Research On VBNC State And Function Analysis Of The Related Gene RpoS In Ralstonia Solanacearum

Bacterial wilt of plants is an important soil borne disease caused by Ralstonia solanacearum.Therelated studies over the past decade indicated R.solanacearum can enter viable but non-culturable?VBNC?state under the stress condition.Although some progress has been made in the field of VBNC of R.solanacearum could be induced by copper sulfate,low temperature,high temperature and VBNC detection,the relationship between the transcriptional regulator RpoS and VBNC,as well as its role in the pathogenesis has not been understood,and PMA-qPCR detection system for all species of R.solanacearum has not been established.In this study,a variety of research methods of bioinformatics,plant pathology,molecular biology and reverse genetics were used to study R.solanacearum strain Z-Aq-1?hereinafter referred to as Aq?isolated from ginger.There are three purposes for us.The first is to establish an accurate and efficient detection system for R.solanacearum,and provide technical support for early diagnosis and epidemiological studies;The second is to explore the inducer of the formation and recovery of the VBNC state of R.solanacearum,and then reveal the role of the VBNC state in the infection cycle of bacterial wilt;The third is to analyze the interrelationship between stress response related transcription factor RpoS and culturable/non-culturable state conversion of R.solanacearum,as well as pathogenicity and other biological phenotypes.1.Development of a PMA-qPCR method for quantitative detection of viable R.solanacearumWe have developed a novel method for specific detection of viable cell of R.solanacearum at species level.We optimized the various parameters of PMA by single factor change test to establish the system of PMA pre-treatment.The optimal conditions for PMA-qPCR were that the final concentration of PMA was 15 ng/mL,the dark incubation time was 10 min,and the exposure time was 5 min.The system is suitable for the detection of bacterial suspension in the range of 5×10²⁵.0×10⁸ cfu/mL.2.Construction of rpoS gene mutant strain and complementary strainBased on the genome sequence R.solanacearum UY031,we successfully constructed rpoS mutant vector and complementary vector by homologous recombination.Mutant strain Aq?rpoS was successfully obtained by natural transformation and resistance screening.However,due to the loss of rpoS,the corresponding complementary strain was failed to obtain by natural transformation and electrotransformation.3.rpoS functional verification in biological phenotypesThe pathogenicity results showed that,compared with wild strains with strong pathogenicity,Aq?rpoS completely lost pathogenicity.The root and stem colonization detection results show that,compared with Aq colonization amount was about 10¹⁰ cells/g,Colonization ability of Aq?rpoS declined sharply in the whole process.NB medium growth curve results showed that compared with wild strain,the growth rate of Aq?rpoS in logarithmic growth phase was significantly slowed down;and in basic culture,Aq?rpoS cannot proliferate.The test results of the Biolog metabolism chip shows that there were obvious differences between the metabolism of Aq?rpoS and Aq in several important carbon source,such as glucose,?-D-glucosamine,pectin,D-fructose.Stress growth assay results showed that in acidic and high osmotic condition survival ability of Aq?rpoS were weaker than wild strains.Copper resistance results showed that five copper fungicides resistance for Aq?rpoS were weaker than wild strain.The determination results of motility and biofilm showed that movement ability and biofilm formation ability of Aq?rpoS were stronger than wild strain.4.VBNC induction and resuscitation of R.solanacearumCopper ion induction results showed that Aq?rpoS completely entered VBNC faster than Aq.Low-temperature induction results showed that Aq?rpoS completely entered VBNC 60 days earlier than Aq.High-temperature induced results showed that by heat shock in 48?for 30 min,Aq?rpoS could fully entered the VBNC,and Aq needed to be treated in 50?for 30 min.Tomato inoculation test showed that Aq inducted into VBNC by above three methods could recovery in tomato tissues,while the Aq?rpoS failed to recover.