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Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species.

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.

SUBMITTER: Palacio-Bielsa A

PROVIDER: S-EPMC3019718 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species.

Palacio-Bielsa Ana A Cubero Jaime J Cambra Miguel A MA Collados Raquel R Berruete Isabel M IM López María M MM

Applied and environmental microbiology 20101029 1

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 protoco ...[more]

PMID: 21037298

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Project description:Comparative studies in Xanthomonas have provided a vast amount of data that enabled to deepen in the knowledge of those factors associated with virulence and Xanthomonas plant interaction. The species of this genus present a wide range of host plants and a large number of studies have been focused to elucidate which mechanism are involved in this characteristic. In this study, comparative genomic and phenotypic analysis were performed between X. citri subsp. citri (Xcc), one of the most studied pathogens within Xanthomonas, and X. arboricola pv. pruni (Xap), a pathogen which has aroused great interest in recent time. The work was aimed to find those elements that contribute to their host divergence despite the convergence in the symptoms that each species cause on Citrus spp. and Prunus spp., respectively. This study reveals a set of genes that could be putatively associated with the adaptation of these pathogens to their hosts, being the most remarkable those involved in environmental sensing systems such as the case of the TonB-dependent transporters, the sensors of the two-component system and the methyl accepting chemotaxis proteins. Other important variants were found in processes related to the decomposition of the cell wall as could be appreciated by their dissimilar set of cell-wall degrading enzymes. Type three effectors, as one of the most important factors in delineating the host specificity in Xanthomonas, also showed a different array when comparing both species, being some of them unique to each pathogen. On the other hand, only small variations could be connected to other features such as the motility appendages and surface adhesion proteins, but these differences were accompanied by a dissimilar capacity to attach on host and non-host leaf surface. The molecular factors found in this work provide the basis to perform a more in-depth functional analyses that unveil those actual factors associated with pathogenesis and host specificity in Xcc and Xap.

Project description:BackgroundXanthomonas arboricola pv. pruni (Xap) causes bacterial spot of stone fruits and almond, an important disease that may reduce the yield and vigour of the trees, as well as the marketability of affected fruits. Xap lies within the Xanthomonas genus, which has been intensively studied because of its strain specialization and host range complexity. Here, we summarize the recent advances in our understanding of the complexities of Xap, including studies of the molecular features that result after comparative phenotypic and genomic analyses, in order to obtain a clearer overview of the bacterial behaviour and infection mechanism in the context of the X. arboricola species.Taxonomic statusBacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species X. arboricola; Pathovar pruni.Host range and symptomsXap infects most Prunus species, including apricot, peach, nectarine, plum and almond, and occasionally cherry. Symptoms are found on leaves, fruits, twigs and branches or trunks. In severe infections, defoliation and fruit dropping may occur.DistributionBacterial spot of stone fruits and almond is worldwide in distribution, with Xap being isolated in Africa, North and South America, Asia, Europe and Oceania. It is a common disease in geographical areas in which stone fruits and almonds are grown. Xap is listed as a quarantine organism in several areas of the world.GenomeThe genomes of six isolates from Xap have been publicly released. The genome consists of a single chromosome of around 5 000 000 bp with 65 mol% GC content and an extrachromosomal plasmid element of around 41 000 bp with 62 mol% GC content. Genomic comparative studies in X. arboricola have allowed the identification of putative virulence components associated with the infection process of bacterial spot of stone fruits and almond.Disease controlManagement of bacterial spot of stone fruits and almond is based on an integrated approach that comprises essential measures to avoid Xap introduction in a production zone, as well as the use of tolerant or resistant plant material and chemical treatments, mainly based on copper compounds. Management programmes also include the use of appropriate cultivation practices when the disease is already established. Finally, for the effective control of the disease, appropriate detection and characterization methods are needed for use in symptomatic or asymptomatic samples as a first approach for pathogen exclusion. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTPR; http://www.cost.eu/COST_Actions/ca/CA16107; http://www.xanthomonas.org.

Dataset Information

Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species.

Publications

Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species.

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