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.