Phenotypical and Molecular Characterization of Local Clavibacter michiganensis subsp. michiganensis, the Causal Agent of Tomato Bacterial Canker Disease

Abstract

Clavibacter michiganensis subsp. michiganensis (Cmm) is a significant bacterial pathogen causing tomato wilt and canker, leading to substantial yield and quality losses in tomato production. This study aimed to characterize Cmm isolates phenotypically and molecularly, assess their genetic variation, and evaluate their pathogenicity. Phenotypic characterization involved biochemical tests, including Gram staining, fluorescence, levan formation, oxidase, pectolytic activity, arginine dehydrolase, tobacco hypersensitivity, gelatin, esculin, and starch hydrolysis, as well as carbon source utilization profiling. Molecular identification employed conventional PCR and qPCR using Cmm-specific primers Cmm5/Cmm6. Genetic variation was assessed using REP-PCR with REP, ERIC, and BOX elements. Results showed that all isolates were Gram-positive, non-fluorescent, and negative for levan formation, oxidase, pectolytic activity, and arginine dehydrolase. They induced a hypersensitive reaction in tobacco and hydrolyzed starch. Variations were observed in gelatin and esculin hydrolysis and carbon source utilization. PCR and qPCR confirmed the isolates as Cmm. REP-PCR revealed that the REP element was ineffective, the ERIC element discriminated only at the subspecies level, while the BOX element differentiated intraspecific genetic variation. Pathogenicity tests demonstrated varying degrees of virulence among isolates. The study highlights the reliability of molecular diagnostic methods and the correlation between genetic diversity and virulence in Cmm isolates, providing insights for developing specific markers to distinguish avirulent and hypervirulent strains.