Abstract
BackgroundPseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii 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 P. savastanoi, 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 P. savastanoi pathovars, in multiplex and in planta.ResultsSpecific PCR primers and probes for the pathovars savastanoi, nerii and fraxini of P. savastanoi 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 P. savastanoi strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars phaseolicola and glycinea of P. savastanoi and bacterial epiphytes from P. savastanoi 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 102 genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants.ConclusionsHere for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of P. savastanoi pv. savastanoi, pv. nerii and pv. fraxini. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and in vivo. Compared with the other methods already available for P. savastanoi, 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 P. savastanoi on olive and oleander propagation materials.
Highlights
Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease
The pathological process depends on the expression of bacterial hrp genes [8], and the development of the spherical knots is caused by phytohormones (3-indoleacetic acid and cytokinins) synthesized by pv. savastanoi (Psv), that trigger uncontrolled proliferation of the cells surrounding the site of infection [9,10,11,12,13]
The strains isolated from olive, oleander and ash can be differentiated according to a series of characteristics concerning host range, production of phytohormones and bacteriocins, assimilation of different carbon sources, monoclonal antibodies, analysis of whole cell fatty acids, DNA relatedness, low molecular weight restriction fragments, restriction fragment length polymorphism (RFLP) and fluorescent amplified fragment length polymorphism (f-AFLP) [3,12,14,15,16,17,18,19,20,21,22,23,24]
Summary
Pseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. The genetic diversity of strains isolated from olive trees was recently deeply investigated [25,26,27,28] According to all these data, the name Psv is used to indicate isolates from olive, while the names P. savastanoi pv. Sources of inoculum for new infections are represented by Psv populations surviving within the young knots, and by Psv naturally resident on healthy olive trees as epiphyte on the phylloplane, on the surfaces of stems and olive fruits. Considering the increasing spread of resistance to copper compounds among P. syringae pathovars and related bacteria [39,40], sensitive and specific methods to monitor Psv natural epiphytic population on olive trees are needed to contribute to the successful preventive control and management of this disease. Psv is among the infective agents of olive, whose absence has to be ascertained for the production of certified olive plants [41]
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