Harischandra Sripathy Prakash

Transcriptome changes in foxtail millet genotypes at high salinity: Identification and characterization of a PHGPX gene specifically up-regulated by NaCl in a salt-tolerant line

Using a macro array filter with 711 cDNA inserts representing 620 unigenes selected from a barley EST collection, we identified transcripts differentially expressed in salt (NaCl)-treated tolerant (cv. Prasad) and sensitive (cv. Lepakshi) seedlings of foxtail millet (Setaria italica L.). Transcripts of hydrogen peroxide scavenging enzymes such as phospholipid hydroperoxide glutathione peroxidase (PHGPX), ascorbate peroxidase (APX) and catalase 1 (CAT1) in addition to some genes of cellular metabolism were found to be especially up-regulated at high salinity in the tolerant line. To analyse this process at the protein level we examined protein expression patterns under various stress conditions. A 25 kD protein with a pI of 4.8 was found to be induced prominently under high salt concentrations (250 mmol/L). This salt-induced 25 kD protein has been purified and identified by peptide sequencing as PHGPX protein. The increase of the PHGPX protein level under salt stress in the tolerant line parallels the PHGPX mRNA results of array analysis but was more pronounced. We cloned and characterized the foxtail millet PHGPX cDNA, which shows 85% and 95% homology at the DNA and protein level, respectively, to one stress-induced member of the small barley PHGPX gene family encoding non-selenium glutathione peroxidases. As shown by Southern blot analysis, a small family of PHGPX genes exists in foxtail millet, too. The specific expression pattern of the PHGPX gene in salt-induced tolerant millet seedlings suggests that its product plays an important role in the defense reaction against salt-induced oxidative damage and that the characterized glutathione peroxidase is one of the components conferring resistance against salt to the tolerant foxtail millet cultivar.

Endophytic Fungal Diversity in Medicinal Plants of Western Ghats, India

Endophytes constitute an important component of microbial diversity, and in the present investigation, seven plant species with rich ethnobotanical uses representing six families were analyzed for the presence of endophytic fungi from their natural habitats during monsoon (May/June) and winter (November/December) seasons of 2007. Fungal endophytes were isolated from healthy plant parts such as stem, root, rhizome, and inflorescence employing standard isolation methods. One thousand five hundred and twenty-nine fungal isolates were obtained from 5200 fragments. Stem fragments harbored more endophytes (80.37%) than roots (19.22%). 31 fungal taxa comprised of coelomycetes (65%), hyphomycetes (32%), and ascomycetes (3%). Fusarium, Acremonium, Colletotrichum, Chaetomium, Myrothecium, Phomopsis, and Pestalotiopsis spp. were commonly isolated. Diversity indices differed significantly between the seasons (). Species richness was greater for monsoon isolations than winter. Host specificity was observed for few fungal endophytes. UPGMA cluster analysis grouped the endophytes into distinct clusters on the basis of genetic distance. This study is the first report on the diversity and host-specificity of endophytic fungal taxa were from the semi evergreen forest type in Talacauvery subcluster of Western Ghats.

Fungal endophytes from the three-leaved caper, Crataeva magna (Lour.) DC. (Capparidaceae)

Fungal endophytes were isolated from Crataeva magna, a medicinal plant growing along the streams and rivers, constituting riparian vegetation in Karnataka, southern India. Fresh bark and twig pieces were used for the isolation using standard methods. Ninety-six endophytic fungal isolates were isolated from 800 bark and twig segments. Mitosporic fungi represented as a major group (85%) followed by zygomycetes (10%) and ascomycetes (5%). Bark samples contained more endophytes than twig samples. Verticillium, Nigrospora oryzae and Fusarium verticilloides were the dominant fungal endophytes.

Dravya, a product of seaweed extract (Sargassum wightii), induces resistance in cotton againstXanthomonas campestris pv.malsvacearum

Dravya, a commercially developed aqueous seaweed extract, was evaluated for its effect on the expression of symptoms of bacterial blight caused byXanthomonas campestris pv.malvacearum (E.F. Smith) Dye in cotton. Seed soaking with Dravya (1:500) followed by foliar spray thrice at intervals of 10 days (10, 20, 30 days after sowing) resulted in a reduction in blight incidence on plants by 66%, 70% and 74% as determined 40, 60 and 80 days, respectively, after sowing. Induction of systemic resistance was associated with increases in plant height, total number of bolls formed, boll weight, stem girth, chlorophyll content, total phenols and peroxidase activity, which intimates that Dravya could be used as an ecofriendly potential input in the integrated management of bacterial-blight of cotton.

Molecular detection and characterisation ofFusarium verticillioides in maize (Zea mays. L) grown in southern India

Fusarium verticillioides causes a wide range of diseases in maize and also produces important mycotoxins like fumonisins which are the major threats to animals and humans. In the present study 14 different isolates ofF. verticillioides were collected from maize seeds represented from different parts of Southern India and the morphological identity was further confirmed by PCR. The isolates were subjected to morphological, biochemical and molecular characterisation. Results showed that the molecular method is the good tool to discriminateF. verticillioides into two different major groups.

Detection and identification of the blackeye cowpea mosaic strain of Bean common mosaic virus in seeds of cowpea from southern India

In different legume-growing regions of India, a total of 136 seed samples of cowpea (Vigna unguiculata (L.) Walp.) were collected and tested for the presence of the blackeye cowpea mosaic strain of Bean common mosaic virus, Cowpea aphid-borne mosaic virus, Cowpea mosaic virus, Cucumber mosaic virus and Bean yellow mosaic virus using growing-on test, enzyme linked immunosorbent assay, differential host test, electron microscopy and immuno-capture reverse transcription polymerase chain reaction. Among the 136 seedlots tested, 43 cowpea seedlots were found to be infected with BCMV-BlCM. The identity of three of these isolates as BCMV-BlCM was further supported by nucleotide sequencing in the 3′ region of the genome. The incidence of seeds carrying transmissible virus ranged from 0.67% to 13.49%. In most cases, only symptomatic seedlings in a growing-on test were found infected with the virus.

Inhibition of TMV multiplication by siRNA constructs against TOM1 and TOM3 genes of Capsicum annuum

The host proteins TOM1 and TOM3 associated with tonoplast membrane are shown to be required for efficient multiplication of Tobamoviruses. In this study, homologous of TOM1 and TOM3 genes were identified in pepper (Capsicum annuum) using specific primers. Their gene sequences have similarity to Nicotiana tabacum NtTOM1 and NtTOM3. Sequence alignment showed that CaTOM1 and CaTOM3 are closely related to TOM1 and TOM3 of N. tabacum and Solanum lycopersicum with 90% and 70% nucleotide sequence identities, respectively. RNA interference approach was used to suppress the TOM1 and TOM3 gene expression which in turn prevented Tobacco mosaic virus replication in tobacco. Nicotiana plants agro-infiltrated with siRNA constructs of TOM1 or TOM3 showed no mosaic or necrotic infection symptoms upon inoculation with TMV. The results indicated that silencing of TOM1 and TOM3 of pepper using the siRNA constructs is an efficient method for generating TMV-resistant plants.

Actinomycete endophytes from the ethno medicinal plants of southern India: antioxidant activity and characterization studies

Abstract Actinomycete endophytes (135) were isolated from Cajanus lineatus W. & A. (Maesen), Leucas ciliata Benth., Rauwolfia densiflora Benth. Ex. Hook. f. and Gomphostemma heyneanum Wall., four ethnomedicinal species of the southern region of the Western Ghats, a hot spot of biodiversity. The isolates were categorized into three supra generic groups on the basis of spore or aerial mycelial characters and confirmed by the partial 16S rRNA gene sequences. The actinomycete strains were subjected to fermentation and the ethyl acetate extracts were screened for the antioxidant assays. Streptomyces globosus (JQ926176) and Arthrobacter sp. (JQ926171) isolated from the stem fragments of R. densiflora and L. ciliata showed remarkable dose-dependent antioxidant activity in the in vitro assays. Among the screened extracts, S. globosus (IC50 88.2±1.03 μg/ml) and Arthrobacter sp. (IC50 = 97.6±1.97 μg/ml) exhibited high DPPH activity. S. globosus and Arthrobacter extracts contained a total antioxidant activity of 52.44±2.03 μM Fe (II)/g and 40.44±1.97 μM Fe (II)/g respectively. A significant correlation between the total phenolic content and total reducing power of S. globosus (R2=0.983) and Arthrobacter sp. (R2=0.987) extracts were noted. GC-MS characterization of the extracts revealed several compounds with antioxidant activity. S. globosus, S. hypolithicus, P. citrea and P. minatonensis are reported as endophytes for the first time in the ethnomedicinal plants.

Genetic diversity and antimicrobial activity of endophytic Myrothecium spp. isolated from Calophyllum apetalum and Garcinia morella.

Calophyllumapetalum and Garciniamorella, medicinal plants are endemic to Western Ghats, Karnataka, India. Sixteen Myrothecium isolates were obtained from the tissues of bark and twigs of these plants. The purpose of this study was to explore the antimicrobial activity and genetic variability of the endophytic Myrothecium isolates. The antimicrobial activity as well as the genetic diversity of endophytic Myrothecium species was investigated through RAPD, ISSR and ITS sequence analysis. Myrothecium isolates were genotypically compared by RAPD and ISSR techniques, 510 and 189 reproducible polymorphic bands were obtained using 20 RAPD and ten ISSR primers respectively. The isolates grouped into four main clades and subgroups using unweighted pair group method with arithmetic mean cluster analysis. rDNA ITS sequence analysis presented better resolution for characterising the isolates of Myrothecium spp. The clustering patterns of the isolates were almost similar when compared with RAPD and ISSR dendograms. The results signify that RAPD, ISSR and ITS analysis can be employed to distinguish the genetic diversity of the Myrothecium species. The endophytic and pathogenic strains were compared by maximum parsimony, maximum likelihood and neighbour joining methods. One isolate (JX862206) amongst the 16 Myrothecium isolates exhibited potent antibacterial and as well as anti-Candida activity.

Bioactive potential of endophytic Myrothecium sp. isolate M1-CA-102, associated with Calophyllum apetalum

Abstract Context: Endophytes colonizing medicinal plants are diverse, constituting a rich bioresource for novel natural products. Objective: Myrothecium sp. isolate M1-CA-102 was the most promising among the 16 Myrothecium isolates screened. The bioactive potential of the crude extract from the Calophyllum apetalum Willd. endophytic Myrothecium sp. (Alb. & Schwein.) Ditmar (Incertae sedis) isolate M1-CA-102 and its thin layer chromatography (TLC) fractions were screened based on antioxidant, anti-inflammatory, antimicrobial activities, and cytotoxicity. Materials and methods: The antioxidant activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical scavenging capacities. Further, 15-lipoxygenase (15-LOX) and human cyclooxygenase-2 (COX-2) inhibition were assessed at different concentrations (25, 50, and 100 μg/mL for the crude extract, 5, 25, and 50 μg/mL for the TLC fractions). DNA-nicking assay as an indicator of the capacity of extracts to scavenge hydroxyl radical was recorded at a concentration of 50 μg/mL. Cell cytotoxicity was recorded by colorimetric 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antibacterial (Bacillus subtilis) and anti-Candida (Candida albicans) assays were performed by the microdilution method. Results: The DPPH and ABTS IC50 values of M1-CA-102 extract were 10 and 6 μg/mL compared with 6.1 and 7.03 μg/mL for the positive control quercetin. The cytotoxicity IC50 value of M1-CA-102 extract was 37 μg/mL, while the M-I TLC fraction was 21 μg/mL. The M1-CA-102 extract gave an IC50 value of 58 and 8 μg/mL for 15-LOX and COX-2, respectively. The MIC values for antimicrobial activity for M1-CA-102 extract ranged from 35 to 54 μg/mL, while for the TLC fractions, it ranged from 91 to 515 μg/mL. Conclusion: The results indicate that Myrothecium M1-CA-102 isolated from C. apetalum is a potential source of natural metabolites of pharmaceutical importance.