Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207) blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira) culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21%) out of 100 culture positive blood samples, three (2.8%) out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001). A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001) signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin

Plant growth promoting rhizobia: Challenges and opportunities

Modern agriculture faces challenges, such as loss of soil fertility, fluctuating climatic factors and increasing pathogen and pest attacks. Sustainability and environmental safety of agricultural production relies on eco-friendly approaches like biofertilizers, biopesticides and crop residue return. The multiplicity of beneficial effects of microbial inoculants, particularly plant growth promoters (PGP), emphasizes the need for further strengthening the research and their use in modern agriculture. PGP inhabit the rhizosphere for nutrients from plant root exudates. By reaction, they help in (1) increased plant growth through soil nutrient enrichment by nitrogen fixation, phosphate solubilization, siderophore production and phytohormones production (2) increased plant protection by influencing cellulase, protease, lipase and β-1,3 glucanase productions and enhance plant defense by triggering induced systemic resistance through lipopolysaccharides, flagella, homoserine lactones, acetoin and butanediol against pests and pathogens. In addition, the PGP microbes contain useful variation for tolerating abiotic stresses like extremes of temperature, pH, salinity and drought; heavy metal and pesticide pollution. Seeking such tolerant PGP microbes is expected to offer enhanced plant growth and yield even under a combination of stresses. This review summarizes the PGP related research and its benefits, and highlights the benefits of PGP rhizobia belonging to the family Rhizobiaceae, Phyllobacteriaceae and Bradyrhizobiaceae

New approximation algorithms for solving graph coloring problem – An experimental approach

Some of the real world applications require the solution to graph coloring problem, an NP-hard combinatorial optimization problem. χ(G), the chromatic number of a graph G, the minimum number of colors required to color the vertex set V(G) with adjacent vertices assigned with different color can also be obtained using evolutionary methods. This paper exhibits two new approximation methods of solving graph coloring based on μ½(G), the median of the degrees V(G). In the first method, a heuristic procedure is designed to color V(G) which works in two stages. In the first stage, to minimize the conflicting edges, the vertices of V(G) whose degrees are ≥μ½(G) are colored. Then the remaining vertices are colored through a heuristic procedure. The second method is implemented using divide & conquer strategy. These new approximation algorithms are exhibited on some of the small, intermediate and large benchmark graphs and the results are compared. The proposed algorithms significantly reduce the computational complexity in obtaining the near optimal solution and also the results are found to be better than the existing approaches

Exploring plant growth-promotion actinomycetes from vermicompost and rhizosphere soil for yield enhancement in chickpea

Abstract The main objective of the present study was to isolate and characterize actinomycetes for their plant growth-promotion in chickpea. A total of 89 actinomycetes were screened for their antagonism against fungal pathogens of chickpea by dual culture and metabolite production assays. Four most promising actinomycetes were evaluated for their physiological and plant growth-promotion properties under in vitro and in vivo conditions. All the isolates exhibited good growth at temperatures from 20 °C to 40 °C, pH range of 7–11 and NaCl concentrations up to 8%. These were also found highly tolerant to Bavistin, slightly tolerant to Thiram and Captan (except VAI-7 and VAI-40) but susceptible to Benlate and Ridomil at field application levels and were found to produce siderophore, cellulase, lipase, protease, chitinase (except VAI-40), hydrocyanic acid (except VAI-7 and VAI-40), indole acetic acid and β-1,3-glucanase. When the four actinomycetes were evaluated for their plant growth-promotion properties under field conditions on chickpea, all exhibited increase in nodule number, shoot weight and yield. The actinomycetes treated plots enhanced total N, available P and organic C over the un-inoculated control. The scanning electron microscope studies exhibited extensive colonization by actinomycetes on the root surface of chickpea. The expression profiles for indole acetic acid, siderophore and β-1,3-glucanase genes exhibited up-regulation for all three traits and in all four isolates. The actinomycetes were identified as Streptomyces but different species in the 16S rDNA analysis. It was concluded that the selected actinomycetes have good plant growth-promotion and biocontrol potentials on chickpea

Radiological characterization of the concrete biological shield of the APSARA reactor

The first Indian research reactor, APSARA, was utilized for various R&D programmes from 1956 until its shutdown in 2009. The biological shield of the reactor developed residual activity due to neutron irradiation during the operation of the reactor. Dose rate mapping and in-situ gamma spectrometry of the concrete structures of the reactor pool were carried out. Representative concrete samples collected from various locations were subjected to high-resolution gamma spectrometry analysis. 60Co and 152Eu were found to be the dominant gamma-emitting radionuclides in most of the locations. 133Ba was also found in some of the concrete structures. The separation of 3H from concrete was achieved using an acid digestion method and beta activity measured using liquid scintillation counting. The depth profile of radionuclide specific activity in the concrete wall of the shielding corner was also studied. Specific activities of the radionuclides were found to decrease exponentially with depth inside the concrete walls. This study would be helpful in bulk waste management during the decommissioning of the reactor

Evaluation of Streptomyces sp. obtained from herbal vermicompost for broad spectrum of plant growth-promoting activities in chickpea

Five strains of Streptomyces sp. (CAI-24, CAI-121, CAI-127, KAI-32, and KAI-90; demonstrated previously to have potential for control of Fusarium wilt disease in chickpea and plant growth promotion [PGP] in rice) were evaluated for their PGP capabilities in chickpea in the 2012–2013 and 2013–2014 post-rainy seasons. The plots inoculated with Streptomyces sp. significantly enhanced number of nodule, nodule weight, root weight, and shoot weight at 30 days after sowing (DAS) and number of pod, pod weight, leaf area, leaf weight, and stem weight at 60 DAS in both seasons over the un-inoculated control plots. At chickpea crop maturity, all of the Streptomyces strains significantly enhanced stover yield, grain yield, and total dry matter in both seasons over the un-inoculated control. In the rhizosphere, the Streptomyces strains also significantly enhanced soil biological and mineral nutrient activities including microbial biomass carbon, dehydrogenase activity, total nitrogen, available phosphorous, and organic carbon in both seasons over the un-inoculated control. All of the five strains were found superior in terms of nodule formation, root and shoot development, and crop productivity; however, KAI-xx had little edge over the other five strains. Scanning electron microscopy (SEM) analysis had revealed the success of colonization by the strains of Streptomyces sp. of the chickpea roots. Quantitative real-time PCR (qRT-PCR) analysis of selected PGP genes revealed overall upregulation of β-1,3-glucanase, indole-3-acetic acid, and siderophore genes in the Streptomyces species studied. This investigation further confirms the broad spectrum of PGP activities by the selected Streptomyces sp

Molecular basis of root nodule symbiosis between Bradyrhizobium and ‘Crack-Entry’ legume groundnut (Arachis hypogaea L.)

Nitrogen is one of the essential plant nutrients and a major factor limiting crop productivity. To meet the requirements of sustainable agriculture, there is a need to maximize biological nitrogen fixation in different crop species. Legumes are able to establish root nodule symbiosis (RNS) with nitrogen-fixing soil bacteria which are collectively called rhizobia. This mutualistic association is highly specific, and each rhizobia species/strain interacts with only a specific group of legumes, and vice versa. Nodulation involves multiple phases of interactions ranging from initial bacterial attachment and infection establishment to late nodule development, characterized by a complex molecular signalling between plants and rhizobia. Characteristically, legumes like groundnut display a bacterial invasion strategy popularly known as “crack-entry’’ mechanism, which is reported approximately in 25% of all legumes. This article accommodates critical discussions on the bacterial infection mode, dynamics of nodulation, components of symbiotic signalling pathway, and also the effects of abiotic stresses and phytohormone homeostasis related to the root nodule symbiosis of groundnut and Bradyrhizobium. These parameters can help to understand how groundnut RNS is programmed to recognize and establish symbiotic relationships with rhizobia, adjusting gene expression in response to various regulations. This review further attempts to emphasize the current understanding of advancements regarding RNS research in the groundnut and speculates on prospective improvement possibilities in addition to ways for expanding it to other crops towards achieving sustainable agriculture and overcoming environmental challenges

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Not AvailableTwo commercially important mussel species are recorded from the Indian coast: green mussel Perna viridis (Linnaeus, 1758) and brown mussel P. indica (Kuriakose and Nair, 1976). Apart from this, a third type referred to as parrot mussel, which has shell shape of brown mussel, but with green shell colouration and suspected to be the hybrid of the above two species has also been reported from Kollam coast of Kerala, where both the species co-occur. In the present work, genetic identity of parrot and sympatric mussel species was determined using protein and genomic DNA markers. Protein markers viz. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and allozymes and the genomic DNA marker Random Amplified Polymorphic DNA (RAPD) were used for determining genetic identity of the three mussel groups. The green and brown mussels could be clearly differentiated using SDS PAGE. The parrot mussel protein pattern was similar to that of brown mussel, except for an additional band of molecular weight 48.7 Kda which is unique to brown mussel. Genus specific protein bands for Perna viz. 66 Kda, 43 Kda and 14.3 Kda, were detected in this study. Allozyme electrophoresis also followed a similar pattern. Of the 10 allozyme loci studied, seven revealed speciesspecific diagnostic differences between P.viridis and P.indica. They were AAT-1* (Aspartate Amino Transferase-1*), AAT-2*, ME (Malic Enzyme)*, PGM-2*(Phospo Gluco Mutase-2*), EST-1* (Esterase- 1*), EST-2*, IcDH* (Isocitrate Dehydrogenase)*. Parrot mussel shared all the alleles of brown mussel, and no hybrid pattern was observed. Species-specific alleles clearly differentiated green mussel from both brown and parrot mussel. The genetic distance of green mussel from brown mussel, estimated from allozyme data was 1.1145 and with parrot mussel it was 1.105. The genetic distance between parrot mussel and brown mussel was negligibly low (0.0005). Using allozyme and RAPD data, the Nei’s Unbiased Measures of genetic distance were calculated and the dendograms prepared based on these values clearly depicted the separation of parrot mussel from green mussel as well as the close resemblance of parrot mussel with brown mussel. The higher gene flow (1.1539) determined using RAPD marker also hints that brown and parrot mussel may be acting as single interbreeding population. Hence this study using molecular tools to test the genetic identity of parrot mussel has helped to conclude that parrot mussel is only a morphotype of brown mussel and not a true hybrid of the two.Not Availabl

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Not AvailableThe in vitro growth inhibitory efficacies of five drug molecules against Theileria equi were evaluated inin vitro cultured parasites. A continuous microaerophilic stationary-phase culture (MASP) system wasestablished for propagation of T. equi parasites. This in vitro culture system was used to assess the growthinhibitory effect of harmaline hydrochloride dihydrate (HHD), hexadecyltrimethylammonium bromide(HDTAB), hesparidin methyl chalcone (HMC), andrographolide and imidocarb dipropionate against T.equi. The 50% inhibitory concentration value of HHD, HDTAB, HMC, and imidocarb dipropionate for T.equi growth were 17.42 M, 14.00 M, 246.34 M and 0.279 M (equivalent to 0.139 g/ml), respec-tively (P < 0.05). The andrographolide was not effective in inhibiting in vitro growth of T. equi in thepresent study. Furthermore, the in vitro cytotoxicity of these five drugs was evaluated on horse PBMC. At2000 M concentration of HHD, HDTAB, HMC, andrographolide and imidocarb dipropionate were 8.34,46.44, 58.53, 31.06, 15.14% cytotoxic on PBMC, respectively. Out of our four tested drug molecules, HHDwas having low IC50value along with least cytotoxicity, as compared to reference drug imidocarb dipro-pionate. The difference in IC50value of HDTAB and HHD was significant, but HDTAB was moderately morecytotoxic on PBMC cell lines. HHD and HDTAB are selective inhibitor for heat shock protein 90 (Hsp90)and choline kinase pathway. It can be concluded that HHD and HDTAB are potential drug moleculesagainst T. equi parasite by acting on Hsp90 and choline kinase pathway.Not Availabl

Evaluation of Streptomyces strains isolated from herbal vermicompost for their plant growth-promotion traits in rice

Six actinomycetes, CAI-13, CAI-85, CAI-93, CAI-140, CAI-155 and KAI-180, isolated from six different herbal vermi-composts were characterized for in vitro plant growth-promoting (PGP) properties and further evaluated in the field for PGP activity in rice. Of the six actinomycetes, CAI-13, CAI-85, CAI-93, CAI-140 and CAI-155 produced siderophores; CAI-13, CAI-93, CAI-155 and KAI-180 produced chitinase; CAI-13, CAI-140, CAI-155 and KAI-180 produced lipase; CAI-13, CAI-93, CAI-155 and KAI-180 produced protease; and CAI-13, CAI-85, CAI-140 and CAI-155 produced ß-1-3-glucanase whereas all the six actinomycetes produced cellulase, hydrocyanic acid and indole acetic acid (IAA). The actinomycetes were able to grow in NaCl concentrations of up to 8%, at pH values between 7 and 11, temperatures between 20 and 40 °C and compatible with fungicide bavistin at field application levels. In the rice field, the actinomycetes significantly enhanced tiller numbers, panicle numbers, filled grain numbers and weight, stover yield, grain yield, total dry matter, root length, volume and dry weight over the un-inoculated control. In the rhizosphere, the actinomycetes also significantly enhanced total nitrogen, available phosphorous, % organic carbon, microbial biomass carbon and nitrogen and dehydrogenase activity over the un-inoculated control. Sequences of 16S rDNA gene of the actinomycetes matched with different Streptomyces species in BLAST analysis. Of the six actinomycetes, CAI-85 and CAI-93 were found superior over other actinomycetes in terms of PGP properties, root development and crop productivity. qRT-PCR analysis on selected plant growth promoting genes of actinomycetes revealed the up-regulation of IAA genes only in CAI-85 and CAI-93