Detection and characterization of virulent, hypovirulent, and nonvirulent Clavibacter Michiganensis subsp. Michiganensis

Abstract

Clavibacter michiganensis subsp. michiganensis (Cmm), a seedborne quarantine pathogen, causes bacterial canker of tomato, which is a serious disease that can severely decrease yields in greenhouse and field production areas. Seed assays are used to prevent dissemination of Cmm through infested seed, but limitations in assay sensitivity and specificity allow canker outbreaks to continue. An assay was developed that detected Cmm when as few as 10 colony forming units (cfu) were present per 50 ml sample. The assay used a three-unit filtration system to capture bacterial cells, followed by a four-day membrane incubation and a colony blot immunoassay using the CMMI monoclonal antibody (MAb). The filtration and immunoassay technique was more sensitive than a standard spread plating assay, and could potentially reduce current assay times by up to 3 weeks. Assays done on nine seed lots yielded a high percentage (81%) of MAb CMMl positive colonies that were hypovirulent or nonvirulent on tomato (Lycopersicon esculentum cv. Kewalo). All strains were confirmed as Cmm using the MicroLog identification system, rep-PCR, two PCR primer sets, and an endoglucanase assay. Of the assays tested, MicroLog and rep-PCR were the most consistent in identifying hypovirulent and nonvirulent MAb CMMI-positive strains as Cmm. The CMMI MAb was further used to quantify in planta movement and multiplication of a nonvirulent Cmm strain when coinoculated with a virulent Cmm strain. In planta coincubation did not significantly alter growth or colonization habits of either strain. Thus, here is no evidence that nonvirulent Cmm strains playa role in bacterial canker epidemiology. However, their continued isolation from diseased and infested tissues, and the importance of nonvirulent strains in other pathosystems, suggest significance and warrants further investigation.