Rapid Detection Technique And Bactericides Activity Evaluation For Pantoea Agglomerans Causing Plum Bacterial Shot-hole Disease

Plum bacterial shot-hole disease was an important bacterial disease in plum production,which occurred widely in the main plum-producing areas in China.Currently,there are no bactericides registered to control bacterial shot-hole.In this study,the occurrence of bacterial shot-hole in different varieties of plum in Guizhou province was investigated.Leaf and branch disease samples were collected to isolate and identify of the disease pathogen.For Pantoea agglomerans,loop-mediated isothermal amplification（LAMP）was developed,and the virulence of bactericides and the inhibition mechanism of tetramycin were studied.Furthermore,the established LAMP technology was applied to the field test of control bacterial shot-hole caused by P.agglomerans.The results will provide a reference for the early diagnosis and accurate control of plum bacterial shot-hole.The main research results are as follows:（1）The incidence of bacterial shot-hole in Guizhou’s main plum producing areas was investigated,and the pathogen was identified as Pantoea agglomerans.From2020 to 2021,the investigation in the main plum-producing areas of Guizhou showed that the incidence of bacterial shot-hole disease in Xiuwen County,Pingzheng Township of Zunyi City and Songtao County was more serious,and the occurrence began in mid-late April or early May every year.Seven representative strains of HXFJ,HSFJ,HSFJ-1,KY,KYY,LY and XWFJ were obtained after isolation,purification and grafting of 545 samples of diseased leaves and branches.Based on morphological observation,physiological and biochemical characteristics,and molecular biological analysis,it was confirmed that Pantoea agglomerans was the pathogenic bacterium of plum bacterial shot-hole in Guizhou province.The growth of P.agglomerans entered a slow stage when it was cultured for 20 h and then entered a decline stage after 36 h.The temperature of 30℃and pH of 7 was favourable for the growth of the pathogen.（2）Based on LAMP,a rapid detection system of P.agglomerans was established.Three pairs of primers were designed based on the gyr B gene sequence of P.agglomerans.The initial reaction system and conditions of LAMP were optimized,and the amplification effect was the best at 65℃for 60 min.The LAMP primer group was confirmed to be positive only for P.agglomerans by specific detection,and the minimum detection limit of LAMP sensitivity was 5 fg/μL,1000 times higher than that of conventional PCR.The established LAMP detection system could detect P.agglomerans rapidly and accurately from indoor inoculated and field infected samples.（3）The toxicity of different bactericides on P.agglomerans was determined,and the antibacterial mechanisms of tetramycin on P.agglomerans were preliminarily explored.Among the ten bactericides,0.3%tetramycin aqueous solution（AS）showed the strongest toxicity to P.agglomerans,with the EC₅₀value of 1.7056μg/mL.Followed by 2%Chunlei·Tetramycin soluble concentrate（SL）,with the EC₅₀value of75.5011μg/mL.P.agglomerans was treated with EC₅₀and EC₇₅of tetracycin to study its antibacterial mechanism.Tetracycin had a stronger protective effect than the therapeutic effect on P.agglomerans.The electrical conductivity of P.agglomerans cultured with tetracycin increased significantly,the leakage of electrolytes became more serious with time.The leakage of macromolecular materials such as DNA and protein also occurred,indicating that the permeability of P.agglomerans cell membrane changed.Meanwhile,biofilm formation and extracellular polysaccharide production of P.agglomerans were significantly inhibited after tetracycin EC₅₀and EC₇₅treated,with the inhibition rate of biofilm formation treated with EC₇₅up to51.26%.In addition,after tetramycin treatment,P.agglomerans cell pyruvate content increased,and the malate dehydrogenase activity and ATP content decreased.The above results indicated that tetramycin could inhibit biofilm formation,destroy the stability of cell membrane,and cause energy metabolism disorder,thus inhibiting the growth of P.agglomerans.（4）Combined with LAMP detection and prevention in advance,applying 0.3%Tetramycin AS could effectively control plum bacterial shot-hole.Field control tests were carried out when plum samples were tested positive.The relative control effect was increased obviously compared with the untested control group which started control after disease symptoms appeared.Among them,the disease index of the LAMP test control group was 36.36%lower than that of the control group without test after two times of 0.3%Tetramycin AS application.This indicated that it is feasible to use LAMP technology to carry out the early diagnosis of bacterial shot-hole disease,which would be conducive to precisely control the disease.At the same time,0.3%Tetramycin AS treatment had the best control effect on plum bacterial shot-hole disease,and the control effect was 78.23%and 72.5%at 7 d and 14 d after 2 times of application in LAMP detection and control group,respectively.The single fruit weight,vitamin C and soluble sugar contents of plums were also increased by 0.3%Tetramycin AS treatment.