Extracellular metabolites of endophytic fungi from Azadirachta indica inhibit multidrug-resistant bacteria and phytopathogens.

Aim: To evaluate antimicrobial activity of extracellular metabolites (EMs) of endophytic fungal isolates (EFIs) from Azadirachta indica. Materials & methods: EFIs were identified by internal transcribed spacer (ITS) sequencing. Antimicrobial activity, and minimum inhibitor concentration (MIC) and minimum bactericidal concentration (MBC) were determined using agar diffusion and microdilution method, respectively. Results: Seventeen EFIs were isolated from different organs of A. indica. Eight of them were identified based on ITS sequencing. The EMs of EFIs inhibited the growth of six multidrug-resistant (MDR) bacterial superbugs and three phytopathogenic fungi. The MDR bacterial superbugs are resistant to six commercial antibiotics of different generations but susceptible to EMs of EFIs. The MIC (0.125-1.0 µg/µl), MBC (0.5-4.0 µg/µl) and minimum fungicidal concentration (1.0-4.0 µg/µl) of the EMs from EFIs are lower enough. Conclusion: The EMs of the EFIs have promising antimicrobial activity against MDR bacteria and phytopathogenic fungi.

Antifungal xanthones produced by the endophytic fungus Paraconionthyrium sp. YM 311593.

During our investigation on the endophytic fungi of Azadirachta indica, the strain YM 311593 was obtained from the fruit of the plant. The culture extract of the strain showed antifungal activities against four phytopathogenic fungi. Based on the morphological features and phylogenetic definition, the strain YM 311593 was identified as Paraconiothyrium sp. Four xanthones and one anthraquinone were obtained from the extract of the fermentation broth of the strain. They were characterized to be globosuxanthone A (1), vertixanthone (2), hydroxyvertixanthone (3), 3,8-dihydroxy-1-methy1-9H- xanthen-9-one (4), and danthron (5), respectively, by spectroscopic elucidation. Furthermore, the absolute configuration of 1 was deduced by X-ray diffraction analysis. Besides, compound 4 was firstly found from natural sources. The antifungal activities of compounds 1-5 towards four phytopathogens were assayed using broth microdilution method. Among them, globosuxanthone A (1) showed obvious antifungal activity towards Fusarium graminearum, Fusarium solani, and Botrytis cinerea with MIC values of 4, 8, and 16 µg/mL, respectively.

Differences in plant metabolites and microbes associated with Azadirachta indica with variation in air pollution.

Mitigation of air pollution by plants is a well-established phenomenon. Trees planted on the roadside are known to reduce particulate matter pollution by about 25%. In an urban ecosystem, especially in a metropolitan city such as Delhi, roadside trees are constantly exposed to air pollution. We, therefore, evaluated the effect of air pollution on a common Indian roadside tree, Neem (Azadirachta indica), and its associated microbes in areas with high and low levels of particulate matter (PM) pollution in Delhi. We hypothesized that alteration in the air quality index not only influences plant physiology but also its microbiome. A 100-fold increase in the number of epiphytic and 10-100 fold increase in endophytic colonies were found with 1.7 times increase in the level of pollutants. Trees in the polluted areas had an abundance of Salmonella, Proteus and Citrobacter, and showed increased secondary metabolites such as phenols and tannins as well as decreased chlorophyll and carotenoid. The number of unique microbes was positively correlated with increased primary metabolites. Our study thus indicates that, alteration in air quality affects the natural micro-environment of plants. These results may be utilized as sustainable tools for studying plant adaptations to the urban ecosystem.

Micromonospora radicis sp. nov., isolated from roots of Azadirachta indica var. siamensis Valenton, and reclassification of Jishengella zingiberis as Micromonospora zingiberis comb. nov.

A novel endophytic actinomycete strain AZ1-13T was isolated from roots of Azadirachta indica, and its taxonomic position was investigated using a polyphasic approach. Pairwise 16S rRNA gene sequence similarities of strain AZ1-13T and its closest species, Jishegella zingiberis PLAI1-1T and Micromonospora endophytica 202201T, were 99.7 and 99.2 %, respectively. Phylogenetic analyses of the family Micromonosporaceae based on 16S rRNA gene sequences indicated strains AZ1-13T and J. zingiberis PLAI1-1Tare located within the genus Micromonospora. The approximate genome size of the strain was 5.96 Mb with 71.9 mol% of G+C content. The strain AZ1-13T exhibited ANIb values of 87.4 % with J. zingiberis PLAI1-1T and 85.1 % with M. endophytica 202201T. Chemotaxonomic characteristics of strain AZ1-13T were consistent within the genus Micromonospora: cell-wall peptidoglycan of the strain contained meso-diaminopimelic acid; glucose, mannose, ribose and xylose are presented as the whole-cell sugars; the predominant menaquinones were MK-9(H4) and MK-9(H6); major cellular fatty acids were iso-C15 : 0, 10-methyl C17 : 0, C17 : 0, anteiso-C17 : 0 and iso-C17 : 1ω8c; diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were detected as distinguished phospholipids. Based on phenotypic properties, phylogeny and genomic data, the strain AZ1-13T could be distinguished from its closest neighbours, representing a novel species of the genus Micromonospora, for which the name Micromonospora radicis sp. nov. is proposed. The type strain is AZ1-13T (=KCTC 39786T=NBRC 112324T=JCM 32147T = TISTR 2404T). This study also proposed that J. zingiberisis transferred to the genus Micromonospora as Micromonospora zingiberis comb. nov. (type strain PLAI1-1T=TBRC 7644T=NBRC 113144T=JCM 32592T).

Production of bioactive compounds with bactericidal and antioxidant potential by endophytic fungus Alternaria alternata AE1 isolated from Azadirachta indica A. Juss.

For combating multidrug-resistant microorganisms, exploration of natural compounds from plant endophytes increases the chance of finding novel compounds. An efficient bioactive metabolites producing endophytic fungal strain AE1 was isolated from leaves of Azadirachta indica A. Juss. The metabolites were found to be thermostable, non-proteinacious and produced prominent zones of inhibition against numbers of Gram positive and Gram negative bacteria. Based on 28S rDNA (D1/D2) sequence homology the isolate AE1 was identified as Alternaria alternata. Malt extract broth was found effective for the maximum production of bioactive metabolites by the isolate and was subjected for solvent extraction. The Ethyl acetate (EA) fraction of AE1 showed MIC values of 300-400 µg/ml against Gram positive and Gram negative bacteria tested. The cidal mode of action of EA fraction was detected by treating bacterial cultures at mid log phase. Scanning electron microscopic study supported morphological disintegration of bacterial cells. Release of nucleic acid, protein and potassium ions (K+) also suggested lysis of bacterial cells or leakage of cell membrane upon treatment. In addition, reduction of the activity of EMP pathway, TCA cycle and gluconeogenic enzymes in all bacteria suggested the interference of antibacterial principles with central carbohydrate metabolic pathways. Thin layer chromatographic separation followed by GC-MS analysis of EA fraction suggested numbers of antimicrobial compound production by AE1. In addition, DPPH free radical as well as superoxide radical scavenging assay also suggested strong antioxidant potential of AE1 with an IC50 value of 38.0±1.7 µg/ml and 11.38±1.2 µg/ml respectively. On the basis of above facts it can be concluded that the strain AE1 will be a good source of bioactive compounds having medicinal importance.

Four limonoids from Bacillus subtilis-fermented neem seeds and their cytotoxic activity.

Four new limonoids, 7,12-dihydroxyvilasinone (1), vilasindione (2), 4-dehydroxynimbandiol (3) and azadiramide B (4), were isolated from extracts of Bacillus subtilis-fermented neem seeds. Their planar structures and relative configurations were elucidated by spectroscopic methods including UV, IR, MS and NMR, and the absolute stereochemistry was determined by comparing their experimental and calculated CD spectra. 4 is a rare salannin-class limonoid alkaloid. In cytotoxic assays, 3 showed inhibitory activity against MDA-MB-231, A375 and Hela cell lines with IC50 values of 21.45 ±â€¯5.41, 17.67 ±â€¯3.96 and 28.13 ±â€¯9.12 µM, respectively, while 4 selectively inhibited growth of MDA-MB-231 cell line with an IC50 value of 15.73 ±â€¯6.07 µM.

Micromonospora azadirachtae sp. nov., isolated from roots of Azadirachta indica A. Juss. var. siamensis Valeton.

A Gram-stain positive actinomycete, strain AZ1-19T, isolated from roots of Azadirachta indica A. Juss. var. siamensis Valeton, collected from Chachoengsao province, Thailand, was characterised taxonomically by using a polyphasic approach. Strain AZ1-19T was found to have characteristics consistent with those of members of the genus Micromonospora. The cell wall peptidoglycan of the strain was found to contain meso-diaminopimelic acid. The predominant phospholipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol. The characteristic whole-cell sugars were identified as glucose, xylose, galactose and mannose. The major menaquinones were found to be MK-9(H6), MK-10(H6) and MK-10(H8), and the major cellular fatty acids were identified as iso-C16:0, iso-C15:0, anteiso-C15:0 and anteiso-C17:0. Comparative analysis of 16S rRNA gene sequences revealed that strain AZ1-19T is closely related to Micromonospora costi CS1-12T (98.75% similarity), Micromonospora avicenniae 268506T (98.75%), Micromonospora haikouensis 232617T (98.68%) and Micromonospora siamensis TT2-4T (98.61%), whilst the corresponding phylogenetic analysis based on partial gyrase subunit B (gyrB) gene sequences indicated that strain AZ1-19T forms a clade with M. avicenniae 268506T with a high bootstrap value. The DNA G + C content was determined to be 69.8 mol%. Moreover, a combination of DNA-DNA relatedness values and some phenotypic and chemotaxonomic properties indicated that the strain could be distinguished from closely related species. Therefore, it is considered that strain AZ1-19T represents a novel Micromonospora species for which the name Micromonospora azadirachtae sp. nov. is proposed. The type strain is AZ1-19T (= KCTC 39941T = NBRC 112784T = JCM 32148T = TISTR 2559T).

Actinomadura rhizosphaerae sp. nov., isolated from rhizosphere soil of the plant Azadirachta indica.

A novel actinomycete, strain SDA37T, belonging to the genus Actinomadura, was isolated from rhizosphere soil collected from Udon Thani Province, Thailand. The taxonomic position of the strain was characterized using a polyphasic approach. Meso-diaminopimelic acid, glucose, ribose, galactose and madurose were detected in cell-wall and whole-cell hydrolysates. The N-acyl type of muramic acid was acetyl. Menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The predominant cellular fatty acids were iso-C16 : 0, C16 : 0, 10-methyl C18 : 0 and iso-C14 : 0. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol. blast analysis of the almost-complete 16S rRNA gene sequence showed 98.8 % similarity to Actinomadura oligospora NBRC 104149T, 98.7 % similarity to Actinomadura gamaensis DSM 100815T and 97.2 % similarity to Actinomadura rupiterrae KCTC 19559T. The DNA G+C content was 73.1 mol%. Strain SDA37T showed low DNA-DNA relatedness (44.3±7.3 to 58.5±8.7 %) to A. oligospora NBRC 104149T, Actinomadura gamaensis DSM 100815T and Actinomadura rupiterrae KCTC 19559T. The new strain could also be distinguished from its closely related strains by the differences in the phenotypic characteristics. The results of taxonomic analysis suggested that strain SDA37T represented a novel species of the genus Actinomadura for which the name Actinomadura rhizosphaerae sp. nov. is proposed. The type strain is SDA37T (=KCTC 39965T=NBRC 112909T=TISTR 2523T).

Cyanobacteria growing on tree barks possess high amount of sunscreen compound mycosporine-like amino acids (MAAs).

The present study describes cyanobacterial species composition and their dominance in biological crusts from barks of different trees, roof top of building and soil of agricultural field. An attempt was also made to explore the presence of photoprotective compounds such as mycosporine-like amino acids (MAAs) in the crust samples. Microscopic examination and growth studies revealed the presence of Oscillatoria species in all the crust samples excluding the crust of roof top of a building. Study on the abundance of dominant genera showed marked differences among various crust samples but Hapalosiphon, Lyngbya, Oscillatoria and Scytonema sp. were the most dominant genera, Oscillatoria being dominant in three crust samples. Screening for the presence of photoprotective compounds showed the presence of major peaks in the range of 308-334 nm thereby pointing to the presence of MAAs in all the crust samples. The highest amount of MAAs was found in the crust of Borassus flabellifer (15,729 nmol g dry wt-1 of bark) followed by crust of roof top (14,543 nmol g dry wt-1 of crust). MAAs were separated and partially purified employing HPLC, the most common MAA present in all the crusts was identified as mycosporine-glycine. Presence of mycosporine-glycine (M-Gly) was further confirmed by FTIR and NMR. Test of in vitro colonization on the bark of Mangifera indica and Azadirachta indica by three isolates namely Hapalosiphon, Oscillatoria and Scytonema sp. showed sign of active colonization. It is felt that identification of all the MAAs other than M-Gly may prove useful in future studies especially for assessing their significance in the protection mechanism of cyanobacteria/algae against various types of abiotic stresses.

Biosynthesis of Anti-Proliferative Gold Nanoparticles Using Endophytic Fusarium oxysporum Strain Isolated from Neem (A. indica) Leaves.

Here we report a simple, rapid, environment friendly approach for the synthesis of gold nanoparticles using neem (Azadirachta indica A. Juss.) fungal endophyte, which based upon morphological and cultural characteristics was eventually identified as Fusarium oxysporum. The aqueous precursor (HAuCl4) solution when reacted with endophytic fungus resulted in the biosynthesis of abundant amounts of well dispersed gold nanoparticles of 10-40 nm with an average size of 22nm. These biosynthesized gold nanoparticles were then characterized by standard analytical techniques such as UV-Visible spectroscopy, X-ray diffraction, Transmission Electron Microscopy and Fourier Transform Infrared Spectroscopy. Cytotoxic activity of these nanoparticles was checked against three different cell types including breast cancer (ZR-75-1), Daudi (Human Burkitt's lymphoma cancer) and normal human peripheral blood mononuclear cells (PBMC), where it was found that our gold nanoparticles are anti-proliferative against cancer cells but completely safe toward normal cells. In addition to this, assessment of toxicity toward human RBC revealed less than 0.1 % hemolysis as compared to Triton X-100 suggesting safe nature of our biosynthesized gold nanoparticles on human cells. Also, our nanoparticles exhibited no anti-fungal (against Aspergillus niger) or anti-bacterial [against Gram positive (Bacillus subtilis & Staphylococcus aureus) and Gram negative (Escherichia coli & Pseudomonas aeruginosa) bacteria] activity thus suggesting their non-toxic, biocompatible nature. The present investigation opens up avenues for ecofriendly, biocompatible nanomaterials to be used in a wide variety of application such as drug delivery, therapeutics, theranostics and so on.

A New Lactone from Chaetomium globosum Strain M65 that Inhibits the Motility of Zoospores.

In a search for endophytes from medicinal plants of Bangladesh, we isolated the M65 fungal strain from the fruit of Azadirachta indica. Following chemical screening, chromatographic purification of the culture extract of strain M65 led to the isolation of the previously reported lasiodiplodin (2), the known derivative 1, and the new derivative 3a, along with two further known compounds (4 and 5). The new (3R,5R)-5-hydroxylasiodiplodin (3a), the enantiomer of the known (3S,5S)-5-hydroxylasiodiplodin (3b), inhibited the motility of zoospores of a devastating late blight phytopathogen Phytophthora capsici by 100% at a concentration of 10 µg/mL. The respective activities of the other metabolites were negligible.

Antimicrobial biosynthetic potential and genetic diversity of endophytic actinomycetes associated with medicinal plants.

Endophytic actinomycetes are one of the primary groups that share symbiotic relationships with medicinal plants and are key reservoir of biologically active compounds. In this study, six selective medicinal plants were targeted for the first time for endophytic actinomycetes isolation from Gibbon Wild Life Sanctuary, Assam, India, during winter and summer and 76 isolates were obtained. The isolates were found to be prevalent in roots followed by stem and leaves. 16S rRNA gene sequence analysis revealed 16 genera, including rare genera, Verrucosispora, Isoptericola and Kytococcus, which have never been previously reported as endophytic. The genus Streptomyces (66%) was dominant in both seasons. Shannon's diversity index showed that Azadirachta indica (1.49), Rauwolfia serpentina (1.43) and Emblica officinalis (1.24) were relatively good habitat for endophytic actinomycetes. Antimicrobial strains showed prevalence of polyketide synthase (PKS) type-II (85%) followed by PKS type-I (14%) encoded in the genomes. Expression studies showed 12-fold upregulation of PKSII gene in seventh day of incubation for Streptomyces antibioticus (EAAG90). Our results emphasize that the actinomycetes assemblages within plant tissue exhibited biosynthetic systems encoding for important biologically active compounds.

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica.

Five new guaiane sesquiterpenes, 1-5, were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss. The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.

Guaiane sesquiterpenes and isopimarane diterpenes from an endophytic fungus Xylaria sp.

Nine oxygenated guaiane-type sesquiterpenes and three isopimarane diterpenes were isolated from the culture broth of an endophytic fungus, Xylaria sp. YM 311647, obtained from Azadirachta indica. The structures of these compounds were elucidated by interpretation of spectroscopic data. The absolute configurations of two of these were confirmed by X-ray crystallographic analysis. All of the compounds were tested for their antifungal activities against five pathogenic fungal cells. The results showed that nine sesquiterpenes were moderately active against Candida albicans and Hormodendrum compactum with MIC values ranging from 32 to 256µg/ml, while the diterpenes were more active; One of these exhibited the most potent inhibitory activity against C. albicans and Pyricularia oryzae with MIC values of 16µg/ml.

Phytoremediation of abandoned crude oil contaminated drill sites of Assam with the aid of a hydrocarbon-degrading bacterial formulation.

Environmental deterioration due to crude oil contamination and abandoned drill sites is an ecological concern in Assam. To revive such contaminated sites, afield study was conducted to phytoremediate four crude oil abandoned drill sites of Assam (Gelakey, Amguri, Lakwa, and Borholla) with the aid of two hydrocarbon-degrading Pseudomonas strains designated N3 and N4. All the drill sites were contaminated with 15.1 to 32.8% crude oil, and the soil was alkaline in nature (pH8.0-8.7) with low moisture content, low soil conductivity and low activities of the soil enzymes phosphatase, dehydrogenase and urease. In addition, N, P, K, and C contents were below threshold limits, and the soil contained high levels of heavy metals. Bio-augmentation was achieved by applying Pseudomonas aeruginosa strains N3 and N4 followed by the introduction of screened plant species Tectona grandis, Gmelina arborea, Azadirachta indica, and Michelia champaca. The findings established the feasibility of the phytoremediation of abandoned crude oil-contaminated drill sites in Assam using microbes and native plants.

Cyclonerol derivatives from Trichoderma longibrachiatum YM311505.

A new sesquiterpene, 10,11-dihydrocyclonerotriol (1), together with two known compounds, catenioblin C (2) and sohirnone A (3), were isolated from Trichoderma longibrachiatum YM311505, an endophytic fungus of Azadirachta indica. Their structures were characterized and identified by spectral analysis. Compounds 1-3 exhibited antifungal activities against Pyricularia oryzae and Candida albicans.

Screening of efficient arbuscular mycorrhizal fungi for Azadirachta indica under nursery condition: a step towards afforestation of semi-arid region of western India.

To optimize nursery practices for efficient plant production procedures and to keep up to the ever growing demand of seedlings, identification of the most suitable species of arbuscular mycorrhizal fungi (AMF), specific for a given tree species, is clearly a necessary task. Sixty days old seedlings of Neem (Azadirachta indica A. Juss) raised in root trainers were inoculated with six species of AMF and a mixed inoculum (consortia) and kept in green house. Performances of the treatments on this tree species were evaluated in terms of growth parameters like plant height shoot collar diameter, biomass and phosphorous uptake capabilities. Significant and varied increase in the growth parameters and phosphorous uptake was observed for most of the AMF species against control. Consortia culture was found to be the best suited AMF treatment for A. indica, while Glomus intraradices and Glomus mosseae were the best performing single species cultures. It is the first time in the state of Gujarat that a wide variety of AMF species, isolated from the typical semi-arid region of western India, were tested for the best growth performance with one of the most important tree species for the concerned region.

Screening of efficient arbuscular mycorrhizal fungi for Azadirachta indica under nursery condition: a step towards afforestation of semi-arid region of western India

To optimize nursery practices for efficient plant production procedures and to keep up to the ever growing demand of seedlings, identification of the most suitable species of arbuscular mycorrhizal fungi (AMF), specific for a given tree species, is clearly a necessary task. Sixty days old seedlings of Neem (Azadirachta indica A. Juss) raised in root trainers were inoculated with six species of AMF and a mixed inoculum (consortia) and kept in green house. Performances of the treatments on this tree species were evaluated in terms of growth parameters like plant height shoot collar diameter, biomass and phosphorous uptake capabilities. Significant and varied increase in the growth parameters and phosphorous uptake was observed for most of the AMF species against control. Consortia culture was found to be the best suited AMF treatment for A.indica, while Glomus intraradices and Glomus mosseae were the best performing single species cultures. It is the first time in the state of Gujarat that a wide variety of AMF species, isolated from the typical semi-arid region of western India, were tested for the best growth performance with one of the most important tree species for the concerned region.

An endophytic fungus from Azadirachta indica A. Juss. that produces azadirachtin.

Azadirachtin A and its structural analogues are a well-known class of natural insecticides having antifeedant and insect growth-regulating properties. These compounds are exclusive to the neem tree, Azadirachta indica A. Juss, from where they are currently sourced. Here we report for the first time, the isolation and characterization of a novel endophytic fungus from A. indica, which produces azadirachtin A and B in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified as Eupenicillium parvum by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Azadirachtin A and B were identified and quantified by LC-HRMS and LC-HRMS(2), and by comparison with the authentic reference standards. The biosynthesis of azadirachtin A and B by the cultured endophyte, which is also produced by the host neem plant, provides an exciting platform for further scientific exploration within both the ecological and biochemical contexts.

Use of plant residues on growth of mycorrhizal seedlings of neem (Azadirachta indica A. Juss.).

BACKGROUND: Owing to its multiple uses in veterinary medicine, biofertilizers, pest control, etc., the commercial cultivation of neem (Azadirachta indica) has been increasing in various countries. The use of arbuscular mycorrhizal fungi (AMF) and plant by-products (composted leaves and residues of neem and sugarcane) for the propagation of seedlings can be an efficient alternative to stimulate plant growth, reducing the propagation time and conferring increased tolerance of plants to biotic and abiotic stresses. Therefore this study aimed to evaluate the effect of plant substrates and inoculation with AMF on the production of neem seedlings. RESULTS: Beneficial effects of the application of neem by-products to neem seedlings were observed on most of the variables analysed. However, the treatment with sugarcane cake did not improve the growth of neem seedlings. In general, the inoculation treatments using Glomus etunicatum in the composted neem substrates improved seedling growth. CONCLUSION: Neem by-products benefit the growth of seedlings of this plant under greenhouse conditions. Inoculation with G. etunicatum enhances plants growth mainly in substrates with residues of neem leaves, providing an alternative for the production of seedlings of this crop under nursery conditions, which can reduce the need for chemical fertilizers that impact the environment.