Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

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.

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Eukaryotes have established symbiotic relationship with microorganisms, which enables them to accomplish functions that they cannot perform alone. In the German cockroach, Blattella germanica, the obligate endosymbiont Blattabacterium coexists with a rich gut microbiota. The transmission of Blattabacterium is vertical, but little is known about how the gut microbiota colonizes newborn individuals. In this study, we treated B. germanica populations with rifampicin, a broad-spectrum antibiotic, during two generations and analyzed gut bacterial composition and the Blattabacterium load in control and rifampicin-treated populations. Rifampicin exerted a drastic effect on gut microbiota composition, which recovered in the second generation in the case where the antibiotic was not added to the diet. Furthermore, we observed that bacterial species present in the diet, and particularly in the feces, contribute significantly to establishing the gut microbiota. Finally, the Blattabacterium population remained unaffected by the antibiotic treatment of adults during the first generation but was strongly reduced in the second generation, suggesting that this intracellular symbiont is sensitive to rifampicin only during the infection of the mature oocytes, when it is in an extracellular stage.

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.

SummaryA PCR-based kit, Probelia TM, for the detection of Erwinia carotovora subsp, atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNAspecific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3• to 1.5x103 cells ml-l). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0xl0 l to 6.2x103 cells ml -I peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method.

An atypical strain of Erwinia amylovora was isolated near an outbreak of fire blight at a nursery in Spain in 1996. It was obtained from a Crataegus plant showing typical symptoms and was identified as E. amy-lovora by biochemical tests and enrichment-enzyme-linked immuno-sorbent assay, but not by polymerase chain reaction using primers based on the pEA29 sequence. Nevertheless, with primers from chromosomal regions, the isolate gave the expected amplification band. This strain carries one plasmid of approximately 70 kb, with no homology with the 29-kb plasmid common to all pathogenic strains, or with a large plasmid present in some E. amylovora strains. Growth of the strain in minimal medium without thiamine was slower compared with cultures in the same medium with thiamine, a characteristic typical of strains cured of the 29-kb plasmid. Nevertheless, aggressiveness assays on pear, apple, and Pyracantha plants and in immature pear fruit showed that this strain exhibited a virulence level similar to other strains containing pEA29. To the best of our knowledge, this is the first report of the isolation from naturally infected plant material of a pathogenic strain of E. amylovora without pEA29, but with a plasmid of approximately 70 kb not previously described.

Eight Erwinia strains, isolated from necrotic pear blossoms in Valè ncia, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovora and E. pyrifoliae. Phylogenetic analysis of 16S rRNA, gpd and recA gene sequences showed that the eight novel strains could not be assigned to any recognized species. On the basis of DNA-DNA hybridization studies, the novel isolates constituted a single group with relatedness values of 87-100 % to the designated type strain of the group, CFBP 5888 T . Depending on the method used, strain CFBP 5888 T showed DNA-DNA relatedness values of between 22.7 and 50 % to strains of the closely related species E. amylovora and E. tasmaniensis. The DNA G+C contents of two of the novel strains, CFBP 5888 T and CFBP 5883, were 51.1 and 50.5 mol%, respectively. On the basis of these and previous results, the novel isolates represent a novel species of the genus Erwinia, for which the name Erwinia piriflorinigrans sp. nov. is proposed. The type strain is CFBP 5888 T (5CECT 7348 T ).Necrotic blossoms were observed in Ercolini (Coscia) and Tendral pear trees growing in Valencia, Spain, from which an unknown bacterium was consistently isolated in 1999 and during the following two years. Infected blossoms were similar in appearance to those affected by the disease fire blight, caused by Erwinia amylovora, but only occurred in spring and the blossoms were the only part of the trees that were affected, unlike the disease caused by E. amylovora (Roselló et al., 2002(Roselló et al., , 2006. The isolated bacterium was subjected to phenotypic and molecular characterization and, although it was recognized as belonging to the genus Erwinia, many of its characteristics differed from those of other pathogenic and non-pathogenic species of the genus Erwinia that have been isolated from pear trees, including E. amylovora (Hauben et al., 1998), Erwinia billingiae (Mergaert et al., 1999), Erwinia pyrifoliae (Kim et al., 1999) and Erwinia tasmaniensis (Geider et al., 2006). The novel isolates provoked a hypersensitivity response when they were allowed to infiltrate tobacco and tomato leaves. Their 3These authors contributed equally to this work.Abbreviations: ERIC, enterobacterial repetitive intergenic consensus; ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbourjoining; REP, repetitive extragenic palindromic; UPGMA, unweighted pair group method with arithmetic mean.