| Pogostemon cablin,commonly known as "patchouli",is an important aromatic herb of the Lamiaceae family.Patchouli is susceptible to lethal bacterial wilt disease,caused by some pathogenic bacteria species.In our previous study,more than ten pathogenic strains,including Ralstonia solanacearum strain PRS-84 and Kosakonia sp.strain Pa82,have been isolated from patchouli plants with typical bacterial wilt symptoms.Tn5 transposon insertion mutants of dominant strain PRS-84 have been constructed and virulence attenuated mutants have been obtained in preliminary screen.In this study,biological characteristics of virulence attenuated mutants PRS-84-4-7 and PRS-84-4-49 were analyzed,including bacterium morphology,flagella,biofilm and extracellular polysaccharides.Different pathogenic strains were individually tested for their pathogenicity to patchouli plants.The virulence attenuated mutant PRS-84-4-49 was then tested for its ability to reduce the severity of patchouli bacterial wilt caused by the wild-type strain PRS-84 or strain Pa82.Then the stability of the virulence attenuated mutants in biological characteristics and potential biocontrol abilities against patchouli bacterial wilt were evaluated.This study aimed to exploit effective biological control agents against patchouli bacterial wilt.1.Review on relevant literatures from domestic and abroadRecent studies on the cultivation techniques and control activity against patchouli bacterial wilt were introduced.Research progress in biological characteristics of plant pathogenic bacteria were summarized.The biological control approaches of plant diseases including bacterial wilt were reviewed.2.Biological characteristics of virulence attenuated mutants from Ralstonia solanacearumThe biological characteristics of virulence attenuated mutants PRS-84-4-7 and PRS-84-4-49 such as bacterium morphology,flagella,biofilm,extracellular polysaccharides and the pathogenicity to patchouli plants were analyzed.There was no obvious variation in the biological characteristics between the mutants and wild-type strain PRS-84.All strains were short-rod shaped with 1 to 4 polarflagella under oil immersion lens of microscope.The biofilm structure and extracellular polysaccharides were analyzed by using confocal laser scanning microscopy(CLSM).The CLSM images revealed that bacterial biofilms were grown in covered glass bottom dishes,while the extracellular polysaccharides attached to the bacterial cell surface and formed complex network structure.Compared to wild-type strain PRS-84,the biofilm formation capability of mutant PRS-84-4-7 and PRS-84-4-49 were impaired,and the secretion of exopolysaccharides of mutant PRS-84-4-7 was increased,but that of the mutant PRS-84-4-49 were not obviously changed.The virulence attenuated mutants were individually tested for their pathogenicity to patchouli plants.Pathogenicity tests revealed that mutants showed less virulence to patchouli plants than that of wild-type strain PRS-84.3.Biocontrol potential of virulence attenuated mutant from Ralstonia solanacearumPathogenicity assays of Ralstonia solanacearum strain PRS-84,Kosakonia sp.strain Pa82 and Ralstonia solanacearum strain GIM1.70 separated from tomato were performed by inoculating in vitro regenerated patchouli plants.Results revealed that the disease incidence of plants inoculated with wild-type strain PRS-84 was highest,while that of plants inoculated with strain GIM1.70 was lowest.Virulence attenuated mutant PRS-84-4-49 was tested for the ability against patchouli bacterial wilt.Biocontrol treatment plants were pre-inoculated with mutant PRS-84-4-49 and then inoculated with wild-type strain PRS-84 or Pa82.Plants mock-inoculated with sterile water and then inoculated with wild-type strain PRS-84 or Pa82 were used as inoculated control.Plants mock-inoculated throughout only with sterile water provided the uninoculated control.Different biocontrol effects of virulence attenuated mutant PRS-84-4-49 against patchouli bacterial wilt caused by wild-type strain PRS-84 and Pa82 were shown,with 27.95%and 62.32%,respectively.4.Effect of serial subcultivation on biological characteristics and biocontrol potential of virulence attenuated mutants from Ralstonia solanacearumBiological characteristics of mutants PRS-84-4-7 and PRS-84-4-49 and their 100th subculture generations such as motility,biofilm formation capability and pathogenicity were analyzed.Results showed that,compared with wild-type strain PRS-84,the ability in motility and biofilm formation of mutant PRS-84-4-7 were reduced,but that of the mutant PRS-84-4-49 were not obviously changed.The disease incidence of plants inoculated with the mutants was significantly lower than that of the wild-type strain PRS-84,There were no significant differences in biological characteristics including motility,biofilm formation capability and pathogenicity between mutants and their 100th subculture generation.The virulence attenuated mutant PRS-84-4-49 and its 100th subculture generation were then evaluated for their stability against patchouli bacterial wilt caused by wild-type strain Pa82.Results showed that the biocontrol treatment of the mutants PRS-84-4-49 before and after serial subcultures could both reduced the severity of bacterial wilt disease,with similar biocontrol effects against patchouli bacterial wilt. |
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