Clavibacter michiganensis subsp michiganensis: Optimization of Detection in Seed and Effect of Seed Treatment on Efficiency of Detection Methods

Clavibacter michiganensis subsp. michiganensis (Cmm) is one of the most important seed-transmitted pathogens in tomato. In recent years, the number of outbreaks has increased. Due to the absence of chemical controls, the most effective way to prevent the spread of this disease is through the use of clean seed, requiring effective seed health testing. A French collaborative program sponsored by the Ministry of Agriculture was launched to study Cmm and improve detection methods. The protocols used routinely in laboratories for seed testing are not highly satisfactory. A program to improve the existing detection/identification methods was implemented; method performance was compared using a collection of 213 isolates (including 141 Cmm, saprophytes, other Clavibacter subspp. and other bacteria pathogenic to tomato). The methods were immunofluorescence, media plating, PCR and a pathogenicity test. Comparison of bacterial growth on two different semi selective media showed that slow and fast growing isolates should be taken into consideration for seed health analysis. The ability of PCR methods to detect the pathogen directly from a seed soaking or after a media growth step was evaluated. A new direct PCR protocol gave the best results. Moreover, efficiency of detection on treated vs. untreated contaminated seed lots was studied and sensitivity of each method was determined. Addition of inhibition controls is suggested to prevent false negative results. This study contributed to the validation of new protocols based on media growth, IF and PCR by the International Seed Federation and the European and Mediterranean Plant Protection Organization.