Screening And Formulation Development Of Biocontrol Strains For Root Rot Of Zanthoxylum Bungeanum

Zanthoxylum bungeanum root rot is a long-standing soil-borne disease that spreads over the years.Once it occurs,it can cause large-scale infection in the Z.bungeanum orchard,seriously affecting its yield and quality,and causing huge economic losses to the Z.bungeanum industry.Currently,chemical pesticides are mainly used for the prevention and control of Z.bungeanum root rot.However,the extensive use of chemical pesticides has caused serious environmental pollution,pesticide residues,soil compaction and other problems,which have aroused people’s high attention.Therefore,it is urgent to develop green and pollution-free prevention and control measures for Z.bungeanum root rot.Biological control is an important way of pollution-free control of plant diseases,and exploring biological control resources is a prerequisite for biological control.Microorganisms are a very important biological control resource.Therefore,this study aimed to isolate and screen Z.bungeanum root rot biocontrol bacteria from the rhizosphere soil of Z.bungeanum and develop its preparation,aiming to provide guidance for pollution-free prevention and control of Z.bungeanum root rot.The following research results were obtained:（1）Three different types of culture media were used to isolate a total of 303 microbial strains from the rhizosphere soil of Z.bungeanum.20 bacterial strains（BS₀）showing significant antagonistic activity against Z.bungeanum root rot were screened using the plate confrontation culture method.Morphological,biochemical,and 16S r DNA sequencing analyses were performed to identify these 20 BS₀ strains,including 5 strains of Bacillus amyloliquefaciens,7 strains of B.velezensis,3 strains of different Burkholderia bacterial species,2 strains of Flavobacterium anhuiense,and the remaining 3 strains were Agrobacterium tumefaciens,Cupriavidus necator,and Delftia tsuruhatensis,respectively.（2）The antagonistic activity of the 20 BS₀ strains was further screened,and 12 strains still showed significant inhibitory activity against Z.bungeanum root rot.These 12 strains were named as BS strains.The inhibitory activity of the 12 BS strains against Z.bungeanum root rot was further tested using the inhibition zone method.Ten BS strains still showed significant inhibitory effects against Z.bungeanum root rot,and among them,B.velezensis P87 had the strongest inhibitory activity,with an inhibition zone diameter of 26.92 mm.（3）Combined with fermentation culture,the inhibitory effect of strain P87 against Z.bungeanum root rot was tested,and it was found that the fermentation broth of P87 had an inhibitory activity of up to 70.86%against Z.bungeanum root rot.Furthermore,through potting prevention and control efficacy tests,it was found that P87 showed a certain preventive and therapeutic effect against Z.bungeanum root rot,with a prevention and control efficacy of 22.5%for the fermentation broth of P87,and a prevention and control efficacy of 46.25%for the fermentation liquid containing bacteriophage of P87.（4）The basic characteristics of P87 were studied and it was found that the colony morphology of P87 formed on different media was different,and it produced a large number of spores.In addition,PCR amplification detected the presence of antibacterial peptide synthesis gene srf AA,srf AC,itu C,itu D,fen D,Bmy B,and bac A in P87.（5）The fermentation conditions for P87 were optimized,and the inhibition activity of the fermentation liquid against Z.bungeanum root rot was used as the detection index.It was found that P87 had the strongest inhibitory activity against Z.bungeanum root rot when the culture medium formula was glucose 5 g,yeast extract 7.5 g,peptone 7.5 g,ammonium sulfate5 g,Na Cl 5 g,water 1 L,p H=7.0,and fermented for 5 days,with an inhibition rate of up to73.6%.（6）The optimal formulation of P87 wettable powder was obtained by screening the carrier silica 25 g/L,wetting agent PEG8000 0.5 g/L,dispersant sodium tripolyphosphate 0.75g/L,protective agent sodium dodecyl sulfate 0.025 g/L,and the quality of P87 wettable powder was 0.36×108 cfu/g.The test results showed that the wettable powder contained 1.36×108cfu/g,suspension rate was 90.52%,wetting time was 43.6 s,p H value was 7.1,fineness was98%,dry weight loss was 0.9%,and the rate of miscellaneous bacteria was 0,all of which were in accordance with the national standard and had strong antibacterial activity.In addition,the potting efficacy test also showed that P87 wettable powder had a significant preventive effect on F.solani,and Z.bungeanum potted seedlings treated with P87 wettable powder had almost no root rot symptoms and could promote the growth of Z.bungeanum roots and the increase of leaf surface area.In this study,we isolated microorganisms from the rhizosphere of Z.bungeanum,and tested their inhibitory activity against the Z.bungeanum root rot pathogen,as well as their disease prevention and control effects.The study not only obtained potential biocontrol strains that have a significant antagonistic effect against Z.bungeanum root rot,but also developed a B.velezensis P87 wettable powder,which exhibits a certain preventive effect against Z.bungeanum root rot.These findings provide important guidance for the implementation of biological control of Z.bungeanum root rot,and ultimately serve the green and pollution-free prevention and control of Z.bungeanum diseases.