Whole genome comparisons provide a quantitative, objective basis for taxonomic classification of bacterial pathogens important to food security.

Dickeya species (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants world‐wide. Dickeya spp. (probably D. dianthicola) were first reported on potato in the Netherlands in the 1970s and have since been detected in many other European countries. However, since 2004–5 a new pathogen, with the proposed name ‘D. solani’, has been spreading across Europe via trade in seed tubers and is causing increasing economic losses. Although disease symptoms are often indistinguishable from those of the more established blackleg pathogen Pectobacterium spp., Dickeya spp. can initiate disease from lower inoculum levels, have a greater ability to spread through the plant’s vascular tissue, are considerably more aggressive, and have higher optimal temperatures for disease development (the latter potentially leading to increased disease problems as Europe’s climate warms). However, they also appear to be less hardy than Pectobacterium spp. in soil and other environments outside the plant. Scotland is currently the only country in Europe to enforce zero tolerance for Dickeya spp. in its potato crop in an attempt to keep its seed tuber industry free from disease. However, there are a number of other ways to control the disease, including seed tuber certification, on‐farm methods and the use of diagnostics. For diagnostics, new genomics‐based approaches are now being employed to develop D. dianthicola‐ and ‘D. solani’‐specific PCR‐based tests for rapid detection and identification. It is hoped that these diagnostics, together with other aspects of ongoing research, will provide invaluable tools and information for controlling this serious threat to potato production.

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5 nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >10 2 cells ml ؊1 was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.Ralstonia solanacearum (Smith) (30) is the agent of bacterial wilt, infecting over 450 plant species, including many economically important crops (12). This species has been subclassified into biovars based on biochemical tests and host-dependent races. Biovar 2A (equivalent to race 3) is adapted to temperate climates, has a narrow host range, and is responsible for recent outbreaks of potato brown rot disease in several countries of Western Europe and elsewhere worldwide (13,27). Although other biovars can also infect potatoes, biovar 2A is the most destructive phenotype in temperate areas.R. solanacearum is listed as a quarantine organism in the European Union (EU) (2), where new legislation has been introduced to control and eradicate the organism (3). Latent infections in seed potato tubers (6) have lead to the spread of the organism, both locally and internationally, and effective control of brown rot is dependent on the reliability of detection of the pathogen at this latent stage. For practical purposes, a detection assay is required which is rapid, specific, and sensitive to levels lower than those occurring in naturally infected potatoes and should be applicable to a crude sample of the specimen of interest (25). Serological techniques such as immunofluorescence (IF) microscopy, the enzyme-linked immunosorbent assay (ELISA) (10,15,21), and molecular techniques involving the PCR (9, 24) have been described for detection of R. solanacearum. An EU control directive (3) allows for a variety of detection methods to be employed. Briefly, a primary screening test (i.e., IF and/or selective isolation) is conducted with extracts from vascular tissue sampled from 200 tubers per 25-tonne lot. To confirm th...

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel‐end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 106–7.4 × 107 colony‐forming units per ml. Following concentration by high‐speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi‐selective media, enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescent‐antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre‐enrichment of samples in semi‐selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi‐selective medium and tomato bioassay could detect fewer than 104 cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.

Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya , previously the Pectobacterium chrysanthemi complex ( Erwinia chrysanthemi ), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi , showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya . The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA–DNA hybridization studies, showed that although related to Dickeya dadantii , these isolates represent a novel species within the genus Dickeya , for which the name Dickeya solani sp. nov. (type strain IPO 2222T = LMG25993T = NCPPB4479T) is proposed.