CRITICAL FEATURES OF THE DIAGNOSIS OF CLAVIBACTER MICHIGANENSIS subsp. MICHIGANENSIS FROM TOMATO SEED

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is one of the most important bacterial disease of tomato (Solanum lycopersicum) worldwide. Seeds are the main pathway for the transmission of the bacterium, listed as an A2 quarantine pest, consequently the reliability of seed detection tests represents a critical point to prevent the introduction and spread of the pathogen. In this study some conventional (isolation and immunofluorescence) and molecular (end-point PCR) methods for the detection of Cmm from tomato seed samples were compared by an inter-laboratory comparison (ITC). Several Italian laboratories, belonging to different institutions, analysed the same panel consisting of 11 Cmm-spiked tomato seed samples for the evaluated methods. The obtained results showed that end-point PCR gave acceptable performance values, even if the choice of enzyme in the PCR reaction was crucial. Currently the EPPO protocol (Standard PM7/42 2) does not contemplate a preliminary screening phase based on molecular methods, but suggests two parallel flow diagrams that use, as first step, the isolation and immunofluorescence, respectively. Since these latter techniques showed low values of performance criteria, the study highlighted the necessity to integrate the EPPO protocol with a preliminary molecular screening test. It is advisable to validate new molecular methods more specific and sensitive than end-point PCR, based on systems such as real time PCR, LAMP and digital PCR.