Screening, Identification Of Multi-functional Trichoderma And Diversity Of Trichoderma In Soil

Trichoderma, an important part of soil microbial flora which can control a variety of plant disease if increase its content in soil, is a kind of biological control fungi of wide application. By analyzing the genetic and functional diversity of Trichoderma in the soil, it can provide the theoretical foundation for developing multi-functional Trichoderma, expanding their application area in effectively controlling plant pathogens. It is possible to find Trichoderma strains of multi-function like nitrogen fixation, phosphate solubilization, except for the ability to control plant pathogens.A number of 244 Trichoderma was isolated from 120 soil samples collected from greenhouse field of tomato, cucumber, leek, chinesevegetable cabbage, garlic and green onions from Linyi, Liaocheng, Shouguang, Zhangqiu of Shandong Province.All of the 244 Trichoderma strains were dually cultured with Rhizoctonia solani which is a pathogen to cotton, and 114 isolates were shown to inhibit Rhizoctonia solani, of which 19 isolates showed better inhibitory ability than the others. All of the 244 Trichoderma strains were detected in their capability to degrade insoluble-phosphate, insoluble-potassium and fix nitrogen by solid plate culture method. The result showed that 74 isolates have obvious insoluble-phosphate solubilization ability, the above mentioned 19 isolates which were better in inhibitory ability showed also the best solubilization ability. The result also showed that no Trichoderma strain solubilize insoluble-potassium, and there is no isolates could grow on nitrogen-free medium.Nineteen Trichoderma isolates with better inhibitory and insoluble-phosphate solubilization ability were detected in their ability to solubilize insoluble-phosphate and insoluble-potassium in liquid culture. The result indicated that some strains did not show the ability of solubilizing the insoluble-phosphate, whereas isolate T13-8 (PO₄^3- content, 1.792μg/mL) and T23 (PO₄^3-content,1.652μg/mL) showed stronger ability in solubilizing the insoluble-phosphate; some isolates solubilized insoluble-potassium in liquid culture, in which isolate T47-4(KCl content, 20.585μg/mL) was the strongest.Besides, the 19 Trichoderma isolates were detected in their phytase activity in solid culture. The result shows that 15 isolates hydrolyse phytin hydrolyzing circle, including isolate T13-8, T9-1 which were the best in phosphate solubilization and T47-4 which was the best in solubilizing potassium in liquid culture.The 19 Trichoderma isolates were identified by morphological and molecular method. The result indicated that isolate T8-4, T9-1, T23, T25, T30-5, T31-3, T38-5, T47-4, and T35 -1 were Trichoderma Longibrachiatum, isolate T80-2, T38-6, T13-8, T44-1, T57, T62-2, T34, and T17 were Trichoderm Harzianum, isolate T61-2 was Trichoderma Virens, isolate T18-1 was Trichoderma citrinoviride. Phylogenetic analysis based on ITS sequence support the identification of these 19 isolates.Combining the soil samples from the same area with the same vegetable, 120 soil samples were mixed into 13 soil samples. ITS conservative sequence amplification primers specific to Trichoderma were used to analyze the diversity of Trichoderma in soil samples with PCR-DGGE technology. It was shown that Trichoderma in soils from Linyi Prefecture was the highest in diversity, followed by Liaocheng, Shouguang and Zhangqiu areas. The result also indicated that similar Trichoderma species occurred in different vegetable crop soils even collected from different areas. Areas with the higher Trichoderma diversity have the relatively lower rate of soil-borne diseases.In order to provide theoretical support to the feasibility of using PCR-DGGE technology to analysis the soil Trichoderma diversity, PCR production amplified from both mixed genomic DNA of 19 Trichoderma strains and genomic DNA of 4 single Trichoderma strain were analyzed by DGGE technology as well as PCR production from corresponding soil genomic DNA. Both studies proved that the method using PCR-DGGE technology to analyze the Trichoderma diversity of soil is feasible.