| Bacillus subtilis Czk1,the so-called endophytic bacteria of rubber tree,proved to have antifungal effect on both the rubber tree root pathogen and Colletotrichum gloeosporioides.Specifically,based on previous pot incubation test conducted by Fan etal.,favorable biocontrol effect the Czkl exerted on Colletotrichum gloeosporioides was observed.Preliminary test results obtained indicated that lipopeptide antibiotics produced by Czkl may have antagonistic effect against pathogen.However,other disease prevention mechanism by Czkl is still unclear.In the present study,the influences that Czkl exerted on both the disease-resistant related defense enzyme activity and plants growth promotion were investigated by the determination of colonization dynamics of Czkl existed in rubber plants or the rhizosphere soil.Furthermore,the colonizing capacity of strain Czkl,the induced generation of systemic resistance related defence enzyme,plant growth promoting effect were identified.Besides,based on the enlargement of disease-prevention mechanism of strain Czkl,solid foundation for the application of strain Czkl in the biological control of plant disease was erected.(1)The marked strain Czk1R selected via antibiotic notion method were immune to both streptomycin(100μg/mL)and cyclomycin(100μg/mL).Compared with the original strain Czkl,the marked strain Czk1R exhibited several characteristics as follows:no significant difference in colonial morphology,similar growth curve,no significant difference in the antagonism against pathogen.Meanwhile,the antibiotic resistance was relatively stable,which absolutely satisfied the colonization test requirements.(2)Varied inoculation methods were employed to detect the invasion routine of marked strain Czk1R into the test rubber plants.Among the methods introduced,strain Czk1R exclusively colonized in the stalk of rubber plants by intravenous injection inoculation method,but colonized either in the root or the stalk of rubber plants by both root-drenching method and root dipping inoculation methods,and the root-drenching method demonstrated optimal colonization results.The colonization dynamics determination results of Czk1R content in root and stalk of rubber plants implied that,after invading the rubber plants,the overall bacteria content first increased up to the maximum colonization content,and then gradually decrease to reach a constant value,specifically,the maximum colonization content in root and stalk of the plant were 8.33×103 CFU/g,8.98×102 CFU/g,respectively.In addition,the Czk1R colonization content in root is always higher than that in the stalk of plant,and the marked strain Czk1R could even be detected after 60 d in the plant stalk and root since inoculation.The detection of strain Czk1R population dynamic in the rubber plants rhizosphere soil revealed that the maximum colonization content of strain CzklR in autoclaved and non-autoclaved soil was 6.44×106 CFU/g,1.20×106 CFU/g,respectively.Moreover,the gradual decrease of bacteria content in soil was noticed after inoculation,particularly,after 60 d of root-drenching inoculation,the marked strain Czk1R determined in autoclaved and non-autoclaved soil were 6.30×102 CFU/g,8.77×101 CFU/g,respectively.(3)To identify the features of induced generation of systemic resistance on rubber seedling plant by Czkl,the influences of Czkl fostered in fermentation liquor with varied concentrations(stock fermentation liquor,10-folds diluted fermen-tation liquor,100-fold fermentation liquor),exerted on 5 types of plant leaf contained systemic resistance related defence enzyme CAT,SOD,POD,PAL and PPO were investigated.The test results indicated that,activity of the 5 types of defence enzyme mentioned above increased by foliage spray of Czkl fermentation liquor,among them,100-fold fermentation liquor group exhibited the optimal effect.in which,the enzyme activity peaks was 3 fold that achieved in the controlled test group.Moreover,Anthracnose resistance of rubber plant can be induced by Czkl strain,generally,inoculation of biocontrol bacteria,Czkl prior to pathogenic bacteria,RC178 could significantly improve activity of the 5 types of defence enzyme mentioned above,and has the least H2O2 content compared with other disposal methods.Such results inferred that the Anthracnose resistance activity could be introduced by the systemic resistance generated by Czkl strain induction.(4)By successively root-drenching treatment of the rubber seedling with Czkl fermentation liquor,60 d later,plant height,plant height growth,leaf number,leaf fresh weight,saturated weight,leaf dry weight were all higher than the controlled group noticeably.However,the relative water content is a bit less than the controlled test group.Meanwhile,by treatment of the rubber plants with Czkl fermentation liquor,drastic increase of chlorophyll a and b content in the rubber seedling leaf was observed,which were 43.76%,42.75%higher than that of the controlled test group,respectively.While the MDA content was almost the same as the controlled test group,which further indicated that treatment of the rubber plant with Czkl fermentation liquor will not lead to the increase of the toxic MDA content,however,the photosynthesis effect was reinforced,which will in turn promote the plant development. |
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