| Soil-borne diseases is a kind of plant disease,which is caused by pathogens in soil,such as fungi,bacteria,nematodes and viruses.When conditions are right,these pathogens begin to destroy the roots or stems of plants,which often have a serious impact on agricultural production.The prevention and cure of this kind of disease mainly adopts chemical control method.However,the long-term use of chemicals will pollute the soil,water and air,destroy the ecological balance.At the same time,the residues of chemical components in crops are also a serious threat to human health.Biological control is the use of antagonistic microorganisms to control soil borne diseases can overcome the above drawbacks of chemical control.Moreover,with the increasing awareness of environmental protection,focus on food safety issues,as well as to the construction of ecological environment,biodiversity conservation and sustainable development of agriculture,biological control of soil borne diseases which has become the focus of research and development.In this study,the common soil borne pathogens were selected as target bacteria to isolate a group of antagonistic microorganisms with strong biocontrol potential.The strongest antagonistic strains were selected as the research object.The strain was identified,and the ability of preventing disease and promoting growth was tested by pot experiment.Furthermore,we optimized the fermentation medium and culture conditions of the strain and defined the biocontrol mechanism of the strain.The main results of this paper are as follows:1.23 strains of antagonistic bacteria were isolated from the rhizosphere soil of pepper by using the method of dilution plate coating and the method of cross confrontation,with Phytophthora capsici as the target pathogen.Among the antagonistic bacteria,one strain,PBS 14 showed the strongest antagonistic activity.The diameter of inhibition zone was 26.0 mm,and it had strong inhibitory effect on several common soil borne pathogens.It has produced indole acetic acid(IAA)and phosphorus,ammonia production capacity,is a biocontrol agent with very good biocontrol potential.And it was identified as Bacillus amyloliquefaciens based on the results of 16S rRNA gene sequence analysis,physiological and biochemical testing,and morphological characteristics.2.The pot experiment was conducted with pepper as the tested plant.The results showed that B.amyloliquefaciens PBS 14 could effectively prevent the occurrence of Pepper Phytophthora Blight.Compared with control group(CK 1),the control effect of strain PBS14 was 77.27%.In addition,the strain PBS14 also significantly promoted the growth of pepper plants,and pepper growth indicators have been improved.Compared with control group(CK a),the plant height increased by 17.4%,the stem diameter increased by 38.8%,the dry weight of above ground increased by a factor of 38.8%,and the dry weight of roots increased by more than 53.8%.3.The diameter of inhibition zone was used as the evaluation index,using single factor test and uniform design method to optimize the B.amyloliquefaciens fermentation medium components,and determined the optimal medium composition were sucrose 0.541%,yeast extract 2%,KH2PO4 0.125%,MgSO4 0.025%.The theoretical value of the diameter of inhibition zone was 21.92 mm.When repeated experiments were done,the the diameter of inhibition zone was 21.47 mm.Using single factor test and response surface methodology to optimize the B amyloliquefaciens fermentation culture condition,and determined the optimal culture conditions were temperature 28?,250 mL tapered bottled liquid 60 mL,shaking speed 210 r/min,time 48 h,inoculation amount 4%.The theoretical value of the diameter of inhibition zone was 32.1292 mm.When repeated experiments were done,the the diameter of inhibition zone was 31.97 mm.4.Lipopeptide extracts were extracted from the sterile fermentation broth of B amyloliquefaciens PBS 14 by acid precipitation and methanol extraction.HPLC chromatography was used to separate the 5 components,of which a No.a had antibacterial activity.The main components were identified by HPLC-ESI-MS analysis,and the main antibacterial substance was identified as C15 bacillomycin.The results of antifungal test of volatile organic compounds flat and solid leaf showed that the volatile organic compounds of the strain PBS 14 had a strong inhibitory effect on most plant pathogens.And it can effectively prevent and control the pathogen of plants.Using headspace solid-phase microextraction method to collect volatile substances,and GC-MS analysis showed that strain PBS 14 produced at least 34 kinds of volatile substances,mainly were benzene,alkanes,alcohols,ketones,aldehydes and lipids.16 kinds of volatile substances were selected to carry out antibacterial experiment,and the results showed that all of them had strong antifungal activity.5.Induced resistance test results show that the simultaneous inoculation of B.amyloliquefaciens PBS 14 and P.capsici compared with the control,single inoculation the strain of PBS14 and single inoculation of P.capsici,the enzyme activity of POD,PPO and PAL can quickly enhance and improve.The enzyme activity of POD and PAL could increase rapidly and reach the maximum value at 48 h,then gradually decreased and maintained at a high level.The enzyme activity of PPO increased rapidly and reached the maximum value at 72 h,then decreased gradually and maintained at a high level.The simultaneous inoculation of B.amyloliquefaciens PBS 14 and P.capsici compared with the control,single inoculation the strain of PBS 14 and single inoculation of P.capsici,The relative gene expression of POD,PPO and PAL was significantly higher than single inoculation of P.capsici,and showed the same trend with the enzyme activity of POD,PPO and PAL. |
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