Crystal Violet Pectate Research Articles

Detection and enumeration of Erwinia carotovora subsp. atroseptica using spiral plating and immunofluorescence colony staining

Immunofluorescence colony staining (IFC) and a new technique using spiral plating combined with IFC were evaluated for the soft-rot pathogen Erwinia carotovora subsp. atroseptica in witloof chicory. Target bacteria could be detected in platings at various dilutions of plant washings. Brilliance of the stained colonies of E. carotovora subsp. atroseptica was high. Spiral plating, used for both the plating of the bacteria and for the delivery of the conjugated antiserum, had a positive effect on the reduction of the background compared with the staining of the bacteria. The combination of spiral plating and IFC proved to be a functional tool for the quantification of target and nontarget bacteria and the isolation of target bacteria as pure culture from IFC-positive colonies. The method uses less conjugated antiserum than traditional IFC and produces results with very small variation within replications. The recovery of the bacteria in both pure culture and plant washing is significantly higher than the recovery using crystal violet pectate medium.Key words: soft rot, witloof chicory, detection, Erwinia carotovora subsp. atroseptica.

Immunomagnetic separation of Erwinia carotovora subsp. atroseptica from potato peel extracts to improve detection sensitivity on a crystal violet pectate medium or by PCR

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw. carotovora subsp. atroseptica was used in combination with a crystal violet pectate (CVP) medium supplemented with 100 micrograms ml-1 of streptomycin to determine the recovery level of the target bacterium. Recovery obtained with a polyclonal antiserum against Erw. carotovora subsp. atroseptica at a concentration of 6 micrograms IgG ml-1 was greater than that obtained with two monoclonal antibodies against lipopolysaccharides of Erw. carotovora subsp. atroseptica at a concentration of 10 micrograms IgG ml-1. A linear relationship was found between particle concentration ranging from 12 to 200 micrograms ml-1 and recovery level. When the Advanced Magnetics (AM) protein A and anti-rabbit IgG particles in the AM separation system and the Dynal anti-rabbit IgG particles in the Dynal separation system were examined, the highest recovery level per microgram of particles (66%) was obtained with the Advanced Magnetics protein A particles, followed by AM anti-rabbit particles (37%). Without IMS, detection of Erw. carotovora subsp. atroseptica in tuber peel extracts on a CVP-medium without streptomycin was impossible when the ratio of Erw. carotovora subsp. carotovora to Erw. carotovora subsp. atroseptica was greater than 100 or when large numbers of other saprophytic bacteria were present, because of overcrowding. IMS, using the AM anti-rabbit IgG particles, ensured that Erw. carotovora subsp. atroseptica could be enumerated in tuber peel extract consistently, to a detection level of 100 cells ml-1. Similarly, the IMS procedure lowered the detection level of Erw. carotovora subsp. atroseptica in a twofold diluted peel extract by PCR to ca 2.0 x 10(3) cells ml-1 or 50 cells per reaction tube. In contrast, positive results in PCR without IMS were obtained only when the peel extract was diluted 100 times and when the concentration of Erw. carotovora subsp. atroseptica was at least 10(5) cell ml-1.

Blackleg potential of potato seed: determination of tuber contamination by Erwinia carotovora subsp. Atroseptica by immunofluorescence colony staining and stock and tuber sampling

SummaryTwo methods to determine numbers of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, in tuber peel extract were compared; (1) growth and cavity formation on crystal violet pectate (CVP) medium (Pérombelon, Lumb & Hyman, 1987) and (2) immunofluorescent colony (IFC) staining (Van Vuurde & Roozen, 1990) using an antiserum against the bacterium conjugated with fluorescein isothiocyanate. Detection, identification and quantification of the bacterium based on the differential effect of temperature on growth in the CVP method were severely restricted and in some cases could not be done at low peel extract dilutions containing > 106 saprophytic bacteria ml“1 and > 103 cells ml‐1 of E. carotovora subsp. carotovora. In contrast, although recovery was c. 50% relative to growth of E.c. atroseptica alone on nutrient agar, numbers of the bacteria could be determined by the IFC method regardless of numbers of saprophytic bacteria and E.c. carotovora present. Moreover, the tedium of counting colonies on a UV microscope could be avoided by automation using an imaging system on photograph film negatives of the microscope fields.Readily accessible tubers from the top layer of one tonne boxes in commercial stores were c. 10 times less contaminated than those from the middle of the boxes. For the two methods of peel extract preparation examined, the estimated sample size needed with an allowable error of log1010 E.c. atroseptica cells ml“1 extract with 95% confidence, was c. five tubers per box and 14 boxes for extract prepared from individual tubers and c. three lots of 10 tubers per box and 10 boxes for extract from 10 pooled tubers. A blackleg potential index for seed stocks was proposed based on the summation of the weighted number of individually tested tubers in different classes of contamination level.

Population dynamics of Erwinia carotovora subsp. atroseptica on the surface of intact and wounded seed potatoes during storage

Population dynamics of Erwinia carotovora subsp. atroseptica (Eca) on the tuber surface during storage (2–4°C) and pregermination, were studied by plating extracts of 6 mm2 point samples on crystal violet pectate medium. To investigate the effect of harvest damage on Eca survival, intact, skinned (epidermis removed), and peeled (complete peel removed) tubers from a dry (pF 3.4) and from a wet (pF 2.0) soil were inoculated either immediately after harvesting or after air drying for 4 h. Eca numbers on intact tuber surface decreased rapidly after inoculation, whereas at the skinned and peeled surface numbers were significantly increased 2 d after harvest. Tubers peeled and inoculated with Eca were rotted by 2 d after harvest while tubers peeled and dried before inoculation did not rot; however, populations were significantly increased 2 d after harvest. For all treatments Eca numbers per point sample decreased to below detection limits 180 d after harvest. Examination of 600 mm2 surface samples of the various treatments 222 d after harvest showed that Eca populations were still present. The number of tubers contaminated with colony‐forming units (cfu) of Eca was significantly lower for intact surface inoculated tubers from dry soil than for those of the other treatments. Drying for 4 h before inoculation resulted in a significant reduction of the number of Eca cfu positive tubers compared to all other treatments. The ELISA OD values of the 600 mm2 surface samples at day 222 were almost all positive and showed only a slight difference between the average of the tubers containing culturable Eca cells and those without culturable Eca cells.

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

Two 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.

An improved formulation and method of preparation of crystal violet pectate medium for detection of pectolytic erwinia

An improved procedure for the preparation of CVP medium is described on which pectolytic erwinia rapidly develop deep, localised pits. The medium can be prepared quickly and easily and forms a smooth, firm gel suitable for streaking.

Comparison of immunofluorescence colony-staining in media, selective isolation on pectate medium, ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in cattle manure slurry

Various isolation and serological techniques were compared for the detection ofErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Ech) in cattle manure slurry containing c. 108 colony forming units (cfu) per ml. The slurry samples could be preserved at −80°C for 8 months without reduction in the number of bacteria but not at −20°C. Samples stored at −80°C were inoculated with concentrations of the target bacterium ranging from 102 to 108 per ml. Only immunofluorescence colony-staining (IFC) in combination with selective media was able to detect the target organism at a concentration of 100 cells per ml. No IFC-positive colonies were found in pour plates of the non-inoculated cattle slurry. The recovery of the target bacterium from slurry inoculated with 102 cfu of Ech per ml was 64% in PT medium (containing polygalacturonic acid) and 19% in crystal violet pectate medium (CVP). Recoveries of Eca were 32% and 82%, respectively. Ech and Eca could be detected at levels of 103 cfu per ml of slurry by isolation on CVP. Crude filtration procedures were necessary for analysis of slurry samples with immunosorbent immunofluorescence (ISIF) cell staining. The detection level of ISIF for Ech was 105 cells per ml of slurry. IF-positive cells were incidentally observed in the non-inoculated slurry. Detection of Ech and Eca with ELISA was only possible in slurry inoculated with 108 cells of the target bacterium per ml.

Detection, quantification and classification of soft rot erwinias associated with potato tubers

An improved method for detection, quantification and classification of soft rot bacteria associated with potato seed tubers, plant material, soil and water has been developed. The method is based on the use of a modified version of the crystal violet pectate selective medium (CVP), enrichment cultures under anaerobic conditions using pectate as the sole carbon source for recovery improvement, and the quantitative estimation ofErwinia spp. by employing a new solid medium - most probable number (MPN) method. The use of this method enabled an improvement in the recovery and identification of specificErwinia spp. in mixed populations. This was done by incubating CVP plates — used for the MPN counting — at three different temperatures (15, 28 and 39°C). These combined techniques were used for estimating low level populations at less than one cell per gram or ml tested ofErwinia carotovora subsp.carotovora, E. carotovora subsp.atroseptica, andE. chrysanthemi.

A Note on the Limitations of Identifying Soft Rot Erwinias by Temperature Tolerances and Sensitivity to Erythromycin on a Pectate Medium

AbstractForty‐five erwinia strains from 14 different hosts have been tested lor growth on a crystal violet pectate medium with or without 70 μ/ml erythromycin at 27, 33.5 and 37°C. On the basis of positive data in the literature it was tried to identify the strains as Erwnia carotovora subsp. atroseptica, E. carotovora subsp. carotovora or E. chrysanthemi using this method.It appeared that relatively few strains reacted differentially, apparently due to strain variation (all strains were identified also by biochemical and physiological tests). The conclusion is that for a reliable identification of field isolates of soft rot crwinias other (biochemical, physiological and serological) methods have to be applied.

Quantitative assessment of tuber contamination by pectolytic erwinia and its possible use in the prediction and control of blackleg

A Most Probable Number Technique based on the use of carbohydrate-free McConkey broth and crystal violet pectate agar for the quantification of tuber-borne pectolytic erwinia has been developed. The population of the organisms on naturally contaminated tubers has been shown to be log-normally distributed. The possibility of using tuber contamination and standards for tuber quality for the prediction and control of blackleg is explored.

A rapid method to identify and quantify soft rot erwinias on seed potato tubers1

Potato tubers are usually contaminated by more than one species or pathovar of soft rot erwinia and, because blackleg incidence is related to the contamination level of seed tubers, the disease potential of seed stocks may be assessed by determining seed‐tuber contamination level. A method is described for identifying and quantifying directly from tubers the three soft rot erwinias commonly associated with potatoes. Replicate lots of 10–15 tubers are peeled by dry abrasion in a commercial potato peeler and the peel weight determined by weighing the tubers before and after peeling. Sap is expressed from the peel, an antioxidant (0.075% dithiothreitol) added, and the sap is dilution‐plated on a diagnostic selective medium (crystal violet pectate [CVP]). After incubating for 24 h at 20°C, the plates are velvet‐replicated onto fresh plates of CVP with or without 35 μg ml‐1 erythromycin and incubated for 48 h at 27°C and 24 h at 33.5 or 37°C. Soft‐rot erwinias typically form deep cup‐like cavities on CVP and they can be identified and enumerated according to the pattern of cavity formation. Cavities are formed by Erwinia carotovora pv. atroseptica only at 27°C, by E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C, whereas E. chrysanthemi forms cavities at all temperatures but fails to grow in the presence of erythromycin. Contamination levels can be expressed as the number of different erwinias per tuber or per g peel.

A rapid method for identifying and quantifying soft rot erwinias directly from plant material based on their temperature tolerances and sensitivity to erythromycin

A method is described for identifying and quantifying three soft rot erwinias directly from plant tissue and from other sources that is particularly useful in epidemiological studies. Colonies of these bacteria form characteristic deep cavities on selective‐diagnostic crystal violet pectate (CVP) medium. Bacteria from individual presumptive erwinia colonies on CVP plates spot inoculated on plates of CVP medium with or without erythromycin (35 μg/ml) added and incubated at 27, 33°5 and 37°C can be identified according to the pattern of cavity formation. Erwinia carotovora pv. atroseptica forms the characteristic cavities only at 27°C and E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C on CVP with or without erythromycin. Erwinia chrysanthemi forms cavities at all temperatures and can also be identified by failure to grow at 27°C on CVP with erythromycin. Similarly, erwinias in mixed populations can be quantified by dilution plating on CVP with or without erythromycin and incubating at the different temperatures. Using this method, ca 80% of 183 erwinia strains in a culture collection were correctly identified, the precision increasing to over 95% when recently isolated erwinia strains were examined.

Survival ofErwinia carotovora in Wisconsin soils

Erwinia carotovora var.atroseptica (van Hall) Dye andE. carotovora var.carotovora (Jones) Dye were detected in agricultural soils in Wisconsin using baiting and enrichment techniques. These soft rot bacteria could not be detected in soils of potato fields prior to planting of crops in the spring period using standard soil dilution plating techniques on a crystal violet pectate medium, however. A procedure involving incubation of samples in pectate enrichment broth followed by preparation of smears on slides and treatment with a fluorescent antibody stain specific forE. carotovora var.atroseptica was the most sensitive of the methods tested for detecting the blackleg pathogen.

Evaluation of procedures for detection of pectolyticErwinia spp. on potato tubers

The effectiveness of a tuber incubation method for detection ofErwinia carotovora var.atroseptica andE. carotovora var.carotovora in potato tubers was compared with a lenticel sampling procedure. In the first method, tubers were injured by puncturing lenticels with sterile toothpicks, then wrapped in moist paper towels and polyvinylidene film, and placed in closed chambers flushed with N2. In later experiments, wrapping tubers in two layers of polyvinylidene film and incubation in air was found to be as effective as the single layer of polyvinylidene and incubation in chambers flushed with N2. Isolations were made on a selective crystal violet pectate (CVP) medium from homogenized samples of tissue removed from soft rot lesions developing around injured lenticels. In the second method, 10 lenticels/tuber were aseptically removed with a scalpel and homogenized in distilled water; the suspension was plated on CVP. The first method was less tedious and slightly more effective than the lenticel sampling method. In a preliminary survey, these methods were used to detectErwinia infestations in small samples of certified seed potato tubers from Maine, Minnesota, Montana, New York, North Dakota, and Wisconsin. PectolyticErwinia spp. were detected in at least one sample from each state except Montana. The percentage of tubers withErwinia infestations varied from 0–100% among samples. Characterization ofErwinia isolates showed that bothE. carotovora var.carotovora andE. carotovora var.atroseptica were present. PectolyticErwinia spp. on symptomless potato seed tubers may serve as inoculum sources for blackleg and soft rot diseases.