Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

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Potato virus M (PVM, Carlavirus) is considered to be one of the most common potato viruses distributed worldwide. Sequences of the coat protein (CP) gene of several Canadian PVM isolates were determined. Phylogenetic analysis indicated that all known PVM isolates fell into two distinct groups and the isolates from Canada and the US clustered in the same group. The Canadian PVM isolates could be further divided into two sub-groups. Two molecular procedures, reverse transcription - polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) were developed in this study for the detection and identification of PVM in potato tubers. RT-PCR was highly specific and only amplified PVM RNA from potato samples. PVM RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 dormant tubers. Restriction analysis of PCR amplicons with MscI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by RFLP analysis may be a useful approach for screening potato samples on a large scale for the presence of PVM.

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.). Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar‐specific region encoding pathogenicity‐related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium, Erwinia and Pantoea. Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant‐associated bacteria. Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.

Aims: To determine the reliable combination of protocols for specific detection and identification of R. solanacearum race 3 biovar 2 (R3bv2) through a comprehensive comparison among currently available techniques. Methods and Results: Sensitivity and specificity of the conventional isolation, bioassay, serological assays, conventional and real-time PCR and multiplex PCR were assessed for the detection of 25 strains of R. solanacearum biovars 1, 2 and 3 (Phylotypes I, II, III and IV) in spiked potato saps. Results indicated that all assays evaluated varied in complexity and sensitivity and should be applied strategically in indexing schemes to maximize efficiency of testing without compromising accuracy of the results. Conclusions: The TaqMan PCR assay, with an internal reaction control and confirmation by melting curve and electrophoretic analysis, achieved best sensitivity at 10 2 -10 3 CFU ml À1 for all eighteen strains of R. solanacearumR3bv2. Selective enrichment on mSMSA medium plates enhanced the detection sensitivity up to 10-100 CFU ml À1 for the conventional PCR-based assays.Significance and Impact of the Study: This is the first time nine different assays were compared side by side for their sensitivity and specificity in detection and identification of R. solanacearum R3bv2. The data accumulated here will provide basis for regulatory applications for low level detection and rapid identification of latently infections caused by R. solanacearum R3bv2.