Anti-MRSA and clot lysis activities of Pestalotiopsis microspora isolated from Dillenia pentagyna Roxb.

There is an urgent call to search for novel natural compounds against developing multidrug-resistant microorganisms. The present work focuses on the characterization of a plant-associated fungus having bioactivity against methicillin-resistant Staphylococcus aureus (MRSA) strains. A fungal strain P31 was isolated from bark of Dillenia pentagyna and identified as Pestalotiopsis microspora. The maximum anti-MRSA activity was observed from extract of P31 grown in sabouraud dextrose broth. The minimum inhibitory concentrations (MIC) values of P31 extract were 14 µg/ml for methicillin-sensitive S. aureus (MSSA) and 32 µg/ml for MRSA strain, respectively. A crude P31 extract showed strong bactericidal activity by killing all treated MRSA cells within 24 h of treatment at their respective MIC value. A scanning electron microscopic study visualized morphological damage of MRSA cells. The membrane permeability of P31 extract-treated MRSA cells gradually increased which caused release of internal cytoplasmic nucleic acids, proteins and potassium ions (K+ ) from cells suggesting cell lysis or leakage from cells. A very low concentration of P31 extract was able to inhibit biofilm formed by MRSA cells. Thin layer chromatographic separation followed by gas chromatography-mass spectrometry analysis of the P31 extract revealed a number of antimicrobial compounds along with an anti-MRSA compound 2,4-di-tert-butylphenol. In addition, the P31 extract also showed in-vitro human blood clot lysis activity at various concentrations. The clot lysis activity of P31 extract was found maximum at 500 µg/ml. These findings suggest that fungal isolate P31 has potential as a source of anti-MRSA compounds useful in staph infections.

Fungal Endophytes as Efficient Sources of Plant-Derived Bioactive Compounds and Their Prospective Applications in Natural Product Drug Discovery: Insights, Avenues, and Challenges.

Fungal endophytes are well-established sources of biologically active natural compounds with many producing pharmacologically valuable specific plant-derived products. This review details typical plant-derived medicinal compounds of several classes, including alkaloids, coumarins, flavonoids, glycosides, lignans, phenylpropanoids, quinones, saponins, terpenoids, and xanthones that are produced by endophytic fungi. This review covers the studies carried out since the first report of taxol biosynthesis by endophytic Taxomyces andreanae in 1993 up to mid-2020. The article also highlights the prospects of endophyte-dependent biosynthesis of such plant-derived pharmacologically active compounds and the bottlenecks in the commercialization of this novel approach in the area of drug discovery. After recent updates in the field of 'omics' and 'one strain many compounds' (OSMAC) approach, fungal endophytes have emerged as strong unconventional source of such prized products.

Harnessing the Phytotherapeutic Treasure Troves of the Ancient Medicinal Plant Azadirachta indica (Neem) and Associated Endophytic Microorganisms.

Azadirachta indica, commonly known as neem, is an evergreen tree of the tropics and sub-tropics native to the Indian subcontinent with demonstrated ethnomedicinal value and importance in agriculture as well as in the pharmaceutical industry. This ancient medicinal tree, often called the "wonder tree", is regarded as a chemical factory of diverse and complex compounds with a plethora of structural scaffolds that is very difficult to mimic by chemical synthesis. Such multifaceted chemical diversity leads to a fantastic repertoire of functional traits, encompassing a wide variety of biological activity and unique modes of action against specific and generalist pathogens and pests. Until now, more than 400 compounds have been isolated from different parts of neem including important bioactive secondary metabolites such as azadirachtin, nimbidin, nimbin, nimbolide, gedunin, and many more. In addition to its insecticidal property, the plant is also known for antimicrobial, antimalarial, antiviral, anti-inflammatory, analgesic, antipyretic, hypoglycaemic, antiulcer, antifertility, anticarcinogenic, hepatoprotective, antioxidant, anxiolytic, molluscicidal, acaricidal, and antifilarial properties. Notwithstanding the chemical and biological virtuosity of neem, it has also been extensively explored for associated microorganisms, especially a class of mutualists called endophytic microorganisms (or endophytes). More than 30 compounds, including neem "mimetic" compounds, have been reported from endophytes harbored in the neem trees in different ecological niches. In this review, we provide an informative and in-depth overview of the topic that can serve as a point of reference for an understanding of the functions and applications of a medicinal plant such as neem, including associated endophytes, within the overall theme of phytopathology. Our review further exemplifies the already-noted current surge of interest in plant and microbial natural products for implications both within the ecological and clinical settings, for a more secure and sustainable future.

Review: Endophytic microbes and their potential applications in crop management.

Endophytes are microbes (mostly bacteria and fungi) present asymptomatically in plants. Endophytic microbes are often functional in that they may carry nutrients from the soil into plants, modulate plant development, increase stress tolerance of plants, suppress virulence in pathogens, increase disease resistance in plants, and suppress development of competitor plant species. Endophytic microbes have been shown to: (i) obtain nutrients in soils and transfer nutrients to plants in the rhizophagy cycle and other nutrient-transfer symbioses; (ii) increase plant growth and development; (iii) reduce oxidative stress of hosts; (iv) protect plants from disease; (v) deter feeding by herbivores; and (vi) suppress growth of competitor plant species. Because of the effective functions of endophytic microbes, we suggest that endophytic microbes may significantly reduce use of agrochemicals (fertilizers, fungicides, insecticides, and herbicides) in the cultivation of crop plants. The loss of endophytic microbes from crop plants during domestication and long-term cultivation could be remedied by transfer of endophytes from wild relatives of crops to crop species. Increasing atmospheric carbon dioxide levels could reduce the efficiency of the rhizophagy cycle due to repression of reactive oxygen used to extract nutrients from microbes in roots. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Induction of Cryptic and Bioactive Metabolites through Natural Dietary Components in an Endophytic Fungus Colletotrichum gloeosporioides (Penz.) Sacc.

Grape skin and turmeric extracts having the major components resveratrol and curcumin, respectively, were used for the induction of cryptic and bioactive metabolites in an endophytic fungus Colletotrichum gloeosporioides isolated from Syzygium cumini. The increase in total amount of crude compounds in grape skin and turmeric extract treated cultures was 272.48 and 174.32%, respectively, compared to the untreated control. Among six human pathogenic bacteria tested, the maximum inhibitory activity was found against Aeromonas hydrophila IMS/GN11 while no inhibitory activity was observed against Enterococcus faecalis IMS/GN7. The crude compounds derived from turmeric extract treated cultures showed the highest DPPH free radicals scavenging activity (86.46% inhibition) followed by compounds from grape skin treated cultures (11.80% inhibition) and the control cultures (1.92% inhibition). Both the treatments significantly (p ≤ 0.05) increased the antibacterial and antioxidant activities of crude metabolites compared to the control. HPLC profiling of crude compounds derived from grape skin and turmeric extract treated cultures revealed the presence of additional 20 and 14 cryptic compounds, respectively, compared to the control. These findings advocate the future use of such dietary components in induced production of cryptic and bioactive metabolites.

Epigenetic Activation of Antibacterial Property of an Endophytic Streptomyces coelicolor Strain AZRA 37 and Identification of the Induced Protein Using MALDI TOF MS/MS.

The endophytic Streptomyces coelicolor strain AZRA 37 was isolated from the surface sterilized root of Azadirachta indica A. Juss., commonly known as neem plant in India. Since only a few reports are available regarding epigenetic modulations of microbial entities, S. coelicolor was treated with different concentrations of 5-azacytidine for this purpose and evaluated for its antibacterial potential against five human pathogenic bacteria (Aeromonas hydrophila IMS/GN11, Enterococcus faecalis IMS/GN7, Salmonella typhi MTCC 3216, Shigella flexneri ATCC 12022 and Staphylococcus aureus ATCC 25923). The crude extract obtained from cultures treated with 25 µM concentration of 5-azacytidine, was found effective against all five pathogenic bacteria tested while the untreated control was only active against 3 pathogenic bacteria. HPLC analysis of crude compounds from treated cultures showed a greater number of compounds than that of the control. Extraction of whole cell protein and its SDS PAGE analysis showed an additional major protein band in 25 µM 5-azacytidine treated culture and MALDI TOF MS/MS analysis revealed that this protein belongs to the porin family.

Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize.

Endophytes are mutualistic symbionts within healthy plant tissues. In this study we isolated Bacillus spp. from seeds of several varieties of maize. Bacillus amyloliquifaciens or Bacillus subtilis were found to be present in all maize varieties examined in this study. To determine whether bacteria may produce antifungal compounds, generally lipopeptides in Bacillus spp., bacterial cultures were screened for production of lipopeptides. Lipopeptides were extracted by acid precipitation from liquid cultures of Bacillus spp. Lipopeptide extracts from Bacillus spp. isolated from Indian popcorn and yellow dent corn showed inhibitory activity against Fusarium moniliforme at 500µg per disk. Using MALDI-TOF mass spectrometry we detected the presence of antifungal iturin A, fengycin and bacillomycin in these isolates. PCR amplification also showed the presence of genes for iturin A and fengycin. B. subtilis (SG_JW.03) isolated from Indian popcorn showed strong inhibition of Arabidopsis seed mycoflora and enhanced seedling growth. We tested for the induction of defence gene expression in the host plant after treatment of plants with B. subtilis (SG_JW.03) and its lipopeptide extract using RT-qPCR. Roots of Indian popcorn seedlings treated with a suspension of B. subtilis (SG_JW.03) showed the induction of pathogenesis-related genes, including PR-1 and PR-4, which relate to plant defence against fungal pathogens. The lipopeptide extract alone did not increase the expression of these pathogenesis-related genes. Based on our study of maize endophytes, we hypothesize that, bacterial endophytes that naturally occur in many maize varieties may function to protect hosts by secreting antifungal lipopeptides that inhibit pathogens as well as inducing the up-regulation of pathogenesis-related genes of host plants (systemic acquired resistance).

Season and tissue type affect fungal endophyte communities of the Indian medicinal plant Tinospora cordifolia more strongly than geographic location.

A total of 1,151 endophytic fungal isolates representing 29 taxa were isolated from symptom-less, surface-sterilized segments of stem, leaf, petiole, and root of Tinospora cordifolia which had been collected at three locations differing in air pollution in India (Ramnagar, Banaras Hindu University, Maruadih) during three seasons (summer, monsoon, winter). Endophytes were most abundant in leaf tissues (29.38% of all isolates), followed by stem (18.16%), petiole (10.11%), and root segments (6.27%). The frequency of colonization (CF) varied more strongly among tissue type and season than location. CF was maximal during monsoon followed by winter and minimal during summer. A species each of Guignardia and Acremonium could only be isolated from leaves, whereas all other species occurred in at least two tissue types. Penicillium spp. were dominant (12.62% of all isolates), followed by Colletotrichum spp. (11.8%), Cladosporium spp. (8.9%), Chaetomium globosum (8.1%), Curvularia spp. (7.6%), and Alternaria alternata (6.8%). Species richness, evenness, and the Shannon-Wiener diversity index followed the same pattern as the CF with the tissue type and the season having the greatest effect on these indices, suggesting that tissue type and season are more influential than geography. Dissimilarity of endophyte communities in regards to species composition was highest among seasons. Colletotrichum linicola occurred almost exclusively in winter, Fusarium oxysporum only in winter and summer but never during monsoon and Curvularia lunata only in winter and during monsoon but never in summer. Emissions of NO(2), SO(2), and suspended particulate matter were negatively correlated with the CF. Ozone did not have any effect. The frequency of most species declined with increasing pollution, but some showed an opposite trend (e.g., Aspergillus flavus). Five unnamed taxa (sterile mycelia) were identified as Aspergillus tubingensis, Colletotrichum crassipes, Botryosphaeria rhodina, Aspergillus sydowii, and Pseudofusicoccum violaceum, using molecular tools. Fifteen of the 29 endophyte taxa exhibited antibacterial activity. B. rhodina (JQ031157) and C. globosum showed activity against all bacterial human pathogens tested, with the former showing higher activity than the latter.

Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant Azadirachta indica A. Juss., and Impact of Culture Media on their Isolation.

Azadirachta indica A. Juss. (neem), native to India, is well known worldwide for its insecticidal and ethanopharmacological properties. Although endophytic microbes are known from this plant as only leaves and stems were the subjects of past reports. Now, a variety of procedures and a number of different media were used to isolate the maximum number of endophytic fungi from unripe fruits and roots. A total of 272 isolates of 29 filamentous fungal taxa were isolated at rate of 68.0% from 400 samples of three different individual trees (at locations-Az1, Az2, Az3). Mycological agar (MCA) medium yielded the highest number of isolates (95, with a 14.50% isolation rate) with the greatest species richness. Mycelia Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes 7.25%, while Hyphomycetes showed the maximum number of representative isolates (81.69%). Mycelia-Sterilia (1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S and 28S rDNA sequences were identified as Fusarium solani (99%), Chaetomium globosum (93%) and Chaetomium globosum (93%) respectively. Humicola, Drechslera, Colletotrichum, and Scytalidium sp. were some of the peculiar fungal endophytes recovered from this plant.

Endophytic actinomycetes from Azadirachta indica A. Juss.: isolation, diversity, and anti-microbial activity.

Endophytic actinomycetes from Azadirachta indica A. Juss. were screened and evaluated for their anti-microbial activity against an array of pathogenic fungi and bacteria. A total of 55 separate isolates were obtained from 20 plants, and 60% of these showed inhibitory activity against one or more pathogenic fungi and bacteria. Actinomycetes were most commonly recovered from roots (54.5% of all isolates), followed by stems (23.6%), and leaves (21.8%). The dominant genus was Streptomyces (49.09% of all isolates), while Streptosporangium (14.5%), Microbispora (10.9%), Streptoverticillium (5.5%), Sacchromonospora sp. (5.5%), and Nocardia (3.6%) were also recovered. Streptomyces isolates AzR 006, 011, and 031 (all from roots) had acute activity against Pseudomonas fluorescens, while AzR027, 032, and 051 (also all from roots) showed activity against Escherichia coli. Meanwhile, an isolate of Nocardia sp. from leaves (AzL025) showed antagonism against Bacillus subtilis. Overall, 32 of the 55 were found to have broad spectrum significant antimicrobial activity, while about 4% of them showed strong and acute inhibition to pathogenic fungi and bacteria. Isolates of Streptomyces AzR031, 008, and 047, Nocardia sp. AzL025, and Streptosporangium sp. AzR 021 and 048 are of particular interest because they showed significant antagonistic activity against root pathogens, including Pythium and Phytophthora sp. Thus, many of the isolates recovered from A. indica in this study may be used in developing potential bio-control agents against a range of pathogenic fungi and bacteria and in the production of novel natural antimicrobial compounds. These results not only further our understanding of plant-microbe interactions but also indicate that there is an untapped resource of endophytic microorganisms that could be exploited in the biotechnological, medicinal, and agricultural industries.

Javanicin, an antibacterial naphthaquinone from an endophytic fungus of neem, Chloridium sp.

The endophytic fungus Chloridium sp. produces javanicin under liquid and solid media culture conditions. This highly functionalized naphthaquinone exhibits strong antibacterial activity against Pseudomonas spp., representing pathogens to both humans and plants. The compound was crystallized and the structure was elucidated by X-ray crystallography. The X-ray structure confirms the previously elucidated structure of the compound that was done under standard spectroscopic methods. The importance of javanicin in establishing symbiosis between Chloridium sp. and its host plant, Azadirachta indica, is briefly discussed.