Saurabh Kulshrestha

Efficiency of plant growth-promoting P-solubilizing Bacillus circulans CB7 for enhancement of tomato growth under net house conditions

P‐solubilizing bacterial isolate CB7 isolated from apple rhizosphere soil of Himachal Pradesh, India was identified as Bacillus circulans on the basis of phenotypic characteristics, biochemical tests, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The isolate exhibited plant growth‐promoting traits of P‐solubilization, auxin, 1‐aminocyclopropane‐1‐carboxylate deaminase activity, siderophore, nitrogenase activity, and antagonistic activity against Dematophora necatrix. In vitro studies revealed that P‐solubilization and other plant growth‐promoting traits were dependent on the presence of glucose in PVK medium and removal of yeast extract had no significant effect on plant growth‐promoting traits. Plant growth‐promoting traits of isolate CB7 were repressed in the presence of KH2PO4. P‐solubilization activity was associated with the release of organic acids and a drop in the pH of the Pikovskayas medium. HPLC analysis detected gluconic and citric acid as major organic acids in the course of P‐solubilization. Remarkable increase was observed in seed germination (22.32%), shoot length (15.91%), root length (25.10%), shoot dry weight (52.92%) and root dry weight (31.4%), nitrogen (18.75%), potassium (57.69%), and phosphorus (22.22%) content of shoot biomass over control. These results demonstrate that isolate CB7 has the promising PGPR attributes to be developed as a biofertilizer to enhance soil fertility and promote plant growth.

Molecular studies on Tomato aspermy virus isolates infecting chrysanthemums

Abstract Tomato aspermy virus (TAV), an important viral pathogen of chrysanthemums, was detected by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) from chrysanthemums exhibiting mottling and deformed inflorescence grown in various states of India. Out of 15 cultivars (cvs.) tested, 11 (73.3%) were found to be positive for TAV. On sap inoculation the virus produced local lesions on Chenopodium spp. and Cucumis sativus. Nicotiana clevelandii, N. glutinosa, N. megalosiphon and N. tabacum reacted systemically to the virus producing severe mosaic, leaf deformation and characteristic leaf enations. Tomato plants produced malformed fruits (2-3/plant) with a few seeds. Myzus persicae and Aphis gossypii transmitted the virus non-persistently. Electron microscopy of partially purified viral preparations revealed polyhedral virions (ca. 29 nm dia). Cytopathology of infected leaves of N. clevelandii showed crystalline inclusions containing virions in the central vacuole of infected cells. The virions showed very good clumping with antiserum to TAV in liquid phase immuno-electron microscopy. Slot blot hybridization was performed to detect the virus in various chrysanthemum cultivars. The virus positivity was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing of coat protein gene (CP). The amplified 657 bp fragment was about 97% identical with respect to CP sequences of other TAV isolates available in the database. In terms of derived amino acid sequence similarity, the homology value was 99%. In order to amplify RNA 1, RNA 2 and RNA 3, multiplex RT-PCR was performed. High genetic similarities of CP gene of TAV Indian isolates with that of other isolates of TAV indicate their probable common ancestry.

A Nano-Au/C-MWCNT based label free amperometric immunosensor for the detection of capsicum chlorosis virus in bell pepper

Accurate and on time diagnosis of plant viruses is an essential prerequisite for efficient control in field conditions. A number of diagnostic methods have been reported with the required level of sensitivity. Here, we propose a label free immunosensor for efficient and sensitive detection of capsicum chlorosis virus (CaCV) in bell pepper. Antigen was immobilized over the surface of gold nanoparticle/multi-walled carbon nanotube (Nano-Au/C-MWCNT) screen printed electrodes using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross linking chemistry followed by interaction with groundnut bud necrosis virus (GBNV)/CaCV specific polyclonal antibody. The electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) using the redox indicator. Electrode surface characterization was done by performing scanning electron microscopy (SEM). Electrochemical studies showed positive results at different antigenic dilutions ranging from 10−2 – 8x10−5. The sensitivity of the immunosensor developed has been compared with direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) and the results showed that the immunosensor developed was 800-1000 times more sensitive, when compared to DAC-ELISA for CaCV detection. The immunosensor we have developed is economical and sensitive and could be used for immediate determination of the presence of virus in extracts from bell pepper leaves.

Molecular characterization and intermolecular interaction of coat protein of Prunus necrotic ringspot virus: implications for virus assembly

Coat protein (CP) and RNA3 from Prunus necrotic ringspot virus (PNRSV-rose), the most prevalent virus infecting rose in India, were characterized and regions in the coat protein important for self-interaction, during dimer formation were identified. The sequence analysis of CP and partial RNA 3 revealed that the rose isolate of PNRSV in India belongs to PV-32 group of PNRSV isolates. Apart from the already established group specific features of PV-32 group member’s additional group-specific and host specific features were also identified. Presence of methionine at position 90 in the amino acid sequence alignment of PNRSV CP gene (belonging to PV-32 group) was identified as the specific conserved feature for the rose isolates of PNRSV. As protein–protein interaction plays a vital role in the infection process, an attempt was made to identify the portions of PNRSV CP responsible for self-interaction using yeast two-hybrid system. It was found (after analysis of the deletion clones) that the C-terminal region of PNRSV CP (amino acids 153–226) plays a vital role in this interaction during dimer formation. N-terminal of PNRSV CP is previously known to be involved in CP-RNA interactions, but our results also suggested that N-terminal of PNRSV CP represented by amino acids 1–77 also interacts with C-terminal (amino acids 153–226) in yeast two-hybrid system, suggesting its probable involvement in the CP–CP interaction.

Potential uses of in vitro expressed and purified recombinant Prunus necrotic ringspot virus coat protein gene.

Abstract Prunus necrotic ring spot virus (PNRSV), an ilarvirus, has a wide host range especially on important commercial crops like almond, apple, apricot, hop, peach, and rose. PNRSV coat protein gene was cloned in expression vector pGEX-2Tk (Amersham Pharmecia, USA) using E. Coli BL 21 strain competent cells. Expression conditions were standardized for maximum recovery of soluble recombinant protein. The in vitro expressed protein was purified and used as an antigen for raising antisera. Both intramuscular and sub-cutaneous routes were used separately for antisera raising. A part of the raised and purified antisera was used in enzyme conjugate preparation. It was then formulated in the form of an ELISA-based diagnostic kit for PNRSV detection. It was also compared with the commercially available kit.

Molecular characterization of tospoviruses associated with ringspot disease in bell pepper from different districts of Himachal Pradesh

Bell pepper (Capsicum annuum L.), an important cash crop for the farmers of Himachal Pradesh was found to be affected with tospovirus like disease. An extensive survey was conducted in the bell pepper grown areas in the five districts of Himachal Pradesh to identify and characterize the causative agent. Hence, 60 symptomatic bell pepper plants exhibiting characteristics symptoms were collected from Solan, Sirmaur, Hamirpur, Kangra and Bilaspur districts. Out of 60 samples, 53 samples were found to be positive by DAS-ELISA with tospovirus group specific antiserum. To confirm the presence of tospovirus, DAC-ELISA was performed using GBNV/CaCV polyclonal antiserum and DAS-ELISA with two monoclonal antibodies i.e. TSWV, GRSV. All the 53 samples were found negative for TSWV and GRSV and positive for GBNV/CaCV. Further, eleven infected isolates from both poly-house and open field conditions were selected for characterization at molecular level. RT-PCR was performed with N gene specific primers for TSWV, GBNV and CaCV. The eleven samples selected for molecular identification were further found to be negative for TSWV and positive for CaCV using RT-PCR. One of the samples from district Sirmaur was found to be positive for mixed infection of GBNV and CaCV. N gene phylogenetic analysis of CaCV/GBNV provided important information about the movement and evolution of tospoviruses in Himachal Pradesh.

Diacylglycerol acyl transferase: A pathogenicity related gene in Colletotrichum gloeosporioides.

To gain more insight into the molecular mechanisms of Colletotrichum gloeosporioides pathogenesis, restriction enzyme‐mediated integration (REMI) mutagenesis identified the mutants of C. gloeosporioides impaired in pathogenicity. Transformants screened for defects in pathogenicity using detached leaves and fruits. Of the 20 REMI transformants tested, two mutants (H4 and H7) showed reduced pathogenicity on leaves of apple, kiwi, mango, peach, and fruits of guava, apple, and capsicum. One tagged gene from the genome sequence of mutant H4 was recovered by inverse PCR. Sequence analysis of the tagged site in mutant H4 revealed insertion in diacylglycerol acyltransferase gene which encodes diacylglycerol acyltransferase enzyme, catalyzing the steps involved in the biosynthesis of triacylglycerol, an important component of biological membranes and source of energy. Therefore, tagging of diacylglycerol acyltransferase gene in mutant H4 resulted in reduced pathogenicity, indicating possible role of this gene in pathogenicity of C. gloeosporioides.

Mycovirus associated hypovirulence, a potential method for biological control of Fusarium species

Fusarium is a large genus of filamentous fungi belongs to the division Ascomycota and was first described as Fusisporium. Innumerable members of this genus act as pathogens, endophytes and saprophytes and can be recovered from plants and soils worldwide. Many of these members are known to be phytopathogens. It is among the most diverse and widely dispersed phyto-pathogenic fungi which cause economically important blights, rots, wilts and cankers of many ornamental, field, horticultural and forest crops both in agricultural commodities and natural ecosystems. Some species, e.g. F. graminearum and F. verticillioides have a narrow host range and mainly infect the cereals, whereas F. oxysporum has effects on both monocotyledonous and dicotyledonous plants. Attempts have been made to control the diseases caused by Fusarium sp. and to minimize crop yield losses. Till date, effective and eco-friendly methods have not been devised for the control of this devastating pathogen. A new potential of using mycovirus associated hypovirulence as biocontrol method against Fusarium species has been proposed. The present review taking into account of worldwide researches to provide possible insights for Fusarium-mycovirus coevolution.

Metagenomics of Fermented Foods: Implications on Probiotic Development

Fermented foods act as delivery vehicles of probiotic cells in human body. These food products boost human health through enhanced nutrition content, digestibility, microbial stability, and detoxification. The importance of fermented foods as probiotics is increasing continuously as they play significant roles in regulating and balancing intestinal microflora. Therefore, efforts are needed toward mining and characterization of microbial communities of fermented foods. The application of metagenomics techniques provides a right way to explore and characterize the unexplored beneficial microbial flora. Furthermore, for commercial development of a probiotic from fermented food, there are some prerequisite, which need to be fulfilled. Here we present the benefits, pitfall, and development of fermented food as probiotic.