The Antagonistic Mechanism Of Pseudomonas Aeruginosa Type ? Secretion System Against Ralstonia Solanacearum

Tomato bacterial wilt is a plant disease caused by Ralstonia solanacearum,which seriously affects the yield and quality of tomatoes.The pathogen of tomato blocked the ducts of plants by secreting exopolysaccharides,polygalacturonase and endoglucanase.They hinder the water transport of plants,thus causing plant wilting.Because of the practical application and environmental protection problems,the traditional control measures are difficult to control Ralstonia solanacearum in China.Biological control has attracted wide attention because of its safety,high efficiency and environmental protection,and has been widely used in practice.At present,biological control mainly uses antagonistic substances of beneficial microorganisms such as bacteria and fungi and induces the expression of resistance-related genes in plant system to control pathogens.When biocontrol bacteria interact with plant pathogens,they can activate protein secretion systems in vivo,and inhibit the growth of plant pathogens depending on effector proteins of protein secretion system.Type ? secretion system is a protein secretion system widely existing in Gram-negative bacteria,which can help bacteria gain growth advantage in competition to adapt to the environment.Pseudomonas aeruginosa secretes toxin proteins to other bacteria through type ? secretory system,inhibits the growth of other bacteria,and makes itself in a dominant position in the competition.Strain Pseudomonas aeruginosa VIH2 with strong antagonism on Ralstonia solanacearum was isolated from the rhizosphere soil of long-term growing tomato plants.The mechanism and regulation of type ? secretion system in Pseudomonas aeruginosa were studied.The feasibility of type ? secretion system against Ralstonia solanacearum was explored.The aim was to provide a new theoretical basis for biological control of type ? secretion system and a new feasible way for sustainable agricultural development.The main results were as follows:1.An antagonistic bacterium Pseudomonas aeruginosa VIH2 was isolated from healthy soil samples collected from tomato planted in Nanjing Vegetable Institute of Jiangsu Province by dilution coating plate method,which could effectively inhibit the pathogen Ralstonia solanacearum.Physiological and biochemical characteristics showed that Pseudomonas aeruginosa VIH2 had strong resistance to stress.It could maintain good growth under acidic and alkaline conditions of pH 5.0 and pH 9.0 and had strong salt stress ability of 7%salt content.At the same time,it has strong resistance to ampicillin,kanamycin and tetracycline and other antibiotics.Strong stress resistance endowed Pseudomonas aeruginosa VIH2 with better colonization ability,which was conducive to obtaining competitive advantage and occupying niche,which was of great significance for biocontrol bacteria.2.After successful colonization of biocontrol bacteria,the plant pathogens were directly inhibited by antibiotics.In this study,common antibiotic genes in Pseudomonas were detected by PCR,and 2,4-DAPG homologous gene was found in the strain Pseudomonas aeruginosa VIH2.However,it was not detected by high performance liquid chromatography(HPLC).The possibility of using secondary metabolites to against Ralstonia solanacearum by Pseudomonas aeruginosa VIH2 was excluded.Contact-dependence experiments showed that direct contact was a necessary condition for Pseudomonas aeruginosa VIH2 to against Ralstonia solanacearum.3.Seven strains of Pseudomonas aeruginosa VIH2 T6SS mutants were constructed by Red/ET method.The effects of these factors on the antagonism were eliminated by measuring the growth curve,swimming ability and biofilm formation ability of mutants.When pathogen Ralstonia solanacearum was co-cultured with wild-type Pseudomonas aeruginosa VIH2,the population of Ralstonia solanacearum decreased obviously,with a decrease of 4 orders of magnitude.However,when any one or more clpV gene were knocked out,the antagonism of mutant strains decreased significantly.The population of Ralstonia solanacearum decreased only 2 orders of magnitude.The antagonism of mutant strains was weaker than the wild-type strain.It was proved that the type ? secretion system of Pseudomonas aeruginosa VIH2 played an important role in the pathogen Ralstonia solanacearum.Meanwhile,C.elegans revealed that H1-T6SS in Pseudomonas aeruginosa VIH2 mainly acted on prokaryotic cells.H2-T6SS and H3-T6SS acted on both prokaryotic and eukaryotic cells.4.In Pseudomonas aeruginosa,the type ? secretion system was regulated by quorum sensing system.Hcp is an important effector protein of T6SS.The activity of T6SS can be proved by detecting protein Hcp.Three strains of Pseudomonas aeruginosa VIH2 quorum sensing mutants were constructed by Red/ET method.The activity of Pseudomonas aeruginosa T6SS was detected by detecting the expression of protein Hcp.The activity of T6SS of different mutant strains decreased significantly,and the antagonism on Ralstonia solanacearum decreased significantly.When pathogen Ralstonia solanacearum was co-cultured with wild-type Pseudomonas aeruginosa VIH2,the population of Ralstonia solanacearum decreased obviously.The antagonistic effect of different Pseudomonas aeruginosa VIH2 quorum sensing mutants was significantly weakened.It was proved that the quorum sensing system in Pseudomonas aeruginosa VIH2 could affect the antagonism on Ralstonia solanacearum through regulating the type VI secretion system.5.Seven strains of Pseudomonas aeruginosa VIH2 H1-T6SS mutants were constructed.Wild-type strain Pseudomonas aeruginosa VIH2 had obvious antagonism on pathogen Ralstonia solanacearum.However,when any one or more tse gene were knocked out,the antagonism of mutant strains weakened significantly.It was proved that Pseudomonas aeruginosa VIH2 H1-T6SS played an important role in the antagonism on pathogen Ralstonia solanacearum.It antagonized pathogen Ralstonia solanacearum mainly through three effector proteins:Tse1,Tse2 and Tse3.6.Pseudomonas aeruginosa VIH2 ppkA mutant strain and supplement strain were constructed.Pseudomonas aeruginosa VIH2 ppkA mutant strain had weak antagonism on the pathogen Ralstonia solanacearum and could not inhibit the colonization of the pathogen in soil and tomato rhizosphere.However,Pseudomonas aeruginosa VIH2 ppkA supplement strain had obvious antagonism on pathogen Ralstonia solanacearum and could effectively inhibit the colonization of the pathogen in soil and tomato rhizosphere.It was proved that the TagQRST system in Pseudomonas aeruginosa VIH2 could affect the antagonism on Ralstonia solanacearum through regulating H1-T6SS.