[Effects of temperature and humidity on infection of Fusarium oxysporum in seedlings of Salvia miltiorrhiza].

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.

[Pathogenicity and induced systemic resistance of Fusarium oxysporum and Verticillium dahlia to Salvia miltiorrhiza].

Salvia miltiorrhiza is a commonly used bulk medicinal material in China. Due to the increasing demand in recent years, the planting area is expanding. In the artificial cultivation of S. miltiorrhiza, continuous cropping obstacles are prominent, which has seriously restrained the growth of S. miltiorrhiza, resulted in serious root diseases, and affected the yield and quality of medicinal materials. The pathogen infection can induce plant resistance. Previously, this research group isolated Fusarium oxysporum and Verticillium dahlia from the roots of diseased S. miltiorrhiza. In this study, 7 days after inoculation of S. miltiorrhiza with F. oxysporum(Foc group) and V. dahlia(Vd group), the incidence rates in S. miltiorrhiza were 48% and 26%, respectively. Both the two pathogens significantly reduced the aboveground biomass of S. miltiorrhiza. Five days after inoculation, the activities of defensive enzymes, such as peroxidase(POD), phenylalanine ammonia-lyase(PAL), superoxide dismutase(SOD), and polyphenol oxidase(PPO) reached the peak. The enzyme activity of the Foc group was significantly higher than that of the Vd group. Three days after inoculation, the expression of defense genes SmPDF2.1 and SmPR10 peaked and then decreased. The results showed that F. oxysporum and V. dahlia showed pathogenicity to S. miltiorrhiza and could strongly induce systemic resistance. In terms of the above indexes, F. oxysporum was superior to V. dahlia.

Comparative Transcriptomics and Metabolites Analysis of Two Closely Related Euphorbia Species Reveal Environmental Adaptation Mechanism and Active Ingredients Difference.

Roots of Euphorbia fischeriana and Euphorbia ebracteolata are recorded as the source plant of traditional Chinese medicine "Langdu," containing active ingredients with anticancer and anti-AIDS activity. However, the two species have specific patterns in the graphic distribution. Compared with E. ehracteolata, E. fischeriana distributes in higher latitude and lower temperature areas and might have experienced cold stress adaptation. To reveal the molecular mechanism of environmental adaptation, RNA-seq was performed toward the roots, stems, and leaves of E. fischeriana and E. ehracteolata. A total of 6,830 pairs of putative orthologs between the two species were identified. Estimations of non-synonymous or synonymous substitution rate ratios for these orthologs indicated that 533 of the pairs may be under positive selection (Ka/Ks > 0.5). Functional enrichment analysis revealed that significant proportions of the orthologs were in the TCA cycle, fructose and mannose metabolism, starch and sucrose metabolism, fatty acid biosynthesis, and terpenoid biosynthesis providing insights into how the two closely related Euphorbia species adapted differentially to extreme environments. Consistent with the transcriptome, a higher content of soluble sugars and proline was obtained in E. fischeriana, reflecting the adaptation of plants to different environments. Additionally, 5 primary or secondary metabolites were screened as the biomarkers to distinguish the two species. Determination of 4 diterpenoids was established and performed, showing jolkinolide B as a representative component in E. fischeriana, whereas ingenol endemic to E. ebracteolate. To better study population genetics, EST-SSR markers were generated and tested in 9 species of Euphorbia. A total of 33 of the 68 pairs were screened out for producing clear fragments in at least four species, which will furthermore facilitate the studies on the genetic improvement and phylogenetics of this rapidly adapting taxon. In this study, transcriptome and metabolome analyses revealed the evolution of genes related to cold stress tolerance, biosynthesis of TCA cycle, soluble sugars, fatty acids, and amino acids, consistent with the molecular strategy that genotypes adapting to environment. The key active ingredients of the two species were quantitatively analyzed to reveal the difference in pharmacodynamic substance basis and molecular mechanism, providing insights into rational crude drug use.

[Effects of interaction between ubiquitin/26S proteasome inhibitor MG132 and mycorrhiza on growth and effective components of Salvia miltiorrhiza].

Ubiquitin/26 S proteasome system(UPS) is one of the main ways to regulate the degradation of proteins in plants, and plays an important role in physiological processes such as secondary metabolism and plant hormone signal transduction. As indicated recently, UPS is involved in plant-microbe interactions, and presumably regulates arbuscular mycorrhizal symbiosis to affect its effects. This study investigated the effects of interaction between Cbz-leu-leu-leucinal(MG132) and the mycorrhiza on the growth and effective components of Salvia miltiorrhiza by inoculation with Glomus intraradices and spraying MG132 solution. The results showed that the inoculation with G. intraradices could promote the growth of S. miltiorrhiza, increase the accumulation of effective components in the aerial and underground parts, and decrease the relative expression level of JMT. Additionally, MG132 could strengthen the growth-promoting effect of G. intraradices. As compared with the control group, the inoculation with G. intraradices could significantly increase aerial and underground fresh weights by 267% and 95%, respectively, under the treatment with MG132 spraying, while under the MG132 spraying-free condition, the increase was 195% and 32%, respectively. Meanwhile, MG132 spraying could enhance the promotion of mycorrhizal fungi on the accumulation of active components of S. miltiorrhiza. On the other hand, regardless of inoculation with G. intraradices or not, MG132 treatment could promote the root division of S. miltiorrhiza, reduce the content of effective components in the aerial parts, and increase the content in the underground part. The inoculation with G. intraradices could alleviate the inhibitory effect of MG132 on the accumulation of effective components in the aerial part of S. miltiorrhiza. The results show that arbuscular mycorrhizal fungi(AMF) can promote the growth of S. miltiorrhiza and the accumulation of effective components, and MG132 treatment can strengthen such promotion effect, which lays a foundation for the application of MG132 in the mycorrhizal cultivation of S. miltiorrhiza in the future.

[Effect of different fungicides on efficiency of mycorrhizal symbiosis on Salvia miltiorrhiza].

Arbuscular mycorrhizal fungi provided is beneficial to Salvia miltiorrhiza for increasing yield, promoting the accumulation of active ingredients, and alleviating S. miltiorrhiza disease etc. However, the application of fungicides will affect the benefit of arbuscular mycorrhizal fungi and there is little research about it. This article study the effect of four different fungicides: carbendazim, polyoxin, methyl mopazine, and mancozeb on mycorrhiza benefit to S. miltiorrhiza by the infection intensity of arbuscular mycorrhizal fungi, the growth of S. miltiorrhiza, and the content of active ingredients. RESULTS:: showed that different fungicides had different effects. The application of mancozeb had the strongest inhibitory effect on the mycorrhizal benefit to S. miltiorrhiza. Mancozeb significantly reduced the mycorrhizal colonization and the beneficial effect of arbuscular mycorrhizal fungi on the growth and the accumulation of active components of S. miltiorrhiza. The application of polyoxin had no significant effect on mycorrhizal colonization. Instead, it had a synergistic effect with the mycorrhizal benefit to promoting the growth and accumulation of rosmarinic acid of S. miltiorrhiza. The inhibitory strengths of four fungicides are: mancozeb>thiophanate methyl, carbendazim>polyoxin. Therefore, we recommend applying biological fungicides polyoxin and avoid applying chemical fungicides mancozeb for disease control during mycorrhizal cultivation of S. miltiorrhiza.

[Cloning and expression analysis of 5-phosphomevalonate kinase gene (CcPMK) in Cinnamomum camphora].

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

[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.