'Candidatus Liberibacter solanacearum' Associated with Bactericera trigonica-Affected Carrots in the Canary Islands.

In 2009 and 2010, commercial carrot (Daucus carota L.) fields located in Tenerife (Canary Islands, Spain) showed symptoms of curling, yellow, bronze, and purple discoloration of leaves, stunting of shoots and tap roots, and proliferation of secondary roots. A large population of the psyllid Bactericera trigonica was noted in those fields. Similar symptoms were reported previously in carrot-production areas of the Canary Islands and mainland Spain that were associated with stolbur and aster yellows (1997 and 1998) (2) and Spiroplasma citri and phytoplasmas (2009 and 2010) (1). These symptoms were also reported in southern Finland in 2008 and associated with 'Candidatus Liberibacter solanacerum' (4). Studies were conducted to investigate whether these pathogens and the psyllid B. trigonica were associated with the observed symptoms in carrot in Tenerife. A total of 18 petiole samples of symptomatic carrots were collected (13 samples in 2009 and 5 samples 2010). Five asymptomatic plants were also sampled. Three samples of psyllids (five individuals grouped) collected from one affected field in 2010 were also included in the assay. Total DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Valencia, CA), and analyzed by nested-PCR assays using primer pairs P1/P7 and R16F2n/R16R2n for phytoplasmas and ScR16F1/ScR16R1 followed by ScR16F1A/ScR16R2 for S. citri detection as described previously (3). PCR was performed using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, for 'Ca. L. solanacearum' (4). S. citri and phytoplasmas were not detected in any of the studied samples. However, a 1,168-bp 16S rDNA fragment and a 669-bp rplJ/rplL fragment were amplified from DNA from 16 symptomatic carrot samples and three psyllid grouped samples using specific primers for 'Ca. L. solanacearum'. No DNA was amplified from the asymptomatic samples. These results indicate the presence of 'Ca. L. solanacearum' in the affected carrot and psyllid samples collected in Tenerife (Canary Islands). Four and one PCR products obtained from DNA of carrot and psyllid samples, respectively, with both primer pairs were sequenced. BLAST analysis of the 16S rDNA sequences obtained from infected carrots (GenBank Accession Nos. HQ454312, HQ454313, HQ454314, and HQ454315) and psyllids (HQ454316) showed 99% identity to those of 'Ca. L. solanacearum' amplified from carrot in Finland (GU373049) and B. cockerelli (EU812557). The rplJ/rplL nucleotide sequences obtained from infected carrots (Accession Nos. HQ454317, HQ454318, HQ454319, and HQ454320) and psyllid (HQ454321) were 98% identical to the analogous rplJ/rplL 'Ca.L. solanacearum' ribosomal protein gene from carrot (GU373051) in Finland and tomato (EU834131) from New Zealand. To our knowledge, this is the first report of 'Ca. L. solanacearum' associated with psyllid-affected carrots in the Canary Islands (Tenerife, Spain) and also the first report of this plant pathogen associated with B. trigonica. References: (1) M. C. Cebrián et al. Plant Dis. 94:1264, 2010. (2) M. I. Font et al. Bol. San. Veg. Plagas 25:405, 1999. (3) I.-M. Lee et al. Plant Dis. 90:989, 2006. (4) J. E. Munyaneza et al. Plant Dis. 94:639, 2010.

Evaluation of the efficacy of immunomagnetic separation for the detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds.

AIMS: To evaluate the effectiveness of the optimized immunomagnetic separation (IMS)-plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed-testing laboratories. METHODS AND RESULTS: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS-plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS-plating allowed the detection of less than 10 CFU ml(-1) of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml(-1) in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 10(3) and up to 10(4) CFU ml(-1) were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 10(3) CFU ml(-1) or more. CONCLUSIONS: Among the evaluated methods, IMS-plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: IMS-plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.

Isolation and characterization of 21 microsatellite markers in the barn owl (Tyto alba).

We report 21 new polymorphic microsatellite markers in the European barn owl (Tyto alba). The polymorphism of the reported markers was evaluated in a population situated in western Switzerland and in another from Tenerife, Canary Islands. The number of alleles per locus varies between two and 31, and expected heterozygosity per population ranges from 0.16 to 0.95. All loci are in Hardy-Weinberg equilibrium and no linkage disequilibrium was detected. Two loci exhibit a null allele in the Tenerife population.

Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation.

The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic separation (IMS), based on the use of immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10(6)IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10(3) to 10(0)CFU/ml. Threshold detection was lower than 10CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.

Taxonomy of Pseudomonas strains isolated from tomato pith necrosis: emended description of Pseudomonas corrugata and proposal of three unnamed fluorescent Pseudomonas genomospecies.

Thirty-three fluorescent Pseudomonas strains isolated from tomato pith necrosis (FPTPN strains) and 89 Pseudomonas corrugata strains were studied by numerical taxonomy. In the dendrogram of distances, the P. corrugata strains constituted a single phenon (phenon 1), whereas 17 of the 33 FPTPN strains clustered in a separate phenon (phenon 2). The other 16 FPTPN strains were included in phena consisting of well-characterized fluorescent Pseudomonas species or were isolated phenotypes. Phena 1 and 2 were distinguished by fluorescence on King B medium, accumulation of poly-beta-hydroxybutyrate, production of levan, and assimilation of sorbitol. DNA-DNA hybridization showed that P. corrugata is a true genomic species (66 to 100% DNA relatedness) and that the FPTPN strains of phenon 2 were divided into three genomic groups. Genomic groups 1 and 2 were not distinct from each other phenotypically, and genomic group 3 could be distinguished from genomic groups 1 and 2 only on the basis of assimilation of citraconate and laevulinate. Genomic groups 1 and 2 are related to P. corrugata (40 to 55% DNA relatedness), whereas genomic group 3 is less closely related to P. corrugata (20 to 23% DNA relatedness). The lipopolysaccharide patterns on electrophoresis gels and fatty acid profiles of strains belonging to genomic group 1 through 3 are different from each other and from the lipopolysaccharide patterns and fatty acid profiles of P. corrugata. However, cross-reactions were observed between P. corrugata and the FPTPN strain genomic groups, indicating that there are common epitopes of the lipopolysaccharides. The three FPTPN strain genomic groups were not named as species but were designated Pseudomonas genomospecies FP1, FP2, and FP3.

Lipopolysaccharides as Determinants of Serological Variability in Pseudomonas corrugata.

The variation in biochemical and serological features of 128 isolates of Pseudomonas corrugata has been studied with 56 isolates from Spain and 72 isolates from other countries. Isolates were analyzed with common diagnostic tests and with the AP150CHE system. Variability among isolates for some standard tests usually listed as positive or negative for this species, such as arginine dihydrolase and gelatin hydrolysis, lipase and lecithinase activities, pigment production, and wrinkled colony morphology, was observed. Three antisera were raised against the type strain and two Spanish isolates from tomato and pepper plants. Serological reactions were studied by indirect immunofluorescence and indirect enzyme-linked immunosorbent assay. Eighty-three isolates reacted with a single antiserum, 6 reacted with two antisera, and none reacted with three antisera. Thirty-nine isolates did not react with any of the three antisera. These results suggest that serology will not be a useful method for routine diagnosis of P. corrugata unless common antigens can be identified. Electrophoresis and immunoelectrotransfer were used to study the antigens involved. Each antiserum reacted with whole-cell lysates, giving two common bands for P. corrugata isolates and other Pseudomonas species and a ladder-like pattern characteristic of lipopolysaccharides (LPS). Common bands were not observed after proteinase K treatment. More than 10 LPS patterns were distinguished in 98 isolates after silver staining of polyacrylamide gels. There was no correlation between the geographical origin or host of the isolates and the LPS patterns. A correlation between LPS groups and serological reaction was observed.