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...
Thirteen Spanish potato isolates of Dickeya chrysanthemi obtained from different cultivars, regions and years have been characterised by biochemical, physiological, serological, molecular and pathogenicity tests. Isolates studied have been classified into biovars 1, 3, 5 and 6 by a microtitre system that correlates with the conventional tests in tubes. This is the first description of the characteristics of D. chrysanthemi isolated from potato in Spain and of the development of a microtitre system for biovar determination.
Understanding how viruses and subviral agents initiate disease is central to plant pathology. Whether RNA silencing mediates the primary lesion triggered by viroids (small non-protein-coding RNAs), or just intermediate-late steps of a signaling cascade, remains unsolved. While most variants of the plastidreplicating peach latent mosaic viroid (PLMVd) are asymptomatic, some incite peach mosaics or albinism (peach calico, PC). We have previously shown that two 21-nt small RNAs (PLMVd-sRNAs) containing a 12-13-nt PC-associated insertion guide cleavage, via RNA silencing, of the mRNA encoding a heatshock protein involved in chloroplast biogenesis. To gain evidence supporting that such event is the initial lesion, and more specifically, that different chloroses have different primary causes, here we focused on a PLMVd-induced peach yellow mosaic (PYM) expressed in leaf sectors interspersed with others green. First, sequencing PLMVd-cDNAs from both sectors and bioassays mapped the PYM determinant at one nucleotide, a notion further sustained by the phenotype incited by other natural and artificial PLMVd variants. And second, sRNA deep-sequencing and RNA ligase-mediated RACE identified one PLMVd-sRNA with the PYM-associated change that guides cleavage, as predicted by RNA silencing, of the mRNA encoding a thylakoid translocase subunit required for chloroplast development. RT-qPCR showed lower accumulation of this mRNA in PYM-expressing tissues. Remarkably, PLMVd-sRNAs triggering PYM and PC have 5ʹ-terminal Us, involving Argonaute 1 in what likely are the initial alterations eliciting distinct chloroses.
Symptoms characteristic of bacterial spot disease of Prunus spp. (4) were observed on almond trees (Prunus dulcis (Mill.) Webb) in 14 localities of Comunidad Valenciana (eastern Spain) and Aragón (northeastern Spain) between 2006 and 2009. Symptoms were first noted in the spring and were observed until leaf fall. Initial infections began on leaves as small, angular, water-soaked spots, which mainly developed toward the tip and along the leaf margins. These water-soaking lesions were surrounded by chlorotic tissue, although chlorosis did not extend more than a few millimeters. Subsequently, the lesions turned light brown, necrotic, and sometimes the necrotic spots fell out. When the lesions coalesced, they produced large necrotic areas. Sometimes premature leaf drop of infected leaves was observed in severely affected trees. Infected fruits initially displayed sunken, corky lesions that oozed gum, which later became raised when the mesocarp dehydrated. Infected fruits either dropped prematurely or remained on trees after harvest. Cankers typical of bacterial spot disease of stone fruit trees were observed on branches and shoots. Isolations from diseased leaves and fruits yielded Xanthomonas-like colonies on YPGA medium (yeast extract, peptone, and glucose agar), which were subsequently purified and characterized. All strains were gram-negative rods, oxidase negative, and strictly aerobic and showed typical biochemical characteristics of the Xanthomonas genus (3). A collection of 70 strains were further identified by PCR with primers Y17CoF/Y17CoR (1) as Xanthomonas arboricola pv. pruni by comparison with reference strains ISPaVe B4 and ISPaVe B6 isolated from Prunus salicina in Italy. A selection of 46 strains were also analyzed by immunofluorescence (IF) and ELISA using commercial polyclonal antibodies from NEOGEN Europe Ltd. (Ayrshire, Scotland, UK) and SEDIAG S.A.S. (Longvic, France), respectively), although ELISA antibodies proved to be not specific for X. arboricola pv. pruni. Pathogenicity was confirmed by inoculation of 70 almond strains and the reference strains on leaves of potted almond trees and/or on detached leaves (2) with bacterial suspensions (107 CFU per ml). One leaf was inoculated at 8 to 10 sites per strain. Characteristic bacterial spot disease symptoms (4) appeared on all inoculated leaves after 1 week of incubation at 25°C and high humidity, but not on the negative controls infiltrated with sterile distilled water. The original pathogen was reisolated from lesions of inoculated leaves and confirmed by biochemical tests, IF and PCR. As observed in Spain, the disease produces serious damage on the most susceptible almond cultivars like Antoñeta, Guara, Marta, Mas Bovera, and Vayro and can be very harmful, with severity of infection depending upon the relative cultivar susceptibility and environmental conditions. Appropriate eradication measures were taken after the causal agent was confirmed as X. arboricola pv. pruni. This pathogen was previously reported on almond in Japan and New Zealand (4). To our knowledge, this is not only the first report on almond in Spain but also in Europe. References: (1) M. C. Pagani. Ph. D. thesis, North Carolina State University, Raleigh, 2004. (2) P. S. Randhawa and E. L. Civerolo. Phytopathology 75:1060, 1985. (3) L. Vauterin et al. Int. J. Syst. Bacteriol. 45:472, 1995. (4) J. M. Young. N. Z. J. Agric. Res. 20:105, 1977.
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