Reclassification of Xanthomonas gardneri (ex Sutic 1957) Jones et al. 2006 as a later heterotypic synonym of Xanthomonas cynarae Trébaol et al. 2000 and description of X. cynarae pv. cynarae and X. cynarae pv. gardneri based on whole genome analyses.

Multilocus sequence analysis of Xanthomonas species revealed a very close relationship between Xanthomonas cynarae, an artichoke pathogen and Xanthomonas gardneri, a tomato and pepper pathogen. Results of whole genome sequence comparisons using average nucleotide identity between representative strains of X. gardneri and X. cynarae were well above the threshold of 95-96 %. Inoculations of X. gardneri strains in artichoke leaves caused mild disease symptoms, but only weak symptoms were observed in the bracts. Both X. cynarae and X. gardneri grew equally and caused typical bacterial spot symptoms in pepper after artificial inoculation. However, X. cynarae induced a hypersensitive reaction in tomato, while X. gardneri strains were virulent. Pathogenicity-associated gene clusters, including the protein secretion systems, type III effector profiles, and lipopolysaccharide cluster were nearly identical between the two species. Based on our results from whole genome sequence comparison, X. gardneri and X. cynarae belong to the same species. The name X. cynarae has priority and X. gardneri should be considered as a later heterotypic synonym. An emended description of X. cynarae (type strain=CFBP 4188T, =DSM 16794T) is given. However, due to the host specificity in artichoke and tomato, two pathovars, X. cynarae pv. cynarae and X. cynarae pv. gardneri, are proposed.

High-Quality Draft Genome Sequences of Two Xanthomonas Pathotype Strains Infecting Aroid Plants.

We present here the draft genome sequences of bacterial pathogens of the Araceae family, Xanthomonas axonopodis pv. dieffenbachiae LMG 695 and Xanthomonas campestris pv. syngonii LMG 9055, differing in host range. A comparison between genome sequences will help understand the mechanisms involved in tissue specificity and adaptation to host plants.

Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis.

The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level.

Draft Genome Sequence of the Flagellated Xanthomonas fuscans subsp. fuscans Strain CFBP 4884.

We report the draft genome sequence of the flagellated strain CFBP 4884 of Xanthomonas fuscans subsp. fuscans, which was isolated in an outbreak of common bacterial blight of beans along with non-flagellated strains. Comparative genomics will allow one to decipher the genomic diversity of strains cohabiting in epidemics.

Draft Genome Sequence of Xanthomonas axonopodis pv. allii Strain CFBP 6369.

We report here the draft genome sequence of Xanthomonas axonopodis pv. allii strain CFBP 6369, the causal agent of bacterial blight of onion. The draft genome has a size of 5,425,942 bp and a G+C content of 64.4%.

High-Quality Draft Genome Sequence of Xanthomonas alfalfae subsp. alfalfae Strain CFBP 3836.

We report the high-quality draft genome sequence of Xanthomonas alfalfae subsp. alfalfae strain CFBP 3836, the causal agent of bacterial leaf and stem spot in lucerne (Medicago sativa). Comparative genomics will help to decipher the mechanisms provoking disease and triggering the defense responses of this pathogen of the model legume Medicago truncatula.

High-Quality Draft Genome Sequences of Xanthomonas axonopodis pv. glycines Strains CFBP 2526 and CFBP 7119.

We report here the high-quality draft genome sequences of two strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule on soybeans. Comparison of these genomes with those of phylogenetically closely related pathovars of Xanthomonas spp. will help to understand the mechanisms involved in host specificity and adaptation to host plants.

A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli.

In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred.

Adhesion and fitness in the bean phyllosphere and transmission to seed of Xanthomonas fuscans subsp. fuscans.

Deciphering the mechanisms enabling plant-pathogenic bacteria to disperse, colonize, and survive on their hosts provides the necessary basis to set up new control methods. We evaluated the role of bacterial attachment and biofilm formation in host colonization processes for Xanthomonas fuscans subsp. fuscans on its host. This bacterium is responsible for the common bacterial blight of bean (Phaseolus vulgaris), a seedborne disease. The five adhesin genes (pilA, fhab, xadA1, xadA2, and yapH) identified in X. fuscans subsp. fuscans CFBP4834-R strain were mutated. All mutants were altered in their abilities to adhere to polypropylene or seed. PilA was involved in adhesion and transmission to seed, and mutation of pilA led to lower pathogenicity on bean. YapH was required for adhesion to seed, leaves, and abiotic surfaces but not for in planta transmission to seed or aggressiveness on leaves. Transmission to seed through floral structures did not require any of the known adhesins. Conversely, all mutants tested, except in yapH, were altered in their vascular transmission to seed. In conclusion, we showed that adhesins are implicated in the various processes leading to host phyllosphere colonization and transmission to seed by plant-pathogenic bacteria.

Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

The Type III secretion system of Xanthomonas fuscans subsp. fuscans is involved in the phyllosphere colonization process and in transmission to seeds of susceptible beans.

Understanding the survival, multiplication, and transmission to seeds of plant pathogenic bacteria is central to study their pathogenesis. We hypothesized that the type III secretion system (T3SS), encoded by hrp genes, could have a role in host colonization by plant pathogenic bacteria. The seed-borne pathogen Xanthomonas fuscans subsp. fuscans causes common bacterial blight of bean (Phaseolus vulgaris). Directed mutagenesis in strain CFBP4834-R of X. fuscans subsp. fuscans and bacterial population density monitoring on bean leaves showed that strains with mutations in the hrp regulatory genes, hrpG and hrpX, were impaired in their phyllospheric growth, as in the null interaction with Escherichia coli C600 and bean. In the compatible interaction, CFBP4834-R reached high phyllospheric population densities and was transmitted to seeds at high frequencies with high densities. Strains with mutations in structural hrp genes maintained the same constant epiphytic population densities (1 x 10(5) CFU g(-1) of fresh weight) as in the incompatible interaction with Xanthomonas campestris pv. campestris ATCC 33913 and the bean. Low frequencies of transmission to seeds and low bacterial concentrations were recorded for CFBP4834-R hrp mutants and for ATCC 33913, whereas E. coli C600 was not transmitted. Moreover, unlike the wild-type strain, strains with mutations in hrp genes were not transmitted to seeds by vascular pathway. Transmission to seeds by floral structures remained possible for both. This study revealed the involvement of the X. fuscans subsp. fuscans T3SS in phyllospheric multiplication and systemic colonization of bean, leading to transmission to seeds. Our findings suggest a major contribution of hrp regulatory genes in host colonization processes.

Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans.

The occurrence of "Xanthomonas axonopodis pv. phaseoli var. fuscans" (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10(5) CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.

Comparison of the phenotypes and genotypes of biofilm and solitary epiphytic bacterial populations on broad-leaved endive.

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.

Pushing the envelope in renal preservation; improved results with novel perfusate modifications for pulsatile machine perfusion of cadaver kidneys.

INTRODUCTION: Novel preservation techniques may diminish ischemia/reperfusion (I/R) injury. Our preservation laboratory has modified Belzer MPS for machine perfusion (MP) with prostaglandin E1 (PGE 1), nitroglycerin (NTG), and polyethylene glycol-superoxide dismutase (PEG-SOD) to attenuate I/R injury. We reviewed our recent experience using this novel formulation (NF) compared with standard perfusates. RESULTS: Between January 1998 and March 2000, 1060 consecutive kidneys were preserved in our laboratory. One hundred forty-eight kidneys (14%) were discarded. Fifty-eight percent of kidneys during this time period underwent MP (n = 532). En bloc kidney pairs were randomly assigned to pulsatile MP using Waters RM3 or MOX-100 perfusion systems using 1 of 3 perfusates; NF (NF; n = 119), Belzer MPS (MPS; n = 201), or Belzer II albumin gluconate (ALB; n = 212) Significant improvements in delayed graft function (DGF) rate were seen with NF versus other perfusates (8% vs 14% vs 19%, respectively; P =.03). At 6 months, graft survival was significantly improved with NF compared with MPS and ALB (96% vs 90% vs 87%, respectively; P =.03). NF also produced a significantly higher percentage of recipients with a serum creatinine level < or = 1.5 mg/dL. CONCLUSIONS: Novel modifications of standard MP perfusate improved outcomes after renal transplantation. Preservation-based interventions targeted to ameliorate I/R injury can improve outcomes and may allow expansion of the donor pool.

The influence of pulsatile preservation on renal transplantation in the 1990s.

BACKGROUND: Unlike simple cold storage (CS), pulsatile machine preservation (MP) of kidneys for transplantation permits pharmacologic manipulation of the perfusate and aids in the pretransplant assessment of the kidney graft. These characteristics of MP may have importance in the era of increasing use of extended criteria donor kidneys. The overall aim of this article is to critically assess practices at our preservation unit with respect to graft function. Specific aims are to (1) compare the influence of MP versus CS on graft function, (2) determine which pretransplant variables have significance in pretransplant assessment, and (3) determine whether pharmacologic manipulation during MP is advantageous. METHODS: There were 650 consecutive kidneys preserved in our laboratory between January 1, 1993 and March 1, 999, by either MP or CS. All MP kidneys were preserved by continuous hypothermic pulsatile perfusion using Belzer-MPS or Belzer II solution. Perfusion parameters and electrolytes were measured serially during pulsatile perfusion. All CS kidneys were stored in University of Wisconsin solution. All kidneys obtained from donors exhibiting extended criteria features underwent pretransplant frozen section biopsies. Transmission electron microscopy (EM) was performed on a subset of kidneys undergoing pharmacologic manipulation. Four agents were assessed prospectively for their ability to influence MP characteristics when added to perfusate: PGE1, trifluoperazine, verapamil, and papaverine. RESULTS: MP was associated with improved immediate, 1-, and 2-year graft function and reduced length of initial hospital stay when compared with CS grafts. Changes in the machine perfusion variables flow and resistance, and the [Ca++] in perfusate, were significantly associated with delayed graft function (DGF) after the transplant. Biopsy information was not predictive of DGF. The addition of PGE1 to perfusate improved MP characteristics, reduced the release of [Ca++] into perfusate, and ameliorated mitochondrial ischemic injury in transmission EM images. Early graft function was improved in the presence of PGE1+MP, compared with function in the presence of other pharmacologic agents or CS alone. CONCLUSIONS: MP is associated with improved early and long term renal function. Moreover, PGE1 augments MP in improving graft function. The combination of MP+PGE1 may be important in optimizing the ability to use extended donor criteria kidneys and, thereby, improve the overall efficiency of cadaveric renal transplantation.