Harnessing fungal endophytes for natural management: a biocontrol perspective.

In the ever-evolving realm of agriculture, the convoluted interaction between plants and microorganisms have assumed paramount significance. Fungal endophytes, once perceived as mere bystanders within plant tissues, have now emerged as dynamic defenders of plant health. This comprehensive review delves into the captivating world of fungal endophytes and their multifaceted biocontrol mechanisms. Exploring their unique ability to coexist with their plant hosts, fungal endophytes have unlocked a treasure trove of biological weaponry to fend off pathogens and enhance plant resilience. From the synthesis of bioactive secondary metabolites to intricate signaling pathways these silent allies are masters of biological warfare. The world of fungal endophytes is quite fascinating as they engage in a delicate dance with the plant immune system, orchestrating a symphony of defense that challenges traditional notions of plant-pathogen interactions. The journey through the various mechanisms employed by these enigmatic endophytes to combat diseases, will lead to revelational understanding of sustainable agriculture. The review delves into cutting-edge research and promising prospects, shedding light on how fungal endophytes hold the key to biocontrol and the reduction of chemical inputs in agriculture. Their ecological significance, potential for bioprospecting and avenues for future research are also explored. This exploration of the biocontrol mechanisms of fungal endophytes promise not only to enrich our comprehension of plant-microbe relationships but also, to shape the future of sustainable and ecofriendly agricultural practices. In this intricate web of life, fungal endophytes are indeed the unsung heroes, silently guarding our crops and illuminating a path towards a greener, healthier tomorrow.

Biodiversity and Functional Attributes of Rhizospheric Microbiomes: Potential Tools for Sustainable Agriculture.

The quest for increasing agricultural yield due to increasing population pressure and demands for healthy food has inevitably led to the indiscriminate use of chemical fertilizers. On the contrary, the exposure of the crops to abiotic stress and biotic stress interferes with crop growth further hindering the productivity. Sustainable agricultural practices are of major importance to enhance production and feed the rising population. The use of plant growth promoting (PGP) rhizospheric microbes is emerging as an efficient approach to ameliorate global dependence on chemicals, improve stress tolerance of plants, boost up growth and ensure food security. Rhizosphere associated microbiomes promote the growth by enhancing the uptake of the nutrients, producing plant growth regulators, iron chelating complexes, shaping the root system under stress conditions and decreasing the levels of inhibitory ethylene concentrations and protecting plants from oxidative stress. Plant growth-promoting rhizospheric microbes belong to diverse range of genera including Acinetobacter, Achromobacter, Aspergillus, Bacillus, Burkholderia, Flavobacterium, Klebsiella, Micrococcus, Penicillium, Pseudomonas, Serratia and Trichoderma. Plant growth promoting microbes are an interesting aspect of research for scientific community and a number of formulations of beneficial microbes are also commercially available. Thus, recent progress in our understanding on rhizospheric microbiomes along with their major roles and mechanisms of action under natural and stressful conditions should facilitate their application as a reliable component in the management of sustainable agricultural system. This review highlights the diversity of plant growth promoting rhizospheric microbes, their mechanisms of plant growth promotion, their role under biotic and abiotic stress and status of biofertilizers. The article further focuses on the role of omics approaches in plant growth promoting rhizospheric microbes and draft genome of PGP microbes.

Purified cellobiose dehydrogenase of Termitomyces sp. OE147 fuels cellulose degradation resulting in the release of reducing sugars.

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and ß-glucosidases when cultivated on cellulose. In order to investigate its effect on cellulose, a highly purified preparation of CDH was obtained from the culture supernatant of the fungus cultivated on cellulose. A combination of ultrafiltration, ion-exchange and gel-filtration chromatography was used to purify CDH by ∼172-fold to a high specific activity of ∼324 U/mg protein on lactose which was used for routine measurement of enzyme activity. The enzyme displayed a pH optimum of 5.0 and stability between pH 5.0 and 8.0 with maximum catalytic efficiency (kcat/Km) of 397 mM-1 s-1 on cellobiose. Incubation of microcrystalline cellulose with the purified CDH led to production of reducing sugars which was accelerated by the addition of FeCl3 during the early stages of incubation. A mass spectrometric analysis revealed fragmentation products of cellulose which were concluded to be cellodextrins, sugars, and corresponding aldonic acids suggesting that CDH can release reducing sugars in the absence of externally added lytic polysaccharide monooxygenases. Polymerized products of glucose were also detected at low intensity.

Next generation sequencing and microbiome's taxonomical characterization of frozen soil of north western Himalayas of Jammu and Kashmir, India

BACKGROUND: Traditionally, microbial genome sequencing has been restrained to the species grown in pure culture. The development of culture-independent techniques over the last decade allows scientists to sequence microbial communities directly from environmental samples. Metagenomics is the study of complex genome by the isolation of DNA of the whole community. Next generation sequencing (NGS) of metagenomic DNA gives information about the microbial and taxonomical characterization of a particular niche. The objective of the present research is to study the microbial and taxonomical characterization of the metagenomic DNA, isolated from the frozen soil sample of a glacier in the north western Himalayas through NGS. RESULTS: The glacier community comprised of 16 phyla with the representation of members belonging to Proteobacteria and Acidobacteria. The number of genes annotated through the Kyoto Encyclopedia of Genes and Genomes (KEGG), GO, Pfam, Clusters of Orthologous Groups of proteins (COGs), and FIG databases were generated by COGNIZER. The annotation of genes assigned in each group from the metagenomics data through COG database and the number of genes annotated in different pathways through KEGG database were reported. CONCLUSION: Results indicate that the glacier soil taken in the present study, harbors taxonomically and metabolically diverse communities. The major bacterial group present in the niche is Proteobacteria followed by Acidobacteria, and Actinobacteria, etc. Different genes were annotated through COG and KEGG databases that integrate genomic, chemical, and systemic functional information.

A hydrolase with esterase activity expressed from a fosmid gene bank prepared from DNA of a North West Himalayan glacier frozen soil sample.

Screening of 20,000 clones of a fosmid gene bank, constructed from DNA extracted from North West Himalaya (NWH) glacier soil sample, using functional approach identified 10 esterase/lipase-producing clones. Of these, a clone designated pFG43 with an insert size of 45 kb which produced the highest concentration of enzyme (467.43 U/mg) was sequenced. Clone pFG43 contained 61 open reading frames (ORF) and of these an ORF of 1155 bp designated ME-003, was found to be closely related to a hydrolase from Acidobacteria sps (77% sequence identity and E value = 1e-164) and subsequently identified as a putative cocaine esterase. ORF ME-003 was amplified and sub-cloned using a TA vector system into E. coli (DH5α). The purified recombinant enzyme with a molecular weight of 43 kDa had optimal activity at 40 °C, pH 6 and the highest activity with shorter chain fatty acids than with higher chain length fatty acids. There is insignificant effect of inhibitors on the enzyme activity of ME-003, except PMSF which completely inhibited its activity. ME-003 activity was also inhibited in the presence of copper oxide but remained stable in presence of other metal ions. The enzyme activity was also inhibited in the presence of organic solvents; however, in the presence of 10% isopropanol, 12% of enzymatic activity was retained. Among various detergents, SDS completely inhibited enzymatic activity. The recombinant enzyme also shows enantio-specific activity against the racemic drug intermediates/precursors and exhibited 90% ee against racemic 1-phenyl ethanol and fluoxetine.

Cloning and expression analysis of chalcone synthase gene from Coleus forskohlii.

Flavonoids are an important class of secondary metabolites that play various roles in plants such as mediating defense, floral pigmentation and plant-microbe interaction. Flavonoids are also known to possess antioxidant and antimicrobial activities. Coleus forskohlii (Willd.) Briq. (Lamiaceae) is an important medicinal herb with a diverse metabolic profile, including production of a flavonoid, genkwanin. However, components of the flavonoid pathway have not yet been studied in this plant. Chalcone synthase (CHS) catalyses the first committed step of flavonoid biosynthetic pathway. Full-length cDNA, showing homology with plant CHS gene was isolated from leaves of C. forskohlii and named CfCHS (GenBank accession no. KF643243). Theoretical translation of CfCHS nucleotide sequence shows that it encodes a protein of 391 amino acids with a molecular weight of 42.75 kDa and pI 6.57. Expression analysis of CfCHS in different tissues and elicitor treatments showed that methyl jasmonate (MeJA) strongly induced its expression. Total flavonoids content and antioxidant activity of C. forskohlii also got enhanced in response to MeJA, which correlated with increased CfCHS expression. Induction of CfCHS by MeJA suggest its involvement in production of flavonoids, providing protection from microbes during herbivory or mechanical wounding. Further, our in silico predictions and experimental data suggested that CfCHS may be posttranscriptionally regulated by miR34.

Evaluation of cellobiose dehydrogenase and laccase containing culture fluids of Termitomyces sp. OE147 for degradation of Reactive blue 21.

This study evaluates culture filtrate, rich in cellobiose dehydrogenase and laccases, of Termitomyces sp. OE 147, in decolouration and degradation of Reactive blue (RB) 21. About 35% decolouration was achieved at low volumes of the culture supernatant without addition of external redox mediators. An optimized dye to culture fluid ratio (75 ppm: 0.1 ml) at a pH of 4-5 resulted in removal of colour by 60%. The degradation products of RB21 were analysed by Electron Spray Ionization-Mass Spectrometry and several small molecules (of m/z 106-199) were detected. These were concluded to be o-Xylene, 2,3-Dihydro-1H-isoindole, Isoindole-1,3-dione, 2,Benzenesulfonyl-ethanol, (4-Hydroxy-phenyl)-sulfamic acid, 2,3-Dihydro-1H-isoindole-5-sulfonic acid and proposed to result from joint action of cellobiose dehydrogenase, laccase, peroxidases and unidentified oxidoreductases present in the culture fluids. Based on the products formed and the known reactions of these enzymes, a degradation pathway was proposed for RB21. The culture fluid was also effective in decolouration (by about 50%) and detoxification (by ∼25%) of the combined effluent collected from a local mill indicating a treatment process that bypasses use of H2O2 and toxic mediators.

Plant Omics: Isolation, Identification, and Expression Analysis of Cytochrome P450 Gene Sequences from Coleus forskohlii.

The omics analyses of plants and the agrigenomics field offer the opportunity to better characterize our ecosystems. In this context, characterization of cytochrome P450 genes (CYP450s), which constitute one of the largest gene families in plants, is important. They play vital roles in biosynthesis of secondary metabolites, phytohormones as well as in detoxification of harmful chemicals. Tuberous roots of Coleus forskohlii accumulate forskolin, a potent and reversible activator of adenylate cyclase, as well as other related diterpenoids. Coleus forskohlii is also known to produce rosmarinic acid, genkwanin (7-O-methylapigenin), and guaiacol glycerin. We report here the isolation of CYP450s from C. forskohlii, expression profiling of CYP450s in different tissues, and how different elicitors/stresses regulate the expression of different CYP450 sequences. Degenerate primers, designed from the conserved regions of CYP450s, were used to amplify fragments from cDNA of C. forskohlii and a library was prepared. Sequences homologous to CYP450s were assembled into seven distinct gene fragments (CfP450C1-C7), belonging to seven CYP450 families. Expression profiling of CYP450s showed that the transcripts of CfP450C1, CfP450C4, CfP450C5, CfP450C6, and CfP450C7 were prominent in aerial tissues (flower, young leaf, and mature leaf), whereas expression of CfP450C3 was dominant in root and root tip. CfP450C2 showed higher expression in flowers and roots as compared to other tissues. Expression profiles of CYP450s, in response to different stresses (abscisic acid, methyl jasmonate, salicylic acid, 2, 4-dichloro-phenoxyacetic acid, UVA, and wounding) were also studied. This study has isolated CYP450s from C. forskohlii, and will help to understand their regulation as well as their functions. This is the first report on the isolation and expression analysis of CYP450s from this herb.

Therapeutic effects of R8, a semi-synthetic analogue of Vasicine, on murine model of allergic airway inflammation via STAT6 inhibition.

This is a follow-up study of our previous work in which we screened a series of Vasicine analogues for their anti-inflammatory activity in a preventive OVA induced murine model of asthma. The study demonstrated that R8, one of the analogues, significantly suppressed the Th2 cytokine production and eosinophil recruitment to the airways. In the present study, we have been using two standard experimental murine models of asthma, where the mice were treated with R8 either during (preventive use) or after (therapeutic use) the development of asthma features. In the preventive model, R8 reduced inflammatory cell infiltration to the airways, OVA specific IgE and Th2 cytokine production. Also, the R8 treatment in the therapeutic model decreased methacholine induced AHR, Th2 cytokine release, serum IgE levels, infiltration of inflammatory cells into the airways, phosphorylation of STAT6 and expression of GATA3. Moreover, R8 not only reduced goblet cell metaplasia in asthmatic mice but also reduced IL-4 induced Muc5AC gene expression in human alveolar basal epithelial cells. Further, R8 attenuated IL-4 induced differentiation of murine splenocytes into Th2 cells in vitro. So, we may deduce that R8 treatment profoundly reduced asthma features by attenuating the differentiation of T cells into Th2 cells by interfering with the binding of IL-4 to its receptor in turn decreasing the phosphorylation of STAT6 and expression of GATA3 in murine model of asthma. These preclinical findings suggest a possible therapeutic role of R8 in allergic asthma.

Ethanolic extract of Alternanthera sessilis (AS-1) inhibits IgE-mediated allergic response in RBL-2H3 cells.

The present study was designed to investigate the anti-allergic effects of ethanolic extract of Alternanthera sessilis (AS-1) in rat basophilic leukemia (RBL-2H3) cells. It significantly reduced the ß-hexosaminidase release from anti-DNP-IgE sensitized RBL-2H3 cells. AS-1also inhibited the IgE antibody-induced increase in Interleukin-6 (IL-6), TNF-α, IL-13 and IL-4 production in these cells. The inhibitory effect of AS-1 on these cytokine was found to be nuclear factor-KB (NF-kB) dependent, as it attenuated the degradation of IKBa and nuclear translocation of NFkB. In addition, AS-1 significantly attenuated the DNP HAS-induced intracellular Ca(2+) release from these cells, which makes us speculate strongly that the decreased intracellular Ca(2+) is involved in the inhibitory effect of AS-1 on ß-hexoaminidase release. Taken together, anti-allergic effects of AS-1 suggest possible therapeutic application of this extract in allergic diseases.

A cold-active esterase of Streptomyces coelicolor A3(2): from genome sequence to enzyme activity.

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30 degrees C and retaining more than 25% of its activity at 4 degrees C. The optimum pH was 8-8.5. The enzyme was active against short synthetic p-nitrophenylesters (C2-C10) with maximum activity towards the acetate ester (C2). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1-1.9).

Molecular cloning of carboxylesterase gene and biochemical characterization of encoded protein from Bacillus subtilis (RRL BB1).

An isolated strain of Bacillus subtilis identified by 16S rDNA sequence analysis produces an enantioselective ester hydrolase. Whole cells of B. subtilis (RRL BB1) and enzyme derived from it was capable of enantioselective hydrolysis of several racemates including drug intermediates with moderate to high enantioselectivity as already reported by us. In this communication, we describe cloning of the gene encoding the enantioselective esterase designated as estBB1. The primary structure of the enzyme determined from the nucleotide sequence indicated that esterase estBB1 has Mw approximately 52kDa and pI approximately 5.2 and belongs to the family of type B carboxylesterases with 50-60% similarity at amino acid level. Alignment studies of sequences of the estBB1 and Pnb esterase 56C8 from B. subtilis showed that estBB1 has an alpha/beta hydrolase fold with catalytic triad formed by Ser190, Glu305 and His394 at active site and Ser190 is located in the conserved motif -G-X-S-X-G-.

Molecular cloning of enantioselective ester hydrolase from Bacillus pumilus DBRL-191.

A gene from Bacillus pumilus expressed under its native promoter was cloned in Escherichia coli. Recombinant B. pumilus esterase (BPE) affects the kinetic resolution of racemic mixtures such as unsubstituted and substituted 1-(phenyl)ethanols (E approximately 33-103), ethyl 3-hydroxy-3-phenylpropanoate (E approximately 45-71), trans-4-fluorophenyl-3-hydroxymethyl-N-methylpiperidine (E approximately 10-13) and ethyl 2-hydroxy-4-phenylbutyrate (E approximately 7). The enzyme is composed of a 34-amino acid signal peptide and a 181-amino acid mature protein corresponding to a molecular weight of approximately 19.2kD and pI approximately 9.4. 3-D the structural model of the enzyme built by homology modelling using the atomic coordinates from the crystal structure of B. subtilis lipase (LipA) showed a compact minimal alpha/beta hydrolase fold.