INTERGENUS PROTOPLAST FUSION STUDIES BETWEEN MARINE STREPTOMYCES SP. ANS4 AND ESCHERICHIA COLI

Objective: To study the intergenus protoplast fusion bioactive marine Streptomyces sp ANS4 with the Escherichia coli for the strain improvement. Methods: Protoplast of both the organisms was prepared by lysozyme treatment and fused using polyethylene glycol (PEG). Fusant colonies were regenerated using regeneration medium where all the regenerated colonies are powdery in consistency. Microscopic, cultural, physiological, enzymatic and antibiotic susceptibility characteristics of one fusant culture and both the parent cultures are studied. Growth curve and bioactive metabolite production by both the parent and fusant cultures was also studied. Results: oval shaped protoplasts of both the parent cultures are clearly seen under the phase contrast microscope. All the colonies developed after protoplast fusion on regeneration medium looked like one of the parent culture Streptomyces sp ANS4. One among the 10 antagonistic colonies selected from the regeneration medium was expressed the characteristics of either or both of the parental cultures and certain new characteristics. The results of growth curve study showed that the fusant culture possess 10 hours reduction in attaining stationary phase compared to parent Streptomyces sp ANS4. The antibiotic activity of fusant culture was also higher than the parent cultures. Conclusion: The intergenus protoplast fusion between the Streptomyces sp ANS4 and the E.coli strain results in the reduction in time for secondary metabolite production and also increase in antimicrobial activity. This suggested that the potential bioactive compounds may be obtained in increased quantity from the fusant culture with lesser fermentation period

ANTIFUNGAL ACTIVITY OF ACTINOBACTERIA WITH A POTENTIAL TO INHIBIT RICE BLAST FUNGUS MAGNAPORTHE ORYZAE (ANAMORPH PYRICULARIA ORYZAE)

Objective: The aims of the present study were to screen the actinobacteria with high potential ability to produce secondary metabolites that have inhibitory activity against plant pathogenic fungi, Magnaporthe oryzae. Production of secondary metabolites was analysis by thin-layer chromatography and bioautography assay. Methods: Screening and selection of potential Streptomyces sp. morphological, cultural, physiological, and biochemical characterization of the screened isolate was carried out. Antifungal compound was confirmed by bioautography assay. Results: Bioautography method use in this study was found to be antifungal fraction from the crude extract. Antifungal secondary metabolites can be readily located on the plates by observing clear zones where active compounds inhibit fungal growth. Conclusion: The bioautography assay shows that this isolates can produce antifungal compound. Therefore, this isolate proves to be a promising microbe which can be further studied for its applications a biocontrol agent against rice blast fungi

Screening, production, and characterization of biologically active secondary metabolite(s) from marine Streptomyces sp. PA9 for antimicrobial, antioxidant, and mosquito larvicidal activity

1319-1326Bioprospecting of actinobacteria from understudied ecosystems is a promising source for extracting novel bioactive metabolites. A study was undertaken to characterize and analyze the bio-efficacy of actinobacterial extract for antimicrobial, larvicidal, and antioxidant activities. Seven morphologically different actinobacterial cultures isolated from mangrove rhizosphere sediment near Parangipettai, South India, were tested for antimicrobial activity. Bioactive metabolites from one potential strain PA9 were produced by submerged fermentation. The selected Streptomyces sp. PA9 was subjected to the production of crude extract for antimicrobial, larvicidal, and antioxidant activity. The actinobacterial compound was characterized by Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC), and gas chromatography–mass spectrometry (GC–MS). The PA9 actinobacterial crude extract showed best antimicrobial activity against clinical bacteria, Salmonella typhi (21.6 ± 0.88 mm) and fungi, Candida albicans (26.6 ± 0.88 mm). The PA9 extract showed significant larvicidal activity against Culex quinquefasciatus with LC50=173.21 µg/ml and r2=0.841. The PA9 extract also exhibited antioxidant activity from DPPH (72%) and nitric oxide free radicals (85%). The characterization of the PA9 extract by FTIR analysis showed the presence of possible functional groups. Active compounds were isolated by HPLC and GC–MS with major and minor peaks observed on the basis of retention time. The bio-efficacy of PA9 has warranted further studies to develop a baseline for the drug development

OPTIMIZATION OF MEDIUM COMPONENTS FOR ANTIBACTERIAL METABOLITE PRODUCTION FROM MARINE STREPTOMYCES SP. PUA2 USING RESPONSE SURFACE METHODOLOGY

Objective: The present study is an attempt to optimize the fermentation conditions for the antibacterial compound production from a newly isolated marine Streptomyces strain PUA2 by adopting response surface methodology as the statistical tool. Methods: Prior to using the Response Surface Methodology, Plackett Burmann (PB) design was used to explore the effect of variables on the antibacterial compound production. In PB method, high and low values were assigned for the eight variables viz., glucose, glycerol, soybean meal, manganese chloride, calcium carbonate, peptone and pH. Calcium carbonate and peptone were used as dummy variables. Based on the results of combined effects glycerol, soybean meal, manganese chloride and pH were investigated by 24 full-factorial central composite design. Results: The results of PB method showed the significant effect of glycerol, soybean meal, manganese chloride and pH on the antibacterial compound production. The results of ANOVA and regression of second order model showed that the linear effects of glycerol and manganese chloride and cross products effects of manganese chloride and pH were more significant. All the critical variables having greatest effect on the production of antibacterial compound from marine Streptomyces species PUA2. Optimization of process parameters resulted in increase in antibacterial activity from 7 mm to 14 mm. Conclusion: The factors optimized in the present study were useful for the increased production of antibacterial metabolite from Streptomyces sp PUA2. The result confirms the feasibility of medium optimization to improve antibiotic production

Bioactive potential of selected actinobacterial strains against Mycobacterium tuberculosis and other clinical pathogens

1307-1311Marine actinobacteria produces diverse array of metabolites with novel chemical structures with potential bioactivities. Exploring the understudied ecosystems may increase the chance of getting novel actinobacteria and new metabolites.The present study explores the bioactive potential of actinobacteria isolated from the marine ecosystem of Andaman and Nicobar Islands, Bay of Bengal, against Mycobacterium tuberculosis and other clinical pathogens. The crude extracts from 15 marine actinobacterial strains were produced through agar surface fermentation using YEME agar and extracted using ethyl acetate. The crude extracts were tested against the standard strain M. tuberculosis H37Rv, clinical drug sensitive M. tuberculosis, and MDR M. tuberculosis strains by luciferase reporter phage (LRP) assay at 500 µg/ml concentration. The anti-microbial activity against other clinical pathogens, namely, Staphylococcus aureus, Escherichia coli, Salmonella paratyphi, Klebsiellapneumoniae, Pseudomonas aeruginosa, Candida albicans, and Cryptococcusneoformans and non-tubercular mycobacteria, M. smegmatis was studied by agar plug method. Among the 15 extracts that were tested for anti-tubercular activity, the crude ethyl acetate extract of the 14 actinobacterial strains showed anti-tubercular activity against at least one of the three M. tuberculosis strains. Exceptionally, the ethyl acetate extract of strain SACC 168 inhibited all three M. tuberculosis strains tested. In anti-microbial screening, the crude extracts of eight strains showed anti-microbial activity including six strains, which were active against the non-tuberculous mycobacteria. Further purification and characterization of the active molecule from the potential extracts will pave way for the potential natural product candidate for tuberculosis and other microbial infections

Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems

Actinomycetes from less explored ecosystems were screened for antibacterial and antimycobacterial activity. Crude bioactive compounds were produced by growing these strains by shake flask fermentation using soybean meal medium. Culture supernatant and mycelia were extracted with ethyl acetate and methanol, respectively. Antibacterial activity of crude extracts was tested by disc diffusion method against gram positive and gram negative bacteria. Actinomycete strains D10, D5, NEK5, ANS2, M104 and R2 showed prominent activity. Culture filtrates and crude extracts were tested against standard strain Mycobacterium tuberculosis H37Rv and drug sensitive and drug resistant clinical isolates of M. tuberculosis by luciferase reporter phage (LRP) assay. Considerable variation was observed in antimycobacterial activity between actinomycete culture filtrates and solvent extracts. Actinomycete strains viz., D10, D5 (desert), CSA14 (forest), CA33 (alkaline soil), NEK5 (Neem plant), MSU,ANS2, R2 and M104 (marine) screened in the present study were found to be highly potent showing good antibacterial and antimycobacterial activity. Five of them such as A3, CSA1, EE9, ANS5 and R9 were exclusively active against M. tuberculosis. Secretary products of actinomycetes of rare ecosystems are meant to antagonize organisms in their respective environments. These are likely to be novel antimycobacterial compounds as they unknown to human pathogens

INFLUENCE OF BIOFERTILIZERS AND IRRIGATION SYSTEMS FOR THE GROWTH AND YIELD OF MULBERRY PLANTS

ABSTRACT : The present study emphasizes the application of various irrigation systems and the effects of chemical and biofertilizers on rhizosphere microorganisms of mulberry plants. The highest total bacterial population was 580x10 3 cfu/gm and fungal population was 39x10 4 cfu/gm. Among the bacterial and fungal population Azotobacter and Aspergillus spp were found predominantly in drip irrigation with green manure (T4) method. Among the four irrigation systems T4 method effectively increases the microbial population in the rhizosphere of mulberry plants. The initial pH of the mulberry garden soil was 8.2 but after the application of chemical (control) and biofertilizer (test), the chemical fertilizer treated soil retained the pH as 8.2 and biofertilizer treated soil pH was found to be 6.8. The rhizosphere microbial population (both bacteria and fungi) was least in the control sample (chemical fertilizer) but in test (biofertilizer) sample, the bacterial and fungal population was gradually increased from 1 st day to 60 th day. Typically, phosphate solubilzing bacteria and Aspergillus spp were present in the biofertilizer treated soil and effectively increasing the growth of mulberry plant's height, branches and leaves

Comparison of established and emerging biodosimetry assays

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3-0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5-4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools