Development of an in vitro protocol to screen Clavibacter michiganensis subsp. michiganensis pathogenicity in different Solanum species

Clavibacter michiganensis subsp. michiganensis (Cmm) is a quarantine organism in Europe and in many other countries. It is one of the most severe bacterial pathogens affecting tomato. Screening tomato plants for their resistance level to Cmm requires a large amount of space under quarantine conditions and is therefore costly. This project developed a new inoculation protocol on in vitro tomato plants to facilitate a more economic and higher throughput disease screening. A new method using the PathoScreen system was tested to localize green fluorescent protein-tagged Cmm in planta and to quantify the pathogen based on the percentage of corrected GFP (cGFP%). The system was sensitive in detecting the GFP-tagged Cmm in the shoots, but in the roots a high autofluorescence masked detection and thus sensitivity of the assay. The in vitro protocol was tested on several wild relatives of tomato, which were previously screened in a greenhouse assay. The correlation between wilt symptoms in vitro and wilt symptoms in the greenhouse was overall moderate (r = 0.6462). The protocol worked well in differentiating the two parents that were used in the mapping studies. This study shows that the in vitro protocol can be efficiently used for resistance breeding in many tomato genotypes.     

Differentiation of Clavibacter michiganensis subsp. michiganensis from seed-borne saprophytes using ELISA, Biolog and 16S rDNA sequencing

Specificity of a monoclonal antibody (MAb), Cmm1, to geographically diverse strains of the seed-borne tomato pathogen, Clavibacter michiganensis subsp. michiganensis (Cmm), was assessed and the MAb was tested for its usefulness as a tool to separate the pathogen from saprophytes in naturally infested tomato seed. Of the 236 international Cmm strains tested, 99% reacted with MAb Cmm1. MAb Cmm1 was also strongly reactive with an additional 32 strains isolated from seed that were later identified as Cmm by the Biolog MicroLog™ microbial identification system (Biolog, Inc., Hayward, CA) and 16S rDNA sequence analysis. It correctly differentiated these strains from 12 MAb Cmm1-negative seed strains that possessed similar colony morphology but were later identified as other Gram-positive genera and species. The specificity of MAb Cmm1 to the pathogen and the near universality of the MAb Cmm1-reactive antigen among diverse Cmm strains make this antibody a useful detection and identification tool. The finding that a large proportion of the Cmm strains associated with naturally infested tomato seed were putatively hypovirulent or non-virulent indicates that such populations cannot be ignored and points to a need for studies to determine their significance in host-pathogen interactions.     

A multiplex PCR method for detection of Clavibacter michiganensis subsp. michiganensis with co-amplification of its host DNA

A multiplex PCR assay system was developed for the detection of Clavibacter michiganensis subsp. michiganensis (Cmm), which combined two tests in one reaction mixture. Cmm-specific primers PSA-4/PSA-R and Solanum lycopersicum-specific primers NS-7-F/NS- 8-R (internal PCR control primer) were combined in one PCR reaction mixture with Cmm and plant DNA as template. The primer sets could amplify the target product successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respectively. The detection level of the optimized multiplex PCR assay was up to 5×10² cfu·mL⁻¹. To verify the applicability of this system, it was employed to detect Cmm in tomato seeds and plantlet samples. Seeds mixed with Cmm and diseased plantlets were detected successfully. The multiplex PCR system will avoid false-negative results and provide a reliable method for the detection of Cmm.