Construction of engineering Trichoderma strains and their characteristics against tomato gray mold

The transformed Trichoderma strains Ttrm31, Ttrm34 and Ttrm55 were obtained from Trichoderma wild strain T21 mutated by REMI technique for more effective biocontrol of tomato gray mold (Botrytis cinerea) with Trichoderma agent. Those transformants appeared much better in biocontrol activity in vitro or in vivo against tomato gray mold were better than that of wild strain T21. The main results were as follow: The transformed Trichoderma strains were detected in their genetic stability by consecutive culture for several generations, growing them on hygromycin-contained medium, and then further detection by PCR. The results showed that the REMI was very useful to construct the transformed Trichoderma strains even though some non-transformants concurrently appeared with positive reaction, but which could be distinguished and avoided by southern blotting. The biological characteristics of transformed Trichoderma strains varied among themselves or differed in parts as compared with wild one. Much changes were detected in the conidia germination, sporulation, tolerance to high-temperature, growth at a certain range of temperature, cultural characteristics under different pH condition as well as utilization of carbon and nitrogen nutrition. Some transformants performed better in the most of mentioned characteristics compared with wild one, others not. For example, conidia germination of strain Ttrm31 was higher than that of wild strain T21. Conidia productivity of Ttrm34 was higher than that of wild strain T21. As the tolerance to high-temperature, Ttrm31 and Ttrm34 were enhanced remarkably relative to wild strain. Meanwhile, it was interested that the growth of transformed Trichoderma strains improved much once treated under high-temperature (50℃) for 1 h. Differently, Ttrm55 grew faster at low-temperature (15℃) ,Ttrm31,Ttrm34 and Ttrm55 grew fast as the wild one at 20-30℃. The transformed Trichoderma strains differed in cultural morphology including color and sporulation at different pH, and even on PDA. Protein and asparagines were screened as better nitrogen, and glucose and maltose as better carbon source. There were several mechanisms expected among transformed Trichoderma strains against Botrytis cinerea. Ttrm31, Ttrm55 and Ttrm111,for instance, had more ability for the nutrition and space competition over pathogen, while Ttrm34 and Ttrm76 inhibited pathogen in the way of mycoparasitism which subsequently confirmed by the higher activities of chitinase and β-glucanase needed for parasitizing pathogen. Similarly Ttrm34 and Ttrm55 were higher in both enzyme activities than those of T21. Additionally a putative role of induced resistance might be involved in the biocontrol mechanism because a series of defensive enzyme in tomato were increased in activities such as PAL, POD, CAT, PPO, SOD, chitinase and β-glucanase once tomato plants treated with transformants, but the level of the induction depended on the transformed Trichoderma strains used. Comparatively, PAL, POD, CAT, PPO and chitinase activities in tomato induced by Ttrm55 were great different from other transformants except SOD and β-glucanase. SOD activity in tomato stimulated by strain Ttrm55 was higher than that induced by others, while β-glucanase activity was lower. The CAT, PAL, PPO activities induced by Ttrm31 were higher than those by other transformants, but lower in SOD, POD and chitinase than those by others. In general POD, PPO, PAL and chitinase activities were higher in tomato induced by Ttrm34 than other enzymes. β-glucanase activities induced by T21 were higher than those of others, but contrary in other enzymes. Transformed Trichoderma strains had the same ability in the colonization on the tomato leaf surface as the wild strain T21, however no much differences were seen between transformants and wild one in the trait. More importantly 3 transformed Trichoderma strains were proved to be very effective in the control of gray mold in flowers and leaves and 14%-18.5% and 7%-9% were increased over wild strain

The transformed Trichoderma strains Ttrm31, Ttrm34 and Ttrm55 were obtained from Trichoderma wild strain T21 mutated by REMI technique for more effective biocontrol of tomato gray mold (Botrytis cinerea) with Trichoderma agent. Those transformants appeared much better in biocontrol activity in vitro or in vivo against tomato gray mold were better than that of wild strain T21. The main results were as follow: The transformed Trichoderma strains were detected in their genetic stability by consecutive culture for several generations, growing them on hygromycin-contained medium, and then further detection by PCR. The results showed that the REMI was very useful to construct the transformed Trichoderma strains even though some non-transformants concurrently appeared with positive reaction, but which could be distinguished and avoided by southern blotting. The biological characteristics of transformed Trichoderma strains varied among themselves or differed in parts as compared with wild one. Much changes were detected in the conidia germination, sporulation, tolerance to high-temperature, growth at a certain range of temperature, cultural characteristics under different pH condition as well as utilization of carbon and nitrogen nutrition. Some transformants performed better in the most of mentioned characteristics compared with wild one, others not. For example, conidia germination of strain Ttrm31 was higher than that of wild strain T21. Conidia productivity of Ttrm34 was higher than that of wild strain T21. As the tolerance to high-temperature, Ttrm31 and Ttrm34 were enhanced remarkably relative to wild strain. Meanwhile, it was interested that the growth of transformed Trichoderma strains improved much once treated under high-temperature (50℃) for 1 h. Differently, Ttrm55 grew faster at low-temperature (15℃) ,Ttrm31,Ttrm34 and Ttrm55 grew fast as the wild one at 20-30℃. The transformed Trichoderma strains differed in cultural morphology including color and sporulation at different pH, and even on PDA. Protein and asparagines were screened as better nitrogen, and glucose and maltose as better carbon source. There were several mechanisms expected among transformed Trichoderma strains against Botrytis cinerea. Ttrm31, Ttrm55 and Ttrm111,for instance, had more ability for the nutrition and space competition over pathogen, while Ttrm34 and Ttrm76 inhibited pathogen in the way of mycoparasitism which subsequently confirmed by the higher activities of chitinase and β-glucanase needed for parasitizing pathogen. Similarly Ttrm34 and Ttrm55 were higher in both enzyme activities than those of T21. Additionally a putative role of induced resistance might be involved in the biocontrol mechanism because a series of defensive enzyme in tomato were increased in activities such as PAL, POD, CAT, PPO, SOD, chitinase and β-glucanase once tomato plants treated with transformants, but the level of the induction depended on the transformed Trichoderma strains used. Comparatively, PAL, POD, CAT, PPO and chitinase activities in tomato induced by Ttrm55 were great different from other transformants except SOD and β-glucanase. SOD activity in tomato stimulated by strain Ttrm55 was higher than that induced by others, while β-glucanase activity was lower. The CAT, PAL, PPO activities induced by Ttrm31 were higher than those by other transformants, but lower in SOD, POD and chitinase than those by others. In general POD, PPO, PAL and chitinase activities were higher in tomato induced by Ttrm34 than other enzymes. β-glucanase activities induced by T21 were higher than those of others, but contrary in other enzymes. Transformed Trichoderma strains had the same ability in the colonization on the tomato leaf surface as the wild strain T21, however no much differences were seen between transformants and wild one in the trait. More importantly 3 transformed Trichoderma strains were proved to be very effective in the control of gray mold in flowers and leaves and 14%-18.5% and 7%-9% were increased over wild strain in