Ashish Mishra

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.

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.

Assessment of diversity, distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant Adenocalymma alliaceum Miers

A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds.

Diversity of endophytic mycobiota of tropical tree Tectona grandis Linn.f.: Spatiotemporal and tissue type effects

Fungal endophytes were isolated from leaf, bark and stem of Tectona grandis Linn.f. sampled at four geographical locations in winter, summer and monsoon seasons. The recovered 5089 isolates were assigned to 45 distinct morphotypes based on morphology. The sequences of the internal transcribed spacers (ITS) of the nrDNA of some morphotypes were identical, but morphological differences were strong enough to consider these morphotypes as separate species. Forty-three morphotypes were assigned to ascomycotina and two to basidiomycotina. Ascomycotina was the predominating group with 99.7% of total isolates followed by basidiomycotina with only 0.3% of total isolates. Diaporthe (Phomopsis) species dominated the communities independently on tissue type, location or season. More than 60% of the examined tissue pieces were colonized by members of this species complex. While these endophytes are ubiquitous others were tissue or location specific. Tissue type had the strongest effect on the species evenness of the endophytic assemblage followed by geographical location and season. However, Shannon-Wiener index (H’) significantly (p ≤ 0.001) varied with all three factors i.e. season, location and tissue type. Leaves supported the highest diversity across all the seasons and locations. In conclusion, all the three factors together determined the structure of endophytic mycobiota assemblage of T. grandis.

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.

Sourcing the Fungal Endophytes: A Beneficial Transaction of Biodiversity, Bioactive Natural Products, Plant Protection and Nanotechnology

Endophytes are the group of microorganisms that reside to internal and healthy tissues without causing negative symptoms to their host plant. Endophytes are extremely diverse and range from fungi, bacteria and actinomycetes. Development of drug resistance to pathogenic forms of bacteria, fungi and other microbes, emergence of lethal viruses, the perpetuating epidemics in developing and under developing countries, and multifold fungal infection, enhancement in human population globally, all shows our inability to overcome these biomedical problems. In addition to this, we are also unable to assure people towards enough food security in specific regions of the earth due to infestation of different plant diseases. Since the fungal endophytes are relatively less studied group of microbial flora, but are responsible for several prospects such as biodiversity, ecology, bioactive metabolites (metabolomics) and nanotechnology, may enable us to overcome the above mentioned problems. Fungal endophytes represent a dependable source of specific secondary metabolites and can be manipulated both physicochemically and genetically to increase yield of desired compounds and to produce novel analogues of active metabolites. In this chapter, we have discussed several bioactive compounds and classified them in to different classes as per their properties such as antifungal, antibacterial, antiviral, antimalarial, anticancer, antioxidants, antidiabetic and immunosuppressive agents derived from fungal endophytes with their hosts and made the chemical structures for 73 compounds using chemdraw 3D ultra version 7.0. These bioactive products are related to human health with MIC/EC/IC50 values less that 50 μg/mL. This article also discusses nematicidal, some antimicrobial volatile compounds (VOCs) that are related to plant protection and faecal disposal. Therefore, this chapter is not very specific and covers almost prospects of fungal endophytes which could be useful in biodiversity, agrochemicals, biotechnology, biomedical and nanotechnology in ecofriendly manner.

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.

Biofabrication of Antibacterial and Antioxidant Silver Nanoparticles (Agnps) by an Endophytic Fungus Pestalotia Sp. Isolated from Madhuca Longifolia

Biofabrication of Antibacterial and Antioxidant Silver Nanoparticles (Agnps) by an Endophytic Fungus Pestalotia Sp. Isolated from Madhuca Longifolia Particles of metals or other materials in nanometre size are called as nanoparticles (NPs). Of ten endophytic fungi screened, seven endophytic fungi namely Alternaria alternata, Botryodiplodia sp., Pestalotia sp., Fusarium sp., Aspergillus sp. Aschersonia sp. and Phomopsis sp. were able to synthesize silver nanoparticles. Silver metal nanopartites were synthesised successfully by both mycelium as well as cell filtrate of Pestalotia sp., which was initially observed by spectrometric analysis. TEM pictures revealed the synthesis of angular shape of silver nanoparticles like trigonal, pentagonal, hexagonal, few of cuboids and some were of spherical in shape too. Most of nanoparticles (76%) were found below the size of 40 nm. FTIR spectra peaks showed the presence of -NH, CO, C=C and C-H containing organic metabolites which might play a role in stabilizing the nanoparticles. Silver nanoparticles solution (0.5 mM) inhibited the growth significantly than control against all pathogenic bacteria tested. Mycosynthesized AgNPs were most active against Aeromonas hydrophila @2 μg/ml IC50 followed by Staphylococcus aureus (6 μg/ml), Escherichia coli (8μg/ml), Morganella morganii (24 μg/ml), Salmonella typhi (42 μg/ml), Enterococcus faecalis (40 μg/ml) and Klebsiella pneumoniae (50 μg/ml) respectively. Silver nanoparticles had also shown antioxidant activity with DPPH free radical scavenging with inhibitory concentration (IC50) at 47 μg /ml. To combat with new emerging pathogenic bacteria that threat our society by causing dreadful diseases, we require the development of effective therapeutics. Thus, eco-friendly synthesised antimicrobial and antioxidant silver nanoparticles allow us to suitably employing their application in nanoparticles mediated drug delivery and therapy of diseases.

Salicylic Acid and its Role in Systemic Resistance Induced by Pseudomonas fluorescens to Early Blight Disease of Tomato

Seventeen isolates of Pseudomonas fluo-rescens (Pf) were examined for growth promotion and the induction of systemic resistance against early blight disease of tomato. A noticeable in-crease in shoot and root length was observed in plants treated with P. fluorescens. However, the root development was reduced by a combination of P. fluorescens and Alternaria alternata (A. alter-nata). P. fluorescens isolates systemically induced resistance against early blight of tomato caused by A. alternata and reduced (approx.18–42%) the dis-ease significantly. All the isolates of P. fluorescens produced salicylic acid (SA) in Kings B (KB) broth and induced its accumulation in tomato leaf within 24 h of bacterial inoculation. SA levels were signifi-cantly increased in plants treated with bacteria compared to the split control from one to five days following inoculation. Low SA concentration (0.1–0.5 mg/ml) did not inhibit mycelial growth of A. alternata in vitro, contrary to inhibition at high concentrations. The exogenously applied SA first accumulates in host tissues and induces resistance, however, endogenous SA accumulation in plant tissue is considered to be involved in induced sys-temic resistance to early blight disease of tomato caused by A. alternata.

Diversity and Biopotential of Endophytic Fungal Flora Isolated from Eight Medicinal Plants of Uttar Pradesh, India

Endophytic fungi are hidden diversity mines of microbes that reside in the healthy and symptomless interior of plant tissues without causing any harmful effects. This chapter focuses on fungal endophytic diversity of eight medicinal plants of Uttar Pradesh, India with their biopotential ability. Total of 4,002 (38.38 % CF) endophytic isolates were recovered from 10,425 segments representing 131 endophytic fungal species belonging to different fungal classes. Out of 4,002 isolates, hyphomycetes were more pronounced with 71.43 % recovery followed by coelomycetes 16.61 %, ascomycetes 6.59 %, mycelia sterilia or unidentified 5.32 % and least from zygomycetes 0.020 %. Among total endophytic fungal species isolated, Cladosporium cladosporioides (3.39 % CF) was found to be the most dominated taxa followed by Alternaria alternata (2.35 % CF), Curvularia lunata (2.13 % CF), Aspergillus niger (1.95 % CF), Chaetomium globosum (1.85 % CF), Nigrospora oryzae (1.57 % CF) and Phoma glomerata (1.09 % CF). From a total of 131 endophytic species, 101 were tested for their antimicrobial and antioxidant activity. Out of 47 active species, 29.78 % displayed antibacterial activity, 27.65 % showed antifungal activity, 38.29 % exhibited antibacterial and antifungal activity both while only 4.25 % displayed antimalarial as well as antioxidant activity. Twenty-one endophytic fungal species were tested for extracellular production of amylase, xylanase and phosphate solubilization where 76.19 % found to produce amylase, 23.80 % for xylanase and 14.28 % exhibited phosphate-solubilization activity.