A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 l of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.

Aims:  To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. Methods and Results:  Citrus fruits with canker‐like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real‐time PCR with SYBR Green or a TaqMan probe, were compared. Canker‐like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real‐time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39–52 lesions depending on the protocol employed. Conclusions:  An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real‐time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. Significance and Impact of the Study:  This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present.

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae was first described in Japan and Korea and is currently an emerging disease that causes major losses in China, Italy, New Zealand, France, Portugal, and Chile. Gold kiwifruit (Actinidia chinensis), especially cvs. Jin Tao and Hort 16A, seem to be more susceptible than green kiwifruit (Actinidia deliciosa) cvs. Hayward and Summer. The bacterium affects male and female woody vines equally, with young vines being more susceptible. The most characteristic symptoms that appear in early spring are reddish orange or white exudates associated with cankers and wounds in branches and/or trunk, as well as brown leaf spots. Buds and fruits were also affected (1). In Spain, 1,132 ha of kiwifruit orchards yielded 25,285 t of fruit in 2009 (2). Most Spanish kiwifruit is cultivated in Galicia (northwest Spain), where the main cultivar is Hayward. In 2010, the first plantation of cv. Jin Tao and one plantation of cv. Summer were established in this area close to Hayward woody vine. In early spring 2011, 80% of the vines in one orchard had twigs with reddish exudates and branches and trunks as well as leaves with angular spots surrounded by yellow haloes. Isolations from both Actinidia spp. were conducted on nutrient agar with sucrose. One hundred and twelve isolates were obtained and seventy-seven were aerobic, gram negative and nonfluorescent on King's B medium. Biochemical tests performed were levan, oxidase, potato rot, arginine didhydrolase, hypersensitivity in tobacco, and utilization of 49 carbohydrates by the API 50 CH system (BioMérieux, Marcy l'Etoile, France). Three PCR protocols were used: two with pathovar-specific primers (PSAF1/PSAR2 and PSAF3/PSAR4) and one with nonspecific primers (PsITSF1/PsITSR2) (3). The results of all biochemical and molecular tests were in agreement with those expected for P. syringae pv. actinidiae. The 16S-23S region of strain EFA 37 isolated from A. deliciosa cv. Summer was sequenced (GenBank Accession No. JF815537) and had 100% sequence identity with P. syringae pv. actinidiae (GenBank Accession Nos. AY342165 and D86357). Pathogenicity tests were performed on 15 plants of A. deliciosa cv. Hayward (five plants per isolate) with the Spanish representative strain EFA 37 and compared with two reference strains isolated from both Actinidia species in Italy and five plants of an untreated control. Three buds per healthy vine were wounded with a sterile needle, inoculated with 30 to 50 μl of each bacterial suspension (108 CFU/ml), sealed, and then covered with plastic. Five leaves per healthy vine were also pierced with a sterile needle and then atomized with the same suspension. Symptoms began to appear after 5 days on inoculated vines, but not on untreated control vines. The bacterium, P. syringae pv. actinidiae, was reisolated from symptomatic plants. The kiwifruit orchard with affected plants was eradicated (25 ha). To our knowledge, this is the first report of P. syringae pv. actinidiae in Spain. References: (1) EPPO Alert List. Online publication. Retrieved from http://www.eppo.org/QUARATINE/Alert_List , June, 2011. (2) Ministerio de Medio Ambiente y Medio Rural y Marino (MARM). Anuario de Estadística, Online Publication. Retrieved from http://www.marm.es/estadistica/pags/anuario/2010 , June 2011. (3) J. Rees-George et al. Plant Pathol. 59:453, 2010.

Aims:  We have examined the intraspecific diversity of a collection of 63 Spanish strains of Erwinia amylovora, isolated from 1995 to 2001, to determine whether or not they could be grouped based on phenotypic or genotypic criteria and to investigate the sources of inoculum for fire blight dissemination in Spain. Methods and Results:  Several biochemical and molecular techniques, such as miniaturized API 20E, API 50CH, ATB G‐5 and API‐ZYM tests, BIOLOG metabolic fingerprinting, PCR ribotyping, pulsed‐field gel electrophoresis (PFGE), minisatellite‐primed PCR (MSP‐PCR), random amplified polymorphic DNA (RAPD) analyses and AFLP were used. We report the first identification in Spain of the PFGE pattern Pt1, already described in other European countries, together with Pt3 and Pt4 patterns. Moreover, PFGE, together with MSP‐PCR, RAPD analyses and AFLP are, until now, the only techniques that have provided information about the possible infection sources and relationships between the different foci in Spain, with AFLP being the most discriminative. Conclusions:  These techniques have allowed grouping of Spanish strains by their geographical origin. Significance and Impact of the Study:  Our results support the hypothesis that some fire blight outbreaks have been caused by the introduction in Spain of infected plant material, or other inoculum sources from different European countries.

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