Wilt disease is a major disease of cultivated Salvia miltiorrhiza, which is caused by Fusarium oxysporum. Since the infection process of F. oxysporum in plants is affected by environment factors, this study was conducted to reveal the relationship between disease severity and concentration of the pathogen in plants in the infection process of F. oxysporum in seedlings of S. miltiorrhiza by pot experiments and to reveal the effects of temperature and humidity on the infection process. The results showed that, after inoculation of S. miltiorrhiza seedlings with F. oxysporum, the pathogen in different parts was detected at different time, and it was first detected in substrates. With the continuous propagation of the pathogen(4-5 d), it gradually infected the roots and stems of the seedlings, and the plants had yellowing leaves and withering. The number of the pathogen reached the maximum in each part after 7-8 d, and then gradually decreased in the later stage of the disease. The concentration of the pathogen in substrates, roots and stems of S. miltiorrhiza showed a trend of decreasing after increasing with the aggravation of the disease and reached the maximum in the samples of moderate morbidity, while the concentration in the samples of severe morbidity decreased. In addition, the infection of F. oxysporum in seedlings of S. miltiorrhiza was affected by temperature and humidity. The suitable temperature was 25-30 ℃ and the suitable humidity was 80%-90%. This study could provide guidance for the experiments on pathogenicity of F. oxysporum, screening of biocontrol bacteria and controlling of wilt.
Seedlings/microbiology* ; Salvia miltiorrhiza ; Temperature ; Humidity ; Fusarium

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.
Abietanes/biosynthesis* ; Ascomycota/growth & development* ; Endophytes/growth & development* ; Plant Roots/microbiology* ; Salvia miltiorrhiza/microbiology* ; Seeds/microbiology*

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.
Bacteria ; classification ; High-Throughput Nucleotide Sequencing ; Rhizosphere ; Salvia miltiorrhiza ; microbiology ; Soil Microbiology

OBJECTIVETo study the characteristics of soil microbial variation during Salvia miltiorrhiza crop rotation.

METHODthe conventional cultivating microbial method was used to study the microbial number and communities structure and soil microbial biomass phosphorus (SMBP) was determined by chloroform vapor extraction method. The data was then analyzed by SPSS software.

RESULTWith the increase of the crop rotation years, the numbers of bacteria and actinomycetes in soil also, but the fungi and SMBP decreased.

CONCLUSIONMicrobial mechanism of crop rotation of the planting S. miltiorrhiza is the regulation of microbial number and bacteria physiological communities, the process rebuilds the soil ecological system balance. Microbial communities in soil need at lest 2 years to get to restore, after planting S. miltiorrhiza, which consisting with traditional planting experience.

Actinobacteria ; isolation & purification ; Bacteria ; isolation & purification ; Plants, Medicinal ; growth & development ; metabolism ; microbiology ; Salvia miltiorrhiza ; growth & development ; metabolism ; microbiology ; Soil Microbiology

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.
DNA, Intergenic ; genetics ; Fusarium ; genetics ; isolation & purification ; physiology ; Plant Diseases ; microbiology ; Polymerase Chain Reaction ; Salvia miltiorrhiza ; microbiology ; Seasons

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.
Chromatography, High Pressure Liquid ; Cluster Analysis ; Denaturing Gradient Gel Electrophoresis ; Endophytes ; chemistry ; Fungi ; chemistry ; Salvia miltiorrhiza ; chemistry ; microbiology

OBJECTIVEIn order to provide a scientific water management for the standardized cultivation, the effects of soil water content on the seedlings growth and active ingredients of S. miltiorrhiza were studied.

METHODA water stress experiment with pot culture was applied to study the effects of different soil water content on the seedlings growth, biomass and the accumulation of tanshinone, salvianolic acid B and mineral nutrition of S. miltiorrhiza.

RESULTSoil water content had serious influence on the growth, yield, outer appearance and inner quality of S. miltiorrhiza when the soil was under severe drought or with too much water. But the shoot and root biomass of S. miltiorrhiza was increased significantly under mild drought. As well as the content and cumulant of dihydrotanshinone I , cryptotanshinone, tanshinone I , tanshinone IIA and salvianolic acid B in root of mild drought were increased. It also enhanced the P, K, Ca, Mg, Mn, Zn, Cu and Fe contents of S. miltiorrhiza under mild drought.

CONCLUSIONThe overall results from the experiment suggest that the appropriate soil water content is 55% to 60% in seedling stage of S. miltiorrhiza. And it will be strongly recommended that the ridge culture and suitable soil moisture management must be carry out in production of S. miltiorrhiza in order to improve the yield and quality of medicinal materials.

Benzofurans ; analysis ; Biomass ; Diterpenes, Abietane ; analysis ; Minerals ; analysis ; Plant Roots ; growth & development ; microbiology ; Salvia miltiorrhiza ; chemistry ; growth & development ; Seedlings ; growth & development ; Soil ; chemistry ; Stress, Physiological ; Water

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.
Antibiosis ; physiology ; Benzofurans ; metabolism ; Diterpenes, Abietane ; metabolism ; Glomeromycota ; physiology ; Host-Pathogen Interactions ; Phenanthrenes ; metabolism ; Plant Diseases ; microbiology ; Plant Roots ; growth & development ; metabolism ; microbiology ; Salvia miltiorrhiza ; growth & development ; metabolism ; microbiology ; Trichoderma ; physiology

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.
Agrobacterium ; physiology ; Benzofurans ; analysis ; metabolism ; Cell Culture Techniques ; instrumentation ; methods ; Cinnamates ; analysis ; metabolism ; Culture Media ; chemistry ; metabolism ; Depsides ; analysis ; metabolism ; Drugs, Chinese Herbal ; analysis ; metabolism ; Plant Roots ; chemistry ; growth & development ; metabolism ; microbiology ; Salvia miltiorrhiza ; chemistry ; growth & development ; metabolism ; microbiology

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.
Aldose-Ketose Isomerases ; genetics ; metabolism ; Diterpenes, Abietane ; biosynthesis ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; enzymology ; genetics ; metabolism ; microbiology ; Reactive Oxygen Species ; metabolism ; Salvia miltiorrhiza ; enzymology ; genetics ; metabolism ; microbiology ; Secondary Metabolism ; Streptomyces ; physiology