Rajesh Kumari Manhas

Purification and characterization of an exoinulinase from Aspergillus fumigatus.

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower Km (0.25 mM) and higher Vmax (333.3 IU/mg) values for inulin.

Antifungal, insecticidal, and plant growth promoting potential of Streptomyces hydrogenans DH16

In the present study, an actinobacterium strain, possessing antagonistic activity against different fungal phytopathogens viz. Colletotrichum acutatum, Cladosporium herbarum, Alternaria brassicicola, Exserohilum sp., Alternaria mali, Colletotrichum gleospoiroides, Alternaria alternata, Cercospora sp., Fusarium oxysporum f.sp. dianthi and Fusarium moniliformae, was isolated from soil and identified as Streptomyces hydrogenans DH16. Application of culture supernatant (5%)/cells (107 cfu ml−1), 2 h post inoculation with A. brassicicola (105 spores ml−1), resulted in 85.95 and 93.75% suppression of black leaf spot of Raphanus sativus, respectively on detached leaves. Whereas cells/culture supernatant (above 5%) completely suppressed the disease incidence when co inoculated with fungal pathogen. The crude extract containing antifungal components was completely stable at 70 °C for 1 h retaining 90 and 67.67% activity after boiling (for 1 h) and autoclaving (121 °C for 30 min), respectively. No loss in activity was observed when treated with proteinase K and on exposure to sun and UV light and found to be active over a wide range of pH (2 to 14). Bioautography of the solvent extract against test phytopathogens revealed the presence of three active components. Ethyl acetate extract of DH16 also demonstrated insecticidal activity against Spodoptera litura, causing 40% larval mortality and extension of larval period. In addition, it produced 30 µg ml−1 of Indole Acetic Acid (IAA) in a medium containing tryptophan which promoted lateral root formation in Vigna radiata (green gram). These results indicate that Streptomyces hydrogenans holds the potential to be used as antifungal, insecticidal, and plant growth promoting agent.

Antagonistic and plant growth promoting activities of endophytic and soil actinomycetes

The objective of this study was the isolation and screening of actinomycete isolates for antagonistic potential and plant growth promoting activities. A total of 321 isolates were recovered from different plants, their rhizospheric soils and non-rhizospheric soils of Punjab and Himachal Pradesh regions. Out of these, 62 were endophytic, 156 were rhizospheric and 103 were non-rhizospheric isolates. In primary screening (dual culture assay), 83 isolates antagonised one or more test phytopathogenic fungi. From these active isolates, 20 were found to be antagonistic in well diffusion assay (secondary screening) and most of them demonstrated broad spectrum inhibitory activity against five to six test fungi. Studies on plant growth promoting activities revealed that 12 showed abilities to produce indole acetic acid, 10 produced siderophores and 12 showed ammonia production. Phosphate solubilisation was observed in five isolates and four fixed atmospheric N2. In addition, production of hydrolytic enzymes such as chitinase, amylase, cellulase and protease was demonstrated by five, twenty, eleven and eleven isolates, respectively. The results of this study indicate that these isolates may be used as biocontrol and plant growth promoting agents. Morphological and chemotaxonomic studies revealed that all the active isolates belonged to the genus Streptomyces

An alpha-glucosidase inhibitor from an endophytic Cladosporium sp. with potential as a biocontrol agent.

This study highlights the importance of alpha-glucosidase inhibitors as mechanisms for endophyte-mediated resistance to insect pests. One of the major benefits which endophytes confer on plants is providing resistance against insect pests. This built-in defense mechanism of the plant can be used for exploring ecofriendly strategies for pest control. In the present study, 34 endophytic fungi were isolated from Tinospora cordifolia and screened for their ability to produce alpha-glucosidase inhibitors. Maximum inhibitory activity was observed in an isolate from T. cordifolia (TN-9S), identified to be Cladosporium sp. The inhibitor was purified using chromatographic techniques. The insecticidal activity of the purified inhibitor was evaluated against Spodoptera litura. The inhibitor induced a significant mortality in the larvae of S. litura and adversely affected its survival and development. It also inhibited the activity of α-glycosidases in vivo in the gut of the larvae. The purified inhibitor was determined to be a phenolic compound with amine groups, demonstrating a noncompetitive type of inhibition in vitro. The production of the inhibitor was optimized. Response surface methodology (RSM) analysis revealed a significant interaction between dextrose and malt extract, with first-order effect of pH.

Insecticidal and growth inhibitory potential of Streptomyces hydrogenans DH16 on major pest of India, Spodoptera litura (Fab.) (Lepidoptera: Noctuidae).

BackgroundDestructive impacts of insecticides on non targeted populations necessitate the development of an eco friendly pest control method. Streptomyces spp. are rich source of bioactive secondary metabolites which may provide valuable alternatives to chemical insect-control agents as they can be less toxic and readily biodegradable. Because of its potent biocontrol attributes, ethyl acetate extract of Streptomyces hydrogenans DH16, a soil isolate, was tested to assess its anti-insect potential against polyphagous noctuid, Spodoptera litura.ResultsThe secondary metabolites in the ethyl acetate extract of S. hydrogenans DH16 exhibited larvicidal and growth inhibitory activities. The results indicated that highest concentration of 1600 μg/ml was significantly effective as 70% larval, 66.66% prepupal and 100% pupal mortality was noticed. The metabolites also prolonged the larval developmental period. The LC50 and LC90 values were 1337.384 and 2070.516 μg/ml, respectively for the insect. Negative effects of S. hydrogenans were also observed on development of the insect. Significant decline in adult emergence, adult longevity, fecundity and % hatching was recorded at higher concentrations along with morphological abnormalities as compared to control. Significant decrease in relative growth and consumption rate, efficiency of ingested and digested food and increase in approximate digestibility in larvae reared on diet supplemented with ethyl acetate extract accounts for the toxic as well as anti-nutritive nature of extract.ConclusionSecondary metabolites in the fermentation broth from S. hydrogenans were toxic to the larvae at higher concentrations whereas lower concentrations significantly reduced the reproductive potential of S. litura. Therefore, these metabolites show considerable potential for incorporation in pest management programmes as new biopesticidal formulation.

Insecticidal potential of an endophytic Cladosporium velox against Spodoptera litura mediated through inhibition of alpha glycosidases

Alpha glucosidase inhibitory activity was exhibited by partially purified fractions obtained from an endophytic Cladosporium velox, isolated from Tinospora cordifolia. Taking into account the increasing importance of digestive enzyme inhibitors as insecticidal agents, the entomopathogenic potential of the fractions obtained was evaluated against Spodoptera litura (Fab.), a polyphagous pest. Considerable mortality was obtained when the larvae were fed on diet supplemented with the partially purified extract. All the concentrations of the extract significantly prolonged the overall developmental period of S. litura. At higher concentrations, the extract influenced the longevity of females as well as their reproductive potential. Phytochemical analysis revealed the presence of phenolic compounds in the active fraction. The phenolic compound responsible for the bioactivities was purified and identified to be chlorogenic acid using HPLC and MS analysis. The content of chlorogenic acid in the extract was quantified to be 250μg/ml. The purified compound also demonstrated inhibition of alpha glycosidases in vivo. The present study indicates that the endophyte imparted resistance to the insects in the plants could be mediated through chlorogenic acid targeting the alpha glycosidases present in the gut of the insect. The isolate obtained can be exploited for the production of chlorogenic acid, which has the potential to be exploited as a biocontrol agent against S. litura.

Evaluation of in vitro and in vivo nematicidal potential of a multifunctional streptomycete, Streptomyces hydrogenans strain DH16 against Meloidogyne incognita

The present work demonstrated the nematicidal potential of Streptomyces hydrogenans strain DH16 (a strain with strong antagonism against fungal phytopathogens and insect pest) against Meloidogyne incognita. The culture supernatant and solvent extract significantly inhibited egg hatching (almost 100%) along with J2 mortality of more than 95% after 96h. The nematicidal activity of 10-(2,2-dimethyl-cyclohexyl)-6,9-dihydroxy-4,9-dimethyl-dec-2-enoic acid methyl ester (SH2; a new antifungal compound) purified from this streptomycete was also evaluated using different concentrations. The juvenile mortality of the nematode increased with increasing concentration and exposure time and reached the maximum (95%) after 96h at concentration of 100μg/ml. After 160h of incubation, egg hatch of 16% was observed at concentration of 100μg/ml as compared to control where 100% egg hatching was achieved. However, at the highest concentration of the compound (200μg/ml), 100% J2 mortality and 0% egg hatching were observed after 72 and 160h of incubation, respectively. In vivo pot experiments further revealed the nematicidal potential of S. hydrogenans where soil drenching with its culture supernatant and cells effectively controlled root galls, egg masses in nematode infested tomato plants and at the same time promoted the growth of tomato plants. Additionally, in the absence of nematodes, soil drenching with culture supernatant and cells significantly enhanced the various agronomic traits of plants as compared to control plants. Thus, the outcomes of the current study endorse the potential of S. hydrogenans strain DH16 and its metabolites to be developed as safe nematicidal and plant growth promoting agents.

Nanoethosomal formulation for skin targeting of amphotericin B: an in vitro and in vivo assessment

Abstract The present study is envisaged to develop nanoethosomal formulation for enhanced topical delivery of amphotericin B (AmB) for the treatment of cutaneous fungal infections. AmB encapsulated nanoethosomes were prepared using mechanical dispersion method in a strength of 0.1% w/w similar to the strength of marketed topical formulation. Vesicle size of nanoethosomal formulations was found to be in the range of 186 ± 2 to 298 ± 4 nm. The optimized nanoethosomal formulation was further incorporated in gel base to form AmB nanoethogel formulation. Rheological characterization study of nanoethogel demonstrated its viscoelastic nature with high elasticity and resistance to deformation at 37 °C. The yield stress value was found to be 108.05 ± 2.4 and 52.15 ± 0.9 Pa for nanoethogel and marketed gel formulation, respectively. The nanoethogel formulation exhibited 2.7- and 3.5-fold higher steady state transdermal flux and skin deposition of AmB, respectively, in comparison to marketed formulation. Confocal laser scanning microscopy (CLSM) study also revealed enhanced skin permeation and deposition with nanoethogel formulation. In vivo study showed that topical application of nanoethogel does not exhibit any skin irritation as tested by Draize test. The developed formulation, in comparison to the marketed gel, demonstrated a remarkable increase in the antifungal activity against Candida albicans. It is thus corroborated from the above results that nanoethosomal formulation represents an efficacious carrier for effective topical delivery of AmB.

Sonochemical synthesis, characterization, antimicrobial activity and textile dyeing behavior of nano-sized cobalt(III) complexes.

Using ultrasonic irradiations, nano-sized cobalt(III) coordination complexes, [Co(NH3)6]Cl3·2H2O (A), [Co(en)3]Cl3·3H2O (B) (en-ethylenediamine) and [Co(dien)2]Cl3·3.5H2O (C) (dien-diethylenetriamine) were synthesized. These complexes were characterized by spectroscopic studies like IR, UV/Visible and NMR. Morphology of these complexes was determined by SEM and particle size with the help of TEM & Zeta-sizer. The comparative thermal stability along with phase difference between nano structures and their respective bulk complexes has been studied by thermal gravimetric analysis (TGA) and X-ray powder diffraction (XRD) study respectively. The dyeing behavior of nano-sized Co(III) complexes and their respective bulks has also been studied (using both exhaust and pad dyeing methods) on cotton and wool fabrics and results shown rationalized dyeing behavior. All these complexes were further tested for antimicrobial activity (against B. subtilis, E. coli, K. pneumoniae, F. oxysporum and A. alternate) and it was observed that nano sized complexes enhanced the activity further.

Biocontrol Potential of Streptomyces hydrogenans Strain DH16 toward Alternaria brassicicola to Control Damping Off and Black Leaf Spot of Raphanus sativus

Biocontrol agents and their bioactive metabolites provide one of the best alternatives to decrease the use of chemical pesticides. In light of this, the present investigation reports the biocontrol potential of Streptomyces hydrogenans DH16 and its metabolites towards Alternaria brassicicola, causal agent of black leaf spot and damping off of seedlings of crucifers. In vitro antibiosis of strain against pathogen revealed complete suppression of mycelial growth of pathogen, grown in potato dextrose broth supplemented with culture supernatant (20% v/v) of S. hydrogenans DH16. Microscopic examination of the fungal growth showed severe morphological abnormalities in the mycelium caused by antifungal metabolites. In vivo studies showed the efficacy of streptomycete cells and culture supernatant as seed dressings to control damping off of Raphanus sativus seedlings. Treatment of pathogen infested seeds with culture supernatant (10%) and streptomycete cells significantly improved seed germination (75–80%) and vigor index (1167–1538). Furthermore, potential of cells and culture supernatant as foliar treatment to control black leaf spot was also evaluated. Clearly visible symptoms of disease were observed in the control plants with 66.81% disease incidence and retarded growth of root system. However, disease incidence reduced to 6.78 and 1.47% in plants treated with antagonist and its metabolites, respectively. Additionally, treatment of seeds and plants with streptomycete stimulated various growth traits of plants over uninoculated control plants in the absence of pathogen challenge. These results indicate that S. hydrogenans and its culture metabolites can be developed as biofungicides as seed dressings to control seed borne pathogens, and as sprays to control black leaf spot of crucifers.