Study On Invasion Process Of Ralstonia Solanacearum To Pogostemon Cablin And Host Resistance Induced By The Pathogenic Bacteria

In this study,Ralstonia solanacearum strain HX6 and strain GIM 1.7 isolated from infected Pogostemon cablin plants and tomato plants respectively were used as tested strains.The adherence and invasion of R. solanacearum to the root of P.cablin were studied.Histopathol ogical changes of the infected plants were also observed.The factors affecting on pathogenicity of R.solanacearum were investigated and discussed.And the cloning and sequence analysis were conducted on the the PilC toxicity related genes of R.solanacearum strain GIM 1.7.Further more,resistant physiologieal responses of P.cablin were explored.The defense-related enzyme activities and their isozymes were analyzed in the host plants induced by different strains of R.solanacearum.The study aimed to reveal the mechanisms of R.solanacearum pathogenicity and disease resistance of P.cablin which might contribute to the integrated control and prevention of bacterial wilt.The main results are as follows:1.Review on relevant literatures in domestic and abroadRecent studies on P.cablin and the growth characteristics,pathogenic mechanism and genomics of R.solanacearum were introduced,and the plant defense mechanism against pathogens on the basis of previous work at home and abroad was also summarized.2.Research on the adherence and invasion of Ralstonia solanacearum to the root of Pogostemon cablinPogostemon cablin plantlets were inoculated with strain HX6 and strain GIM 1.7 of Ralstonia solanacearum.The bacterial adherence quantities on the root surfaces of P.cablin were detected.The results showed that adherent ability of strain HX6 was stronger than that of strain GIM1.7,which revealed that the higher affinity for R.solanacearum to its native-post than that of non-native host.Under electron microscope,the bacteria and extracellular secretion could be observed on section of root at 24h after inoculation,and in the vessel of stem at 48h after inoculation.3.Histopathological study of Pogostemon cablin invaded by Ralstonia solanacearumMorphology changes of the infected plants were observed from the first day to the seventh day.A few leaves started drooping on the first day.And most leaves became wilted and gray-green on the following day.Then,the stems bent down on the third day.Finally,The wilt spread up the plant,leading to its eventual death.The histopathological features of the stem vessel and mesophyll cells of Pogostemon cablin plants invaded by Ralstonia solanacearum were observed via optical microscopy.The results showed that the stem vessels were separated,twisted or even broken,and the leaf mesophyll cells dissolved.This result is consistent with the morphology changes observations of the plants infected with R.solanacearum.4.Study on factors affecting pathogenicity of Ralstonia solanacearum to Pogostemon cablinRalstonia solanacearum strain HX6 was used for infection.The pathogenicity of R.solanacearum on Pogostemon cablin was conducted via inoculations onto wounded or unwounded roots.The disease developed faster on plants with wounded root than those uninjured plants.The disease index of the plants with wounded root was rising rapidly with the extension of inoculation time.And all the plants in both treatments became wilted with the disease index 100 at a later period.The results indicated that after gathering at the surfaces of host plant root,the pathogen can destroy the root cell wall through secreting some extracellular enzymes and then invading the roots.The bacteria cell,fermentation filtrate and fermenting liquid of R.solanacearum were used to induce the test-tube plantlets of P.cablin.The morphology changes of P.cablin in 7 days were observed.The plants cultured on the medium with bacteria cells kept healthy.However,the disease occurring in the plants induced by bacteria fermentation filtrate and bacteria fermenting liquid.The plant stems bent down and the whole plants wilted and then died.The results demonstrated that extracellular secretion is a major virulence factor of R.solanacearum.5.Cloning and sequence analysis of PiIC gene from Ralstonia solanacearum strain GIM1.7Bioinformatics analysis of the Ralstonia solanacearum was performed using the NCBI Database.Type-4 fimbrial biogenesis pilyl-related protein was predicted as virulence factors according to the following statements:1.The gene-encoding protein was determined as the secretory protein for its possession of signal peptide sequence in N-terminal;2.The protein has the similar thaumatin structure domain which may lead to of bacterial wilt in plants through affecting the membranaceous permeability;3.The latter part of this molecular contains analogous Neisseria PilC beta coiled-coil region,which is possibly associated with adherence of pathogenic bacterium to targeted cells.Recognition and adherence to the host cells is an essential step in bacterial pathogenesis or infection.Hence,the PilC gene encoding this protein was selected as the target gene,and cloning and sequence analysis of PilC gene from Ralstonia solanacearum strain GIM1.7 has been conducted 6.Activity analysis of defense-related enzymes in Pogostemon cablin induced by Ralstonia solanacearum Pogostemon cablin plants were induced by strain HX6.strain HX2 and strain GIM1.7 of R.solanacearum.The active changes of defense-related enzymes in induced plants were examined.The result showed that the enzyme activities of POD.PPO and PAL in the control plants was stable and at a fairly low level,while they were observed in a much higher in induced plants with initially increasing and then decreasing.The peak enzyme activities of POD?PPO and PAL in the plants induced by strain HX6 were all apparently higher than those induced by strain GIM1.7 and strain HX2.And the average values of the enzyme activities in plants induced by strain GIM1.7 were mostly a little higher than those induced by strain HX2.The results of Pogostemon cablin plants were induced by different concentrations of bacterial suspensin of strain HX6 revealed that the plants were more sensitive to the pathogens at higher concentration.The enzyme activities in middle or high concentration group increased faster and achieved higher peaks than those in low concentration group.However,the enzyme activity decreased at a later period,with the plants wilting and the plant cells injured.The results indicated that the enzyme activities of POD,PPO,and PAL increased in Pogostemon cablin plants induced by R.solanacearum,which might reduce or delay plant damage caused by R.solanacearum to some extent.7.Analysis on defense-related enzymes isozymes in Pogostemon cablin induced by Ralstonia solanacearumStrain HX6,strain HX2 and strain GIM1.7 of Ralstonia solanacearum were used as tested strains.The isozyme bands changes of POD,PPO and SOD in Pogostemon cablin plants induced by R.solanacearum were analyzed via the polyacrylamide gel electrophoresis.Within 7 days,both the number and strength of these bands increased comparing to the control groups.POD,PPO,and SOD isozymes represented dynamic changes in P.cablin induced by R.solanacearum from the first day to the seventh day.And the occurrence time and the strength of the the isozyme bands were also different among the plants induced by different strains.The results indicated that these enzymes might play important roles in P.cablin defense against R.solanacearum.