http://www.erepo.iihr.ernet.in/handle/123456789/487 2013-05-16T04:34:43Z 2013-05-16T04:34:43Z M J, Pavankumar http://www.erepo.iihr.ernet.in/handle/123456789/496 2012-11-27T20:30:05Z 2010-01-01T00:00:00Z

Title: Development of recombinant protein based diagnosis for chilli Veinal mottle virus infecting chilli Authors: M J, Pavankumar Abstract: Chilli veinal mottle virus (ChiVMV) is a positive sense single stranded RNA virus, with monopartite genome belongs to genus potyvirus of family potyviridae. In this study, specific rabbit polyclonal antibodies against bacterially expressed coat protein of Chilli veinal mottle virus (ChiVMV, genus Potyvirus) were produced using a recombinant DNA approach. The ChiVMV coat protein (CP) gene was cloned in an expression vector pET- 15b (Novagen). Expression of the CP with an N-terminal hexahistidine tag in Escherichia coli BL 21 DE3 cells was induced by adding isopropyl-3-D-1-thiogalactoside (IPTG) to a final concentration of 250 μM. About 4 mg of bacterially expressed CP was purified from 500ml of bacterial liquid culture using a Ni-NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 34 kDa in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against ChiVMV purified particles in Western blots. Expressed and purified CP (SDS-PAGE 34 kDa band) was injected into a white female New Zealand rabbit, approximately 3 month old, four times at weekly intervals by intramuscular injections. The antiserum produced was evaluated for ChiVMV detection in DAC-ELISA. The antiserum raised against the expressed CP (ChiVMV) gave strong ChiVMV specific DAC-ELISA reactions and very weak background reactions with noninfected tissues. Three ChiVMV ELISA-positive samples of chilli, were also confirmed by reverse transcription (RT)-PCR and sequencing. The expected 1.2-kb viral cDNA was amplified from all three samples using ChVMVCPF/CPR primers. Excluding the 3′ poly- A tail, was 1,147 nucleotides (nt) long, comprising the 3′-terminal of the coat protein region (1 to 861 nt), and the 3′-untranslated region (865 to 1,147nt). Comparison of the Coat protein gene sequence with corresponding sequences of potyviruses in GenBank revealed that ChiVMV. Tomato had greatest nucleotide (90.3 to 93.8%) and amino acid (91.6 to 97.2%) identity with pepper isolates of ChiVMV from India, Where as it shared 90.2 to 93.0% nucleotide and 93.7 to 96.1% amino acid identity with ChiVMV isolates from China, Indonesia, Taiwan, Thailand and Vietnam

2010-01-01T00:00:00Z A, Yasotha http://www.erepo.iihr.ernet.in/handle/123456789/495 2012-11-27T20:30:05Z 2008-01-01T00:00:00Z

Title: Morpho-cultural characterization of indigenous Pleurotus isolates Authors: A, Yasotha Abstract: Pleurotus species is a promising mushroom form a group of edible species. Mushroom varieties are at present hard to protect since they are mainly characterized by variable fruit body and yield characteristics. It seems to be morphologically different and is taxonomically discrete. They are greatly influenced by environmental factors. In the present study, six isolates from different geographical regions, Bangalore, Shimoga, Madurai, Udaipur and Banswara (Rajasthan) were studied. All the six isolates showed high degree of variability in terms of colony morphology, media, temperature, mycelial characters and spore production. Pleurotus djamor grew faster than the other Pleurotus species at 25 ºC. Linear growth of mycelium was observed in all the region isolates. The two isolates, P. cystidiosus showed a different kind of growth which produced black heads of coremia on MEA, SSM and RC. Pinkish pigment in the mycelia of P. djamor was produced on MEA, PCA and SSM, which is a characteristic observation. Influence of temperature on the growth of isolates showed variability based on geographical regions. Bangalore isolates showed an optimum of 25 ºC, Shimoga, Madurai and Udaipur isolates of 30 ºC and Banswara (Rajasthan) isolate showed optima of 40 ºC suggesting the existence of variability. Out of six isolates, Bangalore and Udaipur showed a high degree of variability in mycelial thickness in both skeletal and generative hyphal system. There exists variability in the kind of hyphal system also. Bangalore, Shimoga and Udaipur isolates showed Dimitic hyphae, whereas Madurai showed monomitic hyphae. But Banaswara isolate showed trimitic hyphae, which is only showed the existence of variability in all study. Basidiospore and asexual spore characters are considered an important character for species identification. They are similar in structure but size varied. Only P. cystidiosus produced the asexual spore, which is used to distinguish from the other Pleurotus sp. Morphological studies are also used to determine the species. In this, only P. cystidiosus and P. djamor showed a different morphology that is P. cystidiosus showed very large fruiting bodies exists in a single and blackish brown in colour, whereas P. djamor showed pink colour fruiting bodies.

2008-01-01T00:00:00Z V, Venkataravanappa http://www.erepo.iihr.ernet.in/handle/123456789/494 2012-11-27T20:30:05Z 2008-01-01T00:00:00Z

Title: Molecular characterization of bhendi yellow vein mosaic virus Authors: V, Venkataravanappa Abstract: Bhendi/Okra (Abelmoschus esculents (L.) Moench) is one of the important vegetable crops grown extensively throughout the tropical, subtropical and warm regions of the temperate zones of the world. Bhendi yellow vein mosaic virus (BYVMV) is one of the most sever disease which take a heavy toll in India. A roving survey revealed that the occurrence of BYVMV disease incidence ranged from to be 23.0 to 67.67% in Kamataka, 45.89 to 56.78% in Andhra Pradesh, 23 to 75.64% in Tamil Nadu, 42.45 to 75.64% in Kerala, 23 to 85.64% in Maharashtra, 24.85 to 65.78% in Haryana, 35.76 to 57% in Uttar Pradesh, 45.45% in Delhi, 67.78% in Chimdigarh and 45.89 to 66.78% in Rajasthan and The highest disease incidence was recorded in the districts of Gulbarga (75.0%) and least is in Bangalore rural (44.01%). Single whitefly of B biotype and two whiteflies indigenous B. tabaci could able to transmit BYVMV with 30% and 20% efficiency. Nine B biotype and 10 indigenous viruliferous whiteflies required for 100% BYVMV transmission. Minimum AAP and lAP found to be 15 mints, in B biotype and 20 mint. in indigenous whiteflies. 100% transmission obtained in 24 hrs. AAP and 16 hrs. lAP given to B- biotype compared to indigenous whitefly which required 24 hrs. AAP and lAP. Females of B-biotype and indigenous B. tabaci were more efficient in transmitting BYVMV compared to males. Age group of 7-15 days old Bhendi plants were found more susceptible for infection. BYVMV was successfully purified from the systemically infected okra leaves using the modified procedure with addition of sucrose gradient. The UV spectrum observations of 260/280 ratios showed 1.20-1.27 OD with the yield of 12-15 mg/Kg of infected leaves. SDS-PAGE analysis and western blotting of the purified preparation resulted in the presence of band with an approximate molecular weight of 28000 Daltones, which is expected monomeric size of BYVMV coat protein. The direct antigen coating ELISA (DAC-ELISA) was standardized and successfully used to detect virus using polyclonal antibody raised against ACMV and goat anti-mouse IgG-alkaline phosphatase conjugate. The ELISA results showed that BYVMV detect in infected leaf extracts up to 1:10000 antigen dilution. However, optimum reaction has been obtained at 1: 1000 antigen concentrations. Further the BYVMV also detect in infected flowers, pollen, petals, calyx and fruits but not in seed. Dot immuno binding Assay (DIBA) was standardized and successfully used to detect virus using polyclonal antibody raised against ACMV. The result showed that BYVMV detected in non purified sample up to 1: 1000 antigen dilution and 1: 10000 dilution of purified virus with the antibody dilution of 1: 500 from the infected leaf tissues. Dot blot hybridization was standardized using diagoxigenin labeled probes. The methods were found to be equally sensitive in detecting viral nucleic acid up to a concentration of O.OO1 ng from purified BYVMV. Further, the BYVMV isolates were detected using DNA was extracted from the infected leaf tissues. This method could also be used as diagnostic tool for identification of BYVMV with more precision. The complete DNA-A sequence of 113 isolates nucleotide sequence of the DNA-A genome of different isolates size ranged from 27722 to 2793nt. The complete nucleotide sequence of 113 isolates has 84 to 100% similarity among themselves. Ten groups are formed in phylogenetic grouping that have sequence identity of less than 89% with other reported begomoviruses. Ten species were identified as distinct from already known begomovirus species in India. These are Bhendi yellow vein Haryana virus (BYVHV), Bhendi yellow vein Guntur virus (BYVGV), Bhendi yellow vein India virus (BYVIV), Bhendi yellow vein Kamataka Virus (BYVKV), Okra leaf curl India virus (OKLCIV). Bhendi Yellow Vein Kerala Virus (BYVKV), Bhendi Yellow Vein Delhi Virus (BYVDV), Bhendi Yellow Vein Trichy Virus (BYVTV), Bhendi Yellow Vein Bhubaneswar Virus (BYVBV), Okra Enation Leaf Curl Virus (OELCV). Most of isolates of BYVMV showed high percentage of nucleotide sequence and amino acid identity with in ORFs AVl, AV2, ACI AC2 AC3, AC4, AC5 and intergenic region with BYVMV.NOL751, BYVMV-Madurai, OYVMV, BYVMV-Pak301, CLCuVA and (CLCuMV-(Okra] and some isolates showing the nucleotide sequence and amino acid identity ToLCNDV-A VT. The complete DNA-B sequence of 7 isolates nucleotide sequence genome of different isolates size ranged from 2695nt (OY77, OY81, OY174) to 2707nt (OYI64). The complete nucleotide sequence of 7 isolates has 84 to 100% similarity among themselves lead to form four groups which are associated with okra yellow vein mosaic disease namely the isolates OY77, OY142, OY81 and OYT closely with Tomato leaf curl New Delhi luffa isolates (EF620535) and Tomato leaf curl New Delhi AVTl isolate (AY438563 ToLCNDV [Lucknow] and ToLCNDV-sever [Jessor] isolates. the isolate OY164 form separate group with ToLCNDV-AVTl and ToLCNDVluffa. Pk. and one isolate OY131 which shows the nucleotide identity of less than 85% with other okra DNA-B we considered as distinct species of okra yellow vein virus. The full length of 36 isolates Beta DNA molecules ranging in size from 1324 to l403nt in length the sequence of these presumed full length DNA ~ molecules have these conserved 207 features an A-rich region, a satellite conserved region (SCR), and a single open reading frame (putative coding region of gene C 1). The complete nucleotide sequence of 36 Beta DNA molecule revealed the occurrence of four types of Beta DNA molecules were present among the isolates viz., Bhendi yellow vein beta satellite (BYVB), okra leaf curl betasatellite (OLCuB), Ludwigia leaf distortion betsatellite (LuLDB), Croton yellow vein mosaic beta satellite (CroYVMB) and these may are associated with BYVMV. 57 different genotypes were screened for BYVMV under glasshouse and 29 different genotypes under natural conditions. None of genotypes tested was highly resistant or immune. Only 3 genotypes showing resistant reaction, 3 genotypes showing moderately resistant.

2008-01-01T00:00:00Z Sandhya Ravishankar http://www.erepo.iihr.ernet.in/handle/123456789/493 2012-11-27T20:30:05Z 2006-01-01T00:00:00Z

Title: Development of sporeless/low spore shedding strains of Pleurotus species Authors: Sandhya Ravishankar Abstract: Di-mon matings were carried out between monokaryons of commercial Pleurotus species (P.florida and P.sajor-caju) with dikaryotic mycelia of natural sporeless mutant (Psm) yielded 16 (P.florida x Psm) and 18 (P.sajor-caju x Psm) hybrids. Exposure of spore suspension of commercial Pleurotus species (P.florida and P. sajor-caju) to Ultra violet radiation for variable timings resulted in yielding few mutants. Few mutants obtained from P.florida spores even though exhibited spore less character in 1st generation failed to maintain the character (stability) in 2nd generation. The mutant obtained from exposure of spore suspension of P. sajor-caju exhibited sporelessness (low sporing character) with good morphology. The stability of the low sporing character was maintained even after 40th generation. It proved the mutation obtained was permanent. Gill sections of low sporing UV mutant along with its parent P.sajor-caju and commercial species P. florida and sporeless mutant (Psm) observed under microscope, showed P. sajor-caju and P. florida more spore intensity. The biological efficiency of P.florida was 61.09% and P.sajor-caju was 62.02%. Low sporing UV mutant showed lower biological efficiency of 52.67%. Natural sporeless mutant (Psm) showed highest biological efficiency of 70.93%. The shelf life of sporophores of commercial Pleurotus sajor-caju and sporeless strains (Psm and low sporing UV mutant) in pp covers at room temperature was 2 days as compared to only one day in 1.33% ventilation pp cover, commercial perforated cover, pp covers lined with brown paper and air tight container. Whereas sporophores of commercial Pleurotus florida could be stored in good condition only for one day in all the treatments the sporophores of Pleurotus sajor-caju and sporeless mutants (Psm and low sporing UV mutant) showed better shelf life compared to Pleurotus florida. Under cold storage condition (4 ºC) UV mutant and Psm showed 14 days extended shelf life compared to 11 days in Pleurotus florida and 13 days in Pleurotus sajor-caju. As the ventilation increased number of storage days also reduced. The present studies on dehydration of commercial Pleurotus species and two sporeless strains were carried out at room temperature (28-32 ºC) and drier (40 ºC). It took 2-3 days for optimal drying (8- 10% moisture) at room temperature as against 5-6 hours in drier for both commercial Pleurotus and two sporeless strains. RAPD analysis was carried out with random primers to know the polymorphism between the species and strains. The cluster analysis of RAPD markers showed two groups. In first group P. florida and Psm were closely related with 43% similarity, which also reflected in distance measure. Even though P. florida and Psm were closely related, morphological characters of both strains were different. P. florida have white sporophores with high spore content. Whereas Psm have light gray funnel shaped sporophores with no spores. In second group P.sajor-caju and UV mutant were closely related with 64% similarity. However sporophore morphology of both the strains was similar to certain extant. P.sajor-caju having gray sporophores with eccentric stipe and slight wavy margin with high spore content. Whereas UV mutant showed highly lobed (flower type) sporophores with very less spore content. Thus, RFLP and RAPD analysis found to be a suitable technique for observing the polymorphism between the strains.

2006-01-01T00:00:00Z