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.

Metabolic profiling for dissection of late leaf spot disease resistance mechanism in groundnut.

Late leaf spot (LLS) caused by fungi Passalora personata is generally more destructive and difficult to control than early leaf spot. The aim of this study was to decipher biochemical defense mechanism in groundnut genotypes against P. personata by identifying resistance specific biomarkers and metabolic pathways induced during host-pathogen interaction. Metabolomics of non-infected and infected leaves of moderately resistant (GPBD4 and ICGV86590), resistant (KDG128 and RHRG06083) and susceptible (GG20, JL24 and TMV2) genotypes was carried out at 5 days after infection (65 days after sowing). Non-targeted metabolite analysis using GC-MS revealed total 77 metabolites including carbohydrates, sugar alcohols, amino acids, fatty acids, polyamines, phenolics, terpenes and sterols. Variable importance in projection (VIP) measure of partial least squares-discriminant analysis (PLS-DA) showed that resistant and moderately resistant genotypes possessed higher intensities of ribonic acid, cinnamic acid, malic acid, squalene, xylulose, galactose, fructose, glucose, ß-amyrin and hydroquinone while susceptible genotypes had higher amount of gluconic acid 2-methoxime, ribo-hexose-3-ulose and gluconic acid. Heat map analysis showed that resistant genotypes had higher intensities of ß-amyrin, hydroquinone in non-infected and malic acid, squalene, putrescine and 2,3,4-trihydroxybutyric acid in infected leaves. Dendrogram analysis further separated resistant genotypes in the same cluster along with infected moderately resistant genotypes. The most significant pathways identified are: linoleic acid metabolism, flavone and flavonol biosynthesis, cutin, suberin and wax biosynthesis, pentose and glucuronate interconversions, starch and sucrose metabolism, stilbenoid biosynthesis and ascorbate and aldarate metabolism. Targeted metabolite analysis further confirmed that resistant genotypes possessed higher content of primary metabolites sucrose, glucose, fructose, malic acid and citric acid. Moreover, resistant genotypes possessed higher content of salicylic, coumaric, ferulic, cinnamic, gallic acid (phenolic acids) and kaempferol, quercetin and catechin (flavonols). Thus metabolites having higher accumulation in resistant genotypes can be used as biomarkers for screening of LSS resistant germplasm. These results unravel that higher amount of primary metabolites leads to stimulate the accumulation of more amounts of secondary metabolites such as phenolic acid, flavanols, stilbenes and terpenoids (squalene and ß-amyrin) biosynthesis which are ultimately involved in defense mechanism against LLS pathogen. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00985-5.

Transcriptome analysis of sesame-Macrophomina phaseolina interactions revealing the distinct genetic components for early defense responses.

Sesame (Sesamum indicum L.) is an oilseed crop challenged by many biotic stresses. Charcoal rot caused by Macrophomina phaseolina (MP) is one of the most devastating diseases of sesame. Till date, molecular mechanisms of resistance to charcoal rot in sesame is not yet reported. In this study, two sesame variety GT-10 (resistant) and RT-373 (susceptible) were identified with contrasting disease incidence when infected with MP. To get the molecular insight, root samples were collected at 0, 24, 48- and 72-h post inoculation (hpi) with the pathogen and generated RNAseq data was analyzed. A total of 1153 and 1226 differentially expressed genes (DEGS) were identified in GT-10 and RT-373, respectively. During the inoculation with MP, resistant genotype showed high number DEGs at early time point of 24 hpi and when compared to late expression in susceptible genotype at 48 hpi. Distinct clusters were represented for each time period represented by cytochrome P450 83B1-like, single anchor, hypothetical protein C4D60, kirola like and heat shock proteins in the resistant genotype contributing for resistance. Analysis of differentially expressed genes, catalogued the genes involved in synthesis of pathogenesis-related (PR) proteins, MYB, WRKY, leucine zipper protein, bHLH, bZIP and NAC transcription factors, ABC transporters (B, C and G subfamily), glutathione metabolism, secondary metabolites, fatty acid biosynthesis and phytohormones like auxin, abscisic acid, ethylene and gibberellic acid. Additionally, in the resistant response we have found three unique GO terms including ATP binding, ribonucleotide binding and nucleic acid binding in molecular function category. The molecular clues generated through this work will provide an important resource of genes contributing for disease resistance and could prioritize genes for functional validation in the important oil crop. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01039-6.

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.

Distinguish metabolic profiles and defense enzymes in Alternaria leaf blight resistant and susceptible genotypes of groundnut.

Alternaria leaf blight is major fungal disease of summer groundnut, causes significant loss of haulm and pod yield. Aims of this study were to understand the role of metabolites and phenylpropanoid related enzymes in Alternaria leaf blight resistance and to find out metabolic marker for disease resistance. Alternaria leaf blight resistant (GPBD4 and CS186) and susceptible genotypes (GG2 and TPG41) of groundnut were grown in pots during rabi-summer 2015. Groundnut plants were infected with Alternaria alternata (Fr.) Keissler at 40 days after sowing. 5 days after infection, upper second leaves were collected from both control and infected plants for analysis. A total of 67 metabolites comprising sugars, sugar alcohols, amino acids, organic acids, fatty acids, sterols and phenolic were identified using gas chromatography-mass spectrometry (non-targeted metabolomics). Constitutive levels of alpha-d-galactoside, d-mannitol, d-erythropentitol, glycine, and hexadecanoic acid were observed higher in resistant genotypes compared to susceptible genotypes. Moreover, arabinofuranose, cinnamic acid, 2-butendioic acid, and linoleic acid were observed only in resistant genotypes at both control and infected stage. In susceptible genotypes myo-inositol, glucose and fructose content was increased after infection with pathogen while decreased in resistant genotypes. Resistant genotypes had higher constitutive level of cinnamic and salicylic acid compared to susceptible genotypes. Non-infected leaves of resistant genotypes also had higher activities of phenylalanine ammonia lyase and tyrosine ammonia lyase activities. Our results suggest that metabolites specifically present in resistant genotypes impart defense mechanism against Alternaria pathogen and can be used as bio-marker for screening of germplasm.

Nutritional profile and molecular fingerprints of indigenous black jamun (Syzygium cumini L.) landraces.

The indigenous black jamun landraces (Syzygium Cumini L.), found in western Gujarat of Gir forest region (India), produced fruits with different size and shape. Fruit morphology like shape, volume, weight, length, girth were examined and black jamun categorized into six landraces viz., BJLR 1 (big fruit, > 11 g); BJLR 2 (medium to big fruit, 8-11 g); BJLR 3 (medium fruit, 6-8 g); BJLR 4 (medium to small fruit, 5-6 g); BJLR 5 (small fruit, 3-5 g) and BJLR 6 (very small fruit, < 3 g fruit weight). The landraces (BJLR 1 and 2) with larger size fruits were accumulated higher amount of moisture, total fat content, sugars, total protein, starch, free amino acid contents. Smaller fruits (BJLR 6) contained higher amount of ascorbic acid-137 and 132 mg%; anthocynin-47.7 and 2.35 mg%; crude fibre 3.05 and 10.5 g%; and total phenol-21.7 and 45.0 mg g-1 in their fruit pulp and seed part, respectively with better nutritional profile compared with big and moderate fruited landraces. Nutritional profile of six landraces indicated that fruit pulp accumulated higher amount of soluble sugars (6.51-17.6 mg g-1), anthocyanins (29.7-47.7 mg%) and free amino acids (7.54-18.9 mg%) while that of seeds exhibited higher amount of crude fibre (6017-10.5 g%), ascorbic acid (90-137 mg%), starch (22.8-29.4 g%), total protein (4.72-7.17 mg%), phenols (45-56.7 mg g-1). The black jamun landraces were subjected to ISSR based polymorphic finger prints and genetic diversity analysis. Total 144 bands were amplified across six landraces by 18 UBC primers, of which 94 were polymorphic with 64.2% average polymorphism. Cluster analysis demonstrates the BJLR 6 landraces distinguished from other landraces with 53% similarity.

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.

The SRAP based molecular diversity related to antifungal and antioxidant bioactive constituents for biocontrol potentials of Trichoderma against Sclerotium rolfsii Scc.

The study was performed to examine 11 isolates of Trichoderma for their bio-control potentials against Sclerotium rolfsii Sacc. causing stem rot in groundnut. The antagonists Trichoderma were subjected to sequence related amplified polymorphism (SRAP) based molecular diversity analysis and compared with their hardness to S. rolfsii with respect to secretary antifungal and antioxidant profile. T. virens NBAII Tvs 12 evident highest (87.91 %) growth inhibition of test pathogen followed by T. koningii MTCC 796 (67.03 %) at 7 days after inoculation (DAI). Microscopic study confirmed biocontrol mechanism as mycoparasitism for Tvs 12 and antibiosis for MTCC 796. The growth inhibition of test pathogen was significantly negatively correlated with sclerotia formation and lipid peroxidation during antagonism due to release of secretary bioactive antioxidants by antagonists to terminate oxidative burst generated by S. rolfsii and causing inhibition of sclerotium formation. The GC-MS profile identified antifungal and antioxidant constituents hexadecane, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester, 1-hexadecanesulfonyl chloride, and octadecane in potent antagonists Tvs 12; and nonacosane and octadecane in MTCC 796 along with two novel compounds 1-pentadecene and 1-heneicosyl formate for biocontrol activity. Molecular diversity of Trichoderma isolates associated with antagonistic activity was assessed by SRAP markers. The 115 primer combinations generate total 1328 amplified products of which, 1095 are shared polymorphic and 199 are unique polymorphic. The 15 SRAP combinations produced 18 bands to diagnose best antagonist Tvs 12 and 13 SRAP combinations generated 19 unique bands for identification of MTCC 796. The mycoparasitic antagonist Tvs 12 would be the best antagonist and released unique antifungal and antioxidant constituents to combat pathogen infection. The SRAP based genetic diversity indicates Tvs12 strain clustered with T. viride NBAII Tv23 and shared only 52 % similarity with other isolates of Trichoderma. The SRAP similarities explained substantial diversity (19-68 %) across Trichoderma isolates.

Antioxidant defense response induced by Trichoderma viride against Aspergillus niger Van Tieghem causing collar rot in groundnut (Arachis hypogaea L.).

The study was conducted to examine the antioxidant enzymes induced by Trichoderma viride JAU60 as initial defense response during invasion of rot pathogen Aspergillus niger Van Tieghem in five groundnut varieties under pot culture. Seed treatment of T. viride JAU60 reduced 51-58% collar rot disease incidence in different groundnut varieties under pathogen infected soil culture. The activities of the antioxidant enzymes, viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) and ascorbate peroxidase (APX, EC 1.11.1.11), elevated in response to pathogen infection, in higher rate by tolerant varieties (J-11 and GG-2) compared with susceptible (GAUG-10, GG-13, GG-20) and further induced by T. viride treatment. Trichoderma treatment remarkably increased the 2.3 fold SOD, 5 fold GPX and 2.5 fold APX activities during disease development in tolerant varieties and the same was found about 1.2, 1.5 and 2.0 folds, respectively, in susceptible varieties. Overall, T. viride JAU60 treated seedlings (T3) witnessed higher activities of SOD (1.5 fold), GPX (3.25 fold) and APX (1.25 fold) than pathogen treatment (T2) possibly suggest the induction of antioxidant defense response by Trichoderma bio-controller to combat oxidative burst produced by invading pathogen.

Draft Genome Sequence of the Endophytic Bacterium Enterobacter spp. MR1, Isolated from Drought Tolerant Plant (Butea monosperma).

Enterobacter sp. MR1 an endophytic plant growth promoting bacterium was isolated from the roots of Butea monosperma, a drought tolerant plant. Genome sequencing of Enterobacter spp. MR1 was carried out in Ion Torrent (PGM), Next Generation Sequencer. The data obtained revealed 640 contigs with genome size of 4.58 Mb and G+C content of 52.8 %. This bacterium may contain genes responsible for inducing drought tolerance in plant, including genes for phosphate solubilization, growth hormones and other useful genes for plant growth.

Draft Genome Sequence of the Methyl Parathion (Pesticide) Degrading Bacterium Pseudomonas spp. MR3.

Pseudomonas spp. MR3 was isolated from the surrounding soil of pesticide manufacturing industries of Ankleshwar, Gujarat. Under laboratory conditions these microbes were able to degrade up to 500 ppm of methyl parathion within 72 h. Genome sequencing of Pseudomonas spp. MR3 was carried out inIon Torrent (PGM), next generation sequencer. The data obtained revealed 1,268 contigs with genome size of 2.99 Mb and G + C content of 60.9 %. The draft genome sequence of strain MR3 will be helpful in studying the genetic pathways involved in the degradation of several pesticides.

Nontargeted metabolomics approach to determine metabolites profile and antioxidant study of Tropical Almond (Terminalia catappa L.) fruit peels using GC-QTOF-MS and LC-QTOF-MS.

The objective of the present study was to identify the metabolome pattern and study the biological efficacy of Almond (Terminalia catappa L.) peels. In the present study, metabolite profiling was carried out using GC-QTOF-MS and LC-QTOF-MS techniques, from the potent extract showed highest antioxidant efficacy. Antioxidant efficacy of the various extracts derived by six different extraction methods was assessed by different assays viz. 2,2-diphenyl-1-picrylhydrazyl, superoxide, 2,2'-Azino-bis-(3-ethyl)benzothiazoline)-6-sulfonic acid diammonium salt radical cation inhibition assays and ferric reducing antioxidant power. The methanolic maceration extract showed significant antioxidant activity in all assays and richest source of flavonoid content. The results showed that the extraction method significantly altered the antioxidant activity. The correlation between the antioxidant activities and total flavonoid content were ranging between 0.939 to 0.628. Methanolic maceration extraction method proved to be the best extraction method for the extraction of antioxidant. During the metabolite profiling employed by GC-QTOF-MS and LC-QTOF-MS and total 24 and 53 compounds belongs to different groups were detected and identified, will help to uncover the relation with potent antioxidant activity.

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.

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.

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.