H. P. Gajera

Comparison of RAPD and ISSR markers for genetic diversity analysis among different endangered Mangifera indica genotypes of Indian Gir forest region

The genetic variability and relationships among 20 Mangifera indica genotypes representing 15 endangered and 5 cultivars, obtained from Indian Gir forest region, were analyzed using 10 random amplified polymorphic DNA (RAPD) and 21 inter simple sequence repeat (ISSR) markers. RAPD markers were more efficient than the ISSR assay with regards to polymorphism detection. Also, the average numbers of polymorphic loci per primer, average polymorphic information content (PIC) and primer index (PI) values were more for RAPD than for ISSR. But, total number of genotype specific marker loci, Nei’s genetic diversity (h), Shannon’s information index (I), total heterozygosity (Ht), average heterozygosity (Hs) and mean coefficient of gene differentiation (Gst) were more for ISSR as compared to RAPD markers. The regression test between the two Nei’s genetic diversity indexes showed low regression between RAPD and ISSR based similarities but maximum for RAPD and RAPD + ISSR based similarities. The pattern of clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared. Thus, both the markers were equally important for genetic diversity analysis in M. indica.

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.

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.

Lipoxygenase-related defense response induced by Trichoderma viride against Aspergillus niger Van Tieghem, inciting collar rot in groundnut (Arachis hypogaea L.)

Groundnut varieties grown in non-infested soil (T1), pathogen (Aspergillus niger)-infested soil (T2), and seed treatment with Trichoderma viride JAU60 followed by challenge with fungal pathogen (T3), showed significant differences in percent disease incidence of collar rot in pot culture. Percent disease incidence was higher in GG-13, GG-20 (susceptible) followed by GAUG-10 (moderate), and minimum in J-11, GG-2 (tolerant varieties) in A. niger-infested pot culture at 15 days after sowing (DAS). Seed treatment (T3) of T. viride reduced approximately 51% to 58% disease incidence in different groundnut varieties. The lipid peroxidation product – malondialdehyde (MDA) – content was found to be higher in susceptible varieties grown in pathogen-infested soil (T2) and it was significantly reduced to 56% under T. viride treatment (T3) at 12 DAS. However, tolerant varieties reduced the MDA content by 60% in T3 as compared with the T2 at an earlier stage (9 DAS). The lipoxygenase (LOX) activity rose approximately 3.5-fold in tolerant varieties upon infection (T2) and groundnut seed treated with T. viride (T3) at 12 DAS. Trichoderma treatment (T3) maintained a high level of LOX compared with T2, particularly in tolerant varieties. The pathogen infection ability was positively correlated with LOX activity in tolerant and moderately tolerant varieties of T2 seedlings. MDA content was positively correlated with disease incidence in susceptible varieties followed by induction of LOX activity under T. viride JAU60 treatment (T3). T. viride JAU60 signifies induction of a LOX-related defense response to combat the collar rot disease incidence in groundnut seedlings.

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 β-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.

Trichoderma viride induces phenolics in groundnut (Arachis hypogaea L.) seedlings challenged with rot pathogen (Aspergillus niger Van Tieghem)

The study showed significant differences in percent collar rot disease incidence in groundnut varieties grown in non-infested soil (T1), challenged with pathogen – Aspergillus niger (T2), and pathogen + Trichoderma viride 60 (T3) treatments. Total phenols revealed a significantly higher content in tolerant varieties (J-11, GG-2) of groundnut compared with moderately susceptible (GAUG-10, GG-13) and susceptible (GG-20) varieties. The phenol content accumulated at a higher rate (193%) in GG-20, followed by GG-2 (146%) and J-11 (107%) varieties during disease development stages. HPLC analysis detected six major phenolics, viz., hydroquinone, gallic, chlorogenic, ferulic, salicylic and cinnamic acids. Among six peaks, hydroquinone was found highest in GG-2 at 3 days in T3. Gallic and salicylic acids increased up to 9 days, while ferulic acid continued to induce up to 15 days in tolerant varieties (J-11, GG-2) of Trichoderma-treated (T3) seedlings. A correlation study indicated that Trichoderma treatment induced five phenolics – except gallic acid – with a higher level of significance in a susceptible variety to reduce disease incidence compared with tolerant varieties. Results demonstrate the T. viride 60 mediated systemic induction of phenolics for biologic control and their probable role in protecting groundnut against A. niger infection.

Appraisal of RAPD and ISSR markers for genetic diversity analysis among cowpea (Vigna unguiculata L.) genotypes

The genetic variability and relationships among 11 cowpea genotypes representing two cultivars and nine elite genotypes were analyzed using 22 random amplified polymorphic DNA (RAPD) and nine inter-simple sequence repeat (ISSR) markers. ISSR markers were more efficient than RAPD assay with regards to polymorphism detection. But the average numbers of polymorphic loci per primer and resolution power were found to be higher for RAPD than for ISSR. Also, the total number of genotype specific marker loci, Nei’s genetic diversity, Shannon’s information index, total heterozygosity, and average heterozygosity were prominent in RAPD as compared to ISSR markers. The regression test between the two Nei’s genetic diversity indices showed low regression (0.3733) between ISSR and RAPD + ISSR-based similarities but maximum (0.9823) for RAPD and RAPD + ISSR-based similarities. The RAPD- and ISSR-generated cultivar- or genotype-specific unique DNA fingerprints able to identify the most diverse genotypes. A dendrogram constructed based on RAPD and ISSR combined data indicated a very clear pattern of clustering according to the groups (cultivars and elite genotypes). The results of principal coordinate analysis were comparable to the cluster analysis. Cluster analysis showed that most diverse genotypes (GP-125 — small size with good seed quality; GP-129, GP-90L — big size with poor seed quality) were separated from moderately diverse cultivars and genotypes. The genetic closeness among GP-129 and GP-90L, JCPL-42, and JCPL-107 could be explained by the high degree of commonness in these genotypes.

Biocontrol Mechanism of Bacillus for Fusarium Wilt Management in Cumin (Cuminum cyminum L.)

Cumin seeds are used as a spice for their distinctive flavor and aroma. Cumin seeds contain numerous phytochemicals that are known to have antioxidant, carminative and anti-flatulent properties, and are also an excellent source of dietary fibre. Cumin requires a moderately cool and dry climate for its growth, with a temperature between 25 and 30 °C. cumin grows best on well drained sandy loam to loamy soils with a pH range of 6.8–8.3. The yield of cumin is affected by lack of superior varieties, scientific crop production technology and vulnerability to diseases like wilt, blight and powdery mildew incited by Fusarium oxysporum f. sp. cumini, Alternaria burnsii and Erysiphe polygoni, respectively. In these diseases, wilt is most common, results in yield losses up to 35 % in cumin. Fusarium oxysporum is a causative agent of wilt disease in a wide range of economically important crops. Fusarium species is well distributed across many geographical regions and substrates, and also widely distributed in soils, plants, and air. Temperature in different climatic regions also affects the species distribution and virulence.

Metabolomics of groundnut ( Arachis hypogaea L.) genotypes under varying temperature regimes

Various metabolites were analyzed in groundnut genotypes grown under varying temperature regimes (based on date of sowing). Four contrasting groundnut genotypes viz. ICGS44 (high-temperature tolerant), AK159 and GG7 (moderately-high-temperature tolerant), and DRG1 (high-temperature sensitive) were grown at three different temperature regimes i.e., low (early date of sowing), normal (normal date of sowing) and high temperature (late date of sowing) under field conditions. Untargeted metabolomic analysis of leaf tissue was performed by GC–MS, while targeted metabolite profiling was carried out by HPLC (polyamines) and UPLC-MS/MS (phenolics) at both the pegging and pod filling stages. Untargeted metabolomic profiling revealed exclusive expression/induction of beta-d-galactofuranoside, l-threonine, hexopyranose, d-glucopyranose, stearic acid, 4-ketoglucose, d-gulose, 2-o-glycerol-alpha-d-galactopyranoside and serine in ICGS44 during the pegging stage under high-temperature conditions. During the pod filling stage at higher temperature, alpha-d-galactoside, dodecanedioic acid, 1-nonadecene, 1-tetradecene and beta-d-galactofuranose were found to be higher in both ICGS44 and GG7. Moreover, almost all the metabolites detected by GC–MS were found to be higher in GG7, except beta-d-galactopyranoside, beta-d-glucopyranose, inositol and palmitic acid. Accumulation of putrescine was observed to be higher during low-temperature stress, while agmatine showed constitutive expression in all the genotypes, irrespective of temperature regime and crop growth stage. Interestingly, spermidine was observed only in the high-temperature tolerant genotype ICGS44. In our study, we found a higher accumulation of cinnamic acid, caffeic acid, salicylic acid and vanillic acid in ICGS44 compared to that of other genotypes at the pegging stage, whereas catechin and epicatechin were found during the pod filling stage in response to high-temperature stress, suggesting their probable roles in heat-stress tolerance in groundnut.

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

Abstract 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.