A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas

Aims: To develop a sensitive real-time PCR-based protocol for the detection of Xanthomonas campestris pathovars from Brassica seed. Methods and Results: A 5' nuclease real-time PCR assay was developed to screen Brassica spp. seed for the presence of X. campestris pathovars that cause black rot. The assay amplifies a 78-bp segment of the X. campestris hrpF gene and a 100-bp segment of the Brassica spp. 18S-25S internal transcribed spacer region. The Brassica spp. target provides an internal control for the amplification process to prevent false negatives that may arise from inhibitors that are often present in extracts from plant material. Whilst the primers were compatible with SYBR (R) Green I assays, the use of fluorescently labelled probes in a 5' nuclease assay afforded greatest sensitivity and specificity. Seed batches carrying one artificially infected seed among 10 000 were readily detected using the assay. The multiplex real-time PCR assay permitted the rapid detection of pathogenic strains of X. campestris from bacterial colonies, Brassica seed and plants. Conclusions: Strains of X. campestris pathogenic to brassicas were readily detected from seed via a multiplex 5' nuclease real-time PCR assay. The real-time assay offers an improvement in sensitivity and a reduced turn-around time over the conventional multiplex PCR. Significance and Impact of the Study: Real-time PCR can be used to rapidly screen Brassica spp. seed batches for the presence of X. campestris pathovars. This assay provides a means for growers and the seed industry to be aware of the black rot status of their planting material, so that they may more effectively employ disease control measures or seed disinfection.