| Bacterial wilt of plants caused by Ralstonia solanacearum Yabuuchi, is one of the mostdestructive disease worldwide. As a species complex, R. solanacearum has anextremely wide host range including many economically important staple crops andcash crops e.g. potato, bannan, tobacco and tomoto etc. As the main cash crop in thetropics and the fourth major food crops in the world, thebananaproduction in China hasbeen increasing in recent years. By2013, China has been the second largest bananaproduction country. Moko disease of banana, caused by R. solanacearum race2, is acrucial limiting factor in banana production in Cental and South America. Up to now,R. solanacarum race2has not been found in China and listed as a major quarantinepest. Furthermore, Ralstonia solanacearum can enter into the VBNC state (viable butnon-culturable), in which R. solanacearum maintains its metabolic activities andpathogenicity but can not be cultured by conventional culture method, in response toadverse environmental conditions.VBNC R. solanacearum carried by germplasms isconsidered to pose a potential threat to port quarantine due to its nondetectability.As the model system for studying phytobacterium-host interaction, great progreeshave been made in phylogenetics, pathogenomics and virulent regulation network. Lessstudies, however, was focused on the ecological and epidemical aspect of R.solanacearum in soil. The objective of this study is:1) to establish a reliable methodfor dection of R. solanacearum in soil;2) to monitor the population densitiesof fiverepresentative strains belonging to five different race of R. Solanacearumin soil;3) todevelop a molecular method for detection of VBNC R. solanacearum.The results were as follows:1. The effects of SMSA, TZC and modified NA medium on recovery of R.solanacearum from soil were compared. The result indicated that modified NA mediumcan effectly inhibit saprophytic soil bacteria and had better recovery efficiency.Furthermore, the sensitivity of various methods (i. e. immustrip, streak plating, BIO-PCR, Soil DNA extraction-based PCR) for detection of R. solanacearum in soil wascompared. The most sensitive method is BIO-PCR, whose detection sensitivity wasabout102cfu/g soil. The less sensitive method is Agdia immustrip, sensitivity of which was108cfu/g soil.2. By using plate counting method, the survival of five strainscorresponding todifferent races of R. solanacearumwas testedin soils under different conditions. Theresults showed that soil moisture content played the most important role in the survivalof Ralstonia sloanacearumin soil. When soil moisture content was below20%, alltested strains can retain the culturable state for no more than60d. In contrast, whensoil moisture content is above20%, R. solanacearum strain Po41, Z-AQ-2and M7remainedculturable the longest (up to130d), whereas GMI1000remainedculturablefor the shortest time (no more than35d).3. A novel method to differentiate viable/dead cells of R. solanacearum wassuccessfully established by using a DNA dye of propidium monoazide (PMA) incombination with the polymerase chain reaction (PMA-PCR). This method cancompletely inhibit the PCR amplification from dead cells and effieciently detectviableand viable but non-culturable (VBNC) cells at a threshold of250pg/mL of genomicDNA or1×103cfu/mL of bacterium suspension.4. Redefine Po82’S lethal temperature, the lethal temperature increased from52Cthat report from the Australian Government official documents Biosecurity to68C.5. A Real-time PCR fluorescent probes with high specificity was developed and aReal-time PMA-PCR for Ralstonia sloanacearum was explored and preliminarilyestablished. The probes and Real-time PMA-PCR will lay a solid foundation for thedevelopment of detect Ralstonia sloanacearum in the future. |
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