荧光素酶标记无致病力青枯雷尔氏菌的生物学和定殖特性

为示踪青枯雷尔氏菌Ralstonia solanacearum无致病力菌株FJAT1458在番茄植株及根际土壤中的定殖特性,采用电击法对菌株FJAT1458进行荧光素酶(luciferase,LUC)基因标记,并于室内采用生测法测定标记菌株的生物学特性、遗传稳定性、致病力及在番茄植株和根际土壤中的定殖能力。结果表明,成功将luc基因整合至无致病力菌株FJAT1458染色体上,标记菌株FJAT1458-LUC发出强烈的荧光,且PCR扩增出1 612 bp的luc基因片段;与野生型菌株FJAT1458相比,标记菌株FJAT1458-LUC的生长明显滞后,培养8h之后标记菌株FJAT1458-LUC的OD_600nm值均小于野生型菌株FJAT1458;且标记菌株FJAT1458-LUC连续传代20次后菌体浓度显著增加,发光菌体比例显著降低,LUC活性和luc基因表达量均随着传代数增加而显著降低;标记前、后菌株FJAT1458的弱化指数分别为0.90和0.89,且接种番茄植株30 d均未引起植株发病。标记菌株FJAT1458-LUC能在番茄根际土壤、根及茎中定殖,定殖数量呈...

Biology and colonization of a Ralstonia solanacearum avirulent strain tagged with firefly luciferase gene luc

To tracing the colonization characteristic of Ralstonia solanacearum a virulent strain FJAT1458 in the tomato plant and its rhizosphere soil,the strain was tagged with the firefly luciferase(LUC) gene through electroporation.The biological characteristics,genetic stability,pathogenicity,and colonization of LUC-tagged strain FJAT1458-LUC in the tomato plant and rhizosphere soil were studied through bioassay methods.The results showed that FJAT1458 was chromosomally tagged with the marker gene luc,and a 1612 bp fragment of the gene could be amplified by PCR.The strain FJAT1458-LUC could excite strong fluorescence.Compared to wild type FJAT1458,the growth of FJAT1458-LUC was significantly delayed with a significant decrease of the OD_600nm value.FJAT1458-LUC was continuously subcultured for 20 passages.The cell concentration of the subcultures was significantly increased,while the ratio of bacteria with luminescent,activities of luciferase,and relative expression level of the luc gene were all significantly decreased along with the increase of the passage number.The attenuation indexes of FJAT1458-LUC and FJAT1458 were 0.89 and 0.90,respectively.Both FJAT1458-LUC and FJAT1458 could not induce tomato bacterial wilt 30 day post inoculation(dpi).FJAT1458-LUC could colonize in rhizosphere soil,root and stem of tomato plants.The bacterial colonization number tended to increase first and then decrease.The highest colonization number of FJAT1458-LUC in rhizosphere soil,root and stem occurred at 3,5 and 5 dpi,respectively.In addition,FJAT1458-LUC could not be detected in stem and root at 9 and 12 dpi,respectively.The results indicated that FJAT1458 was successfully tagged with the luc gene,the tagged strain could colonize in the tomato plant and rhizosphere soil,showing a good biocontrol potential.