Root-associated endophytic fungi modulate endogenous auxin and cytokinin levels to improve plant biomass and root morphology of trifoliate orange

Root-associated endophytic fungi like Serendipita indica and arbuscular mycorrhizal(AM) fungi can improve plant growth and root construction, but the potential mechanism is unclear. In this study, Funneliformis mosseae(an AM fungus) and S. indica, singly or in combination were inoculated into trifoliate orange(Poncirus trifoliata) seedlings, to assess changes in biomass and root morphological traits, coupled with auxins and cytokinins concentrations in leaves and roots and the expression of auxin synthesis and transporter protein genes. After 20 weeks of inoculation with these fungi, shoot and root biomass, root total length, taproot length, average diameter, surface area, volume, and the number of lateral roots in 1st-, 2nd-, and 3rd-order were improved, and S. indica showed a relatively greater effect than F. mosseae and dual inoculation. Endophytic fungal inoculation also significantly increased the concentration of indoleacetic acid, indole butyric acid, trans-zeatin,dihydrozeatin, and isopentenyl adenine in leaves and roots, whilst F. mosseae and S. indica exhibited relatively greater effects on leaves and roots, respectively. Correlation analysis revealed that both biomass and root morphological traits(except root projected area) were significantly positively associated with endogenous auxins and cytokinins. In addition, the inoculated plants recorded comparatively higher expression levels of indoleacetic acid synthesis genes(PtTAA1, PtTAR2, PtYUC3, PtYUC4, PtYUC6, and PtYUC8) and indoleacetic acid transporter protein genes(PtAUX1, PtLAX1, PtLAX2, PtLAX3, PtPIN1, PtPIN3, PtPIN4, PtABCB1, and PtABCB19) than the non-inoculated plants, among which F. mosseae and S. indica showed better effects in leaves and roots, respectively. These results suggest that root-associated endophytic fungi improved plant growth and root architecture, which were associated with changes in endogenous auxins and cytokinins.

Identification and Bioactivities of Secondary Metabolites Derived from Endophytic Fungi Isolated from Ethnomedicinal Plants of Tujia in Hubei Province:A Review

Tujia is a national minority,inhabiting in the mountainous Wuling area in China.Since 1978,Tujia medicine has been studied,summarized and developed,leading to numerous achievements by Chinese researchers,such as the publishing of approximately 30 monographs of Tujia medicine.These publications are focused on summarizing and improving the theory of Tujia medicine and developing clinical therapies from this system of medicine.The shortage of natural medicinal plants used in Tujia medicine has created the need to discover new resources to replace them and protect endangered natural plant species.Endophytic fungi are one of the conservation options,are considered a source of new bioactive natural products,and are a renewable and inexhaustible source of new drugs and agrochemicals.This review summarizes 260 compounds from endophytic fungi that have been previously isolated from the medicinal plants of Tujia.These compounds include steroids,terpenoids,meroterpenoids,polyketides,alkaloids,peptides,aliphatic compounds,aromatic compounds,and heterocyclic compounds.

A pilot study of antioxidant potential of endophytic fungi from some Sudanese medicinal plants

Objective: To evaluate the total phenolic content and total antioxidant capacity of ethyl acetate extracts of 21 endophytic fungi isolated from five Sudanese medicinal plants: Calotropis procera, Catharanthus roseus, Euphorbia prostrate, Vernonia amygdalina and Trigonella foenumgraecum. Methods: Crude extracts of endophytic fungi and their host plants were tested by classical Folin-Ciocalteu colorimetric method to determine the total phenolic content, also total antioxidant capacity was estimated using 1,1-diphenyl-2-picrylhydrazyl free radical scavenging in vitro method. Results: Among the endophytes, endophytic fungus Aspergillus sp. from Trigonella foenum-graecum seeds demonstrated the highest both total phenolic content in term of gallic acid equivalent(GAE) [(89.9±7.1 mg GAE/g)] and antioxidant activity for 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay [IC50:(18.0±0.1 μg/m L)]. A high positive linear correlation(R2=0.999 1) was found between total antioxidant capacity and total phenolic content of endophytic fungi isolated from Vernonia amygdalina. Conclusions: The present study revealed that some endophytic fungi from the five Sudanese medicinal plants could be a potential source of novel natural antioxidant compounds.

Levels of rhizome endophytic fungi fluctuate in Paris polyphylla var.yunnanensis as plants age

Paris polyphylla var. yunnanensis is an important medicinal plant with abundant saponins that are widely used in the pharmaceuticals industry. It is unclear why the levels of active ingredients increase as these plants age. We speculated that the concentrations of those components in the rhizomes are mediated by fungal endophytes. To test this hypothesis, we took both culture-dependent and-independent(metagenomics) approaches to analyze the communities of endophytic fungi that inhabit those rhizomes in plants of different age classes(four, six, and eight years old). In all, 147 isolates representing 18 fungal taxa were obtained from 270 segments(90 per age class). Based on morphological and genetic characteristics, Fusarium oxysporum(46.55% frequency of occurrence) was the predominant endophyte,followed by Leptodontidium sp.(8.66%) and Trichoderma viride(6.81%). Colonization of endophytic fungi was maximized in the eight-year-old rhizomes(33.33%) when compared with four-year-old(21.21%) and six-year-old(15.15%) rhizomes. Certain fungal species were present only at particular ages. For example,Alternaria sp., Cylindrocarpon sp., Chaetomium sp., Paraphaeosphaeria sporulosa, Pyrenochaeta sp., Penicillium swiecickii, T. viride, and Truncatella angustata were found only in the oldest plants. Analysis of(metagenomics) community DNA extracted from different-aged samples revealed that, at the class level,the majority of fungi had the highest sequence similarity to members of Sordariomycetes, followed by Eurotiomycetes and Saccharomycetes. These results were mostly in accord with those we obtained using culture methods. Fungal diversity and richness also changed over time. Our investigation is the first to show that the diversity of fungi in rhizomes of P. polyphylla var. yunnanensis is altered as plants age, and our findings provide a foundation for future examinations of useful compounds.

Purification of four strains of endophytic fungi from Astragalus and their optimized liquid fermentations

This study was designed to isolate endophytic fungi from A. mongholicus （growing in northeast China） to determine whether they can produce bioactive metabolites. Four strains of endophytic fungi （strains 16, 17, 23 and 75） were successfully isolated from A. mongholicus using the surface disinfection method. According to ITS-rDNA sequences analysis, strains 16 and 75 were identified as Fusarium oxysporum, and strains 17 and 23 were identified as Bionectria ochroleuca. We applied the Box-Behnken design （BBD） to optimize the liquid fermentation conditions and obtain the maximum cell dry weight （CDW） yield. Opti-mal parameters were obtained under the following experimental condi-tions：temperature of 28＆#176;C, potato dextrose agar （PDA） liquid medium of 80 mL and rotation speed of 150 rpm. The four isolated endophytic fungi did not produce astragalosides I-IV, flavonoids or polysaccharides. Iso-lation of additional species of endophytic fungi from A. mongholicus and determination of their capacity to produce biologically active substances are subjects in need of further research.

Non-mycorrhizal fungal endophytes in two orchids of Kaiga forest(Western Ghats), India

We used standard isolation protocols to explore the endophytic fungal communities in three tissue types of two dominant orchids （Bulbophyllum neilgherrense and Vanda testacea） of the Kaiga forest of the Western Ghats. We surface sterilized and assessed 90 segments of each orchid for the occurrence and diversity of endophytic fungal taxa. The 118 fungal isolates were obtained from root, bulb and leaves of B. neilgherrense, consisting of 17 anamorphic taxa （range, 10-15 taxa） with 1.3 fungal taxa per segment （range, 1.2-1.4 taxa）. Four taxa （Aspergillus flavus, A. niger, Penicillium sp. and morpho sp. 1） belonged to the core group （11.1%--32.2%）. The relative abundance of A. flavus and morpho sp. 1 was more than 10%. A total of 130 fungal isolates from roots, stems and leaves of V. testacea yielded 20 anamorphic taxa （range, 11-15 taxa） with 1.4 fungal taxa per segment （range, 1.4-1.5 taxa）. Aspergillusflavus, A. niger, A. ochraceus, Gliocladium viride, Penicillium sp. and morpho sp. 1 belonged to the core group. Relative abundance exceeded 10% for A. flavus, A. niger, and morpho sp. 1. The Simpson and Shannon diversity indices were higher in leaf than root or bulb/stem of both orchids. Jaccard＇s similarity coefficient was higher between root and leaf in both orchids （56.3%-60%） than between other pairs. Our study revealed that the endophytic fungal assemblage and diversity orB. neilgherrense and E testacea of Kaiga forest of the Western Ghats were relatively similar between orchids and their tissues.

Three Novel Endophytic Fungi as Potential Candidates Producing Bioactive Metabolites Isolated from Metasequoia glyptostroboides

[Objectives]This study was conducted to isolate and investigate the endophytic fungi with antimicrobial activity from the healthy leaves and bark of Metasequoia glyptostroboides Hu&W.C.Cheng regarded as an important medicinal plant in China.[Methods]Endophytic fungi with visible antimicrobial activity were isolated from the healthy leaves and bark of M.glyptostroboides,and primarily screened using Escherichia coli and Staphylococcus aureus as test microorganisms.[Results]Three isolates with relatively higher antimicrobial activity,labeled as MG-01,MG-02,MG-09,were selected for study.Molecular and morphological analysis indicated that the three isolated endophytes belong to Penicillium sp.The ethyl acetate(EtOAc)extracts of the three endophytes were obtained and subjected to the disk diffusion assay,using E.coil,S.aureus,Pseudomonas aeruginosa,Enterococcus faecalis,Monilia albicanas,and Bacillus subtilis as test microorganisms.The minimum inhibitory activity(MIC)of the endophyte with relatively highest antimicrobial activity was determined using two-fold broth micro-dilution method.All the endophytes showed a broad spectrum antimicrobial activity.Among the three endophytes,the strain MG-09 exhibited relatively highest antimicrobial activity with the zones of inhibition(ZOI)ranging from 14.15 to 24.07 mm and MIC ranging from 416.67 to 65.11μg/ml.[Conclusions]The endophytic fungi colonized in M.glyptostroboides are capable of producing effective antimicrobial compounds which may be a potential source for drug discovery and biocide production.

Anticancer activity test of ethyl acetate extract of endophytic fungi isolated from soursop leaf(Annona muricata L.)

Objective: To analyze anticancer activity of an ethyl acetate extract of endophytic fungi isolated from soursop leaf(Annona muricata L.). Methods: Anticancer activity of fungal extracts was determined by observing its toxicity against MCF-7(Michigan Cancer Foundation-7) cells in vitro by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay method. At an extract concentration of 100 μg/m L, 4 isolates out of 12 showed high activity against the cancer cell growth. The four isolates were then selected for further IC50 determination, by measuring the inhibition of cancer cell proliferation at extract concentration of 25 μg/m L, 50 μg/m L, 100 μg/m L, 200 μg/m L and 400 μg/m L. Results: Results showed that isolate Sir-G5 had the highest anticancer activity with an IC50 of 19.20 μg/m L. The best isolates were screened again using a normal cell(Chang cells) to determine its toxicity against normal cells. Results indicated that the extracts do not affect the proliferation of normal cells. Molecular identification showed that the fungal isolate Sir-G5 has a close relationship with Phomopsis sp. Conclusions: The endophytic fungi isolated from soursop leaf has the potential to be used as a source of anticancer agents.

Community Structure of Endophytic Fungi in Ginseng and Its Correlation Analysis with Active Components

[Objectives]The paper was to explore the correlation between active components of ginseng and community structure of endophytic fungi in ginseng under different cultivation methods.[Methods]Using ginsengs under two main planting patterns in Huanren County,Liaoning Province as the samples,the community structure of endophytic fungi was studied by molecular means,and the chemical components were identified and the content of chemical markers was measured via LC-MS.Moreover,the correlation between medicinal components of ginseng and diversity of endophytic fungal flora was analyzed.[Results]The diversity and active components of endophytic fungi of ginseng were different.The diversity analysis showed that the medicinal components of ginseng were positively correlated with some fungal groups in ginseng.[Conclusions]Artificial intervention in the planting process would affect the endophytic fungi in medicinal materials,and further affect the chemical components of ginseng medicinal materials.

Endophytic Fungi Associated with a Holoparasitic Plant, Balanophora japonica (Balanophoraceae)

Holoparasitism is a special life cycle of flowering plants. All carbon resources are provided by photosynthetic host plants. A recent study revealed the presence of endophytic fungi in holoparasitic plants, but their ecological and evolutionary roles are still unknown. In this study, we examined endophytic fungi isolated from the holoparasitic plant Balanophora japonica (Balanophoraceae), collected from Kochi, Shikoku in western Japan. We isolated 23 fungal strains on inflorescences and tubers from three B. japonica plants at two locations and on one sample of the host plant (Symplocos lancifolia, Symplocaceae). Predominant isolates were Trichoderma-Hypocrea, Penicillium and Phialemonium. The first group was also predominant in the host plant. Fungal composition revealed in this study differed from the composition on B. harlandii or other root holoparasites with endophytic fungal (Rafflesia cantleyi) data. Those differences might be caused by various factors, including growth habits, location, phylogenetic position or host-parasite relationship.

Isolation, Identification and Pharmacological Activities of Endophytic Fungi from <i>Aponogeton undulatus</i>Roxb.

The objective of this study is to isolate, identify and investigate the pharmacological activities of the endophytic fungi from an aquatic plant Aponogeton undulatus Roxb. (A. undulatus). Endophytic fungi were isolated and identified based on morphological characters. The molecular identification of the fungal isolates was performed using by analyzing the DNA sequence based on mega BLAST program. Spectrums of antibacterial and antifungal activities were studied by Agar diffusion methods. Extracts of the endophytic fungal strains isolated from the plant A. undulatus were screened for probable cytotoxic activities using brine shrimp lethality bioassay. Six endophytic fungi, namely AULE-1, AULE-2, AULE-3, AURE-1, AURE-3 and AURE-4 were isolated and purified from the leaves and roots of the plant such as strains AULE-1 as Trichooderma sp., AULE-2 as Carvularia sp. AULE-3 as Penicillium sp. strains AURE-1 and AURE-4 as Fusarium sp. and AURE-3 as Mucor sp. Strain AURE-4 was further identified as Fusarium solani. At 300 μg/disc concentration, six ethyl acetate extracts of endophytic fungi of the plant A. undulates showed moderate to significant activities against most of the test bacteria and pathogenic fungi. The strain AURE-4 exhibited strong cytotoxic activities (10.18 μg/mL) compared to the standard antitumor agent vincristine sulphate and this positive result suggests that fungal extracts may contain antitumor or pesticidal compounds. This is the first study to report the isolation, identification, antimicrobial and cytotoxic properties of endophytic fungi of Aponogeton undulatus Roxb. in Bangladesh.

Plant growth-promoting characteristics of root fungal endophytes isolated from a traditional Cordillera rice landrace

This study was conducted to isolate and characterize the plant growth-promoting potential of fungal endophytes from the roots of Diket red,a traditional rice plant from the Cordillera,Northern Luzon.Eighteen morphospecies of filamentous endophytes were isolated of which twelve isolates were successfully identified to the species level.These isolates were identified as Aspergillus versicolor,Aspergillus sp.,Chaetosphaeria sp.,Cladosporium cladosporioides,Hypocrea lixii,Microascus murinus,and Trichoderma harzianum.The identified twelve isolates were selected to screen in vitro for their plant growth-promoting characteristics,and evaluated in vivo for their beneficial effects on seedling vigor and early seedling growth.Isolate PPL14 produced the highest IAA at 55.5μg ml^(-1)and M.murinus PPL10 produced the highest amount of IAA at 3.73μg IAA mg^(-1)dry mycelia wt.The seedling vigor assay and in vivo plant growth promotion bioassay indicated overall positive effects of culture filtrate(CF)application of the endophyte isolates.Rice seeds and seedlings grown in aseptic condition and treated with endophyte CFs displayed significantly enhanced levels of germination,seedling vigor,shoot,root,and total plant growth,and biomass compared to non-treated control.Other plant growth-promoting characteristics were also studied including phosphate solubilization,siderophore production,ammonia production,and catalase activity.This study supports the potential use of fungal endophytes as bio-inoculants for plant growth promotion and enhancement of nutrient assimilation of agriculturally important crops.

Diversity of endophytic fungi associated with the medicinally important aromatic plant Gaultheria fragrantissima

A total of thirty three(33)endophytic fungi including sterile mycelia were isolated from the leaf,stem and roots of medicinally important aromatic plant Gaultheria fragrantissima.Majority of the fungi belonged to the phylum Ascomycota.The total percentage colonization frequency(%CF)of each endophytic fungal species was calculated of which Juxtiphoma eupyrena showed highest colonization frequency(20.14%)in the leaf whereas Globisporangium irregulare was found to have highest colonization frequency in the stem(14.58%)and in the roots,Trichoderma viride was observed to have the highest colonization frequency of 21.43%.Diversity of endophytic fungi in the roots was found to be higher as compared to the other plant tissues.

Antioxidant compounds produced by Pseudocercospora sp.ESL 02,an endophytic fungus isolated from Elaeocarpus sylvestris

Objective: To isolate endophytic fungi from Elaeocarpus sylvestris(E. sylvestris) and to isolate antioxidant compounds from a potential source fungus.Methods: Endophytic fungi were isolated from fresh leaves and stems of E. sylvestris and identified based on DNA analysis. 1,1-Diphenyl-2-picrylhydrazyl(DPPH) radical scavenging activity was used to evaluate the antioxidant activity of the fungi. The potential antioxidant fungus was further studied to isolate antioxidant compounds. The isolated compounds were identified by melting point analysis, optical rotation, spectral analysis using a UV spectrophotometer, high resolution fast atom bombardment mass spectrometry, X-ray crystallography analysis,~1H nuclear magnetic resonance analysis and ^(13)C nuclear magnetic resonance analysis. The isolated compounds were evaluated with DPPH radical scavenging, reducing power, and b-carotene bleaching assays.Results: Seven endophytic fungi were successfully isolated from E. sylvestris and identified as Pestalotiopsis sp. EST 01, Pestalotiopsis sp. EST 02, Diaporthales sp. EST03, Meyerozyma sp. EST 04, Diaporthales sp. EST 05, Pestalotiopsis sp. ESL 01, and Pseudocercospora sp. ESL 02. Of the seven fungi, Pseudocercospora sp. ESL 02 had the highest antioxidant activity [IC50=(30.54 ± 0.88) mg/mL]. From that fungus, two compounds identified as terreic acid(1) and 6-methylsalicylic acid(2) were isolated with an IC50 of DPPH radical scavenging activity of(0.22 ± 0.02) mmol/L and(3.87 ± 0.27)mmol/L, respectively. The compounds also had good activities from the reducing power and b-carotene bleaching assays.Conclusions: The Pseudocercospora sp. ESL 02 fungus isolated from E. sylvestris looks promising as a novel source of terreic acid.

Drimane Sesquiterpenoids and Isochromone Derivative from the Endophytic Fungus Pestalotiopsis sp. M-23

Three new drimane sesquiterpenoids(1–3)together with the known 2a-hydroxyisodrimeninol(4),and a new isochromone derivative(5),were obtained from the solid cultures of fungal strain Pestalotiopsis sp.M-23,an endophytic fungus isolated from the leaves of Leucosceptrum canum(Labiatae).Their structures were determined by comprehensive 1D and 2D NMR,and MS analyses.The metabolites were evaluated for their antibacterial activities,and compound 3 showed weak inhibitory activity against Bacillus subtilis.

α-Glucosidase inhibitor produced by an endophytic fungus, Xylariaceae sp. QGS 01 from Quercus gilva Blume

Xylariaceae sp.QGS 01,an endophytic fungus isolated from the stem of Quercus gilva Blume showed high-glucosidase inhibitory activity.α-Glucosidase inhibitor have the role as one of carbohydrate-hydrolyzing enzymes to postpone absorption of glucose in the digestive organs.The α-glucosidase inhibitor constituents were isolated from the ethyl acetate extract of the mycellium of endophytic fungi Xylariaceae sp.QGS 01 using a bioassay-guided fractionation technique.Further separation and purification of the active fraction led to the isolation of constituents with strong inhibitory activities against-glucosidase:8-hydroxy-6,7-dimethoxy-3-methylisocoumarine(1)with inhibitory concentration(IC50)values against-glucosidase from Saccharomyces cerevisiae of 41.75μg/mL,while quercetin as the standard had an IC50 value of 4.80g/mL.The results of the present study showed that the endophytic fungus Xylariaceae sp.QGS 01 is potentially a rich source of antidiabetic medicine.

Transient leaf endophytes are the most active fungi in 1-year-old beech leaf litter

The ecological significance of fungi occurring asymptomatically inside living plant leaves is poorly understood.Given the broad saprotrophic potential of many endophytic fungi,we hypothesized that they persist in decaying litter for an extended period of time after leaf abscission.Fungal assemblages were assessed by highthroughput sequencing in autumn leaves of beech(Fagus sylvatica)and in the corresponding leaf litter in 388 samples from 22 beech forest plots in three widely distant regions of Germany.A considerable proportion of the leafendophytic fungi was also found in 1-year-old litter.Cooccurrence networks revealed that the fungi formed unstructured assemblages inside the living leaves,rather than well-structured communities.Previously endophytic fungi constituted an integral part of the fungal litter community and were by far the most active fungi in 1-year-old litter.We therefore consider these endophytic occurrences to represent transient stages.Composition of the aboveground microbiome appears therefore to be closely connected to the process of litter decomposition.Considering the respective linked fungal habitat will facilitate predicting nutrient and carbon cycling and storage in forest ecosystems as well as elucidating the ecology of leaf microbiomes.

Evaluation of the Antimicrobial Activity of Endophytic Fungus Isolated from <i>Cocos nucifera</i>(L.) Cotyledon against Medically-Important Pathogens

The study aimed to evaluate the endophytic fungi isolated from Cocos nucifera (L.) cotyledon for their possible antimicrobial activity. A total of twelve endophytic fungal species were successfully isolated, from which five were selected and presumptively identified, including Aspergillus niger, Raffaelea sp., Aspergillus flavus, Cladosporium sp. and Pseudallescheria spp. based on their macro and microscopic characters. The agar blocks of actively growing pure culture were assessed for antibacterial activity against five human pathogenic bacterial and fungal strains: Escherichia coli (BIOTECH 1634), Bacillus subtilis (BIOTECH 1697), Staphylococcus aureus (BIOTECH 1592), Serratia marcescens (BIOTECH 1748), and Candida tropicalis (BIOTECH 2085). Among the selected five endophytic used for antimicrobial activity screening, the Raffaelea sp. (CN-EF 2) has shown a high zone of inhibition of 11 mm against Bacillus subtilis, 18 mm against Escherichia coli, 15 mm against Staphylococcus aureus, and 11 mm against Candida tropicalis. Correspondingly, Aspergillus niger (CN-EF 1) has shown a zone of inhibition of 16 mm against C. tropicalis. The endophytic fungus Raffaelea sp. was effective against all test organisms except for Serratia marcescens, while Aspergillus niger was only effective for pathogenic fungi C. albicans. Hence, further in-depth studies of the isolated endophytic fungi are needed to determine the mechanisms behind the endophytes’ inhibitory effects.

Secondary metabolites from an endophytic fungus Aspergillus fumigatus from Solanum insanum L.

An endophytic fungus Aspergillus fumigatus,isolated from the fruits of Solanum insanum L.,was cultured in potato dextrose broth medium.Chromatographic separation of the EtOAc extract led to the isolation of six secondary metabolites,identified as pseurotin A(1),fumigaclavine C(2),monomethylsulochrin(3),trypacidin(4),fumiquinazoline C(5)and guignasulfide(6)by spectral means.Compounds 2 and 3 showed brine shrimp toxicity with IC50 values of 147 and 74.2μM,respectively,while compounds 4 and 5 showed slight inhibition on the growth of fungal pathogen Cladosporium cladosporioides.This is the first report of the isolation of guignasulfide(6)from A.fumigatus,which has been previously isolated only from an endophytic fungus Guignardia sp.

Colletodiphenol A,a new phenol derivative isolated from the endophytic fungus Colletotrichum capsici

A new C_(6)-C_(2)dimer phenol derivative,colletodiphenol A(1),along with two known C_(6)-C_(2)phenol derivatives(2,3)were isolated from the endophytic fungus Colletotrichum capsici related to the traditional Chinese medicine Paeonia lactiflora.Their structures were elucidated based on extensive spectroscopic data analysis,including HRESIMS,UV,IR,1D and 2D NMR.The antioxidant and anti-inflammatory activities of the isolated compounds were evaluated,but none of them displayed significant activity.