Identification Of The Pathogenand Quorum Quenching Study On Bacterial Softrot Of Colored Calla Lily

Recently,colored calla lily is becoming one of the high class flowers and described as "the star of flowers in 21 century".Due to the occurance of soft rot disease of colored calla lily and lack of effective ways to control this disease,the worldwide development of colored calla lily is seriously limited.In this study,isolation and identification of the pathogen of soft rot of colored calla lily was performed.6 isolates were obtained from tissues of different colored calla lily.On the basis of morphological characterization,physiological and biochemical tests,16S rDNA sequence analysis and pathogenicty test,the 6 isolates were all identified as Pectobacterium carotovora subsp,carotovora.Additionally,the pathogen could produce N-acyl-homoserine lactone(AHL) molecular.In order to control soft rot disease of colored calla lily,a biocontrol way was developed by quorum quenching technology using AHL-degrading enzyme ATTM.On one hand,the AHL-degrading enzyme gene attM was amplified from Agrobacterium tumefaciens strain EHA105 by PCR method and cloned into vector pUC19 for attM expression under promoter P_lac.On the other hand,the strong and constitutive promoter P_lpp and hrf1 gene(encode harpin) were amplified from Escherichia coli strain JM109 and Xanthomonas oryzae pv.oryzae strain JXOâ…¢,respectively,and spliced as P_lpp-hrf1 by overlap PCR method.Meanwhile,the promoter P_lpp and attM gene was also spliced as P_lpp -attM by overlap PCR method.Finally,the fragments P_lpp-hrf1 and P_lpp-attM were cloned into vector pUC19 and transformed into E.coli strain JM109,the positive clone was termed as JM109/P_lpp-hrf1-attM.Based on the results of hypersensitive response(HR), AHL-degrading bioassay and pathogenecity test,it was concluded that:(â…°) the attM gene was successfully expressed under the promoter P_lac,and showed AHL degradation activity. (â…±) the hrf1 and attM genes were successfully expressed under strong promoter P_lpp, respectively,in E.coli strian JM109,the clone JM109/P_lpp-hrf1-attM obtained the ability to induce HR in nonhost tobacco together with degrade AHL molecular.(â…²) the expressed AHL-degrading enzyme AttM could reduce the virulence of pathogen in in vivo and in vitro pathogenicity tests,but not affect the growth of pathogen.Moreover,the simultaneous expressed AttM and Harpin could not only reduce the virulence,but also promote the growth of color calla lily,and showed more effective to control this disease.Together,our results provided a new way for control of soft rot disease of color calla lily.