Prem Nath 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.

Isolation and characterization of gut bacteria of fruit fly, Bactrocera tau (Walker)

Gut bacteria of fruit fly, Bactrocera tau (Walker) (Diptera: Tephritidae), were isolated and the isolates attractive to B. tau adults were characterized using morphological, biochemical and 16S rRNA analyses to determine their taxonomic position. Based upon morphological, biochemical and 16S rRNA sequences (on the basis of closest match), five gut bacterial species of B. tau were characterized as Delftia acidovorans, Pseudomonas putida, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp., of which four bacterial isolates, viz., Delftia acidovorans, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp. are new records from guts of the fruit fly species.

An integrated approach of solar photocatalytic and biological treatment of N-containing organic compounds in wastewater

In the present study the integration of TiO2-assisted solar photocatalysis and biological treatment for the degradation of organic nitrogen containing pyrazinamide (antituberculosis drug) synthesis process effluent is investigated. The experimental results showed that a considerable increase in the degradation efficiency of N-containing compounds is obtained by integration of photocatalysis and biological treatment when compared to either biological or photocatalysis treatment alone. The optimization parameter like amount of photocatalyst (TiO2 Degussa-P-25) was performed for photocatalytic treatment under solar irradiation. Furthermore BOD5/COD ratios were also evaluated. The optimum catalyst (wt.%) of 0.35 TiO2, and 40 times dilution are found to be optimum for achieving a maximum of 76, 83, 80% of COD, color and TOC removals respectively and an improved BOD5/COD ratio of 0.3 to 0.7 in a duration of 24 h. Also the formation of increased from 10 to 44 mg L−1 within 20 h of photocatalytic treatment. The effluent subjected to biological treatment for five days (120 h) has resulted 43% COD removal only, whereas the treatment method using integrated approach resulted a 91% COD removal in a duration of 44 h.

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.

Elucidation of biocontrol mechanisms of Trichoderma harzianum against different plant fungal pathogens: Universal yet host specific response.

In the present study, different transcripts of Trichoderma harzianum ThHP-3 were evaluated for their response against four fungal pathogens Fusarium oxysporum, Colletotrichum capsici, Colletotrichum truncatum and Gloesercospora sorghi using RT-qPCR. The time course study of T. harzianum transcripts related to signal transduction, lytic enzymes, secondary metabolites and various transporters revealed variation in expression against four fungal pathogens. In a broader term, the transcripts were upregulated at various time intervals but the optimum expression of cyp3, abc, nrp, tga1, pmk, ech42 and glh20 varied with respect to host fungi. Additionally, the expression of transcripts related to transporters/cytochromes was also observed against Fusarium oxysporum after 96h whereas transcripts related to secondary metabolites and lytic enzymes showed significant difference in expression against Colletotrichum spp. from 72 to 96h. This is first study on transcriptomic response of T. harzianum against pathogenic fungi which shows their host specific response.

Metageographic population analysis of Colletotrichum truncatum associated with chili fruit rot and other hosts using ITS region nucleotide sequences

Colletotrichum truncatum is one of the most economically important fungal pathogen causing anthracnose disease in pre and post-harvest stages of many crops worldwide. Little information is available in the literature on the genetic analysis and demographic history of this fungal pathogen. In the present study nucleotide sequence data of internal transcribed spacer (ITS) region were analyzed for C. truncatum isolates infecting chili and other crops worldwide to determine a metageographic pattern of distribution and evolution of the species. Levels of differentiation (genetic distances and FST values) among sequences of C. truncatum from 23 countries were minimal suggesting the global occurrence of a large and geographically undifferentiated population. Only 11 haplotypes were detected among 98 isolates from 24 geographically distant populations of C. truncatum. Predominant haplotype H1 which occupied a central position in the median joining network was inferred to be ancestral haplotype as it was detected at a high frequency and was shared by multiple populations. Phylogeographic pattern of the species with worldwide presence and predominance of single haplotype suggests human mediated dispersal through domestication and introduction of host plants in different parts of the world, and might have played a significant role in structuring the populations of this devastating pathogen.

Characterization and complete nucleotide sequencing of Pepper Mild Mottle Virus infecting Bell Pepper in India

Capsicum (Capsicum annuum L var. grossum Sendt) commonly known as bell pepper or sweet pepper, is one of the most economical agricultural crop grown under both open and polyhouse conditions. The presence of Pepper mild mottle virus (PMMoV) from different districts of Himachal Pradesh was confirmed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and RT-PCR using coat protein (CP) gene amplification. The first complete genome sequence of Indian isolate of PMMoV (HP-1) was elucidated and compared with other members of Virgaviridae family and PMMoV isolates. Sequence homology, multiple alignment and phylogenetic analysis on the basis of nucleotide and amino acid sequences showed that PMMoV-HP1 isolate is more closely related to the PMMoV-J, the Japanese isolate. Based on CP gene amino acid sequence analysis, the PMMoV-HP1 isolate showed 100 per cent identity with P12 pathotypes (capable of breaking L2 gene mediated resistance in capsicum). This is the first report of the PMMoV complete genome organization intercepted 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.

Molecular and functional diversity of Saccharomyces cerevisiae strains of traditional fermented foods of the North-Western Himalayas

The internal transcribed spacer (ITS) region was used to discriminate 18 Saccharomyces cerevisiae strains isolated from fermented foods of the North-Western Himalayas. ITS sequences were compared with other known lineages available in literature. A phylogenetic tree grouped these strains into two clusters, the first comprising Malaysian, North American, Sake, West African and wine/European strains and the second comprising all Indian strains along with certain other strains from different countries. In both clusters, mixed grouping was observed, with the exception of a few strains with a region-based grouping pattern. The baking and brewing abilities of these strains were studied to expose functional diversity. Four strains, viz. Sc06, Sc11, Sc19 and Sc20, were found promising for baking and three strains, viz. Sc04, Sc05 and Sc24, were found promising for brewing, thereby indicating the existence of functional diversity. At a genetic level, mining of the ADH1 gene of ten randomly selected yeast strains, showed very little variation in the gene sequences irrespective of their alcohol production ability. In contrast, mining of the ATF1 gene showed a lot more variation in the gene sequences, revealing the ATF1 gene to be an appropriate marker to reveal differences in ester formation among these indigenous yeast strains.

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.