Transmission Frequency of Clavibacter michiganensis subsp. insidiosus to Alfalfa Seed and Identification of the Bacterium by PCR

Abstract

A sensitive polymerase chain reaction (PCR)-based identification method was developed for Clavibacter michiganensis subsp. insidiosus, the causal agent of alfalfa bacterial wilt. The method, which targets a high-copy-number insertion element, is rapid and specific for this plant pathogen. The assay was used to determine the frequency of transmission of the pathogen to alfalfa seed. Seed was produced from infected plants grown and pollinated in the greenhouse, from infected plants grown in the field and transplanted to the greenhouse to produce seed, and from diseased 2-year-old field-grown plants. Seed from each infected plant were assayed to identify infected seed lots. Seed were ground to a fine powder and soaked in a liquid medium, after which a portion of the seed slurry was plated on a semi-selective agar medium. The PCR assay was used to identify C. michiganensis subsp. insidiosus colonies on plates. Approximately 6.3 to 7.7% of diseased plants transmitted C. michiganensis subsp. insidiosus to seed. In assays in which individual seed were analyzed from infected seed lots, approximately 2.5 to 8.7% of seed contained the bacterium.