The research on endophytes of medicinal plants mainly relies on the traditional culture and isolation methods. Because of their functions such as promoting host growth, improving stress resistance, promoting the accumulation of medicinal active ingredients or directly producing medicinal active ingredients, the endophytes of medicinal plants have gradually attracted wide attention. However, it was found that the strains isolated by traditional methods were not the true dominant endophytes of medicinal plants by comparing the results of traditional culture isolation with high-throughput sequencing. The blind and random nature of traditional methods leads to the lack of standards in terms of medium selection, culture time and interaction between species. On the contrary, high-throughput sequencing technology is an emerging molecular biology technology developed in recent decades. Due to its high resolution level and indepen-dent culture, it can be used for thorough analysis of the community structure and diversity of environmental microorganisms. Therefore, we proposed the strategy of using high-throughput sequencing technology to guide the traditional culture and isolation of endophytes from medicinal plants. Firstly, the endophytic structure and diversity of medicinal plants were completely clear by high-throughput sequencing technology, and the dominant endophytes of the host were unequivocal. Then according to the characteristics of each dominant endophytes design or query suitable medium for its growth to culture and isolation. Finally, the function of the isolates was studied. This method can prevent researchers from missing out on the important functional strains of the host, expand the research scope of endophytes of medicinal plants, and facilitate the in-depth excavation and utilization of endophytes of medicinal plants.
Endophytes/genetics* ; High-Throughput Nucleotide Sequencing ; Plants, Medicinal ; Research Design

Aquilaria sinensis can generate agarwood, which is closely related with endophyte. Up to now, studies mainly focused on the effects of endophytic fungi on agarwood formation, but studies about endophytic bacteria are rarely reported. In our research, the T-RFs and Shannon index of endophytic bacteria in samples of agarwood increase. The number of distinctive T-RFs fragments of corresponding samples in the same group accounted for more than 60% the number of total T-RFs fragments. In samples of no-agarwood, the dominant bacterial population are Anoxybacillus, Clostridium, Candidatus endobugula, Lysinibacillus. In samples of agarwood, the dominant bacterial population are Clostridium, Lysinibacillus, Luteimonas, phytoplasma. Besides, there are. specific T-RFs fragment in samples of agarwood and no-agarwood respectively. When we perform cluster analysis, we found samples of agarwood highly gather together and samples of no-agarwood highly gather together. This means community of endophytic bacteria emerge significant and regular changes during agarwood formation, which may be result of agarwood production, or maybe it is important reason of agarwood production. In this paper, we obtain more comprehensive and accurate community of endophytic bacteria in Aquilaria sinensis and it's variation during agarwood formation using T-RFLP, which is first study of effects of endophytic bacteria on agarwood formation, and will help to exploit resource of endophytic bacteria more reasonably.
Bacteria ; classification ; genetics ; isolation & purification ; Biodiversity ; Endophytes ; classification ; genetics ; isolation & purification ; Thymelaeaceae ; microbiology ; Wood ; microbiology

A total of 152 strains of endophytic fungi were isolated from the barks of Eucommia ulmoides in three regions (Lueyang country, Zunyi country, Cili country). Based on morphological characteristics and analysis of ITS sequences, these strains were identified into 8 genera. Thereinto Phomopsis, Diaporthe and Alternaria were common genera to Eucommia barks from different sites. But the dominant genus was different: Alternaria was the dominant genus in the barks from Cili country, and Phomopsis was the dominant genus from Zunyi country, then Diaporthe was the one from Lueyang country. According to the similarity coefficient, the composition of the endophytic fungi was distinctly different between the barks from three sites. The diversity and species richness in Lueyang country and Cili country were found higher than those in Zunyi country. The evenness of endophytic fungi was 0.936 5 in Lueyang county, which was higher than 0.737 1 or 0.641 0 in Cili county or Zunyi county, respectively. After phylogenic analysis and calculating the genetic distances of typical strains belong to Phomopsis and its perfect stage--Diaporthe, there was very high genetic diversity in the two genera from our study. In conclusion, the community structure and diversity of endophytic fungi were significant different in Eucommia barks from the three habitats.
DNA, Fungal ; genetics ; DNA, Intergenic ; genetics ; Ecosystem ; Endophytes ; classification ; physiology ; Eucommiaceae ; microbiology ; Fungi ; classification ; genetics ; physiology ; Phylogeny ; Plant Bark ; microbiology

In order to reveal the distribution and population characteristics of endophytic fungi from Zanthoxylum nitidum and the antibacterial potential,this study performed molecular identification and analyzed the genetic diversity and antibacterial activity of endophytic fungi from Z. nitidum in Guangxi. Through culture and molecular identification,35 strains,belonging to 15 genera,12 families,10 orders,4 classes,and 2 phyla,were isolated from various tissues of Z. nitidum,of which Colletotrichum and Fusarium were the dominant genera,respectively accounting for 20% of total strains. The diversity of endophytic fungi was significantly different among roots,stems,and leaves,as manifested by the significantly higher Shannon index( H') in stems( 1. 678) than in roots( 0. 882 1) and leaves( 0. 515 4). The antimicrobial activity analysis showed that 14. 28% of endophytic fungi inhibited at least one indicator pathogen. Among them,Fusarium sp. ZN-34 and Fusarium sp. ZN-26 separately demonstrated the strongest inhibitory effect on Escherichia coli and Staphylococcus aureus. In general,Fusarium sp. ZN-26 and Phialemoniopsis plurioloculosa ZN-35 were advantageous in suppressing the two bacteria owing to the broad spectrum and strong efficacy. In summary,Z. nitidum in Guangxi boasts rich endophytic fungi with the majority showing strong antibacterial activity,which can be used as candidates for the extraction and separation of basic antibacterial substances and the development of natural antibacterial agents.
Anti-Bacterial Agents/pharmacology* ; Anti-Infective Agents ; China ; Colletotrichum ; Endophytes/genetics* ; Fungi/genetics* ; Genetic Variation ; Humans ; Microbial Sensitivity Tests ; Zanthoxylum

OBJECTIVETo identify the endophytic fungal strain PR35 separated from Paeonia delavayi and study chemical constituents of its secondary metabolites.

METHODThe fungal strain PR35 was identified by morphological observation and ITS rDNA sequence analysis. Various chromatographic methods were adopted to separate and purify its secondary metabolites, and their structures were identified by physiochemical properties and spectral data

RESULTThe fungal strain PR35 was identified as Trichoderma longibrachiatum. Five compounds were separated from fermentation products of fungal strain PR35 and identified as 1-(2,6-dihydroxyphenyl)-3-hydroxybutan-1-one (1), 1-(2,6-dihydroxypheny) propan-1-one (2), 1-(2,4,6-trihydroxyphenyl) butan-1-one (3), 4-methoxy-1-naphthol (4), and cerevisterol (5). Among them, compounds 1-3 showed notable antifungal activities against Botrytis cinerea, Fusarium avenaceum and Hormodendrum compactum.

CONCLUSIONThe endophytic fungus T. longibrachiatum was separated from the plant P. delavayi for the first time. Five compounds were first separated from endophytic fungus of P. delavayi. Among them, compound 4 was separated from microbial fermentation products for the first time.

DNA, Fungal ; genetics ; DNA, Intergenic ; genetics ; Endophytes ; classification ; genetics ; isolation & purification ; metabolism ; Paeonia ; microbiology ; Phylogeny ; Trichoderma ; classification ; genetics ; isolation & purification ; metabolism

The aim of the paper is to observe the distribution of the endophytic fungi in leaves of Aquilaria sinensis by using permanent paraffin-cut section, optical microscope photography and histochemistry. Total DNA was extracted with modified CTAB method and rDNA ITS regions of plant and endophytic fungi were amplified with eukaryotic universal primers. The rDNA ITS amplicon was characterized by RFLP analysis, sequencing of rDNA ITS library and phylogenetic analyses using PAUP by maximum parsimony. Fusarium sp. A2 was used to induce the formation of resinous in A. sinensis trees. As a result, endophytic fungi mainly distributed in spongy and phloem in leaf. Endophytic fungi distributed in the phloem in agarwood-producing tree and had a relatively high abundance. Phoma sp. and Collectrotrichum sp. were the absolute advantage species in the leaf tissues of non-resinous and agarwood-producing tree, respectively. Collectrotrichum sp. was the only fungal species detected both in the two types of A. sinensis with different levels of abundance. The culture-independent molecular method can be used to identify fungal species directly and rapidly from the plant tissues. Endophytic fungal communities in non-resinous and agarwood-producing A. sinensis leaf tissues were quite different.
Cluster Analysis ; DNA, Fungal ; genetics ; Endophytes ; classification ; genetics ; physiology ; Fusarium ; classification ; genetics ; physiology ; Phylogeny ; Plant Leaves ; metabolism ; Thymelaeaceae ; microbiology ; Wood ; metabolism ; microbiology

In this paper, 29 endophytes were isolated from different organs and tissues of Lycium barbarum of Ningxia by tablet coating method, 18 of them was fungi, and 11 of them was actinomycetes. The endophytes quantity in the different tissues were leaves > flowers > roots >fruits; The hydroxyl radical scavenging activities of 11 endophytes were investigated by Fenton reaction, and total antioxidant capacities of them were examined by a. total antioxidant capacity test kit; culture features and strain-specific sequence analysis were employed to explore the diversity of the 11 endophytes. The result showed that 5 fungi and 6 actinomycetes that having antioxidant activity could be phylogenetically classified into 3 genera, 3 genera and 3 families, respectively. The total antioxidant capacity and hydroxyl radical scavenging activity of the 11 endophytes showed distinct difference. The antioxidant activity of Aspergillus were stronger, among which total antioxidant capacity of fL1 was (188.5 ± 0.549) U · mL⁻¹ and the IC₅₀ was 0.3 mg · L⁻¹; the IC₅₀ of strain fL1 was 0.42 mg · L⁻¹ and the total antioxidant capacity of fL9 was (113.63 ± 1.021) U · mL⁻¹, all of them were stronger than the positive control Vit C. The experimental results indicated that endophytic fungi of L. barbarum of Ningxia have a great developing and application prospect for the development of antioxidant agent.
Antioxidants ; chemistry ; Bacteria ; chemistry ; classification ; genetics ; isolation & purification ; Biodiversity ; Endophytes ; classification ; genetics ; isolation & purification ; Fungi ; chemistry ; classification ; genetics ; isolation & purification ; Lycium ; microbiology ; Molecular Sequence Data ; Oxidation-Reduction

OBJECTIVETo identify endophytic fungi bn12 from Cinnamomum camphora chvar, borneol and analysis its volatile metabolites.

METHODThe endophytic fungi bn12 was identified by morphological observation. volatile metabolites of endophytic fungi bn12 was analyzed by gas chromatography/mass spectrography (GC-MS).

RESULTVolatile metabolites of endophytic fungi bn12 contain borneol and much indoles. The ITS sequence of endophytic fungi bnl2 is most similar to the ITS sequence of pleosporaceae fungus, particularly C. nisikadoi.

CONCLUSIONEndophytic fungi bn12 is belong to pleosporaceae fungus. It has the ability of producing broneol.

Bornanes ; analysis ; Cinnamomum camphora ; microbiology ; DNA, Ribosomal Spacer ; genetics ; Endophytes ; genetics ; metabolism ; Fungi ; classification ; genetics ; metabolism ; Gas Chromatography-Mass Spectrometry ; Phylogeny ; Volatile Organic Compounds ; analysis

OBJECTIVETo clone calmodulin (CaM) gene in Eleutherococcus senticosus, and study the effect of endophytic fungi on expression amount of CaM gene.

METHODThe CaM full length cDNA sequence was cloned by rapid amplification of cDNA ends (RACE). The gene was analyzed and corresponding structure and functions were predicted by the bioinformatics methods. The expression amount of CaM gene affected of endophytic fungus P116-1a, P116-1b, P1094 and P312-1 was detected by RT-PCR.

RESULTThe full length of CaM cDNA was 856 bp containing an ORF of 450 bp that encoded a protein of 149 amino acids. The homologous of predicted protein was almost 100% with plants like Panax ginseng and Daucus carota. RT-PCR results showed that endophytic fungus improved CaM expression amount significantly (P<0.05). The highest expression amount of CaM occurred 90 d after reinoculated with endophytic fungi P1094, up to 2.96 times of the control.

CONCLUSIONThe CaM gene of E. senticosus was successfully cloned for the first time. The results demonstrated that endophytic fungus of E. senticosus improved CaM expression amount significantly.

Calmodulin ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Eleutherococcus ; classification ; genetics ; metabolism ; microbiology ; Endophytes ; physiology ; Fungi ; physiology ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism

The paper is aimed at studying the diversity of endophytic fungi community from Paris polyphylla var. yunnanensis, and to provide a scientific basis for the utilization value of the endophytic fungi as bioactive material resources. In the present study, endophytic fungi were isolated from roots, rhizomes and leaves of wild P. polyphylla var. yunnanensis collected from Baoshan, Heqing county and Songming city of Yunnan province, and identified and classified by morphological methods together with its ITS sequence analysis. Seven and forty-nine strains of endophytic fungi were isolated from P. polyphylla var. yunnanensis. They were identified belonging to 41 genus. In these 41 genus, 3 genus exist in root only, 12 genus only exist in rhizome and 8 genus only exist in leaf. There was difference in endophytic fungi isolated from different sample sites. Endophytic fungi diversity from rhizomes of Heqing site was the highest. Endophytic fungi similarity coefficient was low among different sites and tissues. Based on these results, it is reasonable to propose that endophytic fungi of P. polyphylla var. yannanensis from different tissue and different sample sites has a certain difference which is possibly relate to their different habitats, different structure and composition of each tissue.
Biodiversity ; Endophytes ; classification ; genetics ; isolation & purification ; Fungi ; classification ; genetics ; isolation & purification ; Liliaceae ; microbiology ; Molecular Sequence Data ; Phylogeny ; Plant Leaves ; microbiology ; Plant Roots ; microbiology ; Plant Stems ; microbiology