Root Endophyte Shift and Key Genera Discovery in Rice under Barnyardgrass Stress

Despite increasing knowledge of barnyardgrass(Echinochloa crus-galli) interference with rice, relatively little is known how endophytes improve the ability of rice against barnyardgrass stress. Here, we provided a detailed temporal characterization of rice root-associated microbiomes during co-cultivation with barnyardgrass and a comparison with the microbiomes of weed-free rice plants. Alpha diversity analysis indicated that barnyardgrass had the opposite effects on endophytic bacteria and fungi in rice roots, in terms of the community diversity, richness and coverage at the rice seedling stage. Principal coordinate analysis showed that barnyardgrass had only a minor effect on the community composition of endophytes in rice roots at the rice seedling stage, but showed a significant and maximum interference at the heading stage. Rice recruited many endophytes to resist biotic stress from barnyardgrass, especially for fungi. PICRUSt(phylogenetic investigation of communities by reconstruction of unobserved states) predictive analysis indicated that 23 metabolic pathways of bacteria were overrepresented in rice. In addition, the main trophic mode of fungi was pathotroph according to FUNGuild analysis. A positive correlation between bacteria and fungi in rice roots was found via network analysis. Anaeromyxobacter, Azospira and Pseudolabrys were the vital bacteria, Phaeosphaeria and Funneliformis were the dominant fungi in maintaining the stability of the ecological network. These results provided data and a theoretical basis for the in-depth understanding of what role endophytes play in rice resistance to barnyardgrass stress and will have implications on improving the resistance of rice against biotic stress using root microbiota.

A New Cytotoxic Sterol Produced by an Endophytic Fungus from Castaniopsis fissa at the South China Sea Coast

A new sterol, ergosta-8(9),22-diene-3,5,6,7-tetraol(3β,5α,6β,7α,22E)(A) together with three known sterols: 3β, 5α,6β-trihydroxyergosta-7,22-diene (B), 3β-hydroxy-5α,8α- epidioxyer-gosta-6,22-diene (C) and ergosterol (D) were isolated from the mycelia of an unidentified endophytic fungus separated from Castaniopsis fissa (chestnut tree). Compound A exhibited potent selective cytotoxicity against Bel-7402, NCI4460 and L-02 cell lines with IC50 values 8.445, 5.03, 13.621 μg/mL, respectively.

Two New Alkaloids from Fusarium tricinctum SYPF 7082,an Endophyte from the Root of Panax notoginseng

Panax notoginseng(Araliaceae)is a famous traditional Chinese medicine mainly cultivated in Yunnan and Guangxi provinces of China.Two new alkaloids,rigidiusculamide E(1)and[-(a-oxyisohexanoyl-N-methyl-leucyl)2-](2),together with two known ones,(-)-oxysporidinone(3)and(-)-4,60-anhydrooxysporidinone(4)were isolated from the mycelia culture of Fusarium tricinctum SYPF 7082,an endophytic fungus obtained from the healthy root of P.notoginseng.Their structures were determined on the basis of extensive spectroscopic analyses.Compounds 1-4 were tested for their inhibitory effects against NO production on Murine macrophage cell line,and the new compound 2 showed significant inhibitory activity on NO production with the IC_(50)value of 18.10±0.16μM.

A new naphthoquinoneimine derivative from the marine algal-derived endophytic fungus Aspergillus niger EN-13

Cultivation of an endophytic fungus Aspergillus niger EN-13 that was isolated from the inner tissue of the marine brown alga Colpomenia sinuosa resulted in the characterization of a new naphthoquinoneimine derivative, namely, 5,7-dihydroxy-2-[1-（4- methoxy-6-oxo-6H-pyran-2-yl）-2-phenylethylamino]-[ 1,4]naphthoquinone. The structure of the new compound was established on the basis of various NMR spectroscopic analyses including 2D NMR techniques, El-MS, and HR-ESI-MS. This compound displayed moderate antifungal activity.

Metabolites from mangrove endophytic fungus Dothiorella sp.

Mangroves are special woody plant communities in the intertidal zone of tropical and subtropical coasts. They prove to be a natural microorganisms and new metabolites storage. In the study of mangrove endophytic fungi metabolites, four new compounds, Compounds 1, 2, 3 and 4, as well as a known octaketide, oytosporone B (5), are isolated from an endophytic fungus, Dothiorella sp., HTF3. They all show cytotoxic activities. The elucidation of these structures is mainly based on 1D/ 2D NMR and ESI-MS spectral analyses.

Secondary Metabolites from the Endophytic Fungus Xylaria sp.hg1009

A detailed chemical investigation of the secondary metabolites produced by the endophytic fungus Xylaria sp.isolated from the stems of Isodon sculponeatus afforded six new compounds,xylariahgins A-F(1-6),two new natural products(7 and 8),along with two known compounds(9 and 10)(Fig.1).The structures of all compounds were unambiguously established by analyzing their spectroscopic data or referring to pertinent literature.Compounds 1-8 were tested for their cytotoxic activity against five human tumor cell lines.

Antimicrobial activity of kojic acid from endophytic fungus Colletotrichum gloeosporioides isolated from Sonneratia apetala,a mangrove plant of the Sundarbans

Objective: To isolate and evaluate the antimicrobial activity of the active principle(s) from the ethyl acetate(EtOAc) extract of endophytic fungus Colietotrichum gloeosporioides(C.gloeosporioides) isolated from Sonneratia apetala. Methods: Water agar technique was used to isolate the fungus, and both microscopic and molecular techniques were used for identification of the strain. Potato dextrose broth was used to grow the fungus in large-scale. Reversed-phase preparative HPLC analysis was performed to isolate the major active compound, kojic acid. The EtOAc extract and kojic acid were screened for their antimicrobial activity against two Grampositive and two Gram-negative bacteria as well as a fungal strain using the resazurin 96-well microtitre plate antimicrobial assay. Results: The fungus C. gloeosporioides was isolated from the leaves of Sonneratia apetala. Initial identification of the fugal isolate was carried out using spore characteristics observed under the microscope. Subsequently, the ITS1-5.8 S-ITS2 sequencing was employed for species-level identification of the fungus C. gloeosporioides. Five litres of liquid culture of the fungus produced approximately 610 mg of a mixture of secondary metabolites.Kojic acid(1) was isolated as the main secondary metabolite present in the fungal extract, and the structure was confirmed by 1 D, 2 D NMR and mass spectrometry. The EtOAc extract and compound 1 exhibited considerable antimicrobial activity against all tested microorganisms.Whilst the minimum inhibitory concentration(MIC) values from the EtOAc extract ranged between 2.4×10^(-4)mg/mL and 2.5 mg/mL, those of kojic acid(1) were between 0.125 mg/mL and1 mg/mL. The EtOAc extract and kojic acid(1) were most active against Pseudomonas aeruginosa(MIC = 2.4×10^(-4). mg/mL) and Micrococcus luteus(MIC = 0.125 mg/mL), respectively. Conclusions:The results revealed that the endophytic fungus C. gloeosporioides could be a good source of commercially important kojic acid, which exhibited antimicrobial properties.

Preliminary study on Salvia miltiorrhiza bung endophytic fungus

Objective To select the strains which can produce tanshinone ⅡA like its host plant Salvia miltiorrhiza bung.Methods A total of 50 strains of endophytic fungi were isolated from healthy,living and symptomless tissues of Salvia miltiorrhiza bung,among which 29 strains were obtained from the root,14 from the stem,3 from the leaf,3 from the flower and 1 from the seed.Their antimicrobial activities against nine different bacteria,including both Gram-negative and Gram-positive bacteria,were measured by Oxford plate agar diffusion bioassay.Results Our data showed that all but four strains had significant antibacterial activities on at least one indicator bacterium to some extent,and five strains(DR1,DR4,DR16,DR18 and DF2)manifested quite prominent antibacterial activities against certain pathogenic bacteria.In some degree,it might indicate that this endophytic fungus isolated from the tissues of Salvia miltiorrhiza bung has a potential value as a natural antibacterial medicine as well.Thin layer chromatography(TLC)and high-performance liquid chromatography(HPLC)were carried out to test selected strains,both inside and outside of the cell to see if any strain can produce tanshinone ⅡA.The result showed that extracts from three strains,labeled as DR12(outside cell),DR21(inside cell)and DF3(inside cell),had a component with the same Rf value in TLC assay as that of authentic tanshinone ⅡA.The extract from DR12(outside cell)and DR21(inside cell)had a peak at retention time identical to that of authentic tanshinone ⅡA in HPLC.Conclusion The fungi appear to produce the bioactive compound tanshinone ⅡA,and they could be used to produce tanshinone ⅡA by fermentation.It provides a new way to synthesize this natural medicine.

Three new pyrrole alkaloids from the endophytic fungus Albifimbria viridis

Three new pyrrole alkaloids albifipyrrols A-C(1-3),were isolated from the endophytic fungus Albifimbria viridis col-lected from the Chinese medicinal plant.Their structures were elucidated by extensive NMR and HRESIMS spectro-metric analyses.All compounds were evaluated for immunosuppressive activity.Fortunately,compound 2 exhibits certain inhibition specifically against the LPS-induced proliferation of B lymphocyte cells with IC50 value 16.16μM.

Arthrinins E-G, Three Botryane Sesquiterpenoids from the Plant Endophytic Fungus Arthrinium sp. HS66

Arthrinins E-G(1-3),three new sesquiterpenoids possessing non-isoprenoid botryane skeleton,were isolated from the fermentation of an endophytic fungus named Arthrinium sp.HS66 which colonized in the stems of Isodon xerophilus.Their structures were determined by extensive spectroscopic methods.Furthermore,the structure of 1 was unambiguously confirmed by X-ray diffraction,while those of 2 and 3 were verified through quantum chemical calculation of NMR data and ECD spectra.

Two New Quinazoline Derivatives from the Moss Endophytic Fungus Aspergillus sp. and Their Anti-inflammatory Activity

Two new quinazoline derivatives versicomides E(1)and F(2),and 10 known compounds(3-12)were isolated from the moss endophytic fungus Aspergillus sp.Their structures were determined on the basis of extensive spectroscopic data analysis and ECD calculations.Among them,the compound 7(6-hydroxy-3-methoxyviridicatin)was first reported as a natural product.Inhibition on LPS-induced NO production in RAW 264.7 murine macrophages found that compounds 5,7 and 8 showed significant inhibitory effects on NO production,with IC50 values of 49.85,22.14 and 46.02μM respectively.

Effect of Endophytic Fungus on Pyrola calliantha H. Andr Responsed to Cold Stress

This study measured the antioxidant enzyme activities of Pyrola under cold and endophytic fungal infection conditions[i.e.,peroxidase(POD),catalase(CAT)and superoxide dismutase(SOD)],and the contents of malondialdehyde(MDA),soluble sugar and soluble protein.In addition,the changes in the anatomical structure of Pyrola vegetable organ during cold stress upon inoculation with a synergistic endophytic fungus were also analyzed.It was found that the soluble sugar and soluble protein contents and POD,SOD and CAT activies of Pyrola all increased,when the plants were exposed to temperature of-5℃.In particular,the MDA contents and the POD activies significantly increased compared with those of the control.In addition,the soluble sugar and soluble protein contents increased during the treatment,which involved in both inoculation with an endophytic fungus and low temperature;however,with a reduction in the concentration of endophytic fungus,those two indicators decreased.The optimal concentration of endophytic fungus was 5×10^(7) cfu·mL^(-1).From the anatomical structure of Pyrola root,endophytic fungi were found to improve the health of.Pyrola in cold weather.These results showed that endophytic fungi played a vital role in the growth of Pyrola,which was an evergreen plant that could survive exposure to low temperature.

Tridepsides from the endophytic fungus colletotrichum gloeosporioides associated with a toxic medicinal plant tylophora ovata

Introduction:Bioactive secondary metabolites from the microbes living in frigid,toxic or other extreme environments are emerging as a new medicinal resource.Here,we report the discovery of new antidiabetic and anti-inflammatory compounds with novel structures from endophytic fungi hosted toxic medicinal plant.Methods:The endophytic fungus isolated from toxic plants was fermented and extracted.The obtained extracts were purified with preparative HPLC to yield pure compounds.The purified compounds were examined by PTP1b inhibition and NO inhibition assays to evaluate their bioactivities.Results:One new tridepsides(Compound 1),one new benzeneacetic acid derivative(Compound 3)and five known compounds(Compounds 2 and 4-7)were isolated from the ethyl acetate extract of Colletotrichum gloeosporioides,an endophytic fungus obtained from a toxic medicinal plant Tylophora ovata.Their structures were determined by spectroscopic data(1D and 2D NMR,HRESIMS)analyses.Compound 2 showed significant inhibitory activity against PTP1b with an IC50 value of 0.84μM.Compounds 2 and 3 exhibited moderate inhibitory activities against the NO(nitric oxide)release in LPS-induced RAW 264.7 cells at 10μM with percent inhibition of 39%and 33%,respectively.Conclusion:The Compound 2 has potent PTP1b inhibitory effect indicating its antidiabetic potential and thus might be considered a lead compound for antidiabetic drug development.

Antimicrobial and cytotoxic activities of endophytic fungus Colletotrichum gloeosporioides isolated from endemic tree Cinnamomum malabatrum

In a survey of endophytic fungi associated with endemic plant Cinnamomum malabatrum leaves harbored a bioactive endophytic isolate CMS 3 was identified as Colletotrichum gloeosporioides through morphological and phylogenetic analysis based on ITS-rDNA.The ethyl acetate extract of fermentation broth of Colletotrichum gloeosporioides CMS 3 displayed antimicrobial activity against gram positive and gram negative bacteria as well as the fungal pathogen,Candida albicans.The ethyl acetate crude extract showed in vitro cytotoxicity against the HeLa,MCF-7 and MG63 cancer cell lines with the IC50 values of 94.2μg/ml,84.3μg/ml and 162μg/ml respectively.Gas chromatography and Mass Spectrophotometry(GC-MS)analysis of crude extract confirmed that CMS 3 was a prolific producer of secondary metabolites,in which nearly 74%of the metabolites not listed in the NIST database.Major compounds were phenol 3,5-dimethoxy acetate(11.82%),4'-isopropylidene-bis-(2-cyclohexyl)phenol,N-Didehydrohexacarboxyl-2,4,5-trimethylpiperazine and 1,2,4-Triazolium ylide.These metabolites may be responsible for its antimicrobial and cytotoxic activities.

New indole derivatives from endophytic fungus Colletotruchum sp. HK-08 originated from leaves of Nerium indicum

Objective:To study secondary metabolites from endophytic fungus Colletotruchum sp.HK-08 originated from the leaves of Nerium indicum.Methods:The compounds were isolated by various column chromatographic techniques,and their structures were elucidated by spectroscopic techniques[high resolution electrospray ionization mass spectroscopy(HRESIMS),one-dimensional(1D)and two-dimensional(2D)nuclear magnetic resonance spectroscopy(NMR)],as well as comparison with literature data.The Ellman method was used to determine the acetylcholinesterase(AChE)inhibitory activity.Results:Four indole derivatives were identified from Colletotruchum sp.HK-08,including 60-hydroxymonaspiloindole(1),2-(2-oxoindolin-3-yl)ethyl 2-(4-hydroxyphenyl)acetate(2),2-(2-oxoindolin-3-yl)ethyl 2-(2-hydroxyphenyl)acetate(3),and monaspiloindole(4).Compound 4 presented weak AChE inhibitory activity with IC50 value of(69.30±6.27)lmol/L[tacrine as the positive control,with IC50 value of(0.61±0.07)lmol/L].Conclusion:Compounds 1–3 were new compounds,and compound 4 had weak AChE inhibitory activity。

Griseofulvin enantiomers and bromine-containing griseofulvin derivatives with antifungal activity produced by the mangrove endophytic fungus Nigrospora sp.QQYB1

Marine microorganisms have long been recognized as potential sources for drug discovery.Griseofulvin was one of the first antifungal natural products and has been used as an antifungal agent for decades.In this study,12 new griseofulvin derivatives[(±)-1-2,(+)-3,(±)-4,10-12,and 14-15]and two new griseofulvin natural products(9 and 16)together with six known analogues[(-)-3,5-8,and 13]were isolated from the mangrove-derived fungus Nigrospora sp.QQYB1 treated with 0.3%NaCl or 2%NaBr in rice solid medium.Their 2D structures and absolute configurations were established by extensive spectroscopic analysis(1D and 2D NMR,HRESIMS),ECD spectra,computational calculation,DP4+analysis,and X-ray single-crystal diffraction.Compounds 1-4 represent the first griseofulvin enantiomers with four absolute configurations(2S,6'S;2R,6'R;2S,6'R;2R,6'S),and compounds 9-12 represent the first successful production of brominated griseofulvin derivatives from fungi via the addition of NaBr to the culture medium.In the antifungal assays,compounds 6 and 9 demonstrated significant inhibitory activities against the fungi Colletotrichum truncatum,Microsporum gypseum,and Trichophyton mentagrophyte with inhibition zones varying between 28 and 41 mm(10μg/disc).The structure-activity relationship(SAR)was analyzed,which showed that substituents at C-6,C-7,C-6'and the positions of the carbonyl and double bond of griseofulvin derivatives significantly affected the antifungal activity.

Crystal Structure of a Marine Natural Compound,Anhydrofusarubin

One marine natural compound, anhydrofusarubin, was isolated from the endophytic fungus No. B77 from the mangrove tree on the South China Sea coast. It was the first time that such a compound was isolated from marine fungus. The structure was identified by NMR data and mass spectrometry （MS）. In addition, its structure was determined by single-crystal X-ray diffraction analysis. It crystallizes in triclinic, space group P1^- with a = 8.3185（15）, b = 12.397（2）, c = 13.767（3） A, α = 65.692（3）, β = 79.266（3）, γ= 75.692（3）°, C15H12O6, Mr= 288.24, V = 1248.0（4） A^3, Z = 4, Dc = 1.534 g/cm^3, F（000） = 600, μ = 0.120 mm^-1, the final R = 0.0459 and wR = 0.1184 for 5360 observed reflections （I 〉 2σ（I））. In the primary bioassay, the title compound shows strong inhibitory activity against Gram-positive bacteria Staphyococcus aureus （ATCC27154） with the MIC value of 12.5 μg/mL.

Production and Partial Characterization of an Extracellular Phytase Produced by Muscodor sp. under Submerged Fermentation

In most of the raw materials of plant origin used in animal feed, a portion of the phosphorus is stored as phytic acid or phytate. Phytate is the main storage form of phosphorus in vegetables but is not readily assimilated into food at low concentrations of the enzyme phytase. In addition to making phosphorous unavailable, phytate binds divalent cations such as calcium, copper, magnesium, iron, manganese and zinc, preventing the absorption of these nutrients in the gut of the animal. Phytase promotes the hydrolysis of the phytate phosphorus-releasing molecule, thereby increasing its bioavailability in feed. Phytase is distributed in plant and animal tissues and it is synthesized by some species of bacteria and fungi. The addition of this enzyme in the diet of animals is essential to promote greater uptake of phosphorus and also contributes to a decrease in the levels of phosphorus excreted by animals, thus reducing the pollution caused by excess phosphorus in the environment. This work aimed to select a fungus that stands out in the production of phytase among 100 isolates from Brazilian caves belonging to the genera Aspergillus, Penicillium and Cladosporium and 13 endophytic fungi of the aerial part of the coffee plant. For selection, the fungi were cultured in medium containing phytic acid as a sole source of phosphorus. After seven days at 25 °C, we evaluated growth and enzyme production by the presence of the phytic acid halo degradation (Enzymatic Index-EI) surrounding the colonies. Forty-seven species produced phytase, and the fungi Penicillium minioluteum (CF279) and Muscodor sp. (UBSX) showed higher degradation halos, 2.41 and 4.46, respectively. Considering the Muscodor sp. as the main source of phytase, high enzymatic levels were obtained when the fungus was grown under submerged fermentation with initial pH of 5.0 using wheat bran as additional carbon source for 144 h, at 125 rpm and 30 °C. Additionally, the enzyme was stable at pH 5.0 and 40 °C, and inhibited (14% - 88%) by all compounds analyzed. Then, this is the first study that reports the production of phytase by the endophytic fungus Muscodor sp.

First Report of Fusarium striatum Causing Root Rot Disease of Panax notoginseng in Yunnan,China

Panax notoginseng is a traditional Chinese medicinal plant.Root rot of P.notoginseng is one of the most serious diseases affecting P.notoginseng growth and causes wilted leaves,fewer lateral roots and rotten roots.Root rot is a soil-borne disease,and mainly occurs from June to August in Yunnan Province when the temperatures are high and the air is humid.In this study,the endophytic fungal genus Fusarium isolate E-2018.1.22-#3.2 was obtained from a P.notoginseng embryo.Fusarium isolate E-2018.1.22-#3.2 was identified as Fusarium striatum based on morphological characteristics and molecular analysis.The fungus was found to have conidiophores and macroconidia,and its ITS,LSU and TEF-1αgenes shared 100%,99.2%and 99%identities with the homologous genes of Fusarium striatum,respectively.Isolate F.striatum E-2018.1.22-#3.2 can cause root rot symptoms,including black,soft roots,fewer lateral roots and leaf wilt,in 93%of the experimental P.notoginseng plants,and could be re-isolated,fulfilling Koch’s postulates.When the P.notoginseng plants were treated with the fungicide pyraclostrobin,isolate F.striatum E-2018.1.22-#3.2 was unable to cause root rot.We have therefore demonstrated that F.striatum E-2018.1.22-#3.2 is able to cause root rot disease in P.notoginseng.This is the first report of root rot disease caused by F.striatum on P.notoginseng in China.