Identification,a Loop-mediated Isothermal Amplification Detection And Prevention Of Rice Bacterial Leaf Brown Spot Disease In Heilongjiang Province

In recent years,rice bacterial leaf brown spot disease has occurred in different regions of the Northeast.This disease has seriously affected rice yield and quality,which caused severe economic losses to local farmers.In order to provide a comprehensive prevention and treatment method of disease,this study identified the pathogen of the rice bacterial leaf brown spot disease,and then developed a rapidly detected method for the pathogen.Moreover,leading rice varieties in Heilongjiang province for identification of resistance and the antibacterial effect of 14 bactericides are also tested in this study.The main results are as follows:?1?In 2017,5 strains were isolated among samples of bacterial leaf brown spot were collected from different areas of four main rice-planting areas in Heilongjiang Province.On the basis of phenotypic properties of morphology,physiology,biochemical and molecula r biology method,and the pathogens were identified as Pseudomonas syringae pv.syringae.?2?A rapid and sensitive system was developed for detection of Pseudomonas syringae pv.syringae based on a loop-mediated isothermal amplification.Specificity of primers was designed for LAMP amplification based on the conserved sequence of hrc N gene.The components in the system were optimized,the results showed that the constant temperature of 63? for 45 min,the optimum concentration of Mg SO₄ was 8 mmol/L,Bst DNA polymerase 8 U,optimal concentration of d NTPs for 1.4 mmol/L,and the ratio of outer primers and internal primers was 1:2 could acquire optimal amplification results.Sensitivity results showed that LAMP allowed detection the lowest concentration for bacterial suspensions was 10⁴cfu/mL.The specificity primers of LAMP could be used to Pseudomonas syringae pv.syringae,and the other strains was negative.The LAMP detection method is more accurate,sensitive and faster,and this is more suitable for field practicability.?3?This report utilizes two methods to test 42 leading rice varieties in Heilongjiang province for identification of resistance.Specifically,the result of inoculating all these rice varieties by artificial acupuncture illustrated that only 14 varieties showed i ntermediate resistance and 2 varieties showed high resistance levels.Besides,by using significant difference analysis of the length of lesions inoculated with rice varieties,the results demonstrated that there were obvious differences among 42 leading rice varieties.Meanwhile,the results of inoculating all these rice varieties by spraying indicated that seven of these rice varieties showed Grade I susceptibility,which accounted for 16.7%.Comparing and analyzing these data,we found t hat two inoculation methods had basically consistent results.Particularly,2 high resistance varieties we found in this study are very significant for the production practice of bacterial leaf brown spot diseases for the sustainable control of rice bacterial leaf brown spot diseases.?4?Whether the bactericides had the antibacterial effect on Pseudomonas syringae pv.syringae,the plate colony counting method was used to determine the virulence of 14 preparatory pesticides to Pseudomonas syringae pv.syringae in vitro.The result of screening bactericide demonstrated that 3% benziothiazolinone,20% zinc thiazole,46% copper hydroxide,30%copper?succinate+glutarate+adipate?etc,all of them have direct antibacterial effect;80% ethylicin,20% thiodiazole-copper,20% thiosen copper,2%kasugamycin etc,all of them have general antibacterial effect;the bacteriostatic effect of 72% streptomycin sulfate were relatively worse.Consquently,benziothiazolinone,zinc thiazole,copper hydroxide and copper?succinate+glutarate+adipate? could be used in the field efficacy,and they can be regarded as ideal control agents for rice bacterial leaf brown spot disease from now and then.