Development of an Efficient Real-Time Quantitative PCR Protocol for Detection ofXanthomonas arboricolapv. pruni inPrunusSpecies

Abstract

Xanthomonas arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as being X. arboricola pv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system in X. arboricola pv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 10 2 CFU ml ؊1 , thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed for X. arboricola pv. pruni strains from different origins as well as for closely related Xanthomonas species, non-Xanthomonas species, saprophytic bacteria, and healthy Prunus samples. The efficiency of the developed protocol was evaluated with field samples of 14 Prunus species and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, and X. arboricola pv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test for X. arboricola pv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.Xanthomonas arboricola pv. pruni (31) (synonym, Xanthomonas campestris pv. pruni [Smith]) is a Gram-negative plant-pathogenic bacterium that causes bacterial spot disease of stone fruits. X. arboricola pv. pruni has been reported to affect a wide range of Prunus species, such as plum, nectarine, peach, apricot, cherry, almond, and ornamental species (19,26,32). The disease was first described for Japanese plum in North America in 1903 (28), and since then, it has been reported to occur in many of the major stone-fruit-producing areas of the world (3, 4). Symptoms occur on leaves, fruits, and twigs, ranging from necrotic angular lesions on leaves and sunken lesions on fruits to cankers on twigs. X. arboricola pv. pruni can be very damaging when severe infections occur on highly susceptible cultivars (27).International trade has led to the dissemination of X. arboricola pv. pruni through contaminated material used for propagation (11). Moreover, the bacterium overwinters in buds and leaf scars, which act as efficient sources of primary inocula for spring infections (34). Because of its negative economic impact, X. arboricola pv. pruni is considered a quarantine organism by European Union phytosanitary legislation (see reference 1 and amendments th...

Plant Disease Diagnosis: Technological Advancements and Challenges

“…citrulli in watermelon seeds, Microdochium nivale in wheat seeds (Taylor et al, 2002), Ralstonia solanacearum race 3, biovar 2, in asymptomatic potato tubers (Ozakman and Schaad, 2003), fungal and oomycete tomato pathogens in plant and soil samples (Lievens et al, 2006), Rhizoctonia solani AG-1 IA in rice (Sayler and Yang, 2007), Fusarium circinatum in pine seed (Ioos et al, 2009), Xanthomonas arboricola pv. pruni in Prunus species (Palacio-Bielsa et al, 2011), Xanthomonas oryzae pv oryzae in rice by Real time Bio-PCR (Cho et al, 2011).…”

Real-time PCR applied to study on plant pathogens: potential applications in diagnosis - a review

“…Control of diseases caused by plant pathogenic bacteria frequently needs precise detection, followed by suitable identification of the causal organism (Palacio-Bielsa et al 2011). Several real-time PCR systems have been validated with significant improvements in speed, specificity, and sensitivity for detection and direct quantification of plant pathogenic bacteria in different environmental samples.…”

Scientific Opinion on pest categorisation ofXanthomonas arboricolapv.pruni(Smith, 1903)

“…pruni has been described by the European and Mediterranean Plant Protection Organization (EPPO, 2006): such detection procedures are currently under revision, as molecular assays were not included. Later, several DNA amplification protocols were developed (Pagani, 2004;Palacio-Bielsa et al, 2011;Bühlmann et al, 2012) and some of them have been evaluated and validated (Lopez et al, 2012;Perez et al, 2013). Therefore, a new EPPO diagnostic protocol is under development.…”