Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

Abstract Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

Antifungal properties of native Trichoderma isolates against Sclerotium rolfsii and Pythium aphanidermatum infecting tobacco.

Isolates of Trichoderma native to tobacco rhizosphere, were evaluated for their antifungal properties against Sclerotium rolfsii and Pythium aphanidermatum, fungal pathogens causing collar rot and damping off diseases in tobacco. Isolate TvJt1 showed maximum HCN production (0.14 OD) followed by ThHt1 and minimum by isolates ThJnt and ThRt1 (0.08 and 0.09 OD). Indole acetic acid (IAA) production among the isolates varied from 6.48 to 14.82 pg ml(-1). Isolate ThJO1 produced maximum siderophore (13.48 m mol ml(-1), whereas isolate TvJt1 produced minimum content (6.24 m mol ml(-1)). Isolate ThJt1 showed maximum and significantly higher chitinase activity (62.12 p kat m;(-1)) as compared to other isolates, followed by isolate TvHt2. Isolate TvHt2 showed maximum and significantly higher ß-1, 3-glucanase activity (9.94 n kat ml(-1)) when compared to all other isolates. Isolate ThJt1 showed second highest activity of ß-1, 3-glucanase (6.75 n kat/ml). In dual culture, inhibition of mycelial growth of Pythium by isolate TvHt2 was maximum (72.45%) followed by ThJt1 whereas inhibition of mycelial growth of S. rolfsii by these two isolates was at a par (78.36% and 76.17%) and significantly higher than some isolates. Isolates TvHt2 and ThJt1 showed maximum and significantly higher inhibition of Pythium and Sclerotium growth (80.24% and 76%) by production of non-volatiles. Inhibition of Pythium and Sclerotium growth by production of volatile compounds by Trichoderma isolates ranged from 54.36 to 72.6% and 66.24 to 84.24% respectively. Hence, further investigation with potential isolates ThJt1 and TvHt2 in tobacco nursery may help to explore these bioagents for control of collar rot and damping off diseases in tobacco.

The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms.