Development of a PCR Assay for the Rapid Detection and Differentiation of 'Candidatus Liberibacter solanacearum' Haplotypes and Their Spatiotemporal Distribution in the United States

Zebra chip, or zebra complex (ZC) has become an important invasive disease of potato in the United States and New Zealand and is caused by a phloem-limited bacterium, 'Candidatus Liberibacter solanacearum' (Lso). A PCR assay using a single pair of simple sequence repeat (SSR) primers was developed for simultaneous detection and genotype differentiation of Lso haplotypes associated with zebra chip disease of potato. The sensitivity of the SSR PCR was similar to a 16S PCR assay, with detection limit of 100 copies of the Lso genome in haplotype A infected potato and psyllid samples and 10 copies of Lso genome in haplotype B potato and psyllid samples. The Lso detection frequency of the SSR PCR assay was 79.1 % in potato and 26.4 % in psyllid samples, respectively; whereas the detection frequency of the 16S PCR assay 59.0 % in potato and 25.9 % in psyllid samples, respectively. Samples of Lso positive potato plants and psyllids from multiple states in the US were demonstrated to have either haplotype A or haplotype B Lso and occasionally both haplotypes were found in individual samples. This is the first report that co-infection of the two haplotypes of Lso exists in potato and potato-psyllid samples. Only haplotype A Lso was detected in North Dakota psyllid samples collected in 2010, in Idaho and Washington ZC potato samples sampled from storage in 2011, and in Idaho ZC potato samples in 2012. Haplotype A Lso was also detected in New Zealand ZC affected potato samples and psyllid samples collected in 2010 and 2011. The PCR assay developed is as sensitive as previously developed assays and has the advantage of simultaneously detecting and differentiating Lso haplotypes of the ZC bacterium, thus making it extremely useful for epidemiological studies.