Pathogen Identification And Control Techniques Of Mustard Black Circle Disease

An unknown root disease of mustard was found at the Northwest Anhui Agricultural University Experiment Station.The pathogenic bacteria were obtained from the rootstock of mustard and verified by colony morphology,physiological and biochemical assays and molecular biology,as well as by Koch’s rule.This disease has not been reported.In this paper,we investigated its biological characteristics and determined the inhibitory effect of seven fungicides and raspberry extracts on the pathogenic fungus,and conducted field screening for efficacy and resistance to the disease.It provides a theoretical basis for further research on the occurrence and prevalence of black ring disease of mustard and its integrated control.1.Symptoms of Mustard black circle Disease.Mustard black circle Disease occurs most often in autumn mustard fields,forming black rings around cortical ducts and darkening of vascular tissue in fleshy roots.In the early stage of the disease,the fleshy roots of mustard had the same appearance as healthy mustard,but radial dots could be observed after crosscutting.In themiddle of the disease,the site of the disease gradually blackened to form a Black circle and radially spread around;Late in the disease,the tissue at the black circle turns dark and the surrounding tissue rots.2.Pathogenic bacteria isolation and identification.The pathogenic bacteria were isolated and purified by dilution scribing method,and the colonies appeared on NA medium were white at the beginning,forming smooth round colonies,and then the color gradually deepened,and later became yellowish brown and accompanied by fishy odor.The bacterium is short rod-shaped,gram-negative bacteria.Pseudocyanin assay,denitrification,gelatin liquefaction,hydrolyzed starch and autotrophic hydrogen formation were negative;while oxidase,esterase（Tween80）and arginine double hydrolysis were positive.Multi-gene clustering analysis of 16Sr RNA and rpo B of the pathogenic bacteria was performed to construct a phylogenetic tree,and the pathogenic bacteria and Pseudomonas putida GQ3523.1 were found to be classified on the same family with 99%support.The combination of morphological observation and molecular identification made it clear that the causative agent of black ring disease in mustard was Pseudomonas putida.Using morphological and physiological-biochemical assays as well as pathogenicity assays,it was shown that the bacterium was Pseudomonas putida.3.Biological properties of Pseudomonas putida,a foul-smelling bacterium of black ring disease of mustardThe colony growth of Pseudomonas putida at different temperatures,different pH and different Na Cl concentrations was determined.The results showed that the bacteria grew better in the range of 26-28℃,the fastest growth rate at 27℃,and could not grow in the water bath at 59℃for 10 min.The OD₆₀₀value of Pseudomonas putida reached the maximum when the pH was 6.5-8.5,and 3%Na Cl had a significant inhibitory effect on the bacteria.4.Virulence of fungicides against Pseudomonas putida in mustard and inhibition of the fungus by raspberry extractThe toxicity of seven agents,namely,selenium fertilizer,sodium trisulfate,metam sodium,dazomet,lime nitrogen,chlorobromoisocyanuric acid and pentachloronitrobenzene,was measured to investigate the inhibitory effect of four crude extracts of raspberry on Pseudomonas putida of mustard.The results showed that metham sodium had the best inhibitory effect with an EC₅₀value of 0.1062μg/m L,followed by dazomet,sodium diquat,pentachloronitrobenzene and lime nitrogen with EC₅₀values of 0.2801μg/m L,0.7119μg/m L,2.0856μg/m L and 6.3384μg/m L,respectively;selenium fertilizer had a lower inhibitory rate with an EC₅₀value of The crude extract of ethyl acetate of raspberry had the best inhibition,the crude extracts of glycolic acid and n-butanol had better inhibition,while the crude extract of ethanol had the worst inhibition.5.Resistance of different varieties of mustard to black ring diseaseThe field resistance of six mustard varieties,"Lin mustard No.1","Lin mustard No.2","Japanese bald mustard No.1","Japanese The results showed that the disease resistance of six mustard varieties,namely,"ASTRI-2"and"ASTRI-2",was not significant.The results showed that the disease resistance of"ASTRI-2"and"ASTRI-1"was more resistant to mustard black ring disease than the other four varieties,and their incidence of mustard black ring disease was lower,4.17%and 15.12%,respectively.6.Seven fungicides field integrated control effectSoil fumigation treatment was carried out with seven agents:35%metham sodium in water,98%dazomet,400 g/L lime nitrogen,97%selenium fertilizer,50%sodium trisulfate soluble powder,50%chlorobromoisocyanuric acid and 40%quintozene,and the overall indexes were measured after the application.The results showed that 35%metam sodium aqueous had the best control effect in the field compared with the other six agents,and the incidence of black ring disease of mustard was significantly reduced compared with the control group,and the health rate of mustard after application was as high as 89.29%,followed by better control effect in the field of dazomet,sodium diquat and pentachloronitrobenzene,and worse control effect in the field of lime nitrogen,selenium fertilizer and chlorobromoisocyanuric acid.