Endophytic Alternaria Alternata Research Articles

Altenuene derivatives produced by an endophyte Alternaria alternata

Two novel aromatic polyketides 1 and 3 and five known compounds, (4S,10S)-talaroflavone (2), altenuene (4), isoaltenuene (5), alternariol (6), and altenusin (7), were isolated from an endophytic strain of Alternaria alternata SI-694. The structures of the new compounds, including their absolute configurations, were elucidated by NMR, IR, UV, and ECD spectroscopies, and the phytotoxicities of the isolated compounds were also evaluated. Altenusin (7) showed moderate cytotoxicity against HL-60 cells, with an IC50 of 6.65 µM, whereas 5, 6, and 7 were phytotoxic against Lactuca sativa, Brassica campestris L., Stellaria aquatica (L.) Scop. and Digitaria ciliaris.

Biocontrol activity of an endophytic Alternaria alternata Aa-Lcht against apple Valsa canker

Apple Valsa canker (AVC), caused by Valsa mali, is the most serious branch disease for apples in East Asia. Biocontrol constitutes a desirable alternative strategy to alleviate the problems of orchard environment pollution and pathogen resistance risk. It is particularly important to explore efficient biocontrol microorganism resources to develop new biocontrol technologies and products. In this study, an endophytic fungus, which results in the specific inhibition of the growth of V. mali, was isolated from the twig tissue of Malus micromalus with a good tolerance to AVC. The fungus was identified as Alternaria alternata, based on morphological observations and phylogenetic analysis, and was named Aa-Lcht. Aa-Lcht showed a strong preventive effect against AVC, as determined with an in vitro twig evaluation method. When V. mali was inhibited by Aa-Lcht, according to morphological and cytological observations, the hyphae was deformed and it had more branches, a degradation in protoplasm, breakages in cell walls, and then finally died completely due to mycelium cells. Transcriptome analysis indicated that Aa-Lcht could suppress the growth of V. mali by inhibiting the activity of various hydrolases, destroying carbohydrate metabolic processes, and damaging the pathogen membrane system. It was further demonstrated that Aa-Lcht could colonize apple twig tissues without damaging the tissue's integrity. More importantly, Aa-Lcht could also stimulate the up-regulated expression of defense-related genes in apples together with the accumulation of reactive oxygen species and callose deposition in apple leaf cells. Summarizing the above, one endophytic biocontrol resource was isolated, and it can colonize apple twig tissue and play a biocontrol role through both pathogen inhibition and resistance inducement.

Purification of ursolic acid and β-sitosterol from endophytic Alternaria alternata for their alpha-amylase inhibitory activity

Fungal endophytes are a known warehouse of bioactive compounds with multifarious applications. In the present investigation two compounds, β-Sitosterol (1) and ursolic acid (2), were isolated from Alternaria alternata, an endophytic fungus associated with Morus alba Linn for the first time. The structure of the compounds was elucidated on the basis of comprehensive spectral analysis (UV, IR, 1 H-, 13 C- and 2D-NMR, as well as HRESI–MS). In the in vitro alpha amylase inhibitory assay both compounds (1) and (2) show potent antidiabetic activity. In support, Docking studies indicate significant binding affinity of the isolated compounds. Hence from the present study, it can be concluded that endophytic fungi in Morus alba Linn can find use in antidiabetic drug development in the medicinal industry. Communicated by Ramaswamy H. Sarma

Promising Endophytic Alternaria alternata from Leaves of Ziziphus spina-christi: Phytochemical Analyses, Antimicrobial and Antioxidant Activities

Fungal endophytes are considered one of the most important reservoirs of bioactive compounds which defeat resistant microbes. In our study, endophytic Alternaria alternata was isolated from Ziziphus spina-christi and identified morphologically and genetically with accession number OM 331,682. Preliminary phytochemical screening of ethyl acetate (EA) crude extract of A. alternata revealed that this extract contains alkaloids, tannins, flavonoids, glycosides, phenols, and terpenoids. Moreover, the extract was analyzed using gas chromatography-mass spectrometry (GC–MS) which verified the presence of numerous bioactive compounds. Antimicrobial results illustrated that EA crude extract exhibited promising antimicrobial activity against Gram-negative bacteria (Escherichia coli ATCC 11229, Proteus vulgaris RCMB 004, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumonia RCMB 003), Gram-positive bacteria (Bacillus subtilis RCMB 015, Staphylococcus aureus ATCC 25923, and Staphylococcus epidermidis ATCC 14990), and unicellular fungi (Candida albicans ATCC 90028). Ultrastructure study of treated K. pneumonia showed remarkably elucidated destruction of the cell wall and cell membrane and leakage of cytoplasmic materials. Furthermore, the extract has potential antioxidant activity where IC50 was 409 µg/mL. Moreover, this extract did not show any toxicity on Vero normal cell line. These findings confirmed that the endophytic A. alternata from Z. spina-christi is a promising source of bioactive compounds which can be used in different biological applications.

Discovery of the anticancer drug vinblastine from the endophytic Alternaria alternata and yield improvement by gamma irradiation mutagenesis.

Several fungal endophytes were isolated from some medicinal plants and screened for their ability to produce the anticancer drug vinblastine. An isolate was found to produce vinblastine (205·38 μg l-1 ), and the identity of the fungal vinblastine was confirmed by UV spectroscopic, high-performance liquid chromatography and electrospray ionization mass spectrometry analyses. Based on both morphological and molecular studies, the vinblastine-producing strain was identified as Alternaria alternata. Cytotoxic activities of the fungal vinblastine were evaluated against CHO-K1, MCF-7 and HepG-2 cell lines by the MTT assay. The proliferation of these cell lines was inhibited after treatment with fungal vinblastine and the recorded IC50 values of the respective cell lines were 12·15, 8·55 and 7·48 μg ml-1 . A strain improvement programme for improving vinblastine productivity by the fungal strain was also used. In addition, 10 broth media were evaluated for further increasing the production of vinblastine. The yield of vinblastine was intensified by 3·98-fold following gamma irradiation at 1000 Gy, and a stable mutant strain was isolated. Among the screened media, M1D broth (pH 6·0) stimulated the highest vinblastine production of 1553·62 μg l-1 by the isolated mutant strain. The present study is the first report on the production and yield improvement of the anticancer drug vinblastine by A. alternata. These findings suggest A. alternata as a viable and potent source with excellent biotechnological potential for the production of vinblastine.

Antioxidant and antimicrobial activity displayed by a fungal endophyte Alternaria alternata isolated from Picrorhiza kurroa from Garhwal Himalayas, India

Picrorhiza kurroa (Kutki) is a medicinal plant of Himalayan region which was exploited for its fungal endophytic association. Endophytic fungi isolated from plants have plant growth promoting attributes and biocontrol activity. We have isolated endophytic fungi to investigate the bioactive compounds which was used for the biocontrol of bacteria of clinical importance. The morphological analysis as well as the molecular characterization using 18s rRNA and phylogenetic analysis, identified the fungus as Alternaria alternata . Investigation using GC-MS of methanolic and ethyl acetate extracts of fungal biomass, led to identification of many bioactive compounds. These chemicals have various medicinal purposes such as antimicrobial activity and antioxidant potential. Our results suggest that these compounds may have clinical importance. The extract from A. alternata was capable of inhibiting B. subtilis and S. aureus. This is the first study conducted about role of secondary metabolites produced by Alternaria alternata isolated from the Picrorhiza kurroa , possessing antibacterial properties and the bioactive compounds may have potential medicinal relevance. • Endophytic fungi isolated from Picorrhiza kurroa medicinal plant. • Characterized as Alternaria alternata . • GC-MS analysis revealed many bioactive compounds. • Fungal extract acted as antimicrobial agents against bacteria of clinical importance.

Novel metabolites from the Cercis chinensis derived endophytic fungus Alternaria alternata ZHJG5 and their antibacterial activities.

Phytopathogenic bacteria, such as Xanthomonas oryzae pv. oryzae (Xoo) and Ralstonia solanacearum (Rs), seriously threaten crop production and are the cause of enormous yield losses. Endophytic fungi are abundant sources of bioactive metabolites that may be potential candidates in the development of new agrochemicals. This work emphasizes the discovery of bioactive polyketides from endophytic Alternaria alternata ZHJG5 and reports their structural elucidation and antibacterial activities in detail. Five novel polyketide derivatives, isotalaroflavone (2), (+/-)-5'-dehydroxytalaroflavone (3a/3b), (+)-talaroflavone (4b), and bialternacin G (7), along with five known compounds (1, 4a, 5, 6, and 8), were obtained from the Cercis chinensis-derived fungus A. alternata ZHJG5. The compounds' structures were characterized using spectroscopic methods and X-ray diffraction. Chiral high-performance liquid chromatography was used to separate the racemates 3 and 4, whose absolute configurations were unambiguously confirmed by comparing their experimental and calculated electron circular dichroism data. All isolated compounds were tested for antibacterial activity against the phytopathogenic bacteria Xoo, Xanthomonas oryzae pv. oryzicola (Xoc) and Rs. Compounds 1, 2 and 8 showed pronounced antibacterial activity against all tested bacteria, with minimal inhibitory concentrations ranging from 0.5 to 64 μg/ml. In addition, compound 1 showed a potent protective effect against rice bacterial leaf blight caused by Xoo with a protective efficacy of 75.1% at a concentration of 200 μg/ml. These findings highlight the practical potential of antibacterial compounds as candidates for the discovery of novel bactericides. © 2021 Society of Chemical Industry.

CYTOTOXIC METABOLITES OF ALTERNARIA ALTERNATA, AN ENDOPHYTE OF THE MEDICINAL PLANT BIDENS BIPINNATA

Objective: Endophytes are widely spread in the plant kingdom and represent a very promising source of biologically active natural products. The medicinal plant Bidens bipinnata Lin. (Asteraceae) which is known for its anti-inflammatory, antifungal and antitumor effects has been chosen for the investigation of its endophyte to search for bioactive metabolites.
 Methods: An endophytic Alternaria alternata species was isolated from the leaves of the plant B. bipinnata Lin. To investigate the metabolic profile of this endophytic fungus it was cultivated in several culture media as static and shaken culture. The antimicrobial and cytotoxic activities of the ethyl acetate extracts of the fungus were examined. Extracts exhibiting highest antimicrobial activities in agar diffusion assay and cytotoxicity against HeLa cancer cell line were subjected to activity-guided chromatographic fractionation for the identification of bioactive metabolites. A cytotoxic assay was performed on the isolated compounds against HeLa cancer cell lines as well as cytostatic activity tests against HUVEC and K-562 cell lines.
 Results: Chromatographic fractionation resulted in the isolation and identification of alternariol and tentoxin from the extract of the fungus cultivated in medium M5 while sterigmatocystin was isolated in addition to alternariol and tentoxin from the extract of the fungus grown in medium M25. Both alternariol and sterigmatocystin proved to be of moderate cytotoxicity and weak cytostatic activity with alternariol showing higher cytotoxic activity than sterigmatocystin. Highest cytotoxicity against HeLa cell lines was observed for tentoxin with a CC50 of 22.5 µg/ml.
 Conclusion: This study presents the isolation and identification of the bioactive metabolites alternariol, sterigmatocystin and tentoxin from the endophyte A. alternata in addition to the antifungal activity of the strain extract as well as the cytotoxic and cytostatic activities of the isolated metabolites against HeLa, HUVEC and K-562 cell lines, respectively.

A new 2H-benzindazole compound from Alternaria alternata Shm-1, an endophytic fungus isolated from the fresh wild fruit of Phellinus igniarius.

Endophytic fungi have been shown in recent years to produce a series of bioactive secondary metabolites. Several endophytic fungi were isolated from the fresh wild body of Phellinus igniarius, and initially evaluated for their antimicrobial activity. Among which, Shm-1 extract showed moderate inhibitory activity against Clavibacter michiganense and the fungus was identified to be Alternaria alternata Shm-1 through the comparison of morphological characteristics and the sequence of the rDNA ITS with those of other Alternaria species. A new 2H-benzindazole derivative, alterindazolin A (1), has been isolated from cultures of the endophyte Alternaria alternata Shm-1. Its structure was characterized as 1-benzyl-5-p-hydroxy-phenyloxygen-benz[e]indazole by spectroscopic data analysis including 1D NMR, 2D NMR and MS spectrum.

Anti-microbial and anti-mycotoxigenic activities of endophytic Alternaria alternata isolated from Catharanthus roseus (L.) G. Don.: molecular characterisation and bioactive compound isolation

ABSTRACTThe present study investigated the anti-microbial and anti-mycotoxigenic activities of the ethyl acetate extract (EA) and a bioactive compound obtained from an endophytic fungus Alternaria alternata isolated from Catharanthus roseus leaves. A. alternata was identified using PCR-based 5.8S rDNA sequencing. The EA and bioactive compound, p-Coumaric acid (PC), showed concentration-dependent broad-spectrum anti-microbial activity against the tested bacteria, yeast, and fungi with MICs ranging from 7.8 to 250 µg/mL. The in vitro production of aflatoxin B1 (AFB1) from Aspergillus flavus and fumonisin B1 (FB1) from Fusarium verticillioides was completely inhibited by EA and PC at 400 µg/mL. The synthesis of the membrane-bound ergosterol from A. flavus and F. verticillioides was strongly inhibited by PC at 200 µg/mL. The EA and PC were found to show significant anti-microbial and anti-mycotoxigenic activities, hence, they could be explored as protective agents for preventing microbial deterioration and mycotoxins accumulation in food and feedstuffs during pre- and post-harvest and storage.

Antiproliferative, antifungal, and antibacterial activities of endophytic alternaria species from cupressaceae.

Recent research has shown the bioprospecting of endophytic fungi from Cupressaceae. Here, we further uncover that the healthy cypress plants such as Cupressus arizonica, Cupressus sempervirens var. cereiformis, and Thuja orientalis host highly bioactive endophytic Alternaria fungal species. Indeed, endophytic Alternaria alternata, Alternaria pellucida, and Alternaria tangelonis were recovered from healthy Cupressaceous trees. Biodiversity and bioactivity of recovered endophytic Alternaria species were a matter of biogeography and host identity. We further extracted such Alternaria's metabolites and highlighted their significant antiproliferative, growth inhibitory, and antibacterial activities against the model target fungus Pyricularia oryzae and the model pathogenic bacteria Bacillus sp., Erwinia amylovora, and Pseudomonas syringae. In vitro assays also indicated that endophytic Alternaria species significantly inhibited the growth of cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. In conclusion, since the recovered Alternaria species were originally reported as pathogenic and allergenic fungi, our findings suggest a possible ecological niche for them inside the foliar tissues of Cupressaceous trees. Moreover, in this study, the significant bioactivities of endophytic Alternaria species in association with Cupressaceae plant family are reported.

Fungal endophyte increases the allelopathic effects of an invasive forb

Endophytic plant symbionts can have powerful effects on the way their hosts interact with pathogens, competitors, and consumers. The presence of endophytes in plants can alter food webs, community composition and ecosystem processes, suggesting that endophyte-plant symbioses may represent unique forms of extended phenotypes. We tested the impact of the fungal endophyte Alternaria alternata (phylotype CID 120) on the allelopathic effect of the invasive forb Centaurea stoebe when in competition with the North American native bunchgrass Koeleria macrantha in a greenhouse competition experiment. The allelopathic effect of C. stoebe on K. macrantha when infected with the fungal endophyte was more than twice that of endophyte-free C. stoebe. However, this allelopathic effect was a small part of the very large competitive effect of C. stoebe on K. macrantha in all treatments, likely because of the priority effects in our experimental design. To our knowledge, these results are the first experimental evidence for a symbiotic relationship between plants and fungal endophytes affecting allelopathic interactions between competing plants, and thus provide insight into the mechanisms by which fungal endophytes may increase the competitive ability of their hosts.

Frequency Distribution and Assessment of Genetic Diversity of Novel Endophyte Alternaria alternata Accessions Isolated from Pongamia pinnata L.

Thepresent study discusses the frequency distribution and genetic diversity of novel fungal endopyte Alternaria alternata within the Pongammia pinnata plant samples. A total of ten plant samples of Pongammia pinnata, Pierre. (Karanja) were collected from specific locations of Sanganer region of Rajasthan for the isolation of fungal endophytes. Of these, maximum frequency of Alternaria alternata (22.29%) were recorded which are morphologically similar but ecologically variant. Efficacy of randomly amplified polymorphic DNA (RAPD), were assessed in seventeen individuals of the primers was GCC 180 where as 10 bands were generated by GCC 181. The similarity coefficient matrix generated for the primers was subjected to algorithm UPGMA (Unweighted Pair Group Method Analysis) and clusters were generated using NTSYS 2.02 pc program. To stabilize the level of relatedness among the seventeen ecologically variant Alternaria alternata accessions, the dendrogram was constructed, which showed that all the isolates were diversified endophytically with in the plant Pongamia pinnata.

Antifeedent and toxic activity of endophytic Alternaria alternata against tobacco caterpillar Spodoptera litura

Fungal endophytes play a significant role in plant resistance to insects by altering the nutritional quality of plants or by production of various alkaloid-based defensive compounds. Ethyl acetate extract of endophytic Alternaria alternata isolated from Azadirachta indica A. Juss was tested for its bioactivity against Spodoptera litura (Fab.). Effect of different concentrations of ethyl acetate extract of A. alternata was assessed on growth and development of S. litura larvae. The results indicate higher larval mortality on diet supplemented with the fungal extract than control. Adverse effects of A. alternata were also observed on development of the insect. Ethyl acetate extract of the fungus also significantly reduced the adult emergence, longevity and reproductive potential of S. litura. Larvae feeding on diet supplemented with fungal extract showed significant reduction in relative growth and consumption rate, as well as efficiency of ingested and digested food. The antifeedant and toxic effects of A. alternata may be due to production of bioactive molecules by this fungus.

Metabolites produced by an endophyte Alternaria alternata isolated from Maytenus hookeri

Endophytes are bacteria or fungi that live in the intercellular spaces of the tissues of host plants without causing discernible manifestation of disease [1]. Recently, endophytes have been recognized as important sources of a variety of structurally novel and biologically active secondary metabolites, including terpenoids, steroids, alkaloids, and isocoumarin derivatives. In the course of our search for new biologically active secondary metabolites from endophytic microorganisms residing in Maytenus hookeri (Celastraceae) [2, 3], we studied chemical substances from an endophytic fungus Alternaria alternata of Maytenus hookeri and isolated and characterized nine compounds, alternariol (1), alternariol monomethyl ether (2), 5 -epialtenuene (3), altenuene (4), uridine (5), adenosine (6), ACTG toxin-E (7), ergosta-4,6,8,22-tetraen-3-one (8), and ergosta7,24(28)-dien-3-ol (9) from the ethyl acetate–methanol–acetic acid extract of the solid-state fermentations of this fungus. Among them, compounds 5 and 6 were isolated for the first time from this genus Alternaria, both 8 and 9 first being obtained from the species. In this paper, we describe the isolation and structure elucidation of these compounds 1–9. With the use of column chromatography over silica gel, and Sephadex LH-20 and reversed-phase RP-18, as well as preparative TLC on silica gel, further separation of methanol and petroleum ether–soluble parts of the ethyl acetate–methanol– acetic acid extract of the cultures of A. alternata afforded nine compounds 1–9. Compound 1 was isolated as a white powder. ESI-MS (negative mode) showed a molecular ion at m/z 257 [M – H]–. From the 13C NMR (DEPT) spectra, the molecular formula was deduced as C14H10O5, with 10 degrees of unsaturation. The 13C NMR and DEPT indicated 14 skeletal C atoms, including one methyl, four methines, and nine quarternary C atoms. In the 1H NMR spectrum, the downfield signals appeared at 6.88 (1H, d, J = 1.69 Hz, H-4), 6.94 (1H, d, J = 2.36 Hz, H-5 ), 6.99 (1H, d, J = 2.48 Hz, H-3 ), and 7.44 (1H, d, J = 1.71 Hz, H-6), which suggested it was a biphenyl-type compounds. The above spectral data were in agreement with values reported in the literature [4] for 3,4 ,5-trihydroxy-6 -methyldibenzo-pyrone. Compound 2, white powder, C15H12O5 was identified as alternariol monomethyl ether by comparison of physicochemical data and spectral data (EI-MS, 1H NMR and 13C NMR) with those reported in the literature [4, 5].

Antifungal activity of diketopiperazines extracted from Alternaria alternata against Plasmopara viticola: An ultrastructural study

Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10 −3, 10 −4, 10 −5 and 10 −6 M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24 h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.