| Bacterial fruit blotch occurred to varying degrees in watermelon and melon planting regions of Liaoning Province,and the disease continued to spread every year.This study used serological and molecular methods to explore the biological characteristics of its pathogens.At the same time,a multiple RT-PCR technique was established for simultaneous detection of two quarantine pests,Acidovorax citrulli and Cucumber green mottle mosaic virus?CGMMV?,in watermelon.In addition,the antimicrobial effect and mechanism of nano copper preparations on Acidovorax citrulli were investigated.The main results are as follows:1.In this study,a total of 11,7 and 9 strains were isolated and purified from diseased watermelon fruit of Youwei Town,diseased melon fruit of Santaizi Town and Cuiyan Town,Linghai City,Liaoning Province,respectively.The pathogens were confirmed by the pathogenicity test of Koch's postulate.The strain JZ17 from the watermelon fruit of Youwei Town,the strain JZX2 from the melon fruit of Santaizi Town and the strain JZT8 from the melon fruit of Cuiyan Town were selected as representative strains for further study.The observation results showed that the culture and biological characteristics of these three strains were consistent with that of the positive strain AAC00-1.The DAS-ELISA serological identification indicated that all of these three strains showed positive reaction.A pair of specific and sensitive primers?YH1/YH2?were designed to amplify the 16S-23 S r DNA ITS sequences of the three strains,and the results showed that these three strain shared more than 99% identity with that of other A.citrulli.These result above revealed that the strains JZ17,JZX2 and JZT8 were clearly identified as Acidovorax citrulli.2.The primers PL1/PL2 were used to identify the groups of A.citrulli,and the strain JZ17 could be identified as group II while the strain JZX2 and JZT8 were identified as group I.In order to analyze the phylogenetic relationship of the three strains,16 S r DNA sequences were amplified by PCR with primers 27F/1492 R.Homology analysis of the obtained sequences was carried out and the results showed that the 16 S r DNA sequences of the strain JZ17,JZX2 and JZT8 shared more than 99% identity with that of other A.citrulli in Gen Bank.Phylogenetic tree analysis based on Neighbor-Joining method showed that the strains JZX2 and JZT8 were clustered with other strains of A.citrulli,and the strain JZ17 was closed to the strain JXZS6.3.Bacterial fruit blotch disease caused by Acidovorax citrulli and watermelon blood flesh disease caused by Cucumber green mottle mosaic virus,are two major quarantine diseases of watermelon and result in considerable losses to global watermelon production.Additionly,these two diseases have emerged in Liaoning Province.In this study,a multiplex RT-PCR was developed for simultaneous detection of CGMMV and A.citrulli in watermelon.A set of three specific primers was designed based on the conserved sequences of the genomic RNA of CGMMV,the internal transcribed spacer?ITS?of A.citrulli,and the elongation factor-1??EF-1??of watermelon.The optimal conditions for multiplex RT-PCR were established: primer concentrations of 0.1,0.1,and 0.2 ?M for A.citrulli ITS,CGMMV and watermelon EF-1?,respectively.The PCR reaction system: an initial denaturation at 94 oC for 5 min followed by 35 cycles of denaturation?94 oC for 30 s?,annealing?54 oC for 30 s?and extension?72 oC for 1 min?,and a final extension at 72 oC for 10 min.The test results showed that 10-4 fold concentration of primitive template could be detected by the multiplex RT-PCR system,and displayed two amplified DNA bands from watermelon leaves only infected by CGMMV?765 bp and 540 bp?or A.citrulli?540 bp and 245 bp?,and only amplified EF-1? fragments?540 bp and 1109 bp?from watermelon leaves infected by other viruses or bacteria,proving the high specificity and sensitivity of detection system.This method was further verified by testing field-collected samples.In addition,the detection technology was used to detect A.citrulli and CGMMV of 184 seed samples from Liaoning province,and the test results were reported to Liaoning plant protection and quarantine station,which provided guidance for the monitoring and quarantine of seeds and seedlings in Liaoning province.4.The antimicrobial activity against A.citrulli was studied by qualitative and quantitative experiments using nano copper hydroxide,nano alkali copper sulfate and industrial alkali copper sulfate prepared by the laboratory.The antimicrobial mechanisms of these three copper preparations agaist A.citrulli were investigated from the cellular level,physiological and biochemical level,and molecular level.The plate diffusion method and turbidimetric method were used to determine the antimicrobial activity of nano copper preparations against A.citrulli.The results showed that the three copper preparations had significant antimicrobial effect against A.citrulli with a low concentration and the bacteriostatic rates were enhanced significantly with the increase of the concentration of the treatment agent.The antimicrobial effect of nano copper hydroxide was twice that of nano alkali copper sulfate and more than three times that of industrial alkali copper sulfate.The MIC of the nano copper hydroxide preparation obtained by the double dilution method was 0.625 ?g/m L,and the MBC was 5 ?g/m L.The MIC values of nano alkali copper sulfate and industrial alkali copper sulfate for the treatment of A.citrulli were 1.33 and 1.55 ?g/m L,respectively,and the MBC values were consistent at 10 ?g/m L.In summary,the nano copper preparations was highly dispersible and stable,which can effectively inhibit the normal growth of A.citrulli,and kill the pathogenic bacteria in a low-concentration.In order to clarify the antimicrobial mechanism of nano copper preparations against A.citrulli,scanning electron microscopy was used to observe the bacterial morphology treated by nano copper preparations.The results showed that the damage of the pathogens treated by these three agents was obvious,and the pathogens were lacunary and distorted.The antimicrobial effect of nano copper hydroxide preparation was stronger than that of nano alkali copper sulfate preparation,which was stronger than that of the industrial alkali copper sulfate preparation.The lactate dehydrogenase activity was about zero after treatments of these three kinds of agents,indicating that these three agents could not alter cell membrane permeability of A.citrulli.The results of DNA degradation experiments showed that these two alkali copper sulfate preparation could degrade the DNA of A.citrulli,whilst the nano copper hydroxide preparation could not.The DNA began to degrade after 25 minutes of treatment,and the nano alkali copper sulfate was more effective and the onset time was shorter.In summary,the results showed that both the nano copper hydroxide preparation and the nano alkali copper sulfate preparation could achieve the bacteriostatic effection by destroying the morphology of the pathogens and making pathogens lacunary and distorted.Moreover,the nano alkali copper sulfate preparation could also effectively degrade the genomic DNA of the pathogens to achieve bacteriostatic effection. |
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