Ravindra N. Kharwar

Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus

AIM To induce the biosynthesis of silver nanoparticles (AgNPs) using Aspergillus clavatus and evaluate their antimicrobial potential. MATERIALS & METHODS Aspergillus clavatus (AzS-275), an endophytic fungus isolated from sterilized stem tissues of Azadirachta indica A. Juss., was challenged with 1 mM AgNO(3) solution. The characterization of the AgNPs was carried out by x-ray diffraction spectrometry, transmission-electron microscopy and atomic force microscopy. RESULTS & DISCUSSION The synthesized AgNPs were found to be extracellular, polydispersed spherical or hexagonal particles ranging from 10 to 25 nm in size. Antimicrobial activity was performed using a disc-diffusion method against Candida albicans, Pseudomonas fluorescens and Escherichia coli. The results showed an average minimum inhibitory concentration of 5.83 microg ml(-1) and minimum fungicidal concentration of 9.7 microg ml(-1) against C. albicans. CONCLUSIONS AgNPs can be mycosynthesized extracellularly using A. clavatus as the fungal system, which is highly advantageous over chemical synthesis not only because it can be synthesized on a large scale, but because of the ease of downstream processing and its biomedical application in antimicrobial activity.

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.

The Endophytic Mycoflora of Bark, Leaf, and Stem Tissues of Azadirachta indica A. Juss (Neem) from Varanasi (India)

A systematic study was made of the endophytes of Azadirachta indica A. Juss (the neem tree) growing in several of its natural habitats in India. A total of 233 isolates of endophytic fungi representing 18 fungal taxa were obtained from segments of bark, stem, and leaves of this tree. Hyphomycetes (62.2%) were the most prevalent followed by the Coelomycetes (27.4%) and Mycelia Sterilia (7.7%). As mathematically determined, the maximum species richness and frequency of colonization of endophytes appeared in leaf segments rather than stem and bark tissues from each location. Endophytic colonization frequency was also greater in leaves (45.5%) than bark (31.5%). The leaf samples from all locations were nearly constant in their endophytic composition, whereas bark samples showed maximum diversity at different locations. Inter-site comparisons for endophytic diversity, however, were not significantly different with Loc1 and Loc2 having a maximum of 66.67% Jc. The smallest similarity was between Loc2 and Loc3 of 54.17% Jc. The dominant endophytic fungi isolated were Phomopsis oblonga, Cladosporium cladosporioides, Pestalotiopsis sp., Trichoderma sp, and Aspergillus sp. Genera such as Periconia, Stenella, and Drechslera are reported here for the first time as endophytes from this host plant. This report illustrates the value of sampling different tissues of a given plant in several locations to obtain the greatest species diversity of endophytes. The rich and sizeable collection of endophytic fungi from this specific plant may represent a unique source of one or more of the interesting and useful bioactive compounds normally associated with A. indica such as the azadirachtins and related tetranortriterpenoids.

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.

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 NO2, SO2, 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.

Erratum to: Biofabrication of anisotropic gold nanotriangles using extract of endophytic Aspergillus clavatus

Biosynthesis of metal and semiconductor nanoparticles using microorganisms has emerged as a more eco-friendly, simpler and reproducible alternative to the chemical synthesis, allowing the generation of rare forms such as nanotriangles and prisms. Here, we report the endophytic fungus Aspergillus clavatus, isolated from surface sterilized stem tissues of Azadirachta indica A. Juss., when incubated with an aqueous solution of chloroaurate ions produces a diverse mixture of intracellular gold nanoparticles (AuNPs), especially nanotriangles (GNT) in the size range from 20 to 35 nm. These structures (GNT) are of special interest since they possess distinct plasmonic features in the visible and IR regions, which equipped them with unique physical and optical properties exploitable in vital applications such as optics, electronics, catalysis and biomedicine. The reaction process was simple and convenient to handle and was monitored using ultraviolet–visible spectroscopy (UV–vis). The morphology and crystalline nature of the GNTs were determined from transmission electron microscopy (TEM), atomic force spectroscopy (AFM) and X-ray diffraction (XRD) spectroscopy. This proposed mechanistic principal might serve as a set of design rule for the synthesis of anisotropic nanostructures with desired architecture and can be amenable for the large scale commercial production and technical applications.

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.

Seed-vectored endophytic bacteria modulate development of rice seedlings

The aim of the present study was to evaluate the effects of the removal of indigenous bacteria from rice seeds on seedling growth and development. Here we report the presence of three indigenous endophytic bacteria in rice seeds that play important roles in modulating seedling development (shoot and root lengths, and formation of root hairs and secondary roots) and defence against pathogens.

Pseudomonas fluorescens and Trichoderma asperellum Enhance Expression of Gα Subunits of the Pea Heterotrimeric G-protein during Erysiphe pisi Infection

We investigated the transcript accumulation patterns of all three subunits of heterotrimeric G-proteins (Gα1 and 2, Gβ, and Gγ) in pea under stimulation of two soil-inhabiting rhizosphere microbes Pseudomonas fluorescens OKC and Trichoderma asperellum T42. The microbes were either applied individually or co-inoculated and the transcript accumulation patterns were also investigated after challenging the same plants with a fungal biotrophic pathogen Erysiphe pisi. We observed that mostly the transcripts of Gα 1 and 2 subunits were accumulated when the plants were treated with the microbes (OKC and T42) either individually or co-inoculated. However, transcript accumulations of Gα subunits were highest in the T42 treatment particularly under the challenge of the biotroph. Transcript accumulations of the other two subunits Gβ and Gγ were either basal or even lower than the basal level. There was an indication for involvement of JA-mediated pathway in the same situations as activation of LOX1 and COI1 were relatively enhanced in the microbe co-inoculated treatments. Non-increment of SA content as well as transcripts of SA-dependent PR1 suggested non-activation of the SA-mediated signal transduction in the interaction of pea with E. pisi under the stimuli of OKC and T42. Gα1 and 2 transcript accumulations were further correlated with peroxidases activities, H2O2 generation and accumulation in ABA in pea leaves under OKC and T42 stimulations and all these activities were positively correlated with stomata closure at early stage of the biotroph challenge. The microbe-induced physiological responses in pea leaves finally led to reduced E. pisi development particularly in OKC and T42 co-inoculated plants. We conclude that OKC and T42 pretreatment stimulate transcript accumulations of the Gα1 and Gα2 subunits of the heterotrimeric G protein, peroxidases activities and phenol accumulation in pea during infection by E. pisi. The signal transduction was possibly mediated through JA in pea under the stimulus of the microbes and the cumulative effect of the co-inoculated microbes had a suppressive effect on E. pisi conidial development on pea leaves.

Soil fungi for mycoremediation of arsenic pollution in agriculture soils.

Soil arsenic (As) contamination of food‐chains and public health can be mitigated through fungal bioremediation. To enumerate culturable soil fungi, soils were collected from the As‐contaminated paddy fields (3–35 mg kg−1) of the middle Indo‐Gangetic Plains.