Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by End Point and Real-Time PCR

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas savastanoi </it>pv. <it>savastanoi </it>is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars <it>nerii </it>and <it>fraxini</it>, respectively. When artificially inoculated, pv. <it>savastanoi </it>causes disease also on ash, and pv. <it>nerii </it>attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including <it>P. savastanoi</it>, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these <it>P. savastanoi </it>pathovars, in multiplex and <it>in planta</it>.</p> <p>Results</p> <p>Specific PCR primers and probes for the pathovars <it>savastanoi</it>, <it>nerii </it>and <it>fraxini </it>of <it>P. savastanoi </it>were designed to be used in End Point and Real-Time PCR, both with SYBR^®Green or TaqMan^®chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four <it>P. savastanoi </it>strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars <it>phaseolicola </it>and <it>glycinea </it>of <it>P. savastanoi </it>and bacterial epiphytes from <it>P. savastanoi </it>host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 10²genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants.</p> <p>Conclusions</p> <p>Here for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of <it>P. savastanoi </it>pv. <it>savastanoi</it>, pv. <it>nerii </it>and pv. <it>fraxini</it>. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and <it>in vivo</it>. Compared with the other methods already available for <it>P. savastanoi</it>, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of <it>P. savastanoi </it>on olive and oleander propagation materials.</p