Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species

High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants

Response of low and medium vigour rootstocks for peach to biotic and abiotic stresses

6 Pags., 6 Tabls. The definitive version is available at: http://www.actahort.org/index.htmNew low and medium vigour Prunus rootstocks compatible with peach cultivars were evaluated under controlled and in field conditions against biotic and abiotic stress factors. Most rootstocks exhibited different levels of resistance to the root-knot nematode Meloidogyne javanica. The plum hybrids ‘PAC 9801-02’ and ‘Krymsk®2’ were the only rootstocks that exhibited resistance to the lesion nematode, although ‘Krymsk®2’ is not compatible with peach. All rootstocks showed a high sensitivity to crown gall. The low vigour plums ‘Evrica’ and ‘Krymsk®1’, and the hybrid peach ‘PADAC 9907-23’ were sensitive to iron chlorosis. The rest showed different levels of tolerance. All plum-based materials were moderately tolerant to tolerant to root asphyxia, whereas peach-based rootstocks were sensitive with the exception of the peach almond hybrid ‘PAC 0009-01’. Most plums tend to emit an excessive number of root and crown suckers. From the agronomic standpoint, ‘PAC 0009-01’ and ‘PAC 9801-02’ present a better overall performance taking into account the combined traits.Peer reviewe

Identification of an Erwinia sp different from Erwinia amylovora and responsible for necrosis on pear blossoms

Necrotic pear blossoms (NPB) were found in several pear orchards of ‘Ercolini’ (‘Coscia’) and ‘Tendral’ in Turís, Valencia (Spain), in April 1999, during a routine survey, in an area free of Erwinia amylovora. The symptoms resembled those of fire blight [E. amylovora] but affected only blossoms and did not progress to other parts of the tree. Erwinia-like colonies were isolated from the necrotic blossoms that year and the following 2 years, and the morphology of the colonies on CCT medium of Ishimaru and Klos, King’s B medium, and sucrose nutrient agar was similar to that of Erwinia amylovora. The isolates were identified as an Erwinia sp. by their microbiological characteristics and showed API 20E, API 20NE, API ZYM, API 50CH patterns and fatty acid profiles similar, but not identical, to those of E. amylovora and Erwinia pyrifoliae. The isolates reacted as E. amylovora in immunofluorescence with several antisera and one monoclonal antibody (MAb) employed for E. amylovora detection, but did not react in the enzyme-linked immunosorbent assay against specific E. amylovora monoclonal antibodies. Polymerase chain reaction with primers from 23S rDNA sequences of E. amylovora were positive, but no signal was obtained with primers from plasmid pEa29 or from chromosomal DNA sequences of E. amylovora. The isolates were able to elicit the hypersensitive reaction on tobacco and tomato leaves and to induce necrosis in pear flowers, but were unable to develop typical fire blight symptoms in other organs of pear trees, and on various host plants of E. amylovora. The isolated Erwinia sp. strains are pathogenic and different from E. amylovora and other described bacterial species affecting pear trees

Chromosomal and Ti plasmid characterization of tumorigenic strains of three Agrobacterium species isolated from grapevine tumours

Fifty-six tumorigenic Spanish grapevine strains of Agrobacterium spp. were tested for biovar classification, pathogenicity on several hosts, opine utilization, 16S rRNA gene sequencing and PCR amplifications using five primer sets targeting chromosomal and Ti plasmid genes. Fifty of them belonged to A. vitis (biovar 3), three to A. tumefaciens (biovar 1) and three to A. rhizogenes (biovar 2). All strains were tumorigenic on grapevines. Most A. vitis strains were also pathogenic on tomato and tobacco plants, while the three A. tumefaciens strains were only pathogenic on grapevine. Although most A. vitis strains used octopine, 12 utilized neither octopine nor nopaline. 16S rRNA gene sequencing clearly distinguished between strains belonging to the three species. Those of A. vitis could be further divided into three chromosomal backgrounds according to their 16S ribosomal RNA gene sequences. No universal primer pair was found for the detection of all three Agrobacterium species isolated from grapevine. DNA from all A. vitis strains was amplified with the chromosomally-encoded pehA primer pair. In both A. vitis and A. tumefaciens a correlation was observed between the amplifications obtained using the tmr and the virA Ti-plasmid-targeting primer pairs. Three types of Ti plasmid were found in A. vitis strains according to their PCR amplifications and opine utilization profiles. A given chromosomal background harboured only one type of Ti plasmid within the strains from each analysed sample, showing a strong association between chromosomal backgrounds and Ti plasmids in A. viti