Biotechnology

A novel immunoassay format employing direct coating of small molecular hapten on microtiter plates is reported for the detection of atrazine and 2,4-dichlorophenoxyacetic (2,4-D). In this assay, the polystyrene surface of microtiter plates was first treated with an acid to generate-NO 2 groups on the surface. Acid treated plates were further treated with 3-aminoprpyltriethoxysilane (APTES) to functionalize the plate surface with amino groups for covalent linkage to small molecular hapten with carboxyl groups. The modified plates showed significantly high antibody binding in comparison to plates coated with hapten-carrier protein conjugates and presented excellent stability as a function of the buffer pH and reaction time. The developed assay employing direct hapten coated plates and using affinity purified atrazine and 2,4-D antibodies demonstrated very high sensitivity, IC 50 values for atrazine and 2,4-D equal to 0.8 ng mL −1 and 7 ng mL −1 , respectively. The assay could detect atrazine and 2,4-D levels in standard water samples even at a very low concentration upto 0.02 and 0.7 ng mL −1 respectively in the optimum working range between 0.01 and 1000 ng mL −1 with good signal reproducibility (p values: 0.091 and 0.224 for atrazine and 2,4-D, respectively). The developed immunoassay format could be used as convenient quantitative tool for the sensitive screening of pesticides in samples.

The chemically sensitized ormosil-modified electrodes were made by encapsulating potassium ferricyanide together with organic-ionophore (dibenzo-18-crown-6/Nafion) within ormosil films. The modified electrodes were studied in details from following angles; (1) the effect of variation in potassium ferricyanide concentration in ormosil films; (2) the effect of variation in dibenzo-18-crown-6 concentration in ormosil films; (3) effect of pH on the electrochemistry of ormosil-modified electrodes, and (4) variation in the microstructure of ormosil's films as a function of chemical sensitizers based on atomic force microscopy. Another remarkable contribution of such films based on the bilayer configuration was investigated and the results justified further enhancement in the selectivity for electrochemical sensing of peroxide. Two approaches of bilayer configuration were adopted based on the absence and the presence of chemical sensitizers (Nafion/crown ether) in the upper layer. Excellent results of peroxide sensing were recorded with remarkable finding as given below: (1) introduction of bilayer configuration reduced the sensing of interfering analytes like ascorbic acid, (2) the presence of crown ether in the upper layer caused enhancement in peroxide sensing, (3) the presence of crown ether in the upper layer and Prussian blue in the lower layer increased the detection limit of peroxide sensing to 0.1 M as compared to that of 0.5 M recorded in earlier publication, (4) the presence of the bilayer resulted in an increase in the linear range of peroxide sensing, (5) the single layer Prussian blue modified electrode showed electrocatalytic oxidation of ascorbic acid whereas the same with the bilayer containing crown ether reduced the electrochemical sensing of ascorbic acid, followed by an increase in peroxide sensing, and (6) the kinetics of peroxide reduction on the bilayer ormosil-modified electrode was reduced as compared to that on the single layer modified-electrode. The results based on cyclic voltammetry showed a linear increase in cathodic and anodic peak currents with increasing the concentration of ferricyanide within the ormosil film. The results on the effect of pH suggested that lowering of pH caused faster reduction as well as oxidation of peroxide whereas the reverse was recorded with increasing the pH. An increase in dibenzo-18-crown-6 concentration resulted higher oxidation current of peroxide, followed by a decrease in reduction of the same. The results of cyclic voltammery and amperometric measurements were also reported in this communication. The calibration curve of peroxide sensing using the bilayer ormosil-modified electrodes was reported.

Several dyes including bromophenol blue are used as tracking dye in gel electrophoresis. The stability of betacyanin extracted from spinach vine fruit (Basella rubra L.) was studied in relation to degradative factors such as pH ranging from 2 to 12 and at 100ºC. Betacyanin pigment was found to be stable at pH range 3-8 while the stability of the pigment at high temperature was found to be moderate. The effectiveness of betacyanin as a tracking dye was checked along with the bromophenol blue for agarose gel electrophoresis. The present study revealed that betacyanin from spinach fruit can be a potential alternative to conventional dye like bromophenol blue used in the loading dye preparation for agarose gel electrophoresis.

The chemically sensitized ormosil-modified electrodes were made by encapsulating potassium ferricyanide together with organic-ionophore (dibenzo-18-crown-6/Nafion) within ormosil films. The modified electrodes were studied in details from following angles; (1) the effect of variation in potassium ferricyanide concentration in ormosil films; (2) the effect of variation in dibenzo-18-crown-6 concentration in ormosil films; (3) effect of pH on the electrochemistry of ormosil-modified electrodes, and (4) variation in the microstructure of ormosil's films as a function of chemical sensitizers based on atomic force microscopy. Another remarkable contribution of such films based on the bilayer configuration was investigated and the results justified further enhancement in the selectivity for electrochemical sensing of peroxide. Two approaches of bilayer configuration were adopted based on the absence and the presence of chemical sensitizers (Nafion/crown ether) in the upper layer. Excellent results of peroxide sensing were recorded with remarkable finding as given below: (1) introduction of bilayer configuration reduced the sensing of interfering analytes like ascorbic acid, (2) the presence of crown ether in the upper layer caused enhancement in peroxide sensing, (3) the presence of crown ether in the upper layer and Prussian blue in the lower layer increased the detection limit of peroxide sensing to 0.1 M as compared to that of 0.5 M recorded in earlier publication, (4) the presence of the bilayer resulted in an increase in the linear range of peroxide sensing, (5) the single layer Prussian blue modified electrode showed electrocatalytic oxidation of ascorbic acid whereas the same with the bilayer containing crown ether reduced the electrochemical sensing of ascorbic acid, followed by an increase in peroxide sensing, and (6) the kinetics of peroxide reduction on the bilayer ormosil-modified electrode was reduced as compared to that on the single layer modified-electrode. The results based on cyclic voltammetry showed a linear increase in cathodic and anodic peak currents with increasing the concentration of ferricyanide within the ormosil film. The results on the effect of pH suggested that lowering of pH caused faster reduction as well as oxidation of peroxide whereas the reverse was recorded with increasing the pH. An increase in dibenzo-18-crown-6 concentration resulted higher oxidation current of peroxide, followed by a decrease in reduction of the same. The results of cyclic voltammery and amperometric measurements were also reported in this communication. The calibration curve of peroxide sensing using the bilayer ormosil-modified electrodes was reported.

In this paper, we report the endophytic microbial diversity of cultivated and wild Oryza sativa plants including their functional traits related to multiple traits that promote plant growth and development. Around 255 bacteria were isolated out of which 70 isolates were selected for further studies based on their morphological differences. The isolates were characterized both at biochemical and at the molecular level by 16s rRNA gene sequencing. Based on 16S rRNA gene sequencing the isolates were categorized into three major phyla, viz, Firmicutes (57.1 %), Actinobacteria (20.0 %) and Proteobacteria (22.8 %). Firmicutes was the dominant group of bacteria of which the most abundant genus was Bacillus. The isolates were further screened in vitro for plant growth promoting activities which revealed a hitherto unreported endophytic bacterial isolate, Microbacteriaceae bacterium RS01 11 as the highest secretor of a phytohormone, IAA (28.39 ± 1.39 μg/ml) and GA (67.23 ± 1.83 μg/ml). Bacil...

Fungi can be broadly classified into three different forms viz., yeast, moulds and macrofungi or mushrooms. Macrofungi are large, conspicuous sporebearing structures and have fleshy, tough umbrella like sporophores that bear holobasidia on the surface of gills or lamellae that hang down from the cap belonging to basidiomycetes and ascomycetes (few under Zygomycota) (Alexopoulus et al., 2000). Macrofungi are found in varied types of habitats, depending on the composition of tree species and other substrates. The distribution of macrofungal species is low in hot and dry seasons while they are abundant in spring and autumn due to favourable climate and abundance of flora during that time (Pilz et al., 2001). Similar to yeast and moulds, macrofungi also possess immense biological and economic impact since they serve as food, medicine, biocontrol agents and produce of bioactive compounds such as anti-oxidant,

Silver nanoparticles (AgNPs) have seen a recent spurt of use in varied fields of science. In this paper, we showed a novel application of AgNP as a promising microbial cell-lysis agent for genomic DNA isolation. We utilized chemically synthesized AgNPs for lysing bacterial cells to isolate their genomic DNA. The AgNPs efficiently lysed bacterial cells to yield good quality DNA that could be subsequently used for several molecular biology works.

Association of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ ...

RNA-seq analysis of B. megaterium exposed to pH 7.0 and pH 4.5 showed differential expression of 207 genes related to several processes. Among the 207 genes, 11 genes displayed increased transcription exclusively in pH 4.5. Exposure to pH 4.5 induced the expression of genes related to maintenance of cell integrity, pH homeostasis, alternative energy generation and modification of metabolic processes. Metabolic processes like pentose phosphate pathway, fatty acid biosynthesis, cysteine and methionine metabolism and synthesis of arginine and proline were remodeled during acid stress. Genes associated with oxidative stress and osmotic stress were up-regulated at pH 4.5 indicating a link between acid stress and other stresses. Acid stress also induced expression of genes that encoded general stressresponsive proteins as well as several hypothetical proteins. Our study indicates that a network of genes aid B. megaterium G18 to adapt and survive in acid stress condition. Bacteria have innate ability to survive and grow in several stress conditions including extreme ecological niches. Acidic stress condition specifically, soil acidic condition has significant relevance and ramification in agriculture, food, and human health. The importance of acid stress has propelled several studies in unraveling the molecular mechanisms of acid tolerance in bacteria especially the pathogenic enteric bacteria and those involved with food and beverages. Neutralophilic bacteria adopt a number of mechanisms in maintaining pH homeostasis including no growth strategy as adopted by Escherichia coli and Salmonella spp., to survive during exposure to pH values that are outside their growth range 1,2. Survival and growth under stress involve changes in cell structure, metabolism, and transport patterns (F1F0-ATPase, ABC transporter). The changes in membrane fatty acid profiles in response to dropping in pH point out the significant role of the cell membrane in acid tolerance 3. The role of amino acid decarboxylases in maintaining bacterial pH homeostasis has also been reported 4. Examples of these systems include lysine, arginine, and glutamate decarboxylases that combine a proton with internalized amino acid (lysine, arginine, or glutamate) and exchange the product for another amino acid 5. Protection or repair of macromolecules such as DNA and proteins as essential acid resistance mechanism has also been described by several researchers 3,6,7. Generation of alkali more specifically, ammonia using the urease and arginine deiminase (ADI) pathways is yet another mechanism adopted by bacteria to tide over acid stress. The acid resistance in gram-positive bacteria is also affected by cell density, as higher cell densities aid in cell to cell communication and formation of biofilm 8. B. megaterium is a Gram-positive, aerobic spore-forming neutralophilic bacterium found in diverse habitats but commonly regarded as a soil bacterium. Its ability to utilize different carbon sources and grow at a wide temperature range (3 °C to 45 °C) makes it an ideal industrial organism 9,10. B. megaterium possesses plant growth promoting activity including biocontrol ability against plant pathogens 11,12. Over the past decade, several studies relating to the genetics of B. megaterium have been reported and a number of stains have been developed and characterized for several features including auxotrophy, recombination, division, sporulation, germination,

Background: The use of chemical fungicides against fungal pathogens adversely affects soil and plant health thereby resulting in overall environmental hazards. Therefore, biological source for obtaining antifungal agents is considered as an environment-friendly alternative for controlling fungal pathogens. Results: In this study, seven endophytic bacteria were isolated from sugarcane leaves and screened for its antifungal activity against 10 fungal isolates belonging to the genera Alternaria, Cochliobolus, Curvularia, Fusarium, Neodeightonia, Phomopsis and Saccharicola isolated from diseased leaves of sugarcane. Among the seven bacterial isolates, SCB-1 showed potent antagonistic activity against the tested fungi. Based on the phenotypic data, Fatty Acid Methyl Esters (FAME) and 16S rRNA gene sequence analysis, the isolate SCB-1 was identified as Bacillus subtilis. The bacterial isolate was screened negative for chitinase production; however, chloroform and methanol extracts of the bacterial culture caused significant inhibition in the growth of the fungal isolates on semisolid media. Volatile component assay showed highest inhibitory activity against Saccharicola bicolor (SC1.4). A PCR based study detected the presence of the genes involved in biosynthesis of surfactin, bacillaene, difficidin, macrolactins and fengycin. Mass spectrometric analysis of the bacterial extract detected the presence of antifungal lipopeptide surfactin, but other metabolites were not detected. The biocontrol activity of the bacterial isolate was established when bacterial pretreated mung bean seeds were able to resist Fusarium infection, however, the untreated seeds failed to germinate. Conclusion: The antifungal potential of isolate Bacillus subtilis SCB-1 was established against taxonomically diverse fungal pathogens including the genera Saccharicola, Cochliobolus, Alternaria and Fusarium. The potent antifungal compound surfactin as well as volatiles produced by the bacterial isolate could be responsible for its bio-control activity against fungal infections.

In nature, species interacts/competes with one other within their surrounding for food and space and the type of interactions are unique to each species. The interacting partners secrete different metabolites, which may have high importance in human welfare. Fungal-fungal interactions are complex mechanisms that need better understanding. Here, 14 fungal isolates were facilitated in 105 possible combinations to interact on potato dextrose agar. Morphologically, no changes were observed when the same fungal isolates were allowed to interact within them. However, 10 interactions between different fungal isolates showed mutual replacement with each fungus; capturing territory from the other. Contrastingly, 35 interactions resulted into complete replacement as one of the fungi was inhibited by rapid growth of the other fungus. In 46 interactions, formation of barrage was observed leading to deadlock type of interaction wherein both fungi have restricted growth. To study in details about the barrage formation, two fungal interactions were taken (i) T. coccinea vs. L. lactinea and (ii) T. coccinea vs. T. versicolor. Microscopic changes in the hyphal growth during interaction were observed. There was significant increase in the enzymatic activities including cellulase, xylanase and chitinase during in-vitro fungal-fungal interaction, suggesting the importance of such interactions for commercial enzyme production.

Fungal-fungal interaction often leads to the change in metabolite profile of both the interacting fungus which may have potential implication in industry or agriculture. In the present study, we performed two sets of fungal-fungal interaction—Trametes coccinea (F3) with Leiotrametes lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) to understand the changes in the metabolite profile during the interaction process and how this process impacts the hyphal/mycelial morphology of the participating fungi. The metabolites produced during interaction of T. coccinea (F3) with L. lactinea (F9) and T. coccinea (F3) with T. versicolor (F1) was analysed through liquid chromatography coupled to mass spectroscopy (LC-MS). Most of the metabolites secreted or produced during interaction are associated with defensive response. Further, visualization with scanning electron microscopy revealed that interaction between the tested fungi led to the changes in the hyphal morphology. The bipartite ...

Three groups of rhizobacteria were isolated using three selective culture media from the rhizosphere of rice grown at fifteen locations in Assam. Four bacteria in each group were selected for taxonomic identification, characterization and also for screening the superior isolates to promote rice growth. The isolates of phosphate-solubilizing and fluorescent bacterial groups were found to be eight different species. The three isolates of azospirilla group were identified as Azospirillum brasilense and the fourth isolate was identified as Azospirillum amazonense. RAPD analysis of the Azospirillum isolates indicated that azospirilla were four distinct genotypes. These bacterial strains showed differences in growth pattern, IAA production level (2.0-21.6 μg/ml of culture supernatant), antibiotic resistance profile and nitrogenase activity (0.33-0.75 nmol C 2 H 4 h - 1 100 - c c root volume) in the inoculated roots. A. amazonense A10 (MTCC 4716), Bacillus pantothenticus P4 (MTCC 4695) and...

Several dyes including bromophenol blue are used as tracking dye in gel electrophoresis.  The stability of betacyanin extracted from spinach vine fruit ( Basella rubra L.) was studied in relation to degradative factors such as pH ranging from 2 to 12 and at 100oC. Betacyanin pigment was found to be stable at pH range 3-8 while the stability of the pigment at high temperature was found to be moderate. The effectiveness of betacyanin as a tracking dye was checked along with the bromophenol blue for agarose gel electrophoresis. The present study revealed that betacyanin from spinach fruit can be a  potential alternative to conventional dye like bromophenol blue used in the loading dye preparation for agarose gel electrophoresis.

Increasing strains with resistance to antibiotics and the resulting failure to treat infectious diseases is a major challenge in the medical and health fields. Drug resistance, especially in recent decades, had led to a search for different approaches and methods for finding new compounds against bacteria and fungi. Scientists have accepted the idea that nanotechnology is a novel area of science that combines biology, chemistry, and physics (Rezaei-Zarchi et al., 2012; Demir et al., 2014). Nanoparticles have dimensions of 100 nm or less. They have gained remarkable attention because of their unusual properties and the various applications they are suited for, when compared to their bulkier counterparts (Kato, 2011; Metzler et al., 2012). These features have helped spread the use of nanomaterials at a faster rate day by day. They can be used in the fight against germs, diagnosis and cure of diseases, water and air purification, food production, cosmetics, and clothing (Aitken et al., 2006). Silver is the most commonly used engineering nanomaterial in all consumer products (Akinoglu et al., 2014). Silver and copper have long been known to display a strong toxicity against a wide range of microorganisms. Thus, silver-and copper-based compounds have been used intensively in medicine to treat burns and infections. These antimicrobial effects have been studied by many researchers (Mirzajani et al., 2011; Yasa et al., 2012). The antimicrobial potential of these metals led to their use in health-related products (Selvaraj et al., 2011). Research showed the strong antibacterial effect of silver nanoparticles on both gram-positive and gram-negative bacteria, mainly multidrug-resistant strains (Ashrafi et al., 2013; Sangiliyandi et al., 2014). It can be used as an antifungal agent, and this effect has been intensively studied (Jo et al., 2009). Antibacterial properties of copper nanoparticles (CuNPs) have been compared with triclosan, and a strong antibacterial effect has been reported for both (Cubillo et al., 2006). The mechanisms causing nanoparticles to act on bacteria have not yet been fully clarified. However, the four most common theories proposed are: (1) the uptake of free silver ions leads to a disturbance in ATP production and DNA replication. (2) Silver nanoparticles interact

Elephant apple fruit, though bestowed with multifarious medicinal properties and available in many Asian countries, has no known commercial processed product due to its tough fibrous calyces, highly astringent juice and brown discolouration. In this study, the juice was blended with concentrated whey in different proportions (v/v) for assessing organoleptic acceptability. Principal Component Analysis of the sensory scores and subsequent evaluation of blends’ antioxidant activity revealed the superiority of 1:1 (v/v) admixture over other counterparts. Further, study focussed on optimization of process variables (maltodextrin concentration and inlet temperature of spray dryer) for recuperating beverage powder from the juice-whey blend (1:1 v/v). Developed powders presented different surface morphologies (wrinkled/shrunk/ellipsoidal/fused particles having 2-30 µm diameters), and were organoleptically favourable with good yield (63.33-88.33%), dispersibility (33.40-56.57%), moderate ant...