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

Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and ß-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and ß-1,3-glucanase, respectively. In vitro production of chitinase and ß-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 ß-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 ß-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.

Molecular diversity in the genus Nicotiana as revealed by randomly amplified polymorphic DNA.

The genus Nicotiana consists of 64 recognized species of which, only two species, tabacum and rustica are cultivated extensively. Wild Nicotiana species are storehouses of genes for several diseases and pests, besides genes for several important phytochemicals and quality traits, which are not present in cultivated varieties. Randomly amplified polymorphic DNA (RAPD) analysis was used to determine the degree of genetic variation in the genus Nicotiana and to develop species specific markers. Twenty two species and two interspecific hybrids were analyzed by using 18 random decamer primers. Genetic polymorphism abounds among the wild species of genus Nicotiana (99.5 %) as evidenced by the high degree of polymorphism in RAPD profiles. The pairwise similarity measures in the species of subgenus Rustica was 0.252 whereas in the subgenus Tabacum was 0.189, suggesting that there was significant diversity among the species of these subgenera. In the species of subgenus Petunioides, the range of pairwise similarity measures was 0.128 to 0.941. The clustering pattern coincided with the traditional classification of Nicotiana species. All the primers generated specific bands in the various species. Thirty six species-specific markers identified in the present study will be useful in interspecific breeding programs.