| Clavibacter michiganensis subsp. michiganensis (Cmm) is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The initial means of bacterial canker entry into a crop is through Cmm-infected seeds or transplants. In order to study the biology of Cmm seed transmission and seedling infection, bioluminescent Cmm strains were constructed by transformation of the reporter gene, lux-operon, into Cmm. A promoterless lux-operon, originally derived from Photorhabdus luminescens, was linked downstream of a chloramphenicol exporter in a Cmm transposon mutagenesis vector, pKGT452Cbeta. Electroporation of the constructed vector pXX2 carrying the Cmx r::luxABCDE::Tn1409 cassette resulted in insertion of lux-operon into the Cmm chromosome. Among 19 bioluminescent Cmm mutants obtained, the virulent, stable, constitutively bioluminescent strain BL-Cmm17 that grew similarly to its parent strain C290 was selected to study seed transmission and seedling infection. Healthy tomato seeds were inoculated with BL-Cmm17 and the dynamics of bacterial colonization on germinating seeds were monitored in real-time using an in vivo imaging system (IVIS). Our results showed that Cmm colonized the hypocotyl and the cotyledon at an early stage of germination. Visualization of seedling systemic infection by BL-Cmm17 through wound showed that Cmm moved towards both root than shoot under both low (45%) and high (83%) relative humidity (RH) regimes. High RH accelerated the systemic movement of Cmm in stem. Luminescent signals were also observed in tomato seedling roots over time. Root development was reduced in inoculated plants maintained under both humidity regimes. The strong positive correlation between light intensity and bacterial population in planta suggested that bioluminescent Cmm strains have additional potential applications such as evaluating the efficacy of bactericides and resistant cultivars.;Because of the importance of seed transmission, seed must be the first point of focus in developing an integrated program to manage this disease. In this study, the effects of seed sanitation treatments combined with post-sanitation treatments of beneficial bacteria Bacillus subtilis and Pseudomonas fluorescens were evaluated. Seed sanitation treatments eliminated the pathogen in seed and reduced disease incidence in seedlings in laboratory and greenhouse grow-out tests. However, beneficial bacteria applied after seed sanitation treatments did not reduce disease incidence or influence seedling growth. Among all sanitation treatments, commercial disinfectants Kleengrow and Virkon most effectively eliminated Cmm from seed and reduced the pathogen populations in greenhouse seedlings. The plant essential oil thymol was phytotoxic at the concentration evaluated, increasing the rate of production of abnormal seedlings. Seed vigor analysis determined using the Seed Vigor Imaging System (SVIS) suggested that thymol, kasugamycin and dry heat treatments decreased seed vigor over long-term storage. |
