No abstract

Xanthomonas campestris is a seedborne bacterium that causes black rot of crucifers. Substantial crop losses may result from the rapid spread of the bacteria under favourable conditions, especially those occurring during seedling production. A PCR-based method has been developed for the rapid and sensitive detection of the pathovars of X. campestris that affect crucifers. Primers were designed to specifically amplify a 619 bp fragment of the hrpF gene from X. campestris . Amplification products were not detected from other Xanthomonas species, or from other pathogenic or epiphytic bacteria occurring on these plants. To avoid false-negative results arising from the presence of amplification inhibitors in plant extracts, primers targeting a 360 bp section of the internal transcribed spacer (ITS) region from Brassica spp. were included in a multiplex PCR. The assay readily detected X. campestris pv. campestris infections in diseased plants and from bacterial colonies isolated on growth media, and was more sensitive and specific than traditional plating methods and a commercially available ELISA. A seed-washing protocol was optimized to allow the detection of a single artificially infected seed among 10 000 healthy seeds using the multiplex PCR.

The Fusarium oxysporum species complex (FOSC) causes disease in plants and animals, but is also widely dispersed in natural ecosystems without evidence of disease. The present study screened a population representing natural ecosystems across the Australian continent for the putative effector genes pisatin demethylase 1 (PDA1), pectate lyase (pelD), secreted gene expression (SGE1) and secreted in xylem (SIX). The genes pelD and SGE1 were prevalent in the natural isolates, PDA1 was present at an intermediate level, whereas SIX genes were detected at low levels. Phylogenies of these putative effector genes were compared to the EF-1a species phylogeny to determine the likely modes of gene transmission: vertical gene transfer (VGT) and horizontal gene transfer (HGT). There was evidence of both modes of gene transmission within the F. oxysporum isolates. PDA1, pelD and SGE1 were likely to be only vertically inherited, whereas the SIX genes had evidence for both VGT and HGT. The phylogenetic relationships of SIX genes in isolates from natural ecosystems and formae speciales from agro-ecosystems were also established. These findings have important implications for the evolution of effectors in the FOSC.

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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® 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.