Xanthomonas arboricola pv. juglandis and pv. corylina: Brothers or distant relatives? : genetic clues, epidemiology, and insights for disease management

Background: The species Xanthomonas arboricola comprises up to nine pathovars, two of which affect nut crops: pv. juglandis, the causal agent of walnut bacterial blight, brown apical necrosis, and the vertical oozing canker of Persian (English) walnut; and pv. corylina, the causal agent of the bacterial blight of hazelnut. Both pathovars share a complex population structure, represented by different clusters and several clades. Here we describe our current understanding of symptomatology, population dynamics, epidemiology, and disease control. Taxonomic status: Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Lysobacterales (earlier synonym of Xanthomonadales); Family Lysobacteraceae (earlier synonym of Xanthomonadaceae); Genus Xanthomonas; Species X. arboricola; Pathovars: pv. juglandis and pv. corylina. Host range and symptoms: The host range of each pathovar is not limited to a single species, but each infects mainly one plant species: Juglans regia (X. arboricola pv. juglandis) and Corylus avellana (X. arboricola. pv. corylina). Walnut bacterial blight is characterized by lesions on leaves and fruits, and cankers on twigs, branches, and trunks; brown apical necrosis symptoms consist of apical necrosis originating at the stigmatic end of the fruit. A peculiar symptom, the vertical oozing canker developing along the trunk, is elicited by a particular genetic lineage of the bacterium. Symptoms of hazelnut bacterial blight are visible on leaves and fruits as necrotic lesions, and on woody parts as cankers. A remarkable difference is that affected walnuts drop abundantly, whereas hazelnuts with symptoms do not. Distribution: Bacterial blight of walnut has a worldwide distribution, wherever Persian (English) walnut is cultivated; the bacterial blight of hazelnut has a more limited distribution, although disease outbreaks are currently more frequently reported. X. arboricola pv. juglandis is regulated almost nowhere, whereas X. arboricola pv. corylina is regulated in most European and Mediterranean Plant Protection Organization (EPPO) countries. Epidemiology and control: For both pathogens infected nursery material is the main pathway for their introduction and spread into newly cultivated areas; additionally, infected nursery material is the source of primary inoculum. X. arboricola pv. juglandis is also disseminated through pollen. Disease control is achieved through the phytosanitary certification of nursery material (hazelnut), although approved certification schemes are not currently available. Once the disease is present in walnut/hazelnut groves, copper compounds are widely used, mostly in association with dithiocarbamates; where allowed, antibiotics (preferably kasugamycin) are sprayed. The emergence of strains highly resistant to copper currently represents the major threat for effective management of the bacterial blight of walnut

Application of PCR-RFLP of 16S rDNA to study the symbionts of tropical EPNs

International audienceTo learn more about the diversity in the Caribbean basin of Xenorhabdus and Photorhabdus in relation with their nematode host and their environment, an extensive survey was conducted in this area. A total of 77 Caribbean isolates, recovered from 72 Heterorhabditis and 5 Steinernema, were studied. ln order to type faster these numerous bacterial isolates, a method previously assessed with reference strains (see Brunel et al., in this book), Restriction Fragment Length Polymorphism analysis of PCR-amplified 16S rRNA genes, was used. The ribosomal genotypic patterns were compared to those of 40 reference strains. Most of the EPN isolates had been previously identified and ecological data about the samples origin were available. The five Xenorhabdus isolates exhibited a high degree of genetic polymorphism and only one was genotypically identical to a reference strain. ln contrast, very little variability was evidenced within the 72 Photorhabdus isolates, as only four closely related genotypes, over the 12 already identified, were detected. When we compared these results with geographical and ecological data, no correlation could be drawn, excepted those directly associated to the environmental habitat of host nematodes. ln fact, a closely relatedness between bacterial genotypes and nematode species was found. This result was particularly obvious within the Photorhabdus-Heterorhabditis couple where two bacterial genotypes were exclusively associated to H. indica and two others to H. bacteriophora. Within the Xenorhabdus-Steinernema association, new symbiont genotypes corresponded to new nematode species. The availability on one hand of a fast molecular identification of bacterial symbionts, and on the other hand the characterization with satellite DNA probes of most EPNs, allowed to compare their respective taxonomie structure on a large scale. Thereby, a congruenee was evidenced which strongly supports an early co-evolution of the symbiotic partners of both couples

Identification of Xenorhabdus and Photorhabdus, bacteria associated with tropical entomopathogenic nematodes by amplified ribosoma DNA restriction analysis

National audienc

Detection of Cytotoxin-Hemolysin mRNA in Nonculturable Populations of Environmental and Clinical Vibrio vulnificus Strains in Artificial Seawater

The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus. For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V. vulnificus and targeted the cytotoxin-hemolysin virulence gene vvhA. Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study. Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4°C. VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively. Our data indicate that seminested RT-PCR is sensitive for the detection of vvhA mRNA in artificial seawater when exclusively nonculturable bacteria are present. This is the first report of the expression of a toxin gene in VBNC V. vulnificus. Moreover, vvhA transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time. This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk

PCR-Ribotyping of Xenorhabdus and Photorhabdus isolates from the Caribbean region in relation to the taxonomy and geographic distribution of their nematode hosts

International audienceThe genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes

Identification of Genomic Species in Agrobacterium Biovar 1 by AFLP Genomic Markers

Biovar 1 of the genus Agrobacterium consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as Agrobacterium ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of Agrobacterium tumefaciens), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely “species-specific” markers tagging the core genomes of the species

Deinococcus deserti sp. nov., a gamma-radiation-tolerant bacterium isolated from the Sahara Desert

International audienceTwo gamma-and UV-radiation-tolerant, Gram-negative, rod-shaped bacterial strains, VCD115 T and VCD117, were isolated from a mixture of sand samples collected in the Sahara Desert in Morocco and Tunisia, after exposure of the sand to 15 kGy gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequences and DNA–DNA hybridizations showed that VCD115 T and VCD117 are members of a novel species belonging to the genus Deinococcus, with Deinococcus grandis as its closest relative. The DNA G+C contents of VCD115 T and VCD117 are 59?8 and 60?6 mol%, respectively. The major fatty acids (straight-chain 15 : 1, 16 : 1, 17 : 1 and 16 : 0), polar lipids (dominated by phosphoglycolipids and glycolipids) and quinone type (MK-8) support the affiliation to the genus Deinococcus. The strains did not grow on rich medium such as trypticase soy broth (TSB), but did grow as whitish colonies on tenfold-diluted TSB. The genotypic and phenotypic properties allowed differentiation of VCD115 T and VCD117 from recognized Deinococcus species. Strains VCD115 T and VCD117 are therefore identified as representing a novel species, for which the name Deinococcus deserti sp. nov. is proposed, with the type strain VCD115 T (=DSM 17065 T =LMG 22923 T)

Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species

Xylophilus ampelinus is the causal agent of blight and canker on grapevine. Only a few data are available on this species implying that the occurrence of this pathogen may be underestimated, and its actual ecological niche may not be understood. Moreover, its genetic diversity is not well known. To improve our knowledge of this species, an analysis of the complete genome sequences available in NCBI was performed. It appeared that several sequences are misidentified. The complete genome sequence of the type strain was obtained and primers designed in order to sequence gyrB and rpoD genes for the strains held in CIRM-CFBP. The genetic barcoding data were obtained for 93 strains, isolated over 35 years and from several geographical origins. The species revealed to be strongly homogenous, displaying nearly identical sequences for all strains. However, the oldest strains of this collection were isolated in 2001 therefore, a new isolation campaign and epidemiological surveys are necessary, along with the obtention of new complete genome sequences for this species