RESEARCHER: Dr. Diane A. Cuppels, Agriculture and Agri-Food Canada, London

PROJECT OBJECTIVE:

To develop a sensitive and reliable assay for detecting tomato and pepper seed lots infested with bacterial pathogens. Once detected, infested material can then be treated with a bactericide or destroyed, thus limiting the spread of disease to non-contaminated plants or seed and reducing crop damage.

METHODOLOGY:

Artificial infestation of germinated tomato seed with the bacterial spot pathogen (Xcv).

Various quantities of germinated seed were weighed and then washed in sufficient buffer to cover the sprouted seed. The buffered washwater was separated from the seed and infested with various concentrations of an antibiotic-resistant strain of Xcv. DNA was then extracted and purified from the washwater by a number of different techniques to determine which one was most suitable for processing large seed quantities.

PCR (polymerase chain reaction) assay for detecting bacterial spot. The presence of bacterial spot in the various batches of seed washwater was determined by PCR using the pathogen-specific AAFC-BSX primers developed by our laboratory and a GeneAmp® PCR System 9700.

RESULTS:

In our previous study, we infested dry, nonpelletized untreated tomato seed with various concentrations of the bacterial spot pathogen (Xcv) ranging from 10⁹ to 1 bacterial cell per gram of seed. We then germinated this seed and tested it for the presence of viable bacterial cells and for the ability to give a positive PCR using our BSX PCR primers. Our limit of detection when assaying 2-g samples of germinated seed was 10 Xcv cells/400-450 dry seeds. By germinating the seed, we allow any living Xcv cells that are on the seed to grow and multiply, thereby increasing our chances of detecting them. In general, the Xcv population appears to increase 10,000-fold after a 4-day germination at 25 C. In our current study, when we tested larger seed samples (5,000, 10,000 and 20,000 seeds), we found that PCR inhibitors naturally present in seed overwhelmed our assay system, giving false negatives. Furthermore, as we infested larger quantities of seed with increasingly smaller populations of Xcv, we could no longer monitor our experiments using selective growth media and conventional methods of bacterial enumeration. As a consequence, we had to develop 1) another method of extracting DNA from our samples of germinated seed and 2) another method of testing the efficacy of our PCR assay. Thus, rather than vacuum-infiltrate dry seed with Xcv, we added bacteria at various concentrations to set quantities of germinated seed and then performed our DNA extraction procedure. At present, our detection limit is 1 viable Xcv cell in 10,000 seeds, We also tested our improved assay method on 20-g quantities of pepper seeds; we were able to detect 4 Xcv cells in 20 g (or 3100 seeds). We should be able to refine our assay to the point where we can find 1 viable Xcv cell in 20,000 (maybe 30,000) tomato or pepper seeds. Because new strains and races of the bacterial spot pathogen continue to appear and are, in fact, inevitable, we also plan to develop a second (and maybe third) set of BSX primers to ensure that we are detecting all forms of this important tomato pathogen.