| Summary: | 碩士 === 中興大學 === 植物病理學系所 === 99 === Diverse endophytic bacteria that grow in the internal tissues of plants are found in a wide variety of plant species. Previous studies showed that some endophytic bacteria have unique metabolic activities that can alter the physiological conditions of the host plants, and some produce antibiotic substances and degradative enzymes that protect host plants from microbial infection. To test if endophytic bacteria have the potential in protecting tomato plants against Ralstonia solanacearum, fourteen endophytic bacteria were isolated from different cultivars of disinfested tomato seedlings in this study. All isolates were identified by FAME and 16S rRNA gene sequences, and one strain was identified as Bacillus cereus (Bce1) that harbors aiiA coding for the quorum quenching enzyme AHL (acyl homoserine lactone) lactonase. The endophytic growth of Bce1 in tomato seedlings was measured by hypocotyl cutting method to show that the bacterium can grow in the vascular tissues to reach a population density of 104 CFU/g at 30 days post inoculation (dpi). In comparison with the untreated plants, tomato seedlings inoculated with Bce1 showed at least one fold of increase in the total weight at 30 dpi, indicating that Bce1 can promote tomato growth. Bce1 had an inhibitory effect on the growth of R. solanacearum strain PS64 that can be readily observed by a dual culture method. When tomato seedlings were inoculated with Bce1 for 30 days and subsequently challenged with R. solanacearum strain PS152 by soil drench, the disease incidence was reduced by 75% in comparing with the 100% wilting symptom of the untreated plants. Knowing the expression of virulence factors in many plant pathogenic bacteria, including R. solanacearum, depends on the quorum sensing (QS) signaling pathways, Bce1 was genetically modified to increase the expression of the AHL-degrading enzyme AiiA to test if the disease severity of tomato bacterial wilt can be further reduced by the removal of QS signaling molecules in planta. Surprisingly, Bce1 with elevated AiiA lost the inhibitory capability on the in vitro growth of R. solanacearum PS64, the beneficial effect on tomato growth, and the protection against tomato bacterial wilt disease, suggesting the bioactive compounds of Bce1 that are involved in promoting plant growth and health may be directly or indirectly regulated by the AHL autoinducers. Genetic studies demonstrated that the extracellular polysaccharide (EPS) of R. solanacearum is an essential virulence factor whose synthesis depends on the other QS signal known as 3-hydroxy palmitic acid methyl ester (3-OH PAME). The feasibility of using genetically engineered endophytic bacteria in the management of tomato bacterial wilt disease will be tested again by expressing the 3-OH PAME degradative enzymes in the endophytic and growth-promoting bacterium Bce1.
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