M. C. M. Perombelon

Two modified crystal violet pectate (CVP) media for the detection, isolation and enumeration of soft rot erwinias

SummaryTwo modifications of the selective-diagnostic crystal violet pectate (CVP) medium for soft rot erwinias were developed using a new formulation of sodium polypectate; these were a single layer medium and a double layer medium consisting of an agar base with a pectate overlayer. Both media contained 0.1% tryptone to promote growth and cavity formation by erwinias and 40 μg ml−1 novobiocin instead of sodium lauryl sulphate to increase recovery rate and selectivity. Colonies of the three erwinias commonly associated with potatoes formed characteristic deep cavities in the single layer modified CVP. Cavities in the double layer CVP although deep were smaller but remained distinct longer. Recovery rate and selectivity with both media were better than with the original CVP.

Modified crystal violet pectate medium (CVP) based on a new polypectate source (Slendid) for the detection and isolation of soft rot erwinias

SummaryThe selective-diagnostic crystal violet pectate (CVP) medium for the detection and isolation of soft rot erwinias was modified and improved to allow the use of a new source of sodium polypectate (Slendid type 440), as the previous polypectate (Bulmer) is no longer available. Two formulations were developed: CVP-S1 medium was less transparent but otherwise similar to the Bulmer polypectate-based CVP medium (CVP-B) except that NaOH was added and CaCl2 concentration reduced. CVP-S2 medium was prepared by mixing equal volumes of two double strength preparations containing 1) polypectate and NaOH, and 2) all other ingredients, both sterilised separately. Although erwinia cavity formation was slower, it showed a number of advantages over CVP-B and CVP-S1 media, thereby facilitating colony/cavity detection and enumeration. These included the absence of precipitate, greater firmness and a reduced risk of liquefaction in the presence of large erwinia numbers, and a reduction in the number of saprophytic bacteria.

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence.

SummaryThe blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi- mutants has allowed us to consider some factors that affect Eca virulence.

Evaluation of a PCR kit for the detection of Erwinia carotovora subsp. atroseptica on potato tubers

SummaryA PCR-based kit, ProbeliaTM, for the detection ofErwinia carotovora subsp.atroseptica (Eca) on potatoes was evaluated at five laboratories in four countries. The kit is based on DNA-specific PCR amplification followed by detection of amplicons by hybridization to a peroxidase-labelled DNA probe in a microplate. Specificity of the PCR primers for Eca, regardless of serogroups, was confirmed by testing against 246 bacterial, fungal and plant species. Detection limits of the assay varied little between six Eca strains in pure cultures (1.3×102 to 1.5×103 cells ml−1). When Eca-free tuber peel extract from four cultivars was inoculated with known numbers of 15 Eca strains, detection limits were more variable (1.0×101 to 6.2×103 cells ml−1 peel extract), attributed probably to inconsistency in the recovery of DNA during extraction. When the PCR assay was compared with three current commercial Eca detection methods, using naturally contaminated tubers, results matched most closely those from viable counts on a selective medium, the most sensitive method (88%), followed by enrichment ELISA (72%) and last ELISA (30%), the least sensitive method.

Evaluation of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora subsp. atroseptica

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Eca) from eight west European countries. These techniques were compared with other fingerprinting techniques, random amplified polymorphic DNA (RAPD) and Ouchterlony double diffusion (ODD), previously used to type this pathogen. Where possible, data were represented as dendrograms and groups/subgroups of strains identified. Simpsons index of diversity (Simpsons D) was used to compare groupings obtained with the different techniques which, with the exception of Biolog, gave values of 0·46 (RFLP), 0·39 (ERIC), 0·83 (phage typng), 0·82 (RAPD) and 0·26 (ODD). Of the techniques tested, phage typing showed the highest level of diversity within Eca, and this technique will now form the basis of studies into the epidemiology of blackleg disease.

Transposon mutagenesis of Erwinia using phage λ vectors

SummaryThe plasmid pHCP2 carries the lamB gene of E. coli. This plasmid was transferred to 4 strains of Erwinia using transformation or conjugal mobilisation. All 4 plasmid-containing strains became sensitive to λ infection. After infection with λ::Tn5 and the “higher-hopper” Tn10 derivatives, a variety of auxotrophic and enzyme-deficient mutants were isolated. Reversion analysis suggested the mutant phenotypes were due to the transposon.

Bacteriophage øP mediated generalized transduction in Erwinia carotovora subspecies carotovora

The bacteriophage oKP is capable of generalized transduction in Erwinia carotovora subspecies carotovora (Ecc) strains SCRI193 and ATCC 39048. oKP is a virulent phage containing double stranded DNA of approximately 46 kb. The frequencies of transduction were established for a number of chromosomal markers and plasmid pHCP2, and conditions for transduction optimized after exposure of the phage lysate to UV irradiation.

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 105 when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro‐colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100‐fold, or 10‐fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 101–102 cells ml−1 and with the latter ca 101 cells ml−1, when different numbers of streptomycin‐resistant Eca strain were added to peel extract from three Eca‐free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.

The resistance of potato plants toErwinia carotovora subsp.atroseptica in relation to their calcium and magnesium content

SummaryThe effect of adding gypsum (CaSO4) to growing media on the resistance toErwinia carotovora subsp.atroseptica (van Hall) Dye of potato (Solanum tuberosum L.) stems and mother and daughter tubers was examined in field and glasshouse experiments. The decayin situ of inoculated mother tubers from field plots treated before planting with gypsum was significantly delayed compared with tubers sampled from non-amended plots. Blackleg development was also significantly delayed in the gypsum-amended plots; however, for both mother tubers and stems the increased resistance associated with the application of gypsum later disappeared. Stem resistance tended to be related to tissue calcium and magnesium concentrations which were higher in plots treated with gypsum than in untreated plots. Daughter tubers produced in plots amended with gypsum generally contained a higher concentration of calcium in their cortex and were more resistant to decay byE. carotovora subsp.atroseptica but the effect was not consistent.

Effects of contamination byErwinia carotovora subsp.carotovora andE. carotovora subsp.atroseptica of potato seed tubers and of cultivar resistance on blanking or nonemergence and blackleg development in Valencia, Spain

SummaryHigh grade potato seed tubers of three cultivars were inoculated in Scotland by vacuum infiltration in suspensions ofErwinia carotovora subsp.carotovora and subsp.atroseptica in different combinations, and transported to Valencia, Spain. They were cut into two to three pieces and planted in January 1984, 1985 and 1986 in fields furrow irrigated before and after emergence. High incidences of blanking and blackleg were associated with seed inoculated with high numbers ofE.c. atroseptica alone or together withE.c. carotovora and not with high or low numbers ofE.c. carotovora alone.E.c. atroseptica predominated at emergence in March in rotting seed pieces which failed to produce a plant, regardless of the seed treatment, and in diseased stems in April but it was replaced byE.c. carotovora in May. The levels of blanking and blackleg after inoculating 16 cultivars with both erwinias were not correlated between years nor were they significantly different within years among most of the cultivars.