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This study investigated the biodiversity, phylogeny, and antifungal activity of endophytic fungi isolated from Zanthoxylum bungeanum. A total of 940 isolates obtained were grouped into 93 morphotypes, 43 species, and 23 genera, which were authenticated by molecular identification based on rDNA internal transcribed spacer (ITS) sequence analysis. A high diversity of endophytic fungi from Z. bungeanum are observed with high species richness S (43), Margalef index D′ (6.1351), Shannon–Wiener index H′ (3.2743), Simpson diversity index Ds (0.9476), PIE index (0.9486), and evenness Pielou index J (0.8705) but a low dominant index λ (0.0524). Significant tissue specificity of the endophytic fungi was observed in Z. bungeanum, and the highest species richness and diversity indexes were obtained in the stem. Phylogenetic analyses of the 93 endophytic isolates were carried out by the neighbor-joining (NJ) method to demonstrate their evolutionary processes. Antifungal activities of endophytic fungi were assayed and eight endophytic isolates showed strong and long-lasting inhibition against host pathogenic fungi Fusarium sambucinum and Pseudocercospora zanthoxyli. Here, for the first time, we systematically demonstrate the biodiversity, phylogeny, and antifungal activity of endophytic fungi associated with Z. bungeanum and reveal the value of sampling different tissues of a given plant to obtain the greatest endophyte species diversity, which might offer a framework for further investigation and utilization of endophytic fungi as aunique source of interesting and useful bioactive compounds.

Four diterpenoid tanshinones and three phenolic acids were isolated from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation. By means of physicochemical and spectrometric analysis, they were identified as tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7). These compounds were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms including eight bacterial and one fungal species. Among the four tanshinones, cryptotanshinone (3) and dihydrotanshinone Ι (4) exhibited stronger antimicrobial activity than tanshinone ΙΙA (1) and tanshinone Ι (2). The results indicated that the major portion of the antimicrobial activity was due to the presence of tanshinones and phenolic acids in S. miltiorrhiza hairy roots, which could be used as the materials for producing antimicrobial agents for use in agricultural practice in the future.

Five spirobisnaphthalenes, namely palmarumycin CP 17 (1), diepoxin κ (2), diepoxin η (3), diepoxin ζ (4), and diepoxin γ (5), were isolated from the endophytic fungus Dzf12 associated with the medicinal plant Dioscorea zingiberensis C. H. Wright. Their structures were identified by physicochemical and spectrometric analysis. Among these spirobisnaphthalenes, 2 was found to have antibacterial activity, and the mixture of 3 and 4 was detected to have both antibacterial and antifungal activities.

Stem canker of Zanthoxylum bungeanum is a devastating disease that seriously affects the plantation and industrial development of Z. bungeanum due to a lack of effective control measures. The objective of this study was to screen out resistant Z. bungeanum varieties and further explore their resistance mechanisms against stem canker. Results showed that the most resistant and susceptible varieties were, respectively, Doujiao (DJ) and Fengxian Dahongpao (FD). Combining transcriptomic and metabolomic analyses, we found that the genes and metabolites associated with the phenylpropanoid metabolism, especially flavonoid biosynthesis, were highly significantly enriched in DJ following pathogen infection compared with that in FD, which indicated that the flavonoid metabolism may positively dominate the resistance of Z. bungeanum. This finding was further confirmed by quantitative real-time polymerase chain reaction analysis, through which higher expression levels of core genes involved in flavonoid metabolism in resistant variety were observed. Moreover, by analyzing the differences in the flavonoid content in the stems of resistant and susceptible varieties and the antifungal activities of flavonoids extracted from Z. bungeanum stems, the conclusion that flavonoid metabolism positively regulates the resistance of Z. bungeanum was further supported. Our results not only aid in better understanding the resistance mechanisms of Z. bungeanum against stem canker but also promote the breeding and utilization of resistant varieties.

The effects of the oligosaccharides from the endophytic fungus Fusarium oxysporum Dzf17 as elicitors on diosgenin production in cell suspension cultures of its host Dioscorea zingiberensis were investigated. Three oligosaccharides, DP4, DP7 and DP10, were purified from the oligosaccharide fractions DP2-5, DP5-8 and DP8-12, respectively, which were prepared from the water-extracted mycelial polysaccharide of the endophytic fungus F. oxysporum Dzf17. When the cell cultures were treated with fraction DP5-8 at 20 mg/L on day 26 and harvested on day 32, the maximum diosgenin yield (2.187 mg/L) was achieved, which was 5.65-fold of control (0.387 mg/L). When oligosaccharides DP4, DP7 and DP10 were individually added to 26-day-old D. zingiberensis cell cultures at concentrations of 2, 4, 6, 8 and 10 mg/L in medium, DP7 at 6 mg/L was found to significantly enhance diosgenin production, with a yield of 3.202 mg/L, which was 8.27-fold of control. When the cell cultures were treated with DP7 twice on days 24 and 26, and harvested on day 30, both diosgenin content and yield were significantly increased and reached the maximums of 1.159 mg/g dw and 4.843 mg/L, both of which were higher than those of single elicitation, and were 9.19- and 12.38-fold of control, respectively.

Three polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharide (SPS), were prepared from the endophytic fungus Fusarium oxysporium Dzf17 isolated from the rhizomes of Dioscorea zingiberensis. The effects of the time of addition and polysaccharide concentration on the growth and diosgenin accumulation in cell suspension culture of D. zingiberensis were studied. Among them, WPS was found to be the most effective polysaccharide. When WPS was added to the medium at 20 mg/L on the 25th day of culture, the cell dry weight was increased 1.34-fold, diosgenin content 2.85-fold, and diosgenin yield 3.83-fold in comparison to those of control. EPS and SPS showed moderate and relatively weak enhancement effects on cell growth and diosgenin accumulation, respectively. The dynamics of cell growth and diosgenin accumulation when WPS was added to the medium at 20 mg/L on the 25th day of culture were investigated, and results showed that dry weight of cells reached a maximum value on day 30 but the maximum diosgenin content was achieved on day 31.

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