P. N. Sharma

Population genetic structure of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) based on mitochondrial cytochrome oxidase (COI) gene sequences

Population genetic structure of melon fly analysed with mitochondrial cytochrome oxidase I gene suggested that melon fly populations across the globe is homogeneous with non-significant variation of 0.000–0.003 base substitutions per site. Test isolates representing various geographic situations across the world were placed in 26 mitochondrial haplotypes based on variations associated with a maximum of three mutational steps and the predominant haplotype i.e. H1 was present in all melon fly populations except Hawaiian population. Evolution of mtCOI gene suggested that the fly could have originated some 0.4 million years ago. The present study also indicated that the B. cucurbitae population expansion is an event of post Pleistocene warm climatic conditions with small number of founder population. The invasion of B. cucurbitae in Hawaii was associated with the large population size and the global presence of the fly is associated with human mediated dispersal. The very low genetic variation suggested that the fly management might be possible by large scale sterile insect techniques programme.

Evaluation of resistance sources and inheritance of resistance in kidney bean to Indian virulences of Colletotrichum lindemuthianum Evaluation of resistance in bean to anthracnose

SummaryForty nine common bean lines comprising of exotic accessions and locally grown cultivars evaluated against Colletotrichum lindemuthianum exhibited differential resistance to its races in Himachal Pradesh, a north-western Himalayan state of India. Some exotic accessions like G 2333, Cornell 49242, PI 207262, Mexique 222, TO, Perry Marrow, Kaboon and Widusa were resistant to more than five Indian races, whereas two Indian accessions KRC-5 and Hans showed resistance to six and four races, respectively. However, nine accessions KRC-8, KR-40, KR-43, KR-81, KR-62-2, KR-90, KR-142, KR-148, and KR-216 were resistant to three races. Race specific resistance has been observed in different bean cultivars. Studies on inheritance of resistance in exotic accession G 2333 and Indian accession, KRC-5 showed that two independent dominant genes conferred resistance in G 2333 to race 3 and 515 and a single dominant gene controlled resistance in KRC-5 to race 775, indicating resistance from these sources is easily transferable to the locally adapted susceptible cultivars.

Pathogenic and molecular variability in bean common mosaic virus infecting common bean in India

Pathogenic variability studies in bean common mosaic virus (BCMV) infecting common bean (Phaseolus vulgaris L.) revealed the existence of two pathogroups PG-I and PG-II and four strains (NL-1, NL-1n, NL-7 and NL-7n) in Himachal Pradesh, a North-Western Himalayan state of India. Two strains, NL-1 and NL-7 were identical to the previously described NL-1 and NL-7 strains from Europe and USA, whereas the other two designated as NL-1n and NL-7n differed from earlier identified strains with respect to their necrotic reaction on cultivar Jubila at high temperature (>30°C). Reverse phase HPLC peptide profiling of tryptic digests of coat protein of these strains further confirmed that NL-1, NL-1n, NL-7 and NL-7n are distinct from each other. This study constitutes the first record of pathogenic variability in BCMV infecting common bean in India.

Virulence and RAPD data—A tool to study the evolutionary trends of Colletotrichum lindemuthianum virulences in the North Western Himalayan region of India

Abstract Bean anthracnose pathogen (Colletotrichum lindemuthianum) known to display high pathogenic variability, also explains the existence of large number of races in Himachal Pradesh. An evolution model based on virulence data of 29 C. lindemuthianum races and RAPD patterns revealed the existence of four evolutionary groups (EG I – EG IV) in Himachal Pradesh, accommodating 12, 14, 2 and 1 races, respectively. Some races viz., 935, 643, 529, 647 and 613, opted more than two evolutionary routes and races like 598, 707, 935, 631, 639, 615, 115 and 119 harbouring more than six virulence genes may pose a threat to bean cultivation in this part of the world as they can break many resistance genes present in the locally grown beans. However, two exotic accessions G 2333 and AB 136 resistant to all the Indian pathotypes could be exploited as resistance donors in developing anthracnose resistant cultivars suitable for cultivation in this region.

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.

Identification and Genetic Diversity Analysis of Ascochyta Species Associated with Blight Complex of Pea in a Northwestern Hill State of India

Forty isolates of Ascochyta spp. infecting pea collected from different commercial pea growing areas of Himachal Pradesh were characterized into three species—Ascochyta pinodes, A. pisi and A. pinodella—based on symptoms, morphological and cultural traits. The identity of the isolates was confirmed by studying the rDNA sequence patterns. Based on various cultural and morphological trait combinations, 36 isolates were identified as A. pinodes, one isolate as A. pisi and one isolate as A. pinodella. Whereas two isolates, As3 and As14, could not be placed in any of the species as equal number of characters matched with A. pisi and A. pinodes. However, sequencing of ITS region of these isolates resolved the ambiguity and placed them in A. pinodes. This necessitates ITS sequencing as one of the criteria to establish the taxonomic status of Ascochyta species infecting pea. RAPD, RAMS, and Rep-PCR analysis with 10, 4 and universal ERIC, and BOX-AIR primers categorized different isolates according to their geographical locations. This study established the predominance of A. pinodes in Himachal Pradesh where edible pea is one of the remunerative off-season vegetable crops. This also constitutes first report of genetic variability of Ascochyta species infecting pea in India.

Identification of Colletotrichum spp. associated with fruit rot of Capsicum annuum in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.

Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli

Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production.

ENHANCEMENT OF SOLUBILITY AND DISSOLUTION RATE OF SIMVASTATIN BY USING SOLID DISPERSION TECHNIQUE ALONG WITH DIFFERENT COMBINATION OF POLYMERS

The solubility and dissolution rate of simvastatin, a drug used for the treatment of hyperlipidaemia. Simvastatin is a selective competitive inhibitor of HMG Co A reductase. However its absolute bioavailability is 5%. To increase the solubility of drug solid dispersion was prepared. Solid dispersion preliminary solubility analysis was carried out for the selection of the carrier and solid dispersion was prepared with Hydroxy Propyl Methyl Cellulose (HPMC) and Methyl Cellulose (MC). These solid dispersions were analyzed for the solubility and in-vitro dissolution profile solid dispersion of drug with polymer has shown enhanced solubility with improved dissolution rate. Further FTIR, X-Ray studies were carried out. Solid dispersion prepared with polymer in 1:5 ratios shows the presence of amorphous form confirmed by the characterization study. The study also shows that dissolution rate of simvastatin can be enhanced to considerable extent by solid dispersion technique with Polymer. Keywords: Solubility enhancement, Solid dispersion, Low aqueous solubility

Population Structure of Colletotrichum truncatum in Himachal Pradesh and Identification of Broad-Spectrum Resistant Sources in Capsicum

Anthracnose disease of Capsicum caused by Colletotrichum truncatum (syn. C. capsici) is a catastrophic disease causing huge loss in productivity worldwide. Consequently, interest in elucidating its genetic structure has grown in recent decade. In the present study, six random amplified microsatellites were used to access the population structure of 94 isolates of C. truncatum from three agro-climatic zones of Himachal Pradesh. Among the 104 reproducible fragments, 81.73% were polymorphic. Cluster analysis grouped 94 isolates into four groups with no congruence with geographical origin of isolates. Average gene diversity ranged between 0.38 and 0.45, and expected heterozygosity ranged from 0.39 to 0.44. Analysis of molecular variance showed high genetic variability within the zones (99.53%). Screening of 194 genotypes of Capsicum against 94 C. truncatum isolates showed low-level resistance to the fungus. Some promising lines possessing resistance to more than 40 C. truncatum isolates were identified. These genotypes may harbor diverse resistance specificities and can be used to develop anthracnose-resistant genotypes in the State. This is the first resistance evaluation study where Capsicum genotypes were screened individually against a large number of isolates. The results suggest considerable evolutionary potential of pathogen that has enabled it to acquire pathogenicity genes (virulence factors) for infecting diverse host varieties over time and space.