| Pinus armandii Franch is an endemic species of China. It is one of the main trees for afforestation in the mountain regions of western China and the Upper and Middle Protection Forests of Yangtze River. However, their growth of diameter, height and volume, wood mechanical properties, the value of technologic process are declining for over ten years because of destroying by the blister rust. It is difficult to control, because the pathogen(Cronartium ribicola J.C Fischer)has a complicate life cycle and lots of alternate hosts. Nowadays, developing biocontrol pesticides from antagonistic microorganism, mycoparasite and Plant Growth-Promoting Rhizobacteria(PGPR) has become a heated topic. Three strains of Trichoderma spp. which had strongly destructive effects on C. ribicola have been screened by our group members of"Research on mechanism of Acremonium eryuanensis infecting Cronartium ribicola". They were proved to destroy aeciospores by chitinase andβ- 1,3 -glucanase.Based on the taxonomy and identification of three strains of Trichoderma spp.(TR1,TR2,TR3), the biological characteristics and enzymology properties of crude enzyme of these three strains were researched, and their controlling effect on infected (by C. ribicola) Armandii Pine was also studied in this paper. It will provide a theory basis for the application of biocontrol pesticides in the future. The main results were listed as the follows:(1)Cultural characters of TR1on PDA medium were obviously different from those of TR2 and TR3, and variation occurred after three generations. According to the microscopic features and cultural characters, TR1 was identified as Trichoderma atroviride P. Karsten. The microscopic features and cultural characters between TR2 and TR3 were very similar, and they were identified as T. viride Pers ex Fr. It was shown that the homology of ITS sequences between TR1 and T. atroviride was 100%; ITS sequences of TR2 and TR3 were completely the same to those of the T. atroviride. (2) Studies on the biological characteristics showed that: conidiospores of these three strains of Trichoderma spp. could germinate on all of these culture medium which were agar plate, glucose, PDA, modified Fries, bark juice of armandii pine and aeciospores. The germination rate and germination speed of TR1 were the highest on those culture medium; few conidiospores of TR2 and TR3 could germinate on the agar plate and glucose medium. The optimum temperature for conidiospore germination of these three mycoparasites ranged from 20℃to 25℃; The optimum pH value range was 4.0 to 5.0. The conidiospores of TR1 could germinate well when the relative humidity (RH) over 65%; those of TR2 and TR3 were over 50%. The conidiospores of TR1 germinated much better than those of TR2 and TR3 when the RH ranged from 90% to 100%. Darkness and nature light could stimulate conidiospore of TR1 to germinate. However, continuous exposure to light was benefit to conidiospore germination of TR2 and TR3. The optimum nutrient combination for mycelium growth of these three strains was made up of barley flour, soybean flour, K2HPO4, compound VB with 120r/min of rocking bed rate and pH 6.0. The maximum mycelia production of TR1 occurred at an inoculation volume of 5%, 25~30℃,-2.32 MPa of water potential; while maximum mycelia production of TR2 reached at an inoculation volume of 5%~9%, 20~25℃,-4.54 MPa of water potential. Light could stimulate the TR1 and TR2 to sporulate.It was the first time to study on the biological characteristics of T. atroviride as a mycoparasitic and biocontrol fungus.(3)Enzymology properties: Theβ-1,3-glucanase optimum temperature of TR1 and TR2 was 35℃; both were stable below 45℃; Ca2+ was their activator; Cu2+, Fe3+ and Al3+were their inhibitor. Acid-base stability range of TR1 was from pH 3.0 to 6.0 and the optimum was pH4.0; the Km was 43.82μg/ml. Acid-base stability range of TR2 was from pH 3.0 to 5.0 and the optimum was pH5.0; the Km was 71.28μg/ml. The highest enzyme activity of chitinase from TR1 reached at 40℃, while chitinase of TR2 kept high activity at 30℃~50℃. Acid-base stability range of chitinase from both strains was from pH 4.0 to 6.0. The optimum pH of TR1 was 6.0; the Km was 92.49μg/ml. The optimum pH of TR2 was 5.0; the Km was 71.11μg/ml. Mn2+ and Ca2+ could stimulate both enzyme activity, but Cu2+ and Fe3+ had inhibitory effects on them.(4) The indoor controlling effects on the C.ribicola showed that conidiophores and mycelium of TR1 and TR2 could grow and colonize on the accium of infected armandii pine, and caused the aeciospore's cell-wall or verrucae collapsing to die. It also indicated that the crude enzyme solution of these two strains had the same effect on the aeciospores. All of these proved that TR1 and TR2 achieved controlling effects by destroying aeciospores with the cell wall-degrading enzymes(CWDE).(5) The distribution of the mycelium and haustorium of C.ribicola in the infected stem have been detected by fuchsine acid staining. It showed that the mycelium and haustorium in the diseased pine mainly distributed at the cortex of stems and branches, and they ranged from 20 cm above the disease speckles to 15 cm below them. However, they were not found in the tissues of shoots and needles.(6) The field control trial by TR1 and TR2 showed that TR1 and TR2 could greatly decrease the disease incidence. The preventing and controlling effects was remarkable. Being treated by the TR1 at pycnia stage, the controlling effect could reach 53.7% each time. Continuing to be controlled two years by TR2, the best effect could reach 100%; the average controlling effect of the hillside sample plots was 99.1%; the average controlling effect of the ditch-side sample plots could reach 91.5%. Obviously, TR1 and TR2 would be an effective biocontrol agents on C.ribicola.
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