted with 70% methanol containing 1% formic acid and determined by HPLC. As a result, three kinds of A. rhizogenes A4, LBA9402, 15834 could induce hairy roots and Ri plasmids were integrated into the genome of S. miltiorrhiza by PCR. Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid, which were (3.27 ± 0.37)% [including (1.04 ±0.36)% of RA and (2.22 ± 0.29)% of SalB] and (3.17 ± 0.20)% [including (0.92 ± 0.31)% of RA and (2.25 ± 0.26)% of SalB], respectively. Hairy roots induced by A. rhizogenes LBA9402 when they were cultured in MSOH liquid media produced much more salvianolic acid, which was (4.56 ± 0.36)%, including (1.12 ± 0.26)% of RA and (3.44 ± 0.23)% of SalB. Hairy roots induced by A. rhizogenes LBA9402 produced the most salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81, which was 4.85%, including 1.16% of RA and 3.69% of SalB. So Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81. The research had established the foundation on genetic engineering to improve the quality of S. miltiorrhiza.

[Roles of reactive oxygen species in Streptomyces pactum Act12-induced tanshinone production in Salvia miltiorrhiza hairy roots].

Our previous research indicated that the Streptomyces pactum Act12 (Act12) had a certain promotional effect on tanshinone accumulation and up-regulated the expression of genes 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in Salvia miltiorrhiza hairy roots. This study focuses on the roles of reactive oxygen species in S. pactum Act12-induced tanshinone production in S. miltiorrhiza hairy roots. The 4% Act12, 4% Act12 + CAT and 4% Act12 + SOD were added to S. miltiorrhiza hairy root and subcultured for 21 days, the dry weight, contents of reactive oxygen species, contents of tanshinones and expression of HMGR and DXR were determined at different harvest-time. The generation of reactive oxygen species (ROS) in S. miltiorrhiza hairy roots was triggered by 4% Act12 treatment. The relative expressions of genes HMGR and DXR in 4% Act12 treatment were 32.4 and 4.8-fold higher than those in the control. And the total tanshinone in the hairy roots was 10.2 times higher than that of the control. The CAT and SOD could significantly inhibit the ROS accumulation and relative expressions of genes HMGR and DXR in 4% Act12 treatment, which induced the total tanshinone content was decreased by 74.6% comparing with the 4% Act12 treatment. ROS mediated Act12-induced tanshinone production. The Act12 may be via the ROS signal channel to activate the tanshinone biosynthesis pathways. Thereby the tanshinon content in hairy roots was increased.

[Effect of Glomus versiforme and Trichoderma harzianum on growth and quality of Salvia miltiorrhiza].

The present study aimed to investigate the effect of Glomus versiforme and Trichodema harzianum on the growth and quality of Salvia miltiorrhiza continuous cropping under field conditions. The field plot experiment was conducted, these active components in the plant were analyzed by HPLC, the root diseases incidence rate of S. miltiorrhiza determined by observation and counting, and relative parameters were measured. The data was statistically processed. The result showed that inoculation of G. versiforme and combined inoculation of G. versiforme with T. harzianum significantly decreased the root diseases incidence rate of S. miltiorrhiza, and combined inoculation of G. versiforme with T. harzianum was better than other treatments. All treatments improved accumulation of active ingredients in root. Inoculation of G. versiforme and combined inoculation of G. versiforme with T. harzianum significantly increased the content of salvianolic acid B and cryptotanshinone of root (P < 0.05), Inoculation of G. versiforme, T. harzianum and combined inoculation of G. versiforme with T. harzianum significantly enhanced the content of tanshinone I and tanshinone II(A) of the root (P < 0.05). It may conclude that inoculation of G. versiforme and combined inoculation of G. versiforme with T. harzianum can effectively reduce the root diseases incidence of continuous cropping S. miltiorrhiza, and improve the quality of S. miltiorrhiza.

Characteristics of foliar fungal endophyte assemblages and host effective components in Salvia miltiorrhiza Bunge.

Salvia miltiorrhiza Bunge, a well-known medicinal plant, has more than 20 effective components. The aim of this study was to comprehensively investigate foliar fungal endophyte communities of S. miltiorrhiza and explore the inside relationship between host-specific fungal endophytes and effective components accumulation. Five plant samples were collected from four geological different provinces in China. Foliar fungal endophyte communities were determined by terminal restriction fragment length polymorphism (T-RFLP) of the ITS region. Effective components were analyzed with high-performance liquid chromatography. The results showed that S. miltiorrhiza foliage harbored a large diversity of fungal endophytes. Principal component analysis revealed similar T-RFLP profiles and the characteristics of the 24 effective components among the five samples, which could be clustered into three groups. In foliar T-RFLP profiles derived from the restriction digestion by CfoI, HaeIII, MspI, or TaqI, there were identical 45, 42, 38, and 34 terminal restriction fragments (T-RFs) from the five samples. We consider these T-RFs as host-specific fungal endophytes. Correlation analysis of these T-RFs' area and 24 effective components contents revealed a significant correlationship between some host-specific fungal endophytes and foliage or root effective components accumulation.

[Research wilt disease of Salvia miltiorrhiza and its pathogen].

Salvia miltiorrhiza is a highly valued traditional chinese medicine for the treatment of atherosclerosis-related disorders in china, such as cardiovascular and cerebrovascular diseases in China. The wilt disease is serious in the culture of S. miltiorrhiza. Wilt disease cause biomass of plant shoots and roots is lessened, active components are decreased. To solve these problems, we research the pathogen causing wilt disease of S. miltiorrhiza. The suspected pathogen is identified by morphology and etiological test. The identification was further confirmed by alignment the sequences of internal transcribed spacer (ITS) amplified by PCR. Our result show the wilt disease of S. miltiorrhiza mostly occurred in July and August, which is hot and wetter. The wilt disease rate of S. miltiorrhiza continuous cropping for one year in S. miltiorrhiz stubble is 10%, but the wilt disease rate of S. miltiorrhiza continuous cropping for three years in S. miltiorrhiz stubble is 60%-70%. The root rot of S. miltiorrhiz caused by the wilt disease, so the wilt disease was mistaken for the rot root in production. Morphological characteristics show the pathogen is Fusarium oxysporum. The sequence of ITS wes determined and found by BLAST shared 99% identity to that of F. oxysporum f. sp. cucumerinum. So it comes to the conclusion that the causing agent of wilt disease on S. miltiorrhiza belongs to F. oxysporum.