Fungal endophytes as prolific source of phytochemicals and other bioactive natural products: A review.

ion; it is believed to have antioxidant activity 11 times greater than trolox, a vitamin E derivative (Strobel et al. 2002, Harper et al. 2003). The endophytic Cephalosporium sp. IFB-E001 and Microsphaeropsis olivacea were isolated from host plants Trachelospermum jasminoides and Pilgerodendron uviferum respectively; both produced a phenolic metabolite graphislactone A (Fig18), which displayed potent in vitro antioxidant and free radical-scavenging activity stronger than the standards, butylated hydroxytoluene (BHT) and ascorbic acid (Hormazabal et al. 2005, Song et al. 2005). Huang et al. (2007) investigated the antioxidant capacities of endophytic fungal cultures of medicinal Chinese plants and its correlation to their total phenolic contents. They suggested that the phenolic content were the major antioxidant constituents of the endophytes (Huang et al. 2008a). The methanol extract of an endophyte Xylaria sp. isolated from the medicinal plant Ginkgo biloba exhibited strong antioxidant capacity due to the presence of “phenolics” and “flavonoids” among identified compounds (Liu et al. 2007). Recently, the endophytic Corynespora cassiicola was found to produce potent antioxidant compounds corynesidones A and B, and corynether A, together with a known diaryl ether (LL-V125α) (Fig 18), as revealed by oxygen radical absorbance capacity (ORAC) assay with units ranging between 4.35.9. Corynesidone B could also scavenge 2,2diphenyl-1-picrylhydrazyl (DPPH) free radicals with an IC50 22.4 μM, at the same activity as that of ascorbic acid. Beside antioxidant activity of corynesidone A, its exhibit aromatase inhibitory activity with an IC50 value of 5.30 μM; this activity magnitude is comparable to the first generation aromatase inhibitor drug, aminoglutethimide. Both antiaromatase and antioxidant activities of corynesidone A are interesting functions because this dual biological activity may be useful for cancer chemoprevention, particularly for breast cancer (Chomcheon et al. 2009). Also, the endophyte fungus Phyllosticta sp. isolated from Guazuma tomentosa was also found to exhibit strong antioxidant activity (Srinivasan et al. 2010). Zeng et al. (2011) Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/3 60 reported many of endophytic fungi as potential novel source of natural antioxidants from medicinal plant Scapania verrucosa. Antiviral Activities of Endophytic Fungi Many reports demonstrated the importance of endophytic fungi in production of antiviral agents, such as, cytonic acids A and B, novel human cytomegalovirus (hCMV) protease inhibitors, which had been isolated from solid-state fermentation of the endophytic fungus Cytonaema sp., )Guo et al. 2000). Investigation of endophytes associated with leaves of Quercus coccifera lead to isolation of the endophyte with the ability to synthesize hinnuliquinone, a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Singh et al. 2004). Endophytic isolates (582) with 360 morphologically distinct fungi were obtained from 81 Thai medicinal plant species. Extracts of 92 isolates could inhibit Mycobacterium tuberculosis, while 6 extracts inhibited Plasmodium falciparum, and strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (Wiyakrutta et al. 2004). Mellisol and 1,8-dihydroxynaphthol 1O-a-glucopyranoside were isolated from endophytic fungus Xylaria mellisii, which possess activity against herpes simplex virustype 1 (Pittayakhajonwut et al. 2005). Florke et al. (2006) reported antihepatitis C virus (HCV) activity of dihydroisocoumarin (R)-(-)-mellein (Fig 19). It inhibits HCV protease with an IC50 value of 35 mM. This compound had been isolated from a number of endophytic fungi, such as Pezicula livida, Plectophomella sp., and Cryptosporiopsis malicoticis (Krohn et al. 1997). Pullularins A (Fig 19), which had been isolated from ethyl acetate extract of endophytic fungus Pullularia sp., was also shown to have antiviral activity against herpes simplex virus type 1(HSV-1) with IC50 3.3 mg/ml (Isaka et al. 2007). Pestalotheol C (Fig 19), which was isolated from the endophyte Pestalotiopsis theae, was found to have anti-HIV properties (Li et al. 2008b). The aryl tetralin lignans, such as podophyllotoxin and its analogs showed antiviral and cytotoxicity activities and used as the precursor for many drugs for treatment cancer and viral infections, like etoposide, teniposide, and etopophos phosphate. Podophyllotoxin was found to produced by many endophytes: Trametes hirsute, Aspergillus fumigates, Phialocephala fortinii, and Fusarium oxysporum ( Eyberger et al. 2006, Puri et al. 2006, Kour et al. 2008, Kusari et al. 2009a). Arunpanichlert et al. (2010) investigated the secondary metabolites of endophytic fungus Penicillium sclerotiorum, and isolated the known compound (+)Sclerotiorin. (+)-Sclerotiorin (Fig 19) was evaluated for its inhibitory effect on human immunodeficiency virus HIV-1 integrase and protease and for antifungal activity, and found to exhibit anti-HIV-1 integrase and protease activities with IC50 values of 45.88 and 198.41μM, respectively, and showed weak anti-fungal activity against Candida albicans and Cryptococcus neoformans with MIC values of 202.53 and 101.26 μM, respectively. The endophyte Phomopsis sp., isolated from Musa acuminata, was found to produce hexaketide γ-lactones. Oblongolides Z, and 2deoxy-4α-hydroxyoblongolide X (Fig 19) showed anti-herpes simplex virus type 1 (HSV1) activity IC50 values of 14 μM and 76 μM, respectively. Oblongolides Z exhibited comparable cytotoxic activity against KB, BC, NCI-H187, and nonmalignant Vero cell lines with IC50 values of 37, 26, 32, and 60μM, respectively (Bunyapaiboonsri et al. 2010). New chlorinated pupukeananes possessing a unique spiroketal peroxide skeleton, named chloropupukeanolides A (Fig 19), were isolated from endophytic Pestalotiopsis fici. This compound was found to inhibit HIV-1 replication in vitro in C8166 cells with an IC50 value of 6.9 μM, and showed cytotoxicity against human cancer cell lines HeLa, MCF-7 and MDA-MB-231 with IC50 values of 16.9, 15.5 and 15.9μM, respectively (Liu et al. 2010a). Zhang et al. (2011) reported isolation and structure elucidation of Emerimidine A, and B (Fig 19) from culture of endophytic fungus Emericella sp., both of them showed moderate inhibition to Influenza virus H1 N1 with IC50 values of 42.07 mg/ml and 62.05 mg/ml respectively. Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/3 61 Fig. 19 – Structure of Antiviral Compounds isolated from some Endophytic Fungi Other Biological Activities of Endophytic Fungi Endophytic fungi are also known as producers of many other metabolites of biological interest, such as anti-inflammatory, anti-diabetic, anti-malarial and immunesuppressant agents, as well as insecticidal and antinematodes agents. Immunosuppressive drugs are used to prevent allograft rejection in organ transplantation, and could be used to treat autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes. Lee et al. (1995a) reported endophytic Fusarium subglutinans can produce compounds (Subglutinol A and B) that can influence the immune system of animals. Subglutinol A (Fig 20) and B are noncytotoxic diterpene pyrones; both of compounds have IC50 values of 0.1 μM and were roughly as potent as the immunosuppressant drug cyclosporin A. The lack of toxicity associated with subglutinols A and B suggests that they could be use as immunosuppressant agents. Recently, the endophytic Pestalotiopsis leucothes isolated from Trypterygium wilfordii was found to produce compounds which have variable effects on Tand B-cells and monocyte, these compounds may represent a new source of immunomodulatory agents or for treatment of human immune mediated diseases (Kumar et al. 2005). A new compound named Collutelin A, has been isolated from endophytic Colletotrichum dematium and was shown to exhibit strong immunosuppressive activity as it inhibited CD4 (+) Tcell activation of Interleukin 2 production (Ren et al. 2008). Gliocladicillins A and B were reported as effective antitumor agents in vitro and in vivo, since they induced tumor cell apoptosis and showed significant inhibition on proliferation of melanoma B16 cells implanted into immunodeficient mice (Chen et al. 2009). Zhang et al. (1999) investigated the anti-diabetic activity of endophytic fungi by isolation of nonpeptidal L-783, 281 from an endophytic Pseudomassaria sp., this compound was found to act as insulin, with Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/3 62 advance that it is not destroyed in the digestive tract and may be given orally. Oral administration of L-783,281 in two mouse models of diabetes resulted in significant lowering of blood glucose levels, these results may lead to a new therapeutic agent for diabetes. Insulin-mimetic compound demethyl asterriquinone B-1 (Fig 20) has been isolated from culture of endophytic Pseudomassaria sp. (Salituro et al. 2001, Strobel 2002). Many reports indicated that endophytic fungi are capable of producting of antiinflammatory compounds, such as phomol and mevinic acid (Fig 20), which had been isolated from culture of the endophytic fungus Phomopsis. (Weber et al. 2004a). Both compounds showed strong anti-inflammatory activity. Phenylpropanoids compounds were reported to be isolated from endophytes; they have received more interest for medicinal applications as they have multifold activities, such as anticancer, antioxidant, antimicrobial, anti-inflammatory and immunosuppressive properties (Korkina 2007). Wang et al. (2010) reported isolation of four compounds (Epicoccins M & R, Entepicoccin G, and Diketopiperazine) that exhibited potent anti-inflammatory activities from cultures of endophytic Epicoccum nigrum. The compounds were found to inhibit the platelet activating factor-induced release of β-glucuronid

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Biology of Endophytic Fungi

Rhizophora mucronata is an important ecosystem entity of the Malaysian mangrove forest. Since the species grows in a harsh environment, any organism that is isolated from this species would be of huge interest due to its potential in having novel bioactive compounds. In the present work, we isolated, identified and characterized, a total of 78 fungal isolates harboring inside the leaf tissues of R. mucronata. Molecular identification using the nuclear ribosomal DNA internal transcribe spacer (ITS) sequences returned with high similarity matches to known sequences in the GenBank. Maximum likelihood analysis revealed the phylogenetic relationship of all isolates from this study. Most of the dominating fungal endophytes were from the genera Pestalotiopsis, followed by Alternaria and Cladosporium. Six isolates representing the genera Alternaria, Fusarium, Nigrospora, Pestalotiopsis, Phoma, and Xylaria, were further screened for their antagonism activities. Dual culture test assay revealed their inhibition percentages against the phytopathogenic fungus Fusarium solani between 45-66%, and 0.8-23% when using non-volatile test assay. Of the six isolates, only Fusarium lateritium and Xylaria sp. showed antibacterial activities against the pathogenic bacteria, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, with the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) ranging from 0.5 to 2 mg/mL. The DPPH radical scavenging assay recorded a high level of antioxidant activity in Xylaria sp., 3-fold above that of F. lateritium. We demonstrate for the first time, two members belonging to the endophytic fungal community in the tropical mangrove species that have potential use as antagonists and antibacterial agents for future biotechnological applications.

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Diversity and Characterization of Endophytic Fungi Isolated From the Tropical Mangrove Species, Rhizophora mucronata, and Identification of Potential Antagonists Against the Soil-Borne Fungus, Fusarium solani.

Diversity and heavy metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in China

Aims: To isolate endophytic fungi from vegetable plants and examine their in vivo anti-oomycete activity against Phytophthora infestans in tomato plants.



Methods and Results:  Endophytic fungi were isolated from surface-sterilized plant tissues and anti-oomycete activity was measured by in vivo assay using tomato seedlings. Endophytic fungi showing potent anti-oomycete activity were identified by morphological characteristics and nuclear ribosomal ITS1-5.8S-ITS2 sequence analysis. A total of 152 isolates were obtained from 66 healthy tissue samples of cucumber, red pepper, tomato, pumpkin and Chinese cabbage and the fermentation broths of 23 isolates showed potent in vivo anti-oomycete activity against tomato late blight with control values over 90%. The Fusarium oxysporum strain EF119, which was isolated from roots of red pepper, showed the most potent disease control efficacy against tomato late blight. In dual-culture tests, it inhibited the growth of Pythium ultimum, P. infestans and Phytophthora capsici.



Conclusions:  Among endophytic fungi isolated from healthy tissues of vegetable plants, F. oxysporum EF119 showed the most potent in vivo anti-oomycete activity against tomato late blight and in vitro anti-oomycete activity against several oomycete pathogens.



Significance and Impact of the Study:  Endophytic fungi showing anti-oomycete activity in vitro and in vivo may be used as biocontrol agents particularly of tomato late blight.

Some fungal endophytes from vegetable crops and their anti-oomycete activities against tomato late blight.

One hundred and thirty endophytic fungi isolated from 12 Chinese traditional medicinal plants collected at Yuanmou county and Dawei Mountain, Yunnan province, southwest China, were tested for antitumour and antifungal activities by MTT assay on human gastric tumour cell line BGC-823 and the growth inhibition test against 7 phytopathogenic fungi. The results showed that fermentation broths from 9.2% of the isolates exhibited antitumour activity and 30% exhibited antifungal activity, moreover, some of them exhibited broad-spectrum antifungal activity. The active isolates were identified to 32 taxa. The results indicate that the endophytic fungi of Chinese traditional medicinal plants are promising sources of novel bioactive compounds.

Haiyan Li

Papers

Screening for endophytic fungi with antitumour and antifungal activities from Chinese medicinal plants