| Camellia oleifera(oil-tea)is a unique and important woody edible oil tree species in China,which is widely distributed in the south of Qinling-Huaihe region of China.Athracnose and leaf blight are common diseases of Oil-tea,which seriously restrict the development of Oil-tea industry.At present,chemical pesticides are mainly used for disease control.However,the large-scale use of chemical pesticides will not only make the pathogenic fungi of Oil-tea resistant,but also pollute the environment and be harmful to human body.Microbial control of plant diseases has the characteristics of high quality,high efficiency,low toxicity,low residue,and environmental safety.Therefore,microbial control of Oil-tea diseases is an effective measure to achieve green and sustainable control of Oil-tea diseases.In order to screen out new biocontrol bacteria resources,taking the pathogenic fungi of oil-tea anthracnose as indicator fungi,the endogenous antagonistic bacteria in healthy leaves of Oil-tea were isolated and their fungistasis properties were studied in order to provide new strain resources for the development of microbial pesticides.In this study,the antagonistic bacteria were isolated and screened from the leaves of healthy Oil-tea,and the strain with the best fungistasis was selected.The strain was identified,the fermentation medium was optimized,and the antifungal activity was determined.Based on the existing biological control research foundation of our laboratory,the antagonistic strains with high quality,high efficiency and compoundability were selected scientifically.Finally,the preparation of water dispersing agent and the field control effect test were performed.The results of this study are as follows1.Five strains with stable antagonistic effect were screened out in this experiment.The bacterium HBMC-B05 with the best antagonistic effect was selected as the object of further research.Through colony and cell morphology observation,biochemical characteristics and physiological,gyrA gene and 16S rDNA gene Sequence analysis was used to identify the species,and it was identified as Bacillus velezensis.2.The single-factor test determined that the optimal basal medium of B.velezensis HBMC-B05 strain was YSP medium,the sucrose was the best carbon source,and the best nitrogen source was yeast extract and tryptone;the results of response surface analysis showed that the optimal fermentation medium of the strain is:sucrose 20.12 g/L,tryptone 9.96 g/L,yeast extract 5.2 g/L and magnesium sulfate 2.22 g/L;Under the control of single fermentation condition,the optimal culture temperature is 28?,the optimal initial pH of medium is 7.0,and the optimal rotating speed of shaking table is 180 r/min.3.The antifungal test showed that the antifungal effect of B.velezensis HBMC-B05 on the main pathogenic fungi of oil tea anthracnose can reach more than 70%,it was the best for Colletotrichum fructicola,with a inhibitory rate of 81.31%.It also has good antifungal effect on the other 4 pathogenic fungi of oil-tea.The effect of HBMC-B05 fermentation broth on the mycelial growth and spore germination of C.fructicola,an experiment was further investigated.The results showed that the fermentation liquid had a great influence on the hyphae growth and spore germination of C.fructicola.The specific manifestations were as follows:the hyphae bulge,the branches of the hyphae decreased,dysmorphic and the top expanded,and only a very small number of spores were observed,which obviously hindered the normal formation of the colony,while the mycelium in the control group could produce spores normally,forming dense mycelium colony normally.The spore germination was dysmorphic to varying degrees,the germ tube was enlarged as a whole,abnormal branching occurred,and the number of spore germination was less.According to the research basis of the biological control of Oil-tea disease in the early stage of the laboratory,Bacillus subtilis Y13,which has good biological control effect on Oil-tea disease and is also an endophyte of Oil-tea,was selected as the target compound strain,and experiments showed that the compound control effect It is greater than the control effect of a single strain,and the two have affinity,so Y13 and HBMC-B05 form a BY10 complex.4.The dosage form of the compound microbial agent is water dispersing granule.This paper mainly studies the selection and the best usage of the carrier,wetting agent,dispersing agent,disintegrating agent and binder of BY 10 compound bacterial water dispersing agent Through single factor test and orthogonal test,the formulation of BY10 compound bacteria water dispersing agent was determined as:10%-90%BY10 compound bacteria original drug,5%polyvinyl alcohol,6%sodium polyphosphate,6%sodium alginate,3%polyethylene glycol,carrier diatomite to 100%.The field control effect test showed that the compound bacterial agent had good inhibitory effect and preventive effect on Oil-tea disease,and the control effect of BY10 compound water-dispersible granule diluted 100 times could reach 59.05%. |
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