The Control Efficacy Of Bacterial Wilt In Solanaceae Crops By Antagonistic Bacterium And Avirulent Mutants Of Ralstonia Solanacearumand Mechanisms

Plant bacterial wilt (BW) disease causes large damage and is becoming more and more serious in plant production around world. Many researchers focus on the study to control BW disease. BW disease is caused by Ralstonia solanacearum which is a soil-borne gram-negative bacterium and can live for a long time in soil as pythogenesis or parasitism. R. solanacearum can disseminate in various ways in environment. When conditions are suitable, the pathogen can burst into multiplication and hardly be controlled. Because of the limitation of crop rotation and intercropping and the environmental problems and little efficacy of chemical methods in control of plant bacterial wilt, biocontrol is received more attentation.Antagonistic bacteria to R. solanacearum and avirulent mutants of R. solanacearum are most commonly used as biocontrol agents. Improving host resistance is also considered as a good strategy to control BW disease, but breeding for resistant plants is expensive and time-consuming. So it is a good choice to induce plant resistance by engineering bacteria as plant vaccine.R.solanacearum uses a very complex system to regulate its pathogenicity, of which PhcA (phenotype conversion A) is the core factor. PhcA, activated by signal molecular 3-OH PAME (3-OH palmitic acid methyl ester) positively regulates the production of exopolysaccharides (EPS), Pme and acyl homoserine lactone (AHL)-dependent quorum-sensing system. On the other hand, PhcA represses siderophore synthesis, motility and environmental tolerance. When R.solanacearum multiplies rapidly in the vascular of plants, PhcA is accumulated with high activity and activates the expression of many other genes including some virulent genes and promotes the multiplication of R.solanacearum. PhcA is encoded by phcA which is located in the chromosome of R.solanacearum.The hrp (hypersensitive reaction and pathogenicity) cluster locates on the megaplasmid in R.solanacearum and includes 23 correlative genes mainly encoding the type 3 secretion system which is the crucial virulence factor of R. solanacearum. Avirulent mutants of R. solanacearum are constructed by genetic engineering method and their abilities to induce resistance of tomato plants to BW disease were investigated in this paper. And the potential biocontrol effect of mutants against tobacco BW disease was also studied. The main results are as follows:(1) Effect of Bacillus amyloliquefaciens S20 as biocontrol agent against eggplant bacterial wilt.A pot experiment demonstrated that S20 could control eggplant bacterial wilt and the control efficacy could up to 70.7% when S20 was applied with organic fertilizer. The results showed organic fertilizer could improve the survival of strain S20 in the soil.An HPLC-ESI-MS analysis indicated that iturins A were the major antibiotic compounds in strain S20, mainly including C13-C16 iturins A.(2) Construction of phcA- and hrp- mutants of R. solanacearum ZJ3721A phcA- mutant with a 700bp-fragment deletion in phcA and a hrp- mutant with a 2500bp-fragment deletion in hrp cluster in R. solanacearum ZJ3721 were constructed by homologous recombination in the genome. Their avirulence was confirmed by potting experiment.(3) Characters of the phcA- mutant and the hrp- mutant.Comparision of growth and carbon source utilization between the mutants and the wild type of R. solanacearum ZJ3721 showed that the both mutants grew slowly in the middle of exponential-phase and had a lower population in stationary-phase compared with wild type of R. solanacearum ZJ3721. Both of the mutants could utilize twenty-three carbon sources faster than the wild type strain, such as α-D-Glucose, D-Mannitol, Sucrose et al.. In addition, the phcA- mutant could utilize Dextrin, D-Fructose and D-Gluconic acid faster than both the hrp- mutant and the wild type strain. But the both mutants lost the ability to utilize the y-amino-butyric acid.Expressions of lots of virulence-related genes in mutants were suppressed compared with wild type. Expressions of xpsR, egl, tek and epsE in the mutants were suppressed more than 95% than in wild type.(4) Effect of the phcA- mutant as biocontrol agent against tomato bacterial wilt.The phcA- mutant as biocontrol agent against tomato BW was invesigated in potting experiment and the results showed there was no significant biocontrol effect except delaying the wilt development by about one day when phcA- mutant was applied with the pathogen at the same time. But when the phcA- mutant was pre-inoculated three days earlier than pathogen, the biocontrol efficacy of B W by phcA- mutant could get 90% at the twenty fifth day.Investigation of populations of the pathogen and the mutant in the rhizosphere soil showed that pre-inoculation of the mutant three days earlier than pathogen can inhibit the populations of the pathogen more effectively than inoculation with the pathogen at the same time.(5) The phcA-mutant can induce systemic resistance of tomato plants.We investigated the gene expression related to defense pathways in tomato plants at three days after inoculation with the phc A" mutant or WT in hydroponic experiment. The results indicated that phcA-mutant could induce systemic resistance of tomato by activating salicylic acid (SA) pathway, jasmonic acid (JA) pathway and Ethylene (ET) pathway in different degree. The expressions of GluA and PR-1a (SA) induced by the phcA- mutant were 66-fold and 7.5-fold respectively higher than that induced by the wild type strain. But the induction effect reduced at fifteen days after inoculation.Investigation of populations of the pathogen and the mutant colonizing in the surface of the tomato roots showed the populations of wild type strain could keep steady in fifteen days after inoculation, but the populations of the phcA- mutant could only keep steady in ten days after inoculation and decreased significantly after fifteen days later(6) The phcA- mutant and the hrp- mutant can also induce systemic resistance of tobacco plants.Investigation of the ability to induce resistance of tobacco by mutants showed that the both of phcA- mutant and hrp- mutant could activate salicylic acid (SA) pathway and jasmonic acid (JA) pathway of defense response in tobacco.Comparing with antagonistic bacterial strains Bacillus amyloliquefaciens SQR-7 and Pseudomonas brassicacearum J12 which showed weaker abilities to induce systemic resistance of tobacco plant, both of the phcA- mutant and the hrp- mutant could induce much stronger resistance in tobacco.(7) Colonization of the phcA- mutant in soil.It was found that organic fertilizer containing fermented pig manure and fermented rapeseed meal could promote the growth of the phcA- mutant in soil. While the organic fertilizer incorporation with some dried pig manure could promote their growth more efficiently.(8) Isolation of the 3-OH PAME degrading strain.One strain capable of degrading 3-OH PAME was isolated from soil, which was named PE3. When R. solanacearum ZJ3721 was cocultured with it, the expressions of xpsR, epsE, egl and tek were repressed.