L. J. Ward

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil.

Aims:  To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia.

Pectobacterium spp. Associated with Bacterial Stem Rot Syndrome of Potato in Canada

Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.

Molecular characterization of Canadian populations of potato cyst nematodes, Globodera rostochiensis and G. pallida using ribosomal nuclear RNA and cytochrome b genes

Abstract The mitochondrial cytochrome b gene (cytb), the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rRNA gene and D2-D3 expansion segments of the 28S rRNA gene were amplified, sequenced and used to characterize several populations of potato cyst nematodes, Globodera pallida and G. rostochiensis, collected from different areas in Canada. Diagnostic PCR-ITS-RFLP profiles with three restriction enzymes are provided for identification of both species. Sequences of ITS rRNA and cytb genes were compared with those in Genbank of other potato cyst nematode populations originating from Europe, South America, USA, Australia and New Zealand. The ITS rRNA sequences of Canadian G. rostochiensis were similar to those of all previously sequenced populations of this species. Sequence divergence of ITS rRNA for G. rostochiensis varied from 0 to 1.6%, whereas for G. pallida sequence divergence among populations reached 1.95%. Sequence and phylogenetic analysis of cytb and ITS rRNA genes using Bayesian inference revealed that Canadian G. pallida is almost identical to European and USA populations and formed a large clade with all these populations on the phylogenetic trees. The present molecular result with cytb confirmed the hypothesis that there are possibly several sibling species within G. pallida. Our study also supports a previously proposed hypothesis regarding introduction of G. pallida from a restricted area in Peru, firstly into Europe with subsequent spread to other continents including North America. Our sequence analysis revealed that several newly obtained sequences cannot be translated into functional cytb protein, because point indels disrupt the reading frame. Poly(T) variation in mtDNA genes in G. pallida might be explained by post-transcriptional editing mechanisms in Globodera mitochondria as well as by errors during PCR amplification of mononucleotide repeats within these genes.

Comparative genomics‐guided loop‐mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop‐mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean (Phaseolus vulgaris L.).

Hsp90 gene, an additional target for discrimination between the potato cyst nematodes, Globodera rostochiensis and G. pallida, and the related species, G. tabacum tabacum

The heat-shock gene, Hsp90, was targeted as a new variable genomic region to supplement other DNA-based tests for identification and discrimination of Globodera pallida, G. rostochiensis and G. tabacum tabacum. Populations of the potato cyst nematodes, G. pallida and G. rostochiensis (PCN), originating from Canada, France, Belgium and USA, together with two populations of G. tabacum tabacum from the USA and France were used for the amplification of a fragment of the Hsp90 gene. General and specific primers and probes for each species were derived from the consensus and non-consensus regions of the aligned sequences, respectively. A triplex conventional PCR assay, using a general forward and reverse or three specific reverse primers, as well as a real-time PCR using general primers and specific TaqMan probes, were developed. Melting curve analysis and restriction fragment polymorphisms using high resolution electrophoresis were explored for identifying PCR amplicons that characterized and discriminated the three Globodera species in both pure and mixed samples. Results from the different molecular assay strategies confirmed the usefulness of Hsp90 as a new additional gene target and showed that several different test options could be used for discrimination of PCN.

Molecular detection of Clavibacter michiganenis ssp. insidiosus in alfalfa seed

Bacterial wilt of alfalfa (Medicago sativa), caused by Clavibacter michiganensis ssp. insidiosus (Cmi), is a serious disease that spreads to new production areas mainly by infected seed. Hence, detection of Cmi in seedlots is an important disease mitigation measure. A conventional polymerase chain reaction (PCR) assay targeting the internal transcribed spacer region of the rrn operon and a real-time Taqman PCR assay based on published primers targeting a genomic insertion element were evaluated for detection and identification of Cmi. Detection of Cmi in alfalfa seed was facilitated by incubation of seed samples in a nutrient medium that promoted growth of Cmi followed by extraction of DNA from the bacterial fraction for use as template in PCR reactions. A real-time Taqman BIO-PCR assay and subsequent confirmation of amplicon identity by melting peak analysis made possible by addition of EvaGreen™ to the reaction mix, was deemed most useful for routine analysis in a diagnostic laboratory setting. The potential of confirming the presence of Cmi in PCR-positive samples by bioassay on alfalfa seedlings was also explored and involved verification of Cmi infections in root-inoculated seedlings by the conventional and real-time PCR assays. Key words: lucerne, Medicago sativa, seed testing, PCR, Taqman assay, melting point analysis. Le flétrissement bactérien de la luzerne (Medicago sativa), causé par Clavibacter michiganenis ssp. insidiosus (Cmi), est une maladie grave qui se répand dans les nouvelles superficies en production par l’intermédiaire de semences infectées. Par conséquent, la détection de Cmi dans les lots de semences est une mesure d’atténuation importante de la maladie. Afin de détecter et d’identifier l’agent pathogène, une PCR traditionnelle, visant la région de l’espaceur intergénique de l’opéron rrn, et une autre de type Taqman en temps réel, basée sur des amorces publiées et visant un élément d’insertion génomique, ont été évaluées. La détection de Cmi dans les semences de luzerne a été facilitée par l’incubation d’échantillons de graines dans un milieu nutritif favorisant la croissance de Cmi, suivie de l’extraction de l’ADN de la fraction bactérienne afin de l’utiliser comme matrice pour les réactions de la PCR. Une épreuve biologique de type Taqman en temps réel, ainsi que la confirmation subséquente de l’identité de l’amplicon par l’analyse des points de dissociation rendue possible par l’ajout d’EvaGreen au mélange, s’est avérée des plus utiles lors d’analyses de routine effectuées au laboratoire de diagnostic. La possibilité de confirmer la présence de Cmi dans les échantillons positifs à la PCR lors d’épreuves biologiques sur des plantules de luzerne a également été explorée et a nécessité la vérification, à l’aide des PCR traditionnelle et en temps réel, de la présence d’infection par Cmi chez les plantules dont les racines avaient été inoculées. Mots-clés : luzerne, Medicago sativa, évaluation des semences, PCR, épreuve Taqman, analyse des points de dissociation.

Persistence of Clavibacter michiganensis subsp. sepedonicus in Sterilized Soil but Failure to Confirm It’s Survival Overwinter in Field Soil

Bacterial ring rot is caused by Clavibacter michiganensis subsp. sepedonicus (Cms). This gram-positive coryneform pathogen survives well in potato tubers during storage providing the major source of inoculum each growing season (1). However the bacterium grows relatively slowly on artificial media and is easily out competed by other microorganisms commonly found in environmental samples. Although it does not survive well in soil, it is not known whether it may persist at low levels. In this study we investigated the persistence of Cms in sterilized soil and naturally infected field soil using both serological and nucleic acid based technologies for detection.