Isolation Of Biocontrol Bacteria For Citrus Canker And Its Colonization On Navel Orange Leaves

Citrus is an important economic crop in the world,which is planted in more than 130countries and regions around the world,with an annual output of 10.282.2 million tons and an area of 107.3 million acres,ranking first among all the fruits.China is one of the important places of origin of citrus,where citrus is rich in resources and has many excellent varieties.It has a cultivation history of more than 4,000 years.The planting areas are mainly distributed in various areas in the south.Citrus canker disease,caused by Xanthomonas campestris pv.Citri(Xcc)is an important quarantine disease that harms the world's citrus industry and affects the yield and quality of citrus.In areas where the disease is endemic,an integrated management approach,including chemical control and cultural practices is used.Chemical pesticides can reduce the incidence to a certain extent.However,increased application led to copper toxicity in soils,as well as resulted in resistant strains in xanthomonad populations copper-tolerant phytobacterial strains.In addition,repeated application of copper-containing fungicides had adverse effects on microbial biomass and bacterial community diversity of citrus soils and at the same time cannot meet people's requirements for green and healthy food.Biological control has attracted widespread attention due to its small impact on the environment,low toxicity,and low residue,and has carried out a series of research work.Therefore,it is of great significance to study the biological control of citrus canker.In this study,navel orange leaves were used as materials to isolate and screen biocontrol bacteria that have a good inhibitory effect on citrus canker.Species classification and identification,optimization of fermentation conditions,resistance to 14 common fungicides and insecticides were screened.The colonization law on the leaves has been systematically studied,and the following research results have been obtained:A total of 30 bacterium were isolated from navel orange leaves by the dilution gradient method.Six strains with inhibitory effect on citrus canker were screened out by the diffusion method,and then re-screened with in vitro leaves.Finally,two biocontrol strains QC-Y,QC-J was obtained.Based on the morphological,physiological and biochemical characteristics analysis and16S rDNA sequence analysis results,the biocontrol strains QC-Y and QC-J were classified and identified.The identification results showed that the strain QC-Y had 100%homology with Bacillus amyloliquefaciens,and the colony morphology,culture characteristics,and physiological and biochemical characteristics were also consistent with those of typical Bacillus amyloliquefaciens.The strain QC-J was identified as Bacillus subtilis based on morphological observation,physiological and biochemical analysis and 16S rDNA sequence analysis.The optimal fermentation conditions of producing antibiotic products of the biocontrol strains QC-Y and QC-J were given out by single factor experiment.The optimal culture conditions of the strain QC-Y for the antibacterial protein producing are pH 8.0,in SPA liquid medium of 75 mL/250mL with shaking for 48h.The optimal culture conditions of the strain QC-J for the antibacterial protein producing are pH 6.0,in NYD liquid medium of 50mL/250mL with shaking for 24h.Biocontrol strains were tested for resistance to 14 common bactericidal/insecticides for citrus.Based on the field application concentration,increase or decrease the drug concentration appropriately.The study found,Mancozeb demonstrated strong inhibitory effect on the growth of QC-Y and QC-J;Chlorpyrifos at high concentrations inhibited QC-Y and QC-J.Pyraclostrobin had a weak inhibitory effect on the growth of QC-J only at a high concentration(the concentration of the drug solution was 2010 mg/mL),but had no effect on the strain QC-Y.Thiophanate-methyl,Bordeaux mixture,Kasugamycin,Imidacloprid,amino acid,Difenoconazole,Etoxazole,Alphacypermethrin,Buprofezin,Spirodiclofen,Avermectin,and Pyraclostrobin had no effect on the growth of QC-Y and QC-J.The antibiotic control method was used to label the biocontrol strains with streptomycin resistance,and the leaf spray method was used to inoculate,and the plate was diluted and separated to determine the colonization on citrus leaves.Strains QC-Y and QC-J were able to colonize effectively on navel orange leaves.The maximum amount of bacteria on the first day after inoculation was 2.413×10~5 CFU/cm~2,4.375×10~5 CFU/cm~2,and both were marked with time.The colonization number of the strains showed a general decline.After 12 days of inoculation,it showed a stable trend,and the bacteria amount was 2.188×10~4 CFU/cm~2,3.281×10~4 CFU/cm~2,respectively.