Salicylic acid-functionalised chitosan nanoparticles restore impaired sucrose metabolism in the developing anther of cotton (Gossypium hirsutum) under heat stress.

Nanotechnology provides tremendous potential in agriculture, mitigating climate change impact and improving abiotic stress management strategy. Chitosan nanoparticles (NCS) were synthesised using the ion gelation method and characterised for size (75.5nm in particle size analyser), shape (spherical under scanning electron microscopy) and stability (132.2mV zeta potential). Further, salicylic acid was incorporated into NCS to craft salicylic acid-functionalised chitosan nanoparticles (SA-NCS) and illustrated for size (517nm), shape (spherical) and stability (197.1mV). The influence of the exogenous application of SA-NCS (0.08%) was studied at the reproductive stage of three genotypes of cotton (Gossypium hirsutum ): (1) heat-tolerant Solar-651 BGII; (2) moderately heat-tolerant Solar-701 BGII; and (3) heat-susceptible Solar-805 BGII, exposed to different temperature regimes: (1) H1 (optimal), 32/20±2°C; (2) H2 (sub-optimal), 38/24±2°C; H3 (supra-optimal), 45/30±2°C. Heat stress significantly reduces carbon-fixing Rubisco, enzymes related to sucrose metabolism and pollen tube length. Considering three genotypes and reproductive stages (sepal and anther tissues), activities of Rubisco (sepals), invertase (sepals), sucrose phosphate synthase (anthers), sucrose content (sepals) and pollen tube length were elevated under high-temperature regimes, signifying better source to sink transposition of sucrose influenced by SA-NCS. The study provides new insights into SA-NCS to improve source-sink imbalance and restore sucrose metabolism for better growth of reproductive structure under heat stress in cotton.

Biochemical and molecular depictions to develop ech42 gene-specific SCAR markers for recognition of chitinolytic Trichoderma inhibiting Macrophomina phaseolina (Maubl.) Ashby.

Trichoderma isolates were inhibited variably in-vitro growth of soil-borne phytopathogen Macrophomina phaseolina (Maubl.) Ashby causes root rot in cotton. The growth inhibition of test-pathogen was found to be higher (90.36%) in T. viride NBAIITv23 followed by T. koningii MTCC796 (85.77%) under dual culture antagonism. The microscopic examination suggested that the antagonists Tv23 and MTCC796 adopted mycoparasitism as a strong mode of action to restrain pathogen growth. However, antagonists T. harzianum NBAIITh1 (77.89%) and T. virens NBAIITvs12 (61.74%) demonstrated strong antibiosis action for growth inhibition of the test pathogen. A significant positive correlation was established between the growth inhibition of M. phaseolina and the release of cell wall degrading enzymes- chitinase (p = 0.001), ß-1,3, glucanase (p = 0.01), and protease (p = 0.05) under the influence of pathogen cell wall. The chitinase and ß-1,3, glucanase activities were elevated 2.09 and 1.75 folds, respectively, in potent mycoparasitic Tv23 strain influenced by a pathogen cell wall compared to glucose as a carbon source. The three unique DNA-RAPD fragments OPA-07(1033), OPA-16(983), and OPO-15(239), amplified by potent mycoparasitic Tv23 strain, were subjected to DNA sequencing and derived functional 864 bp from OPA-16(983) and have sequence homology to ech42 gene with partial CDs of 262 amino acids (nucleotide accession No. KF723016.1 and protein accession No.AHF57046.1). Novel SCAR markers were developed from a functional sequence of OPA-16 fragments and validated across the genomic DNA of eleven Trichoderma antagonists. The novel SCAR markers evolved from the RAPD-SCAR interface to authenticate chitinolytic Trichoderma associated with mycoparasitic action for eco-friendly biocontrol activity.

Intracellular metabolomics and microRNAomics unveil new insight into the regulatory network for potential biocontrol mechanism of stress-tolerant Tricho-fusants interacting with phytopathogen Sclerotium rolfsii Sacc.

The present study employed microRNA (miRNA) sequencing and metabolome profiling of Trichoderma parental strains and fusants during normal growth and interaction with the phytopathogen Sclerotium rolfsii Sacc. In-vitro antagonism indicated that abiotic stress-tolerant Tricho-fusant FU21 was examined as a potent biocontroller with mycoparasitic action after 10 days. During interaction with the test pathogen, the most abundant uprising intracellular metabolite was recognized as l-proline, which corresponds to held-down l-alanine, associated with arginine and proline metabolism, biosynthesis of secondary metabolites, and nitrogen metabolism linked to predicted genes controlled by miRNAs viz., cel-miR-8210-3p, hsa-miR-3613-5p, and mml-miR-7174-3p. The miRNAs- mml-miR-320c and mmu-miR-6980-5p were found to be associated with phenylpropanoid biosynthesis, transcription factors, and signal transduction pathways, respectively, and were ascertained downregulated in potent FU21_IB compared with FU21_CB. The amino benzoate degradation and T cell receptor signaling pathways were regulated by miRNAs cel-miR-8210 and tca-miR-3824 as stress tolerance mechanisms of FU21. The intracellular metabolites l-proline, maleic acid, d-fructose, Myo-inositol, arabinitol, d-xylose, mannitol, and butane were significantly elevated as potential biocontrol and stress-tolerant constituents associated with miRNA regulatory pathways in potent FU21_IB. A network analysis between regulatory miRNA-predicted genes and intracellular metabolomics acknowledged possible biocontrol pathways/mechanisms in potent FU21_IB to restrain phytopathogen.

Exploring conserved and novel MicroRNA-like small RNAs from stress tolerant Trichoderma fusants and parental strains during interaction with fungal phytopathogen Sclerotium rolfsii Sacc.

The study investigated potential microRNA-like small RNAs (milRNAs) from multi-stress-tolerant Tricho-fusants and parental strains (P1- Trichoderma virens NBAIITvs12 and P2- Trichoderma koningii MTCC796) for antagonistic activity during interaction with phytopathogen Sclerotium rolfsii. The Trichoderma was cultured in-vitro, with and without antagonism, against the pathogen and total RNA was extracted followed by small RNA library construction and sequencing. The milRNAs were identified by mapping high-quality unique reads against a reference genome. The milRNAs were recognized higher in antagonist Trichoderma during interaction with test pathogen compared to normal growth. The novel milRNAs candidates were found to vary during interaction with the pathogen and normal growth. The gene ontology and functional analysis illustrated that a total of 5828 potential targeted genes were recognized for 93 milRNAs of potent Fu21_IB and 3053 genes for 62 milRNAs of least fusant Fu28_IL. Functional annotation of milRNA-predicted genes integrating KEGG pathways indicates new insights into regulatory mechanisms, by interfering with milRNAs, associated with signal transduction, amino sugar metabolism, benzoate degradation, amino acid metabolism, and steroid and alkaloid metabolism for potential biocontrol of stress-tolerant Tricho-fusant FU21 during interaction with S. rolfsii. The present investigation is the first report of conserved and novel milRNAs from Tricho-fusants and parental strains interacting with S. rolfsii.

Comparative Appraisal of Leaf Proteomic and Mass Spectrometry Analyses During Fusarium Wilt Infection in Resistance and Susceptible Genotypes of Castor (Ricinus communis L.).

The resistant and susceptible genotypes of castor were utilized for leaf proteomic study during Fusarium wilt infection. The histopathological study was observed under SEM and it confirmed that the infection of Fusarium oxysporum f. sp. ricini was higher in the root of susceptible JI-35, while incompatible interaction is observed in resistant SKI-215 genotype. The acidic and neutral proteins were maximally up-expressed with 2 to 171 kDa in treated resistant and 2 to 150 kDa in treated susceptible interactions. In resistant genotype, the leaf proteins were recognized with 3.0- and 5.8-fold higher at infection stage and post-infection stage, respectively, as compared to susceptible genotype. The highly up expressions of leaf acidic (4.76 pI) and basic (8.77 pI) proteins were found with 224.94- and 61.68-fold change, respectively during the post-infection stage in treated resistance compared to its control. The protein spots at 4.76 pI and 8.77 pI were characterized with nanoLC-MS Triple TOF and were recognized as signalling molecules small GTP binding protein (23 kDa) and actin (8 kDa), respectively, on the basis of mass spectrometry and peptide sequences. However, basic and neutral proteins were up regulated as 30.11- and 20.30-fold changes in treated susceptible compared to its control. These proteins were identified as HSP90 (10 kDa) and LEA (27 kDa) proteins. The 148 kDa protein is recognized as histidine kinase in incompatible resistant interaction compared to compatible susceptible (serine threonine protein kinase, 65 kDa) as common acidic protein at 3.80 pI during infection stage. Some acidic proteins were maximally up-regulated in the leaf of resistant castor genotype and played a significant role in defense response.

Biosynthesis and characterization of extracellular metabolites-based nanoparticles to control the whitefly.

The Beauveria spp. were isolated from soil and insect cadavers and confirmed as Beauveria bassiana by molecular identification using a specific primer. The bioefficacy of 14 B. bassiana against whiteflies indicated the highest percent mortality in JAU2, followed by JAU1. The LC50 and LC90 values were found to be 0.043 × 105 and 0.05 × 1014 conidia.ml-1, respectively, in JAU2. Extracellular metabolites of B.bassiana are derived and used for the green synthesis of silver nanoparticles (AgNPs). The synthesized green AgNPs were characterized for size (24.8 nm), shape (scanning electron microscopy), stability (200 mV zeta), and purity (energy-dispersive X-ray spectroscopy, 3 keV). A total of 63 extracellular metabolites were identified using LC-MS/QTOF in potent JAU2 with recognition of alcohols, phenols, carboxylic acids, amines, alkynes, and amides as functional groups. The functional groups of green AgNPs were also confirmed in Fourier transforms infrared spectroscopy (FTIR) with the specific spectra in the electromagnetic spectrum. The relationship between identified metabolites of antagonist and the FTIR spectrum of the functional group indicated the involvement of extracellular novel compounds, viz., homoisocitrate, aconitine, phodexin A, capillone, solanocapsine, and anethole in the synthesis of green AgNPs. The efficacy of green AgNPs on whiteflies suggested that corrected percent mortality was observed at 60 µg Ag.ml-1 at 120 h, which corresponds to the LC50 value (66.42 µg Ag.ml-1). Results were interpreted to show that green AgNPs synthesized from extracellular metabolites of B.bassiana JAU2 gave better insecticidal activity at LC50 as compared to live antagonist JAU2.

Characterization and bio-efficacy of entomopathogenic Beauveria associated with cuticle-degrading enzymes to restrain sucking pest Bemisia tabaci.

The entomopathogenic Beauveria spp. were acquired from insect cadavers and soil rhizosphere of cotton, groundnut, and castor. Among Beauveria, five spp. derived from infected insects, eight Beauveria found from soil, and one strain of Beauveria bassiana collected from MTCC 9544. Beauveria were characterized for morphology and cuticle-degrading enzyme activity associated with virulence against Bemisia tabaci. The colony morphology, conidial arrangement, size, and shape confirmed all isolates as Beauveria. The chitinase (EC 3.2.1.14) and lipase (EC 3.1.1.3) activities were observed the highest in Beauveria JAU2, while higher protease (EC 3.4.21.4) activity found in JAU4 followed by JAU2 at 240 h. The bio-efficacy of Beauveria (1 × 107 conidia.ml-1) illustrated that potent JAU2 was examined with the highest % mortality and corrected mortality of B. tabaci at 144 h followed by JAU1. The LC90 and LC50were determined from potent (JAU1 and JAU2) and weak (JAU6), and it was found the lowest in JAU2. The most potent Beauveria JAU2, isolated from insect cadaver (Harmivora armigera), was illustrated higher virulence than other isolates. The Beauveria JAU2 were recognized as Beauveria bassiana based on the shape of conidia and size (2.00 to 2.09 µm dia) as examined in SEM. Study insight into recognition of potent Beauveria bassiana JAU2 was linked with cuticle-degrading enzyme activity for insecticidal action. The JAU2 isolate established the most positive correlation (P0.01: 0.864) between chitinase activity and corrected mortality of insect.

Green synthesis and characterization of nanosilver derived from extracellular metabolites of potent Bacillus subtilis for antifungal and eco-friendly action against phytopathogen.

The potent antagonist Bacillus isolated from the soil rhizosphere elucidated the highest antagonism against the phytopathogen Fusarium oxysporum f. sp. cumini and was identified as Bacillus subtilis strain JSD-RSCu-8D based on molecular recognition by 16S rRNA sequencing (NCBI Accession No. KT894724). Live Bacillus may not work as effectively against phytopathogen under unfavorable environmental conditions like temperature, humidity, or other abiotic stresses. The extracellular metabolites, obtained from culturing potent B. subtilis, were exploited for the creation of green nanosilver for proficient actions in a changing climate. The synthesized green nanosilver was illustrated for shape (spherical with 65.21 ± 3.71 nm under SEM), size (70.9 nm in PSA), purity (2.69 keV peak corresponded to the binding energy of silver under EDAX), and stability (44.2 mV as ZETA). The formation of green Ag-NPs from extracellular metabolites was confirmed by a comparative appraisal of the electromagnetic peak of the metabolite's functional groups, silver nitrate, and green nanoparticles in Fourier transform infrared spectroscopy. The novel mode of action of pathogen mycelium degradation was elucidated by the minimum inhibitory concentration (MIC) of green nanosilver as 40 µg Ag ml-1 to diminish F. oxysporum (SEM morphology). The green nanosilver at 2 DAI renowned the leakage of sugars from mycelia of the cell membrane and defeated the activity of respiratory chain dehydrogenases, followed by lipid peroxidation and the highest leakage of proteins at 3 DAI on MIC. The in-vivo study might allow for novel insight to utilize green nanosilver at MIC (40 µg Ag ml-1) as an eco-friendly and fungicide alternate way for antifungal action to demolish Fusarium wilt infection under harsh conditions.

Characterization and bioefficacy of green nanosilver particles derived from fungicide-tolerant Tricho-fusant for efficient biocontrol of stem rot (Sclerotium rolfsii Sacc.) in groundnut (Arachis hypogaea L.).

An efficient and eco-friendly bioefficacy of potent Tricho-fusant (Fu21) and its green nanosilver formulation against stem rot (Sclerotium rolfsii) in groundnut was established. Fu21 demonstrated higher in-vitro growth inhibition of pathogen with better fungicide tolerance than the parental strains. The green nanosilver particles were synthesized from the extracellular metabolites of Fu21 and characterized for shape (spherical, 59.34 nm in scanning electron microscope), purity (3.00 KeV, energy dispersive X-ray analysis), size (54.3 nm in particle size analyzer), and stability (53.7 mv, zeta). The field efficacy study exhibited that the seedling emergence was high in seeds treated with green nanosilver (minimum inhibitory concentration-[MIC] 20 µg Ag/ml), and a low disease severity index of stem rot during the crop growth was followed by the live antagonist (Fu21) in addition to seed treatment with a fungicide mix under pathogen infestation. The seed quality analysis of harvested pods revealed a high oil content with balanced fatty acid composition (3.10 oleic/linoleic acid ratio) in green nanosilver treatment under pathogen infestation. The residual analysis suggested that green nanosilver applied at the MIC level as seed treatment yielded similar effects as the control for silver residue in the harvested groundnut seeds. The green nanosilver at MIC has a high pod-yield under S. rolfsii infestation, demonstrating green chemistry and sustainability of the nanoproduct.

Metabolomics of extracellular compounds and parasitic enzymes of Beauveria bassiana associated with biological control of whiteflies (Bemisia tabaci).

The Beauveria spp. were isolated from soil and insect cadavers of crop rhizosphere and characterized for parasitic enzyme activity and virulence against whiteflies (Bemisia tabaci). The colony morphology and molecular identification using ITS specific marker were carried out and confirmed entomopathogenic fungi as Beauveria bassiana. The bioefficacy of B. bassiana against whiteflies demonstrated highest corrected mortality and lowest LC50 in isolate B. bassiana JAU2 (SEM morphology) followed by JAU1 on 6th days. Parasitic enzymes chitinase and lipase were determined highest in JAU2 and protease activity examined higher in isolate JAU4 followed by JAU2 isolate on 6th days after inoculation. Comparative extracellular metabolomics carried out from potent (JAU1 and JAU2), moderate (JAU4 and JAU14) and weak (JAU6) B. bassiana isolates in normal suborder dextrose agar with yeast extrect (SDAY) and chitin induced media. Results illustrated that total 105 metabolites identified common for all five B. bassiana isolates differing in virulence. However, the color intensity of the metabolites changes in heat map showing differential concentration of that extracellular compound compared to other isolates. The volcano plot analysis illustrated 58 compounds significanlty diverse between potent JAU1 and JAU2 under two different culture conditions of which 34 compounds recognized up regulated in most potent JAU2 under chitin induced media. Out of 34 metabolites, ten compounds viz., fumaricine, resazurin, N-methyldioctylamine, penaresidun B, tetralin, squamocin B, oligomycin C, pubesenolide, epirbuterol and gentamicin C1a were recognized significantly upregulated in most potent JAU2 and reported for antimicrobial, nematicidal, larvicidalor insecticidal activities. The mass spectra and fragment structure were elucidated under LCMS-QTOF for some novel and unique compounds recognized in most potent B. bassiana JAU2, involved in parasitic activity against whiteflies.

The impact of bacterial size on their survival in the presence of cationic particles of nano-silver.

BACKGROUND: Microbial surface area is one of the battlegrounds for invading microbes and host defense. Hence, infectious diseases caused by drug resistant microbes with large surface area are more difficult to treat than small size microbes. Nanobiology offers opportunities to re-explore the biological properties of conventional drugs at molecular level to combat these microbes. The purpose of the present study was to examine size depended susceptibility of Gram-positive bacteria towards nano-silver particles. METHODS: This study investigated the growth, surface charge, and morphology of emerging B. megaterium MTCC 7192 and re-emerging S. aureus MTCC 3160 cells in order to observe the susceptibility of these bacteria towards cationic nano-silver particles. Nano-silver particles were applied into wells formed on the Nutrient agar plates containing 108 CFU/mL of the bacteria. Surface potential of normal and treated cells was measured by Microtrac and the effects of nano-silver particles on bacterial cells were assessed by Scanning Electron Microscopy (SEM). RESULTS: In this work, synthesized nano-silver particles were found to be more effective against B. megaterium MTCC 7192 than S. aureus MTCC 3160. For B. megaterium MTCC 7192, a 0.30 fold increase in inhibition zone was observed after the addition of nano-silver particles in the wells. From our studies, it is reasonable to state that alternation of zeta potential may affect the cell morphology, which was further confirmed by SEM. CONCLUSION: The present study concluded that nano-silver particles appears to interact with a larger surface area more effectively.

Bacterial membrane destabilization with cationic particles of nano-silver to combat efflux-mediated antibiotic resistance in Gram-negative bacteria.

AIMS: With the purpose of exploring combinatorial options that could enhance the bactericide efficacy of linezolid against Gram-negative bacteria, we assessed the extent of combination of nano-silver and linezolid. MAIN METHODS: In this study, we selected Escherichia coli MTCC 443 as a model to study the combinatorial effect of nano-silver and linezolid to combat efflux-mediated resistance in Gram-negative bacteria. The acting mechanism of nano-silver on E. coli MTCC 443 was investigated by evaluating interaction of nano-silver with bacterial membrane as well as bacterial surface charge, morphology, intracellular leakages and biological activities of membrane bound respiratory chain dehydrogenase and deoxyribonucleic acids (DNA) of the cells following treatment with nano-silver. KEY FINDINGS: The alternation of zeta potential due to the interaction of nano-silver towards bacterial membrane proteins was correlated with enhancement of membrane permeability, which allows the penetration of linezolid into the cells. In addition, the binding affinity of nano-silver towards bacterial membrane depressed biological activities of membrane bound respiratory chain dehydrogenases and DNA integrity. SIGNIFICANCE: Our findings suggested that nano-silver could not only obstruct the activities of efflux pumps, but also altered membrane integrity at the same time and thus increased the cytoplasmic concentration of the linezolid to the effective level.

Bactericidal assessment of nano-silver on emerging and re-emerging human pathogens.

With the threat of the growing number of bacteria resistant to antibiotics, the re-emergence of previously deadly infections and the emergence of new infections, there is an urgent need for novel therapeutic agent. Silver in the nano form, which is being used increasingly as antibacterial agents, may extend its antibacterial application to emerging and re-emerging multidrug-resistant pathogens, the main cause of nosocomial diseases worldwide. In the present study, a completely bottom up method to prepare green nano-silver was used. To explore the action of nano-silver on emerging Bacillus megaterium MTCC 7192 and re-emerging Pseudomonas aeruginosa MTCC 741 pathogenic bacteria, the study includes an analysis of the bacterial membrane damage through Scanning Electron Microscope (SEM) as well as alternation of zeta potential and intracellular leakages. In this work, we observed genuine bactericidal property of nano-silver as compare to broad spectrum antibiotics against emerging and re-emerging mode. After being exposed to nano-silver, the membrane becomes scattered from their original ordered arrangement based on SEM observation. Moreover, our results also suggested that alternation of zeta potential enhanced membrane permeability, and beyond a critical point, it leads to cell death. The leakages of intracellular constituents were confirmed by Gas Chromatography-Mass Spectrometry (GC-MS). In conclusion, the combine results suggested that at a specific dose, nano-silver may destroy the structure of bacterial membrane and depress its activity, which causes bacteria to die eventually.

Interruption in membrane permeability of drug-resistant Staphylococcus aureus with cationic particles of nano­silver.

The increasing drug-resistance pathogens among gram-positive bacterial species are becoming a major health concern nowadays. Over the past few years, the bactericidal efficacy of nano­silver against some drug-resistant gram-positive bacteria has been established, however further investigation is needed to determine whether nano­silver could be an option for the treatment of drug-resistant gram-positive microbial infections. The purpose of the present study was to determine the bactericidal efficacy of nano­silver with its membrane destroying property using drug-resistant Staphylococcus aureus MTCC 3160. In the present study, bactericidal assessment of nano­silver with different antibiotics was determined by agar well diffusion method. Interaction of nano­silver towards bacterial membrane was carried to understand the probable bactericidal actions of nano­silver, which was further confirmed by respiratory chain dehydrogenase, zeta potential, Scanning Electron Microscopy (SEM) and Gas Chromatography-Mass Spectrometry (GC-MS). The effect of nano­silver on bacterial Deoxyribonucleic Acids (DNA) was evaluated by agarose gel electrophoresis. Bactericidal assessment of nano­silver showed a very strong bactericidal action compare to antibiotics. The binding affinity of nano­silver towards bacterial membrane induced loss of catalytic activity for respiratory chain dehydrogenases. Zeta potential, SEM and GC-MS analysis also revealed extensive damage to the bacterial cell membrane. Moreover, the analysis of agarose gel electrophoresis revealed that nano­silver can enhance the decomposability of bacterial DNA, which was directly attached to the bacterial cell membrane. The present findings suggested that nano­silver directly interact with the bacterial cell surface without the need to penetrate; and this distinctive property raises the hope that nano­silver will remain an important bactericide in bacteria than antibiotics.

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc.

The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management.

Molecular heterozygosity and genetic exploitations of Trichoderma inter-fusants enhancing tolerance to fungicides and mycoparasitism against Sclerotium rolfsii Sacc.

Protoplast fusion is an imperative tool to develop Trichoderma inter-fusants having desire traits through genetic manipulation. Study designed to develop diverse Trichoderma fusants for fungicide tolerance (Mancozeb, Thiram, Tebuconazole, and Carbendazim) and enhanced mycoparasitic activity against Sclerotium rolfsii sacc. The mycoparasitic T. virens NBAII Tvs12 and fungicide tolerant T. koningii MTCC 796 were utilized for protoplast fusion. The derived inter-fusants were subjected to diploidization using d-camphor in minimal media followed by successive three sub culturing onto potato dextrose agar to obtain 36 stable fusants. The stable fusants were employed for conidial size, fungicide tolerance, mycoparasitism, gene specific SSR amplification and molecular heterozygosity analysis. The results explained that 22 homozygous mutants illustrated characteristic of either one parental strain and 14 heterozygous recombinants depicted traits of both parental strains. The antagonistic activity of fusants against S. rolfsii depicted highest growth inhibition (87.91%) by potent inter-fusant (Fu 21) with improved fungicide tolerance capacity. The molecular study revealed highest observed heterozygocity (0.544), coefficient of gene differentiation (0.526) and gene flow (0.387) by Fu 21 indicating better genetic exploitation of parental strains into that fusant with good genetic purity. Principal coordinate analysis of fusants and parental strains exhibited 65.07% total variation and confirmed the scattering pattern matched with UPGMA clustering pattern. The stable heterozygous Fu 21 derived from inter-fusion between Tvs 12 and MTCC 796 might be useful to practice eco-friendly bioformulation tolerance to fungicides for effective integrated stem rot disease management in groundnut.

Antidiabetic and antioxidant functionality associated with phenolic constituents from fruit parts of indigenous black jamun (Syzygium cumini L.) landraces.

ABSTRACT: Fruit phenolics are important dietary antioxidant and antidiabetic constituents. The fruit parts (pulp, seed, seed coat, kernel) of underutilized indigenous six black jamun landraces (Syzygium cumini L.), found in Gir forest region of India and differed in their fruit size, shape and weight, are evaluated and correlated with antidiabetic, DPPH radical scavenging and phenolic constituents. The α-amylase inhibitors propose an efficient antidiabetic strategy and the levels of postprandial hyperglycemia were lowered by restraining starch breakdown. The sequential solvent systems with ascending polarity-petroleum ether, ethyl acetate, methanol and water were performed for soxhlet extraction by hot percolation method and extractive yield was found maximum with methanolic fruit part extracts of six landraces. The methanolic extracts of fruit parts also evidenced higher antidiabetic activity and hence utilized for further characterization. Among the six landraces, pulp and kernel of BJLR-6 (very small, oblong fruits) evidenced maximum 53.8 and 98.2% inhibition of α-amylase activity, respectively. The seed attained inhibitory activity mostly contributed by the kernel fraction. The inhibition of DPPH radical scavenging activity was positively correlated with phenol constituents. An HPLC-PDA technique was used to quantify the seven individual phenolics. The seed and kernel of BJLR-6 exhibited higher individual phenolics-gallic, catechin, ellagic, ferulic acids and quercetin, whereas pulp evidenced higher with gallic acid and catechin as α-amylase inhibitors. The IC50 value indicates concentration of fruit extracts exhibiting ≥50% inhibition on porcine pancreatic α-amylase (PPA) activity. The kernel fraction of BJLR6 evidenced lowest (8.3 µg ml-1) IC50 value followed by seed (12.9 µg ml-1), seed coat (50.8 µg ml-1) and pulp (270 µg ml-1). The seed and kernel of BJLR-6 inhibited PPA at much lower concentrations than standard acarbose (24.7 µg ml-1) considering good candidates for antidiabetic herbal formulations.

Inhibition coefficient and molecular diversity of multi stress tolerant Trichoderma as potential biocontrol agent against Sclerotium rolfsii Sacc.

Trichoderma is one of the most exploited biocontrol agent for the management of plant diseases. Twenty strains of Trichoderma (six of T. harzianum, four of T. viride, three of T. virens, three of T. koningii, each one of T. hamatum, T. reesei, T. parceramosum and Trichoderma spp.) subjected to in vitro antagonism up to 12days after inoculation against Sclerotium rolfsii Sacc. causing stem rot in groundnut. A new concept was developed to determine inhibition coefficient representing pathogen biology and biocontrol related biophysical variables. Results explained differential inhibition coefficient of test pathogen by Trichoderma antagonists. The inhibition coefficient of test pathogen was examined highest (91.13%) by T. virens NBAII Tvs12 followed by T. virens MTCC 794 (89.33%) and T. koningii MTCC 796 (62.39%). Microscopic study confirmed biocontrol mechanism as mycoparasitism for Tvs12 and antibiosis for T. koningii MTCC 796. The sclerotial biogenesis of test pathogen was elevated during weak antagonism and diminished in interactions with strong antagonists. The inhibition coefficient of S. rolfsii was significantly negatively correlated with sclerotia formation and lipid peroxidation during the antagonism. Trichoderma strains were screened for fungicides (carbendazim and tebuconazole, thiram and mancozeb) and abiotic stress (drought and salt) tolerance. Results indicated that T. koningii MTCC 796 efficiently grew better than the other strains with maximum radial growth under adverse conditions. The genetic variability among the Trichoderma was determined using 34 gene specific markers which amplified 146 alleles. The SSR similarities explained substantial diversity (15 to 87%) across Trichoderma strains and pathogen S. rolfsii. Principal coordinates analysis (PCA) were comparable to the cluster analysis and first three most informative PC components explained 64.45% of the total variation. In PCA, potent antagonists appear to be distinct from other strains. Five SSR markers T1F/T1R(311), TvCTT56f/TvCTT56r(387), TvGAT18f/TvGAT18r(364), TvCA39f/TvCA39r(196) and TvAG29f/TvAG29r(418) found to be unique to distinguish best antagonist strain Tvs12. However, MTCC 796 was examined most stress tolerant strain with better inhibition coefficient which might be useful to control the disease under adverse conditions or as a part of integrated pest management.

Antipathy of Trichoderma against Sclerotium rolfsii Sacc.: Evaluation of Cell Wall-Degrading Enzymatic Activities and Molecular Diversity Analysis of Antagonists.

The fungus Trichoderma is a teleomorph of the Hypocrea genus and associated with biological control of plant diseases. The microscopic, biochemical, and molecular characterization of Trichoderma was carried out and evaluated for in vitro antagonistic activity against the fungal pathogen Sclerotium rolfsii causing stem rot disease in groundnut. In total, 11 isolates of Trichoderma were examined for antagonism at 6 and 12 days after inoculation (DAI). Out of 11, T. virens NBAII Tvs12 evidenced the highest (87.91%) growth inhibition of the test pathogen followed by T. koningii MTCC 796 (67.03%), T. viride NBAII Tv23 (63.74%), and T. harzianum NBAII Th1 (60.44%). Strong mycoparasitism was observed in the best antagonist Tvs12 strain during 6-12 DAI. The specific activity of cell wall-degrading enzymes - chitinase and ß-1,3-glucanase - was positively correlated with growth inhibition of the test pathogen. In total, 18 simple sequence repeat (SSR) polymorphisms were reported to amplify 202 alleles across 11 Trichoderma isolates. The average polymorphism information content for SSR markers was found to be 0.80. The best antagonist Tvs 12 was identified with 7 unique SSR alleles amplified by 5 SSR markers. Clustering patterns of 11 Trichoderma strains showed the best antagonist T. virens NBAII Tvs 12 outgrouped with a minimum 3% similarity from the rest of Trichoderma.

Molecular evolution and phylogenetic analysis of biocontrol genes acquired from SCoT polymorphism of mycoparasitic Trichoderma koningii inhibiting phytopathogen Rhizoctonia solani Kuhn.

The biocontrol agent Trichoderma (T. harzianum, T. viride, T. virens, T. hamantum, T. koningii, T. pseudokoningii and Trichoderma species) inhibited variably (15.32 - 88.12%) the in vitro growth of Rhizoctonia solani causing root rot in cotton. The T. koningii MTCC 796 evidenced highest (88.12%) growth inhibition of test pathogen followed by T. viride NBAII Tv23 (85.34%). Scanning electron microscopic study confirmed mycoparasitism for MTCC 796 and Tv23 which were capable of completely overgrowing on R. solani by degrading mycelia, coiling around the hyphae with hook-like structures. The antagonists T. harzianum NBAII Th1 and, T. virens NBAII Tvs12 exhibited strong antibiosis and formed 2-4 mm zone of inhibition for 70.28% and 46.62%, respectively growth inhibition of test pathogen. Mycoparasitism is a strong mode of action for biocontrol activity compared with antibiosis. The antagonists Trichoderma strains were performed for start codon targeted (SCoT) polymorphism to acquire biocontrol genes from potent antagonist. The six unique SCoT fragments amplified by genomic DNA of best mycoparasitic antagonist MTCC 796 strain are subjected to DNA sequencing resulted to confirm two functional sequences for activity related to biocontrol genes. The phylogenetic and molecular evolution of functional 824 bp of SCoT-3(920) and 776 bp of SCoT-6(806) fragments signify sequence homology with biocontrol genes endochitinase (partial cds of 203 amino acids) and novel hmgR genes (partial cds of 239 amino acids), respectively and the same were annotated and deposited in NCBI GenBank database. The hmgR gene is liable to be express hmg - CoA reductase which is a key enzyme for regulation of terpene biosynthesis and mycoparasitic strains produced triterpenes during antagonism to inhibit growth of fungal pathogen as evidenced with GC-MS profile. The biocontrol genes are found in best antagonist T. koningii MTCC 796 for mycoparasitic activity to restrain the growth of test pathogen R. solani.