Analysis of Antimicrobial Peptide Metabolome of Bacterial Endophyte Isolated From Traditionally Used Medicinal Plant Millettia pachycarpa Benth.

Increasing prevalence of antimicrobial resistance (AMR) has posed a major health concern worldwide, and the addition of new antimicrobial agents is diminishing due to overexploitation of plants and microbial resources. Inevitably, alternative sources and new strategies are needed to find novel biomolecules to counter AMR and pandemic circumstances. The association of plants with microorganisms is one basic natural interaction that involves the exchange of biomolecules. Such a symbiotic relationship might affect the respective bio-chemical properties and production of secondary metabolites in the host and microbes. Furthermore, the discovery of taxol and taxane from an endophytic fungus, Taxomyces andreanae from Taxus wallachiana, has stimulated much research on endophytes from medicinal plants. A gram-positive endophytic bacterium, Paenibacillus peoriae IBSD35, was isolated from the stem of Millettia pachycarpa Benth. It is a rod-shaped, motile, gram-positive, and endospore-forming bacteria. It is neutralophilic as per Joint Genome Institute's (JGI) IMG system analysis. The plant was selected based on its ethnobotany history of traditional uses and highly insecticidal properties. Bioactive molecules were purified from P. peoriae IBSD35 culture broth using 70% ammonium sulfate and column chromatography techniques. The biomolecule was enriched to 151.72-fold and the yield percentage was 0.05. Peoriaerin II, a highly potent and broad-spectrum antimicrobial peptide against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 was isolated. LC-MS sequencing revealed that its N-terminal is methionine. It has four negatively charged residues (Asp + Glu) and a total number of two positively charged residues (Arg + Lys). Its molecular weight is 4,685.13 Da. It is linked to an LC-MS/MS inferred biosynthetic gene cluster with accession number A0A2S6P0H9, and blastp has shown it is 82.4% similar to fusaricidin synthetase of Paenibacillus polymyxa SC2. The 3D structure conformation of the BGC and AMP were predicted using SWISS MODEL homology modeling. Therefore, combining both genomic and proteomic results obtained from P. peoriae IBSD35, associated with M. pachycarpa Benth., will substantially increase the understanding of antimicrobial peptides and assist to uncover novel biological agents.

Bacterial rhizosphere community profile at different growth stages of Umorok (Capsicum chinense) and its response to the root exudates.

The role of microflora is an indispensable part of the living organisms. Plants actively recruit specific microbial community to establish favorable habitat with the distinct microbiome, essentially unique for each species, offering new opportunities for plant growth and productivity. Umorok, an indigenous chili variety of northeastern India, production is highly affected by various factors; therefore, rhizosphere bacteria and their relationship with the root exudates released were analyzed to demonstrate rhizosphere bacterial impact on plant growth and health. Culturable and metagenomic bacterial DNA was characterized and the chemical nature of the root exudate was analyzed using chemotaxis assay after its basic analysis in HPLC. Juvenile stage exhibited diverse bacterial species of gammaproteobacteria, alphaproteobacteria, and actinobacteria but lacked the betaproteobacteria while the microbial diversity was reduced in flowering and fruiting stages. However, every growth stage maintained a similar amount of bacterial population regardless of diversity. The population of Pseudomonas, Bacillus, and Burkholderia species was increased several folds in flowering and fruiting stage. Further, the chemotaxis assay unveiled the advantage of root exudate chemical composition for specific microbial recruitment. The chemical composition analysis of root exudates showed substantial variation in the concentration of organic acids, phenolics, and flavonoids that are favoring unique bacterial species. Thus, root exudates confer and limit the related microbial population besides typical plant-bacterial synergetic association. This study emphasized information about the type of microbial load present in each growth stage, which is essential to develop a microbial consortia package for Umorok overall crop improvement.

Synthesis and characterization of ZnO:CeO2:nanocellulose:PANI bionanocomposite. A bimodal agent for arsenic adsorption and antibacterial action.

In the present study we report the generation of a bimodal, ZnO:CeO2:nanocellulose:polyaniline bionanocomposite having an appreciable remediation efficiency for dissolved Arsenic along with a noticeable antibacterial activity. The microstructural analysis of the synthesized bionanocomposite was carried out by TEM, XRD and FTIR studies, which confirmed the incorporation of the nanoscaled ZnO and CeO2 in the polymeric nanocellulose:polyaniline matrix. The bionanocomposite exhibited a remediation efficiency above ∼95% against As under different adsorbent concentrations and pH conditions. The biosorption mechanism of As on the nanobiosorbent was found to conform to the Freundlich and Dubinin-Radushkevich isotherms. Antibacterial assays for the bionanocomposite showed a high antibacterial activity with MIC50 values of 10.6µgml(-1) against the Gram-positive Bacillus subtilis and 10.3µgml(-1) against the Gram-negative Escherichia coli. Thus, the bionanocomposite shall be of high interest as a novel and sustainable matrix for the design of coats/devices that effectuate arsenic adsorption and microbial control, to generate contaminant free potable water.

Mitochondrial DNA-based genetic diversity of Anopheles nivipes in North East India.

Malaria is a major public health problem in north-east India mainly transmitted by Anopheles baimai and An. minimus while Anopheles nivipes plays an important supportive role. The genetic diversity of An. nivipes in north-east India was investigated by employing two mitochondrial DNA markers namely NADH dehydrogenase 5 (ND5) and cytochrome oxidase sub unit II (COII). High genetic diversity in An. nivipes was observed by the detection of 16 haplotypes among 30 sequences of ND5 gene and 29 haplotypes among 29 COII sequences. Anopheles nivipes of north-east India was significantly differentiated genetically with that of neighboring South-east Asian An. nivipes as revealed by pairwise FST values of 0.127 (p < 0.01) and 0.044 (p < 0.001) for ND5 and COII genes, respectively, suggesting geographical barriers to gene flow in this species between the two geographical areas resulting in significant population structuring.

Unveiling the optimal parameters for cellulolytic characteristics of Talaromyces verruculosus SGMNPf3 and its secretory enzymes.

AIMS: Elucidation of different physico-chemical parameters and the secretory enzymes released by Talaromyces verruculosus SGMNPf3 during cellulosic biomass degradation. METHODS AND RESULTS: We determined the optimal pH, temperature and time course parameters for the efficient degradation of different natural and commercial cellulosic substrates by T. verruculosus SGMNPf3, previously isolated from a forest soil. The optimal growth of the fungus and production of its cellulases were obtained when the culture condition was maintained at pH 3·3 and temperature 30°C. Activity of the crude cellulases was maximum at 60°C. Activity of cellulase enzymes produced on natural cellulose substrates was higher than that on commercial cellulose substrates. A continuous increase in cellulase activity at different time points indicated no apparent end product inhibition. This might be attributed to the high individual cellulases, notably ß-glucosidase (316·1 µmol g(-1) ) production. Zymogram of extracellular crude proteins showed two dominant extracellular protein bands of molecular weight 72·3 and 61·4 kDa, indicating their cellulolytic nature. MALDI-TOF and LC-MS/MS analysis of the 2DE spots also identified several enzymes including ß-glucosidase involved in the process of cellulose degradation. CONCLUSIONS: Based on its optimal parameters for cellulolytic activities, we suggest that the fungus is acido-mesophilic. There was apparently no end-product inhibition of the cellulase activity and this is attributed to the ability of the fungus to produce sufficient ß-glucosidase. The dominant proteins secreted by the fungus were confirmed to be cellulases. SIGNIFICANCE AND IMPACT OF THE STUDY: The high individual cellulase activities, better cellulase production on natural substrates and apparent absence of end-product inhibition are characteristics of T. verruculosus SGMNPf3 for use in harvesting naturally endowed energy in cellulosic biomass.

Molecular analysis of drought tolerance in tea by cDNA-AFLP based transcript profiling.

A cDNA-AFLP approach was used to identify transcript and/or genes specifically expressed in response to drought in tea. Drought was artificially induced and whole genome transcript profiling was done at three different stages-6 days before wilting, 3 days before wilting and at wilting stage of both tolerant and susceptible cultivars, and genetic differences was thus visualized as polymorphisms in the transcriptome. The cDNA-AFLP technique allowed genes and transcripts to be identified in the tolerant genotype (TV-23) whose expression is responsive to drought stress. The cluster analysis revealed two types of clustering-type I separated the tolerant and susceptible cultivar, whereas type II separated the time point of sample and this may be grouped as early and late responsive transcripts. 108 transcript derived fragments were identified as differentially expressed in tolerant genotypes of which 89 sequences could be obtained. Fifty-nine of them showed homology in the public databases. Functional ontology showed genes related to carbohydrate metabolism, response to stress, protein modification process and translation. Cluster I includes five fragments and cluster II includes 25 fragments. Other genes strongly expressed in response to drought in tolerant genotype would help us in identifying and determining the genetic basis of mechanisms involved in conferring drought tolerance in tea.

Identification of drought tolerant progenies in tea by gene expression analysis.

Understanding the genes that govern tea plant (Camellia sinensis) architecture and response to drought stress is urgently needed to enhance breeding in tea with improved water use efficiency. Field drought is a slow mechanism and the plants go through an adaptive process in contrast to the drastic changes of rapid dehydration in case of controlled experiments. We identified a set of drought responsive genes under controlled condition using SSH, and validated the identified genes and their pattern of expression under field drought condition. The study was at three stages of water deficit stress viz., before wilting, wilting and recovery, which revealed a set of genes with higher expression at before wilting stage including dehydrin, abscissic acid ripening protein, glutathione peroxidase, cinnamoyl CoA reductase, calmodulin binding protein. The higher expression of these genes was related with increase tolerance character of DT/TS-463 before wilting, these five tolerant progenies could withstand drought stress and thus are candidates for breeding. We observed that physiological parameter like water use efficiency formed a close group with genes such as calmodulin related, DRM3, hexose transporter, hydrogen peroxide induced protein, ACC oxidase, lipase, ethylene responsive transcription factor and diaminopimelate decarboxylase, during wilting point. Our data provides valuable information for the gene components and the dynamics of gene expression in second and third leaf against drought stress in tea, which could be regarded as candidate targets potentially associated with drought tolerance. We propose that the identified five tolerant progenies on the basis of their drought tolerance can thus be utilised for future breeding programmes.

Spatial distribution and molecular characterization of Anopheles nivipes and Anopheles philippinensis (Diptera: Culicidae) in north-east India.

Anopheles philippinensis-nivipes species of mosquitoes are considered secondary malaria vectors in the north-east India. However, difficulty in separating An. philippinensis and An. nivipes accurately on the basis of morphological identification, due to the overlapping female wing characters, has led to the paucity of information on their spatial distribution and vectorial importance, essential for understanding malaria disease epidemiology in areas under their influence. In view of this a study was undertaken to generate authentic information on distribution of An. philippinensis and An. nivipes in the north-east India using molecular tools. Between the two, An. nivipes was recorded as the dominant species in Assam, and Nagaland states whereas An. philippinensis was found predominant in the state of Mizoram and Arunachal Pradesh. Species differentiation based on female wing characters proved wrong in about 74% specimens of An. philippinensis but found accurate in case of An. nivipes. No intra-specific variation in second internal transcribed spacer (ITS2) region of ribosomal DNA(r-DNA) in either An. nivipes or An. philippinensis from different geographical areas of north-east India was noted.

Understanding Darjeeling tea flavour on a molecular basis.

Darjeeling teas are the highest grown teas in the world and preferred for its flavour, aroma and quality. Apart from the genetic makeup of the plant, earlier reports suggest that insect infestation, particularly jassids and thrips triggers the aroma and flavour formation in Darjeeling tea. The present work encompasses the identification of the genes/transcriptomes responsible for the typical flavour of Darjeeling tea, besides understanding the role of jassids and thrips in particular, in producing the best cup character and quality. The quantitative real time PCR analysis was based on a suppression subtractive hybridisation forward library of B157 (tea clone infested with thrips), providing us transcripts related to aroma and flavour formation. We observed the expression of genes like leucine zipper, ntd, nced, geraniol synthase, raffinose synthase, trehalose synthase, amylase, farnesyl transferase, catalase, methyl transferase, linalool synthase, peroxidases, elicitor responsive proteins, linamarase, nerolidol linalool synthase 2, 12-oxophytodienoate reductase, glucosidase, MYB transcription factor, and alcohol dehydrogenase, highly regulated due to insect infestation, manufacturing stresses and mechanical injury. The first report on gene expression dynamics in thrips infested Darjeeling tea leaves can be extrapolated with increase in volatiles which is responsible for enhancing the quality of Darjeeling tea, specially the flavour and aroma of the infusion. We hope to model these responses in order to understand the molecular changes that occur during Darjeeling tea flavour formation.

Molecular evidence for the involvement of Anopheles nivipes (Diptera: Culicidae) in the transmission of Plasmodium falciparum in north-eastern India.

The Anopheles philippinensis and An. nivipes mosquitoes that form part of the An. annularis species group are morphologically very similar and difficult to differentiate as adults. In consequence, researchers generally refer to all field-collected individuals of these species simply as the An. philippinensis-nivipes species complex. Although this species complex is understood to play a role in the transmission of parasites causing human malaria in north-eastern India, the identity of the exact species involved, and the relative importance of the local An. philippinensis and An. nivipes, are far from clear. To settle this issue, house-frequenting female adults of the An. philippinensis-nivipes species complex were collected from 23 localities in the six north-eastern states, identified to species (using an allele-specific PCR to explore the insects' ribosomal-DNA internal-transcribed-spacer-2 sequences) and checked for Plasmodium DNA (using a nested PCR based on the 18S subunit of the parasite's ribosomal DNA). Of the 337 females of the An. philippinensis-nivipes species complex that were investigated, 275 were identified as An. nivipes and 62 as An. philippinensis. Malarial infection was detected in the heads/thoraces of just two specimens, with P. falciparum DNA detected in one An. nivipes from Nagaland state and one An. nivipes from Assam state. These results provide unambiguous evidence of the presence of both An. philippinensis and An. nivipes in the north-eastern region of India and the involvement of An. nivipes in transmitting P. falciparum in this area.

Variability in symbiotic effectiveness of native Rhizobia in acid stress.

In vitro experiments had been conducted to assess the effect of pH on different native isolates of Rhizobium from the lower Brahmaputra valley region of Assam, India. The growth rate of all the Rhizobium isolates were compared growing at three different pH levels viz. 4.5, 5.5 and 6.5. All the slow-growing Rhizobium isolates (AR1, BR5, BR8, BR12, GR13, AR10, BR13, GM16, GR21) were showed better growth at all the pH levels, whereas three fast-growing Rhizobium isolates (PR7, PR12 and PR16) failed to grow at pH 4.5 and 5.5 and could show growth only at pH 6.5. Experiments were also conducted in vitro to determine the symbiotic effectiveness of all the Rhizobium isolates with test legume, green gram (Phaseolus aureus Roxb.) at three different pH levels for 42 days and significant differences were observed on nodulation, nodule dry weight and nitrogenase activity. The acid-tolerant isolates could be used as bioinoculant for enhancement of growth of leguminous plants in the acid soils.

Prevalence of anaemia in pregnant & lactating women in India.

BACKGROUND & OBJECTIVES: The prevalence of anaemia during pregnancy and lactation was significantly lower in the National Family Health Survey 1998-1999 (NFHS-2), using the hemocue method for haemoglobin estimation compared to earlier surveys. The present study selected seven States and used the same districts and villages studied in the NFHS-2, to see if the reported reduction in prevalence of anaemia was due to health and nutrition inputs and/or due to a different method for haemoglobin estimation. METHODS: A total of 1,751 women (1,148 pregnant and 603 lactating- exclusively breastfeeding up to 3 months of age), from seven States- Himachal Pradesh and Haryana in north; Assam and Orissa in east; Kerala and Tamil Nadu in south and Madhya Pradesh in central India, were selected. Haemoglobin was estimated by the cyanmethaemoglobin method, so that comparison was possible with earlier studies. Data on socio-demographic characteristics, pregnancy, nutritional status and dietary intakes were collected. RESULTS: Prevalence as well as severity of anaemia was significantly higher in the present study as compared to the NFHS-2 study data. The difference could be due to haemocue method, which gives higher haemoglobin values. The contributing factors found on multiple regression analysis for anaemia in pregnancy and lactation were: literacy, occupation and standard living index of the study women; their awareness about anaemia, its prevention by regular consumption of ironfolate tablets and increase in food intake. Maternal height, age of marriage, parity and foetal loss also contributed to haemoglobin level. There were interstate differences; lower fertility, higher literacy and better diet was observed in Himachal Pradesh as compared to Haryana. The literacy and nutritional status of women in Tamil Nadu was lower than Kerala. The remaining 3 states had poor fertility, lower social living index and nutritional status with >90 per cent women being anaemic in pregnancy and lactation. Low prevalence of severe anaemia in Orissa as compared to Assam was due to availability and consumption of iron folate tablets. The antenatal services in the first trimester and checkup by a doctor, along with availability and consumption of iron folate tablets over 3 months in all the States influenced haemoglobin levels. INTERPRETATION & CONCLUSION: Despite the measures taken to control anaemia in pregnancy and lactation in the last two decades, the severity of nutritional anaemia continues to remain a public health issue of great magnitude, suggesting that these measures have been largely ineffective. The present findings also showed interstate differences particularly in fertility, women education, nutrition status and occupation; availability of antenatal services and iron folate tablets as possible factors responsible for differences in prevalence of anaemia.

Phytopesticidal and repellent efficacy of Litsea salicifolia (Lauraceae) against Aedes aegypti and Culex quinquefasciatus.

Litsea salicifolia is one of the many plants used as phytopesticide, traditionally by various tribes of Assam. Of the five extracts of L. salicifolia tested for the bioactivity against Aedes aegypti and Culex quinquefasciatus, the aqueous extract was more effective compared to other extracts exhibiting bioactivity at 72 ppm against A. aegypti. The hexane extract (2000 ppm) exhibited 70% repellent activity for 3 hr against A. aegypti and 46% activity for 3 hr against C. quinquefasciatus. This is the first report on the insecticidal properties of L. salicifolia which can be further developed as an eco-friendly biopesticides of the future.