| The potato belongs to the family Solanaceae,an annual dicotyledon herb,and is the fourth largest food crop in the world.But the potato late blight caused by Phytophthora infestans(Mont.)de Bary is recognized as the world’s largest crop disease.At present,the occurrence and damage of diseases are mainly reduced by selecting disease-resistant varieties,spraying chemical pesticides and strengthening cultivation management.chemical pesticides are still the main means of controlling late blight in production.Due to the long-term excessive use of chemical pesticides,P.infestans has developed strong resistance and polluted the environment,and even threatened human health.In addition,in recent years,due to the accelerated physiological and biochemical differentiation and mutation of P.infestans,especially the emergence of A2 mating types,the frequency of genetic recombination of P.infestans has increased,and a full-spectrum physiology race that can overcome all 11 single late blight resistance genes in the entire set of identification hosts has emerged.At the same time,the resistance of the P.infestans to chemical pesticides is also increasing,as a result,late blight is more and more difficult to control.Therefore,the search for new,safe and friendly control measures has become a research hot spot for many scholars.In recent years,the use of microorganisms and their metabolites to inhibit the P.infestans and control potato late blight has been attended greatly by many researchers.But,exploring biocontrol preparations of microorganisms and their metabolites to inhibit P.infestans and control potato late blight is relatively rare.In our laboratory,many antagonistic strains against P.infestans have been screened in the early stage,but in the process of using the antagonistic bacteria to continuously inhibit the growth of P.infestans mycelium and sporangia germination,and to control late blight on in vitro detached leaf,tuber slices and potted plants,it was found that the anti-oomycetes activity and disease prevention effect of a single bacterial strain were often not stable enough.However,if two or more antagonistic bacteria are used for compound fermentation(CF)or their fermentation liquids were mixed together(MF)according to a certain ratio after cultured alone,the anti-oomycetes activity and disease prevention effect of CF and MF were significant better than that of single strain.It was conjectured that there was a synergism among different strains.In particular,the anti-oomycetes activity and disease prevention effect of CF from five strains of bacterial SR13-2,HT-6,ST-1,W-7,and actinomycete Sy11,on P.infestans mycelial growth and sporangia germination,as well as late blight were significant better than that of the single strain HT-6.However,the CF is a suitable solution for ready-to-use,and has strict requirements on storage environmental conditions,short storage time,and is not convenient to use in the field.Based on the previous work in our laboratory,this thesis intends to make the CF liquid of these five strains into a biological preparation to control potato late blight in pot and field plants,and to clarify the mechanism of promotion plant growth and disease prevention as well as its stability,in order to provide reference for controlling potato late blight in actual production.The research contents of this thesis mainly include:(1)five antagonistic compound fermentation liquids of bacteria SR13-2,HT-6,ST-1,W-7 and actinomycete Sy11 were made into a compound bacterial agent(CBA),and to investigate the effects of different temperature,UV,and storage time on the number of live bacteria in CBA;(2)To clarify the effect of the CBA treatment on the potato tissue in vitro,seedling germination stage and pot plants to prevent late blight in potato;(3)To understand the physiological changes of the plant in the process of promoting the potato plant growth and enhancing potato resistance to late blight,including soluble protein,soluble sugar,MDA,chlorophyll content,and related resistance enzyme activities;(4)preliminary effect evaluation of using CBA for preventive potato late blight in field.Completion of the above content can provide a foundation for development and utilization of CBA of these five stains of bacteria to effectively prevent potato late blight in future.The main results obtained in this experiment are as follows:1.The CBA of five antagonistic strains of was obtained.This agent was resistant to high temperature and ultraviolet radiation and could be stored at room temperature for more than 6months.The composite fermentation liquids of these five strains of bacteria were centrifuged,mixed with the carrier,dried,ground,and filtered and made into a CBA.The number of alive bacteria was 2.61×10~9 cfu/g in CBA.With a strong environmental stability,it could be stored at room temperature for at least 6 months,and resistant to high temperature of 90℃and direct UV irradiation at 2 pm for 60 min.Under the above conditions,the number of viable bacteria in CBA could be maintained between 1.06×10~6 cfu/g and 3.37×10~7 cfu/g.2.There was no adverse effect on potato tuber slices after treatment with CBA,but the significant control effects on the occurrence of late blight on tuber slices,seed potato germination,potted plants and detached leaves were observed.Three ways including disease prevention and cure of CBA,as well as CBA treatment with inoculation pathogen simultaneously for control disease were tested in this experiment.Among them,the preventive effect was significantly better than that of cure and simultaneously treatments(P<0.05).The disease index(DI)was 0(DI 85 in control),and the relative protection rate was100%,and significantly better than that of metalaxyl manganese and zinc treatment(DI 30)(P<0.05),on potato tuber slices;the disease prevention effect(DI 30)after seed soaking of CBA in seed germination stage was significantly better than metalaxyl manganese and zinc(DI 46.7),which is significantly better than LBL control(DI 73.3);the control effect of soaking seeds combining with spraying leaves on potted plants(DI 24)is significantly better than that of soaking seeds(DI 33.2)and spraying leaves(DI 46.5)individual treatment,as well as metalaxyl manganese and zinc treatment(DI 36)(P<0.05);On in vitro leaves,the disease prevention effect of CBA soaking seeds combining with spraying leaves were also the best,and the DI was 20,while DI of seed soaking and leaf spraying treatment alone were 43and 55,respectively,which were significantly better than those of LBL(DI 86.7)and metalaxyl manganese and zinc(DI 36.7)(P<0.05).3.The CBA could significantly promote the growth of potted potato plants,the plant height,stem thickness,leaf number,leaf area,and root growth and tuber yield were significantly increased.Of them,the effect of promoting growth after soaking combining with spraying the leaves was the most obvious.On the 67th day of sowing,the plant height,stem thickness,leaf number,leaf area,root length,fresh root weight,dry root system,and tuber yield treated after CBA soaking seed tuber combining with spraying the leaves were 45.3 cm,2.2 cm,54 pieces,53.27 mm~2,17.9 cm,37.6 g,5.79 g,and 215.7 g,respectively;there was a significant difference between the leaf area(53.27 mm~2)of CBA soaking seed tuber combining with spraying the leaves and that(54.43 mm~2)of spray treatment alone(P<0.05).Plant height,stem thickness,leaf number,root growth and yield of CBA soaking seed tuber combining with spraying the leaves were all superior to seed soaking alone,but there were no significant difference between them.4.Content of soluble sugar,protein,resistant enzymes and total chlorophyll in the leaves of potted potato plants were significantly increased after different treatments.In comparison with other treatments,the largest increase was found in that of CBA soaking seed tuber combining with spraying the leaves.After emergence of seedlings,the 7-8th compound leaves were completely unfolded(leaf spraying for 5 days),soluble sugar,protein,PPO,POD,PAL,MDA and total chlorophyll content in leaf were 27.5 mg/g,14.0 mg/g,60.7 U/g min,372.6U/g min,438.4 U/g min,0.89 mmol/g,3.56 mg/g,respectively,which was significant better than the treatments of seed soaking,spraying leaves,and metalaxyl manganese and zinc alone(P<0.05).5.The promotion effect of potato plant growth and a good practical disease prevention effect of CBA were preliminarily verified in the field plots.Compared with the LBL control,CBA had a obvious promotion effect on potato plant height,leaf area,leaf number and tuber yield.In the Zhang bei test site,the DI of the late blight after CBA treatment was 26.3,and that of metalaxyl manganese and zinc as a control was 33.3,there was a significant difference between them(P<0.05). |
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