Assessment of coliform contamination in drinking water from source to point of use in Mysore city of Karnataka, India

Drinking water supply of Mysore city was assessed for coliform contamination. A 277 drinking water samples were randomly collected from different water sources such as bore wells, taps of consumer points and stored household water samples. The samples were analyzed for microbial parameters like heterotrophic plate count and coliform count. Out of 226 samples from consumer points, 80 samples were contaminated with enteric bacteria. Nearly 325 isolates of coliform were identified of which there were 79 E. coli, 26 Salmonella, 92 Klebsiella and 98 Citrobacter isolates. From the study, the isolation of pathogenic microorganisms indicated that the stored household water was unsafe. Coliform contamination in household water was high even when source water was of good quality. The present study highlights the population’s hygiene, health behavior and environmental sanitation. Coliform in stored household water suggests that improvement in behavior and water hygiene practices can improve the household water quality

Biochemical characterization of Fusarium oxysporum f. sp. cubense isolates from India

The Fusarium wilt caused by Fusarium oxyspoum f. sp. cubense (Foc) is a major biotic constraint for banana production. The characteristics of F. oxyspoum f. sp. cubense isolates were investigated using electrophoretic studies of isozyme and whole-cell protein. The morphological characteristics of the isolates were very similar to each other. All the Foc isolates were pathogenic to banana cultivar 'Nanjangud Rasabale' but they did not induce any disease symptoms on cultivar 'Cavendish'. F. oxyspoum (Isolate 6) did not induce wilt symptoms on either 'Nanjangud' or 'Cavendish' cultivar. Isozyme banding patterns showed 46 scoreable markers and cluster analysis with UPGMA using genetic distance showed that the isolates belonged to three main groups. Group 1 contained isolates 1, 2, 4, 5, 7 and isolate 3 and 6 were placed in group 2 and 3. Results indicated that the estimated intra-specific variation may be more pronounced with isozyme analysis than with protein markers. The level of isozyme variability detected within F. oxysporum f.sp. cubense suggested that it is reliable, efficient and effective in determining genetic relationships among Foc isolates

Reference gene selection and validation for gene expression studies in downy mildew infected pearl millet by quantitative real-time PCR

Pearl millet is considered as one of the future crops in terms of food security due to its drought tolerant nature. However, the plant's susceptibility to the evolving Sclerospora graminicola causing downy mildew disease has enforced researchers to identify genes in pearl millet associated with disease resistance. This could be achieved by analyzing the dynamics of gene expression with quantitative real-time PCR (qPCR). qPCR is a sensitive and reliable approach that requires suitable reference genes (RGs) as internal controls for normalizing transcript abundance of genes of interest (GOI). In the current study, six RGs i.e. ACT, TUB, UBQ, GAPDH, PP2A and EF-1α were evaluated for their expression stability in two experimental datasets: treatment and time-point. The former dataset analyzed the effect of elicitor treatment while the latter analyzed the effect of time-point on the expression stability of RGs in pearl millet during post pathogen inoculation. Three statistical softwares-geNorm, NormFinder and BestKeeper facilitated identification of PP2A, TUB and UBQ as stably expressing RGs for treatment dataset and PP2A and EF-1α for time-point dataset. Validation of these stable RGs for both the datasets was performed by comparative analysis of normalization strategies on the transcript abundance of GOI involved in host resistance against downy mildew. Altogether, the present study forms a basis for RG selection during qPCR analysis of pearl millet-downy mildew or other similar plant- pathogen interaction

Partial purification and characterization of polygalacturonase-inhibitor proteins from pearl millet

Polygalacturonase-inhibitor proteins (PGIPs) are plant cell wall glycoproteins, involved in the inhibition of microbial endo-polygalacturonases (EPGs). The present study involved activity guided partial purification of pearl millet [Pennisetum glaucum (L.) R.Br.] protein extract by cation exchange chromatography, which resulted in two pooled protein peaks -Peak-A and Peak-B, both of which showed inhibitory activity against the Aspergillus niger EPG. Protein separation of the two peaks by gel electrophoresis showed prominent bands between 29 and 43 kDa, consistent with the molecular weights of the known plant PGIPs. The two PGIP peaks were further studied for their inhibitory activities with respect to three parameters viz., inhibitor concentration, pH and temperature effects. Enzyme inhibition was partial and increased with inhibitor concentration. The Peak-B was found to be the more active inhibitor of the two. The results indicate the presence of at least two isoforms of PGIP in pearl millet. This is the first such study to be undertaken in understanding the presence of the PGIPs in millets

Induction of β-1,3-glucanase in Seedlings of Pearl Millet in Response to Infection by Sclerospora graminicola

Differential resistance of pearl millet cultivars to downy mildew disease was correlated with the levels of β-1,3-glucanase in their seeds. Higher activity of the enzyme in highly resistant cultivars and lower activity in the highly susceptible ones suggested the possible use of β-1,3-glucanase as a biochemical marker for screening pearl millet cultivars for downy mildew disease. Inoculation of seedlings with the downy mildew pathogen Sclerospora graminicola resulted in increased enzyme levels in resistant cultivars. Mesocotyl and shoot regions of seedlings recorded higher levels of enzyme than the root. Isoelectric focusing revealed four basic isoforms with pI 9.6, 9.0, 8.9 and 8.2 and two acidic isoforms with pI 4.9 and 6.2 of β-1,3-glucanase in pearl millet. The pI 9.6 isoform was a major isoform of the enzyme in the pearl millet seedlings with a probable developmental function. Isoforms pI 6.2 and pI 8.2 appeared to be involved in resistance and pI 4.9 isoform seemed to be involved in pathogenesis of pearl millet-downy mildew

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen Sclerospora graminicola

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris-SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3' untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.</p

Spore cell wall components ofAspergillus niger elicit downy mildew disease resistance in pearl millet

Elicitors derived from the cell wall of fungi are shown to be active in eliciting resistance in plants against a wide range of pathogens. In the present study carbohydrate components from the autoclaved spore cell wall ofAspergillus niger were prepared as aqueous suspensions and tested for defense response in pearl millet (Pennisetum glaucum (L.) R.Br.) against the oomycetous downy mildew pathogenSclerospora graminicola (Sacc.) Schroet. The aqueous suspension derived from the spore cell wall ofA. niger was used as a seed soak treatment at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg ml−1 for time intervals of 3, 6, 9 and 12 h. The concentration of 0.5 mg ml−1 for a 6 h soaking period offered 94% seed germination and seedling vigor index increased to 1526. The seed germination and the seedling vigor were significantly higher than the untreated check. Spore cell wall suspension as seed treatment at a concentration of 0.5 mg ml−1 required a 3-day time interval to provide 67% protection against downy mildew. Histological and biochemical studies were conducted to elucidate the mechanism of defense response in treated seedlings uponS. graminicola infection. Resistance host response was detected in the form of lignin and callose deposition in the epidermal cell wall of pearl millet seedlings, which is the site ofS. graminicola infection. A time course study showed rapid and localized deposition of lignin and callose in epidermal cell wall of carbohydrate components-treated pearl millet seedling coleoptiles. Increased levels of the defense-related enzyme peroxidase were detected in the treated seedlings. Peroxidase activity in elicitor-treated samples reached a peak at 8 h post-infection, which was 45% more than in their respective uninoculated control. Characterization of peroxidase isoforms by isoelectric focusing revealed 16 different isoforms, of which pI 6.8, 7.2 and 8.7 increased in elicitor-treated samples uponS. graminicola infection

Isolation and characterisation of a NBS-LRR resistance gene analogue from pearl millet

Plant resistance (R) proteins belonging to nucleotide-binding site-leucine-rich repeat (NBS-LRR) family are mainly involved in recognition of effectors secreted by pathogens. Pearl millet [Pennisetum glaucum (L.) R.Br] is one of the most drought tolerant cereals, staple food crop of the semi-arid tropics but is highly susceptible to the downy mildew disease caused by oomycetous Sclerospora graminicola (Sacc) schroet. Earlier studies have identified several resistance gene analogues (RGAs) in pearl millet which may be involved in resistance against downy mildew. Of these, a clone RGPM213 was shown to have more than 60% identity with R-proteins coding for NBS-LRR-like protein kinase. The exact nature and function of the R-protein encoded by this gene was not known. In the present study, the cDNA of RGPM213 encompassing NBS-LRR region was inserted into an expression vector pRSET-A and transformed into BL21 E.coli cells. The expressed recombinant fusion protein with a His tag was purified using nickel affinity purification and it had a molecular weight of 35 kDa on SDS-PAGE. Immunoaffinity purification using antibodies raised against this recombinant R-protein identified two proteins of molecular weights 55 kDa and 66 kDa from pearl millet seedling extracts. Peptide mass fingerprinting of these proteins followed by homology search in database revealed similarity of the 55 kDa protein with a protein kinase from Brassica oleracia containing serine/ threonine kinase domain

Potential anti-inflammatory bioactives from medicinal plants of Western Ghats, India

Natural products have long been a thriving source for the discovery of new drugs because of their chemical diversity. With increased use of herbal remedies, traditionally used medicinal plants are receiving increased attention from scientific and pharmaceutical communities. The newer work on medicinal plants is mostly the rediscovery of traditional effects at cellular and molecular levels. Development of standardized, safe and effective herbal formulations as multi-target therapeutics and prophylaxis could be a tenable approach for the future. Hundreds of plant metabolites are reported to have many pharmacological activities although most of these reports are of academic interest and very few find entry at clinical trials. Compilation of the information would help promote wider acceptance and use of these plant based drugs in main stream of medicine. The present review is directed towards compilation of the pharmacological attributes of medicinal plants of Western Ghats, India in the drug discovery and development process as it could be a driving force to identify lead molecules providing an attractive strategy for novel and improved therapeutics