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.

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.

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.

Trichoderma viride induces pathogenesis related defense response against rot pathogen infection in groundnut (Arachis hypogaea L.).

The study examine induction of defense enzymes involved in phenylpropanoid pathway and accumulation of pathogenesis related proteins in rot pathogen (Aspergillus niger Van Tieghem) challenged groundnut seedlings in response to Trichoderma viride JAU60. Seeds of five groundnut varieties differing in collar rot susceptibility were sown under non-infested, pathogen infested and pathogen+T. viride JAU60 seed treatment. Collar rot disease evident between 31.0% (J-11, GG-2) and 67.4% (GG-20) in different groundnut varieties under pathogen infested which was significantly reduced from 58.1% (J-11, GG-2) to 51.6% (GG-20) by Trichoderma treatment. The specific activities of polyphenol oxidase (EC 1.14.18.1) and ß-1,3 glucanase (EC 3.2.1.6) elevated 3.5 and 2.3-fold, respectively, at 3 days; phenylalanine ammonia lyase (EC 4.3.1.5) evident 1.6-fold higher at 6 days; and chitinase (EC 3.2.1.14) sustained 2.3-2.8 folds up to 9 days in Trichoderma treated+pathogen infested seedlings of tolerant varieties (J-11, GG-2) compared with moderate and susceptible (GAUG-10, GG-13, GG-20). T. viride JAU60 induces defense enzymes in a different way for tolerant and susceptible varieties to combat the disease. This study indicates the synergism activation of defense enzymes under the pathogenic conditions or induced resistance by T. viride JAU60 in a different groundnut varieties susceptible to collar rot disease.