The microecology of endophytic fungi in special habitats, such as the interior of different tissues from a medicinal plant, and its effects on the formation of metabolites with different biological activities are of great importance. However, the factors affecting fungal community formation are unclear. This study is the first to utilize "mini-community" remodeling to understand the above phenomena. First, high-throughput sequencing technology was applied to explore the community composition and diversity of endophytic fungi in the above-ground tissues (Ea) and below-ground tissues (Eb) of Ephedra sinica. Second, fungi were obtained through culture-dependent technology and used for "mini-community" remodeling in vitro. Then, the effects of environmental factors, partner fungi, and plant tissue fluid (internal environment) on endophytic fungal community formation were discussed. Results showed that environmental factors played a decisive role in the selection of endophytic fungi, that is, in Ea and Eb, 93.8% and 25.3% of endophytic fungi were halophilic, respectively, and 10.6% and 60.2% fungi were sensitive to high temperature (33 °C), respectively. Meanwhile, pH had little effect on fungal communities. The internal environment of the plant host further promoted the formation of endophytic fungal communities.

Ephedra sinica , a well-known Chinese medicinal plant, is characterized as having the opposite medicinal effect among its root and stem. However, there is a lack of understanding to differentiate the active components present in the root and stem of E. sinica, as well as the molecular mechanisms underlying the formation of the differential compounds, which has significantly hampered the further development and utilization of E. sinica resource. In this study, forty-five differential metabolic markers are affiliated to alkaloids, flavonoids, terpenoids, and organic acids between root and stem of E. sinica , and sixty genes of key enzymes are involved in their biosynthesis distributed in metabolic pathway branches such as phenylalanine metabolism, flavonoid biosynthesis and phenylpropane biosynthesis, based on combination non-targeted metabolome with transcriptome technologies. The finding revealed that the expression activity changes of these enzyme genes had a direct impact on the distinction of differential metabolic markers in the root and stem of E. sinica . This study will help to understand the molecular mechanism of the differentiation and biosynthesis of the primary active metabolites in the root and stem of E. sinica , providing a theoretical foundation for its quality control and promotion in cultivation.

Phytoremediation potential of Azolla in removal of nitrogen from wastewater has been promising. However, little is known about the response of Azolla to high concentrations of nitrogen. In this study, the responses of four Azolla species to different concentrations of total nitrogen ranging from 0 to 180 mg L-1 were examined. The responses varied among different species, and the high nitrogen-tolerant species A. caroliniana and A. microphylla could remove nitrogen from aqueous solutions with higher efficiencies. We further performed transcriptome analysis to explore the molecular mechanism underlying the response to high nitrogen stress in Azolla. RNA-seq analysis revealed a synergistic regulatory network of differentially expressed genes (DEGs) involved in nitrogen transport and metabolism in A. microphylla, mainly in the roots. Under high nitrogen treatment, the DEGs encoding nitrate transporters or nitrate transporter 1/peptide transporters (NRTs/NPFs), ammonium transporters (AMTs), nitrate reductase (NIA), nitrite reductase (NIR) and glutamine synthetases/glutamate synthases (GSs/ GOGATs) were down-regulated, and the DEGs encoding glutamate dehydrogenases (GDHs) were up-regulated, suggesting that A. microphylla possessed high tolerance against excess nitrogen through down-regulation of nitrate and ammonium uptake and fine regulation of nitrogen assimilation in the roots. Our results provided a theoretical foundation for better utilization of Azolla for wastewater treatment.

以沙漠来源的锁阳-唐古特白刺(简称白刺)寄生植物体为对象,通过高通量测序分析寄生植物体内生细菌分布和组成及功能.结果表明,锁阳与寄生白刺、锁阳与非寄生白刺之间内生细菌OTU(Operational taxonomic units)组成相似性分别为11.50％和2.33％;锁阳内生细菌物种丰富度显著高于非寄生白刺(P＜0.05),而锁阳和寄生白刺内生细菌丰富度差异不显著;锁阳和非寄生白刺组间UniFrac距离为0.75,而锁阳和寄生白刺组间UniFrac距离为0.35,说明锁阳与寄生白刺间的菌群结构差异显著减小.随着寄生行为的发生,寄生双方内生细菌群落组成发生显著变化,寄主白刺相对丰度较高的属为糖霉菌属和Lepidoceras;锁阳中为Lepidoceras、伯克氏菌属、鞘氨醇单胞菌属和苍白杆菌属;非寄生白刺中为Lepidoceras和伯克氏菌属.Lepidoceras和伯克氏菌属等7株菌在锁阳中的相对丰度显著高于寄生白刺(P＜0.05),仅有糖霉菌属和Olivibacter在寄生白刺中相对丰度显著高于另外两种样本(P＜0.05).内生细菌功能分析表明,分解木质纤维素的主要功能基因如NADH脱氢酶(K05575)、淀粉磷酸化酶(K00688)、α-淀粉酶(K01176)和环葡聚糖水解酶(K01208)等在寄生白刺中的相对丰度显著高于锁阳(P＜0.05),这对认识沙漠条件下锁阳植物的成功寄生具有重要意义.

There are many species of Chinese traditional leguminosae family plants that are well known for their medicinal applications, such as Astragalus membranaceus, Catsia tora, Glycyrrhiza uralensis, Sophora flavescens and Albacia acacia. Their unique bioactive composition and internal phenological environment contribute to the formation of specific and unique endophytic fungal communities, which are important resources for new compounds used in a variety of pharmacological activities. Nonetheless, they have not been systematically studied. In the last decade, nearly 64 genera and thousands of species of endophytic fungi have been discovered from leguminosae plants, as well as 138 secondary metabolites (with 34 new compounds) including flavonoid, alkaloids, phenol, anthraquinone, macrolide, terpenoid, phytohormone and many more. These were shown to have diverse applications and benefits, such as antibacterial, antitumor, antioxidative, immunoregulatory and neuroprotective properties. Here, we provide a summarized overview with the aim of raising awareness of endophytic fungi from medicinal leguminosae plants and providing a comprehensive review of the discoveries of new natural products that may be of medicinal and pharmaceutical importance.

BACKGROUND The contents of some its crucial metabolites tend to decrease when Rhodiola crenulata is cultured under low altitude. Interestingly, an endophyte Phialocephala fortinii showed that it could alleviate this problem. RESULTS There were 16 151 differential genes including 14 706 up-regulated and 1445 down-regulated uni-genes with significant differences (P < 0.05), and a total of 1432 metabolites exhibited statistically significant (P < 0.05) metabolic differences comprising of 27 different marker metabolites which showed highly significant value's of VIP > 5 and P < 0.01. Results highlight differential regulation of 20 enzymatic genes that are involved in the biosynthesis of five different marker metabolites including acetaldehyde, homocysteine, cyclopropylamine, 1-pyrrolinium and halistanol sulfate. CONCLUSION The positive physiological effect of P. fortinii on R. crenulata encompasses differential regulation in carbohydrate metabolism, lipid metabolism and secondary metabolite synthesis. This article is protected by copyright. All rights reserved.

Cynomorium songaricum Rupr. (CSR) has a long history of being widely used as a functional food and medicine by different ethnic groups worldwide; CSR is used to treat impotence and premature ejaculation and traditionally used as natural source of tonic food. Numerous bioactive compounds linked with human health were recently identified. In this work, all available information on CSR were retrieved from NCBI PubMed and CNKI database, and the recent findings about the compositions and functions of CSR were summarized. At least 76 bioactive compounds, including flavonoids, terpenoids, steroids, organic acids, saccharides, glycosides, and phloroglucinol adducts, were isolated and identified in CSR. These compounds render CSR with pharmacological functions, such as anti-aging, anti-oxidation, anti-fatigue, and anti-HIV activities, and the effects of CSR on immune system, nervous system, reproductive system, and other bioactivities were well reviewed; results serve as valuable data for further research and for development of human health-related products. However, studies on CSR were far from perfect, and further research is warranted. This paper highlights the components and pharmacological functions of CSR, with the aim to provide a comprehensive review and useful data for future studies and for the development of relevant food products.

药用植物发育和代谢受到多种因素的影响,而内生真菌对植物的影响是近年来才得以重视并逐步成为热点.为探索内生真菌Phialocephala fortinii对大花红景天基因及代谢途径的影响,本实验通过RNA转录组测序筛选差异表达基因;并用Real-time PCR对基因表达进行验证,利用2-△△Ct的方法分析脂代谢、信号转导和环境适应性等重要代谢途径差异基因的相对表达量.结果表明,验证结果与测序结果一致,内生真菌P.fortinii能够促进大花红景天脂代谢途径中的差异基因表达量;它对信号转导途径中相关基因表达的影响不同,但控制Notch信号通路途径的基因表达量显著提高,为对照组的34倍;对适应性代谢途径差异基因表达上调,进而促进红景天环境适应能力.研究结果旨在为揭示内生真菌与红景天的互作机制、红景天道地性形成原因、内生真菌功能的开发等提供分子基础数据.

Objective:Identification and genetic relationship of Cynomorium Songaricum and its host Nitraria tangutorum were investigated by ITS2 molecular marker analysis.Methods:The genome DNA of plant was extracted by modified CTAB method.Interual transcribed spacer of rDNA-ITS2 was amplified by universal premiers of ITS2,then was aligned and jointed by DNAstar.The secondary structure of ITS2 was predicted in professional web.Intraspecific and intergenic genetic distances of C.songaricum and N.tangutorum were detected with Kimura-2-parameter by MEGA 6.06 software.DNA barcode analysis system and neighbor-joining method were used to create polygenetic tree to analyze genetic relationships.Results:ITS2 sequences of C.songaricum and N.tangutorum were 229 bp and 232 bp,respectively.GC contents were 48％ and 63％,respectively.Intraspecific genetic distances of C.songaricum and N.tangutorum were 0-0.004 4 and 0-0.022,respectively.Interspecific genetic distance of C.songaricum and N.tangutorum was 2.234 0-2.443 3.Conclusion:ITS2 barcode was an effective method for specific identification and evaluation of genetic relationship to C.songaricum and N.tangutorum.

Endophyte is a factor that affects the physiology and metabolism of plant. However, limited information is available on the mechanism of interaction between endophyte and plant. To investigate the effects of endophytic fungus ZPRs-R11, that is, Trimmatostroma sp., on salidroside and tyrosol accumulations in Rhodiola crenulata, signal transduction, enzyme gene expression, and metabolic pathway were investigated. Results showed that hydrogen peroxide (H2O2), nitric oxide (NO), and salicylic acid (SA) involved in fungus-induced salidroside and tyrosol accumulations. NO acted as an upstream signal of H2O2 and SA. No up- or down-stream relationship was observed, but mutual coordination existed between H2O2 and SA. Rate-limiting enzyme genes with the maximum expression activities were UDP-glucosyltransferase, tyrosine decarboxylase (TYDC), monoamine oxidase, phenylalanine ammonialyase (PAL), and cinnamic-4-hydroxylase sequentially. Nevertheless, the genes of tyrosine transaminase and pyruvate decarboxylase only indicated slightly higher activities than those in control. Thus, TYDC and PAL branches were the preferential pathways in ZPRs-R11-induced salidroside and tyrosol accumulation. Trimmatostroma sp. was a potential fungus for promoting salidroside and tyrosol accumulations. The present data also provided scientific basis for understanding complex interaction between endophytic fungus and R. crenulata.

This is the first report of the distribution and genetic relationships of endophytic fungi from the parasitic plant Cynomorium songaricum and its host Nitraria tangutorum. Endophytic fungi from the root of natural N. tangutorum, parasitic plant C. songaricum and its host N. tangutorum were isolated by tissue culture, and they were identified by morphology combined with molecular barcoding based on ITS-rDNA sequence. The isolation rates, colonization rates, isolation frequency, diversity index, evenness index, similarity coefficient and genetic relationships among fungal taxa were estimated by phylogenetic analysis, and differences in fungal endophyte distribution were investigated. The results showed that a total of 49 isolates were obtained belonging to 18 different taxa. 95.9% of these taxa were in Ascomycota, and the remaining was in Basidiomycota (4.1%). The isolation rate and colonization rate of endophytic fungi were 15.3% and 25.0%, respectively. The Shannon biodiversity index was the highest in the root of natural N. tangutorum at 2.13. The simila-rity coefficient was highest between the stem of C. songaricum and the flower of C. songaricum at0.50. Fusarium was the dominant genus in N. tangutorum, while Penicillium was the primary genus in C. songaricum. The differential distribution of fungal taxa between N. tangutorum and C. songaricum suggested that the parasitic relationship influences the endophytic fungal community.

研究内生真菌ZPRa-R -1 对大花红景天组培苗中信号分子NO,SA和H 2 O 2 ,及主要次生代谢物酪醇（p-tyrosol）和红景天苷（salidroside）积累的影响。分别采用Greiss试剂法、RP-HPLC荧光法、硫酸钛法检测NO,SA和H 2 O 2 等信号分子;采用分光光度法检测关键酶PAL和CA4H的活性;采用RP-HPLC检测酪醇和红景天苷。ZPRa-R -1 接种于红景天,首先引起宿主信号分子NO含量增高,其次是SA和H 2 O 2 ,它们达到最大值的时间分别是共生后第8,10和12 d,含量分别为0.193μmol·g -1 ,0.062μg·g -1 ,0.092μmol·g -1 ;关键酶PAL和CA4H活性最大值分别为166.400 U·g -1 和0.625 U·g -1 ·h -1 ,分别是对照组的8和1.5倍,最终引起酪醇和红景天苷的积累效应,在第8和10 d达到最大值,含量分别为8.656和0.498 mg·g -1 ,是对照组的7和2.8倍。研究结果表明内生真菌ZPRa-R -1 能通过诱导激活宿主信号分子网络系统,引起关键酶活性的变化,进而调控酪醇和红景天苷等次生代谢物的积累。

2-(4-Hydroxyphenyl)ehyl-β-d-glucopyranoside (salidroside) and 4-(2-hydroxyethyl)phenol (p-tyrosol) are famous food and medicine additives originally derived from alpine Rhodiola plants. Salidroside or p-tyrosol production by the endophytic fungus Rac56 (Phialocephala fortinii) was confirmed by UPLC/Q-TOF-MS and 1H-NMR. The fermentation conditions were optimized by orthogonal design using data processing system software. The broth fermentation results showed that salidroside and p-tyrosol extraction yields from Rac56 were stable and reached 1.729 ± 0.06 mg and 1.990 ± 0.05 mg per mL of culture medium, respectively. The optimal conditions for salidroside and p-tyrosol production in fermentation culture of Rac56 were determined to be 25 °C, pH values of 7 and 5, Czapek-Dox culture medium volumes of 150 mL and 50 mL in 250 mL flasks, rotation speeds of 100× g and 200× g, and fermentation durations of 7 and 15 days, respectively. Under these optimal conditions, stable yields of 2.339 ± 0.1093 mg and 2.002 ± 0.0009 mg per mL of culture medium of salidroside and p-tyrosol, respectively, were obtained, indicating that the P. fortinii Rac56 strain is a promising source of these compounds.

目的 采用RAPD和ISSR分子标记进行野生红景天种间亲缘关系及种内的遗传多样性分析.方法 用CTAB法提取基因组DNA,然后通过筛选得到的11个RAPD及11个ISSR引物对不同采集地的4种野生红景天进行遗传多样性分析.结果 11条RAPD引物共扩增出96条条带,多态性百分比为90.62％;11条ISSR引物共扩增出102条条带,多态性百分比为100％.ISSR多态性的检测能力优于RAPD;聚类分析结果表明,ISSR法、RAPD和ISSR联用法均将17个样品聚为3大类;RAPD将样品聚为4大类.结论 2种标记均可用于红景天属植物种间亲缘关系与种内遗传多样性研究;4种红景天种内存在一定的遗传差异,种间基因流较小.

通过发酵培养真菌,对发酵液的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、?OH?自由基、O2??自由基、亚硝酸盐清除能力以及Fe2+螯合能力进行系统测定,综合评价长白红景天内生真菌的抗氧化能力.结果发现,对DPPH自由基、?OH?自由基、O2??自由基、亚硝酸盐清除率以及Fe2+螯合率大于50％的内生真菌菌株分别占供试菌株的84％、74％、58％、4％和30％.菌株Ra-R-32抗氧化活性较高,在5种评价中各清除率均大于50％.实验表明,长白红景天内生真菌具有良好的抗氧化活性,是珍贵的化合物筛选资源库.

对柴胡红景天中一株具有抗氧化活性的内生真菌Rb-R-1进行研究,获得其分类地位并评价抗氧化活性强弱.结果表明:这株真菌为Ascomacota门Hypocreales目Neonectria属的N.ramulariae真菌,具有较强的1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、羟自由基(·OH)、超氧阴离子自由基(O2-·)、NO2-清除能力和Fe2+螯合能力,IC50值分别为8.86、4.40、9.65、16.53、1.91 mg/mL;其发酵产物中总酚和总黄酮含量分别达18.12、22.48 mg/g.发酵条件优化结果表明,在以乳糖为碳源、牛肉膏为氮源、装液量90 mL/250 mL、pH 7.0、25℃条件下培养10d时,内生真菌Rb-R-1发酵产物对DPPH自由基的清除率最大,达到93.12％;总酚和总黄酮含量最高,分别为19.14 mg/g和28.25 mg/g.