Erwinia uzenensis is a plant-pathogenic bacterium, recently described in Japan, which infects pear trees, causing the 'bacterial black shoot disease of European pear' (BBSDP). Like other Erwinia pear pathogens, E. uzenensis causes damp, black lesions on young shoots resembling those of E. amylovora, but not blossom blight, fruitlet blight or wilting of the shoot tip. The distribution of E. uzenensis seems restricted to the country where it was reported up to now, but it may spread to other countries and affect new hosts, as is the current situation with E. piriflorinigrans and E. pyrifoliae. Fast and accurate detection systems for this new pathogen are needed to study its biology and to identify it on pear or other hosts. We report here the development of a specific and sensitive detection protocol based on a real-time PCR with a TaqMan probe for E. uzenensis, and its evaluation. In sensitivity assays, the detection threshold of this protocol was 101 cfu ml-1 on pure bacterial cultures and 102-103 cfu ml-1 on spiked plant material. The specificity of the protocol was evaluated against E. uzenensis and 46 strains of pear-associated Erwinia species different to E. uzenensis. No cross-reaction with the non-target bacterial species or the loss of sensitivity were observed. This specific and sensitive diagnostic tool may reveal a wider distribution and host range of E. uzenensis initially considered restricted to a region and will expand our knowledge of the life cycle and environmental preferences of this pathogen.

Antibiotic resistance is recognized as one of the major challenges in public health. The global spread of antibiotic resistance is the consequence of a constant flow of information across multi-hierarchical interactions, involving cellular (clones), subcellular (resistance genes located in plasmids, transposons, and integrons), and supracellular (clonal complexes, genetic exchange communities, and microbiotic ensembles) levels. In order to study such multilevel complexity, we propose to establish an experimental epidemiology model for the transmission of antibiotic resistance with the cockroach Blatella germanica. This paper reports the results of five types of preliminary experiments with B. germanica populations that allow us to conclude that this animal is an appropriate model for experimental epidemiology: (i) the composition, transmission, and acquisition of gut microbiota and endosymbionts; (ii) the effect of different diets on gut microbiota; (iii) the effect of antibiotics on host fitness; (iv) the evaluation of the presence of antibiotic resistance genes in natural- and lab-reared populations; and (v) the preparation of plasmids harboring specific antibiotic resistance genes. The basic idea is to have populations with higher and lower antibiotic exposure, simulating the hospital and the community, respectively, and with a certain migration rate of insects between populations. In parallel, we present a computational model based on P-membrane computing that will mimic the experimental system of antibiotic resistance transmission. The proposal serves as a proof of concept for the development of more-complex population dynamics of antibiotic resistance transmission that are of interest in public health, which can help us evaluate procedures and design appropriate interventions in epidemiology.

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.

Citrus bacterial canker (CBC), or asiatic citrus canker, is a serious disease of most commercial citrus cultivars and some citrus relatives. The disease is widely distributed and endemic in many tropical and subtropical citrus-growing regions. CBC is now known to be caused by two different species, Xanthomonas citri subsp. citri (also known as X. axonopodis pv. citri) and Xanthomonas fuscans subsp. aurantifolia. The latter infects lime and lemon but not orange. CBC is spread mainly by wind and rain over short distances. Long-distance spread usually occurs by the movement of diseased propagating materials. Disease symptoms, management methods, and a protocol for pathogen detection are included in this chapter.

To molecularly and phenotypically characterize a selection of Agrobacterium‐like isolates from grapevine canes, crowns, soil and tumours in plants grown under cold conditions.

The genus Dickeya contains bacteria causing soft rot of economically important crops and ornamental plants. Here, we report the draft genome sequences of two Dickeya sp. isolates from rotted leaves of Phalaenopsis orchids.

The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

Erwinia piriflorinigrans is a new pathogenic species of the bacterial genus Erwinia that has been described recently in Spain. Accurate detection and identification of E. piriflorinigrans are challenging because its symptoms on pear blossoms are similar to those caused by Erwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detect E. piriflorinigrans and to differentiate it from E. amylovora and other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains of E. piriflorinigrans analyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of all E. piriflorinigrans strains tested, whereas 304 closely related pathogenic and nonpathogenic Erwinia strains and microbiota from pear trees were not amplified. In sensitivity assays, 10(3) cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 10(2) cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detecting E. piriflorinigrans in 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necrotic Pyracantha sp. blossoms, and in necrotic pear and apple tissues infected with both E. amylovora and E. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle of E. piriflorinigrans in different hosts and plant tissues and its interaction with E. amylovora.

Erwinia piriflorinigrans is a newly described pathogen causing necrosis of pear blossoms. Complete sequencing of the 37‐kb plasmid pEPIR37 common to 27 E. piriflorinigrans strains revealed homology to sequences of the ubiquitous plasmids pEA29 of the fire blight pathogen E. amylovora, plasmid pEP36 of E. pyrifoliae, plasmid pEJ30 of Erwinia sp. from Japan, and genomic regions of the related Rosaceae epiphytic Erwinia species E. tasmaniensis and E. billingiae. A second 5·5‐kb cryptic plasmid pEPIR5, found in 12 E. piriflorinigrans strains, was also sequenced revealing mobilization and replication proteins with similarities to many small ColE1‐type plasmids in Erwinia spp. and other enterobacteria. Functional analyses of pEPIR37 introduced into a strain of E. amylovora cured of pEA29 plasmid, which has a reduced virulence, showed a role in increasing symptom development similar to that observed in E. amylovora carrying plasmid pEA29.

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco.

Forty Erwinia amylovora strains isolated from the Middle Atlas Mountains in Morocco have been characterized using sequences from tandem repeat of variable numbers (VNTR) as chromosomal markers. Three VNTR sequences were identified and used for studying the genetic diversity of the strains using a PCR fingerprinting method. Thirty nine strains showed high genetic homogeneity, but one strain displayed a profile quite different. We were able to obtain reproducible and characteristic profiles in strains from remote geographical areas, and associated them with each of the primers. Comparative analyses of Moroccan strains with three reference strains from Spain, France and England, were performed. The results revealed some polymorphisms compared with the English and the French strains, but notable similarities were observed with a Spanish strain obtained from plants imported from Belgium. The developed methodology has demonstrated its usefulness for being applied in epidemiological studies.

The number of described pathogenic and non-pathogenic Erwinia species associated with pome fruit trees, especially pear trees, has increased in recent years, but updated comparative information about their similarities and differences is scarce. The causal agent of the fire blight disease of rosaceous plants, Erwinia amylovora, is the most studied species of this genus. Recently described species that are pathogenic to pear trees include Erwinia pyrifoliae in Korea and Japan, Erwinia spp. in Japan, and Erwinia piriflorinigrans in Spain. E. pyrifoliae causes symptoms that are indistinguishable from those of fire blight in Asian pear trees, Erwinia spp. from Japan cause black lesions on several cultivars of pear trees, and E. piriflorinigrans causes necrosis of only pear blossoms. All these novel species share some phenotypic and genetic characteristics with E. amylovora. Non-pathogenic Erwinia species are Erwinia billingiae and Erwinia tasmaniensis that have also been described on pome fruits; however, less information is available on these species. We present an updated review on the phenotypic and molecular characteristics, habitat, pathogenicity, and epidemiology of E. amylovora, E. pyrifoliae, Erwinia spp. from Japan, E. piriflorinigrans, E. billingiae, and E. tasmaniensis. In addition, the interaction of these species with pome fruit trees is discussed.

The genus Erwinia includes plant-associated pathogenic and non-pathogenic species. Among them, all species pathogenic to pome fruit trees (E. amylovora, E. pyrifoliae, E. piriflorinigrans, Erwinia sp. from Japan) cause similar symptoms, but differ in their degrees of aggressiveness, i.e. in symptoms, host range or both. The presence of plasmids of similar size, in the range of 30 kb, is a common characteristic that they possess. Besides, they share some genetic content with high homology in several genes associated with exopolysaccharide production and hence, with virulence, as well as in some other genes. Knowledge of the content of these plasmids and comparative genetic analyses may provide interesting new clues to understanding the origin and evolution of these pathogens and the level of symptoms they produce. Furthermore, genetic similarities observed among some of the plasmids (and genomes) from the above indicated pathogenic species and E. tasmaniensis or E. billingiae, which are epiphytic on the same hosts, may reveal associations that could expose the mechanisms of origin of pathogens. A summary of the current information on their plasmids and the relationships among them is presented here.

The purpose of this study was to assess the stability of mRNA and rRNA for evaluation of viability for Xanthomonas citri subsp. citri (Xcc). Total RNA from Xcc suspensions subjected to different stress treatments (high temperature or chemical treatment with sodium orthophenylphenate at different concentrations) was extracted at different time periods post‐treatment (0, 3, 24 and 48 h) and analysed by quantitative real‐time reverse transcription PCR (Q‐RT‐PCR). Primers were designed from selected fragments of rRNA and mRNA from genes involved in bacterial fitness, virulence or general metabolic mechanisms (gumD, rpfB, avrBs2 and gyrB). After stress treatment, only a 445‐bp fragment from the gumD mRNA was detected in live Xcc cells specifically, whereas other RNA fragments, as well as DNA targets, were detected in both viable and nonviable cells. Statistical analyses demonstrated that the amount of some transcripts from genes involved in xanthan synthesis, pathogenicity factor regulation and DNA processing was significantly reduced after lethal treatments. The amplification of the 445‐bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc; the product was detected specifically from viable bacteria on leaf and citrus fruit surfaces and in citrus canker lesions. Instability of long RNA fragments can be used as a practical tool for the study of survival of citrus canker bacteria or for diagnostic purposes when the presence of viable bacteria needs to be confirmed.

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.

Fire blight has a two-decade history in Bulgaria and intensive field observations and laboratory analyses have led to the collection of a representative collection strains of E. amylovora. Within the period 1989-2008, the pathogen was isolated from numerous plants of rosaceous species located throughout the whole country. In order to define the plasmid status of 324 strains from 11 hosts in 24 out of 27 districts, a duplex PCR protocol that recognizes pEA29 and pEI70 in a single assay was employed. The PCR test clearly showed that all strains tested carry the pEA29 plasmid but are negative for pEI70. These results, added to those obtained in other European countries, provide valuable information for large-scale epidemiological analysis of fire blight spread and for mapping of the pEI70 plasmid.

Eight Erwinia strains, isolated from necrotic pear blossoms in València, 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) (=CECT 7348(T)).

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.

We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor.