The Applications Of Bacillus Subtilis Bac1 Against The Few Important Diseases Of Walnut

Bacillus spp.,a class of aerobic or partly anaerobic Gram-positive bacteria that can produce budding spores and have rich physiological characteristics.They are widely distributed on the surface of plants and roots.Walnuts are the first of the"Four Dried Fruits"in the world and contain medicinal and commercial values,but the walnut industry in China still faces many problems,such as many kinds of diseases,wide distribution and heavy hazards.Therefore,it is very important to improve the quality of walnuts and promote the green development of the walnut industry through the green control of major walnut diseases.In this paper,we investigated the broad-spectrum resistance of Bacillus subtilis strain Bac1 and TasA,which was previously stored in the laboratory in advance,to determine the inhibition spectrum,optimize fermentation conditions,prepare and validate the wettable powder and the structure prediction and prokaryotic expression of TasA protein.The following results were obtained:1.The inhibition spectrum of the strain Bac1 is determined.The results of the plate confrontation test between the strain Bac1 and the main pathogens bacteria of walnut showed that the strain Bac1 inhibition activity was broad-spectrum,and the strongest inhibition effect is on Boeremia exigua.The results of qualitative enzyme production tests using the hydrolysis loop method showed that the strain Bac1 can secrete and produce chitinase and protease.srf AA,fen D,bmy B,and bac A gene fragments were obtained by PCR amplification,indicating that the strain Bac1 may have the potential to produce these kinds of lipopeptides.2.Optimization of the strain Bac1 fermentation conditions.We investigated the effects of the interaction between fermentation broth p H,incubation temperature and rotational speed on the density of the strain Bac1 and the total protein content of fermentation broth,and concluded that p H 7,temperature 30℃and rotational speed 200 r·min^-1 were the optimal fermentation conditions for the strain Bac1,according to the prediction of response surface methodology and verified by experiments.3.Preparation of the strain Bac1 microbial wettable powder.Diatomaceous earth was selected as the best carrier,and Tween-80,PEG6000 and dextrin were selected as the best additives.The quality ratio of the wettable powder was 60%diatomaceous earth,10%Tween-80,15%PEG6000 and 15%dextrin,and the quality of the agent meet a criterion of the national standard.The results of plate confrontation and field prevention tests showed that the powder had a better inhibitory effect on pathogenic fungi and prevention effect on walnut diseases,which was significantly better than the control,and thereafter could be further promoted for use in practices.4.TasA protein properties and gene expression characteristics.tas A gene amplification resulted in a 786 bp sequence,the number of encoding amino acid number is 261,the weight of relative molecular is 28128.65 Dalton,and isoelectric point is 6.35,which is a hydrophilic stable protein localized to the extracellular or cell wall.In an antagonistic culture with B.exigua,and Fusarium solani in the antagonistic medium,the relative expression of tas A showed a decreasing trend and was highest at 12 h.In an antagonistic test in the fermtation with Dothiorella viticola,Colletotrichum gloeosporioides,and Botryosphaeria dothidea,the Bac1 relative expression of tas A showed a trend of increasing and then decreasing,with the highest at 18 h.5.Prokaryotic expression of the repressor protein TasA.The recombinant plasmid expression p ET-30a（+）:tas A was successfully constructed,transferred to expression strain BL21,and the expression was induced using IPTG.By screening the IPTG concentration and induction time of TasA expression conditions,it was determined that the prokaryotic expression conditions were 0.05 mmol·L^-1 IPTG induced at 20°C for 2 h.The protein purification was performed using Ni columns,and TasA purified protein showed a different degree of inhibition activity against pathogenic fungi,with the best inhibition effect against C.juglandis.This study showed that the strain Bac1 and TasA have a broad-spectrum inhibitory effects.This study also provides a theoretical and technical basis for the research and utilization of the strain Bac1 and the inhibitory protein TasA of B.subtilis,and the green control of walnut diseases.