Screening And Evaluation Of Biocontrol Bacteria Against Citrus Canker

Citrus canker is a bacterial disease caused by pathogen Xanthomonas axonopodis pv.citri,which seriously affects the productivity and product quality of citrus orchards and brings huge economic losses to the citrus industry.The prevention and treatment of citrus canker mostly depends on spraying copper preparations and antibiotics.Long-term use will not only cause drug resistance of pathogenic bacteria,but also lead to environmental and personal safety problems such as pesticide residues.Biological control has the advantage of green environmental protection,which is the focus of research at home and abroad in recent years,but the lack of scientific evaluation of biocontrol bacteria leads to the problems of low colonization rate and poor stability after application.therefore,the screening of biocontrol strains with excellent characteristics is of great significance to improve the stability of biological control.In this study,the leaves of Qiongzhong green orange and Chengmai Fu orange from Hainan Province were used as materials to isolate the pathogen of citrus canker and the biocontrol bacteria with good antagonistic effect.The biocontrol bacteria were evaluated based on the abundance,antagonistic circle and colonization ability of Bacillus in the leaves.The effects of the target strain on citrus defense enzymes and the prevention and control effects of citrus canker were further analyzed,and the results were as follows:1.A bacterial strain was isolated from leaves with typical citrus canker spots collected from the citrus base in Fushan Town,Chengmai County,Hainan Province.Based on morphological observation,molecular identification and Koch’s rule verification,the isolated bacteria was identified as the pathogen of citrus canker(Xanthomonas axonopodis pv.citri)and named as XC.2.The endophytic bacillus was isolated from the leaves of green orange and Fu orange by coating plate method.Then,39 isolated strains of endophytic bacteria were screened by plate antagonism preliminary screening and fermentation liquor antagonism re-screening,combined with the antagonism effect of fermentation liquor and the population abundance of Bacillus in the leaves.Two strains of biocontrol bacteria FS1-2 and QZ2-1 with significant antibacterial effect were selected for follow-up study.Species identification of biocontrol bacteria FS1-2 and QZ2-1 was conducted by morphological,physiological and biochemical tests and molecular identification.The results showed that both FS1-2 and QZ2-1 were Bacillus velezensis.3.Biocontrol bacteria FS1-2 and QZ2-1 were labeled with tetracycline resistance by antibiotic labeling method,and their colonization in green orange leaves was detected by plate coating method after leaf spray inoculation.The results showed that after spraying biocontrol bacteria for 3 days,the number of biocontrol bacteria FS1-2 and QZ2-1 in green orange leaves treated with auxiliaries was 4.65×10~4 CFU/g and 3.53×10~4 CFU/g,respectively,which was 25.10 and 70.60 times higher than that without auxiliaries,indicating that auxiliaries could significantly improve the ability of biocontrol bacteria to enter leaves,and the colonization number of biocontrol bacteria decreased gradually with time.On the 27th day,the colonization numbers of the two biocontrol bacteria in green orange leaves were 3.38×10~4 cfu/g and 0.90×10~4 cfu/g,respectively,indicating that strain FS1-2 was significantly better than QZ2-1.4.The effect of spraying biocontrol bacteria FS1-2 on defense enzymes of green orange was tested.The results showed that SOD activity,POD activity,CAT activity,PAL activity and PPO activity of green orange were 1.86,2.47,2.33,1.60,1.63 times higher than those of the control,respectively.The SOD activity,POD activity,CAT activity,PAL activity and PPO activity of green orange sprayed with biocontrol bacteria QZ2-1 were 1.75,2.06,2.12,1.59,1.47 times higher than those of the control,respectively,and reached a significant difference compared with the control.5.In vitro leaf and plant control tests,the control efficiency of biocontrol bacteria FS1-2 and QZ2-1 against Xanthomonas axonopodis pv.citri in vitro leaves was 53.53%and43.43%,respectively,after being diluted 50X,indicating that the biocontrol ability of strain FS1-2 was significantly better than that of QZ2-1.The control efficiency of FS1-2 strain inoculating with 5X dilution was 29.09%,and the control efficiency of FS1-2 strain inoculating with 5X dilution was 43.64%,which was 1.50 times of that without additive.The results indicated that adding additives could improve the control efficiency of biocontrol bacteria against citrus canker.In this study,two biocontrol strains were screened from citrus leaves with high antagonistic activity and good leaf colonization ability.Both strains could significantly improve the enzyme activities of SOD,POD and other defense enzymes in citrus leaves,and had good control effect on Xanthomonas axonopodis pv.citri in vitro leaves or plants.It laid a good foundation for further research on prevention and control mechanism of citrus canker pathogen.