Immunofluorescence Colony Staining Research Articles

Deciphering the function of Xiangsha-Liujunzi-Tang in enhancing duodenal mucosal barrier by inhibiting MC/Tryptase/PAR-2 signaling pathway in functional dyspepsia rats

Ethnopharmacological relevanceXiangsha-Liujunzi-Tang (XSLJZT) is a classical formula for treating the diseases of digestive system, which can effectively and significantly improve the symptoms of functional dyspepsia (FD) patients. The main function of XSLJZT is to benefit Qi and spleen, and harmonize stomach. Aim of the studyThe purpose of this study was to investigate the intervention effect of XSLJZT on duodenal mucosal injury in FD rats and the response mechanism of MC/Tryptase/PAR-2 signal pathway. Materials and methodsUltra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed to qualitatively and quantitatively identify the chemical component of XSLJZT. A comprehensive modeling method (iodoacetamide infusion + irregular diet + swimming exhaustion) was used to construct the FD rat model. XSLJZT decoction was given to intervene FD rats for 2 weeks. The indicators of digestive function including body mass, 3-h food intake, visceral sensitivity, gastric emptying rate and intestinal propulsion rate were routinely measured for FD rats. The pathological changes of duodenum and microstructure of intestinal epithelial cells were observed by HE staining and transmission electron microscopy respectively. The inflammatory factors (VCAM-1, IL-6, TNF-α, and ICAM-1) and histamine content were evaluated by enzyme-linked immunosorbent assay (ELISA). The expression levels of Tryptase, PAR-2, ZO-1, β-catenin, p–NF–κBp65 and p-ERK1/2 in duodenal tissues were measured by Western blot (WB) and immunofluorescence colony-staining (IFC). ResultsXSLJZT administration significantly improved the survival of FD rats, increased body mass and 3-h food intake, improved visceral sensitivity, and restored gastric emptying rate and intestinal propulsion rate. HE staining showed that XSLJZT recovered the structure of duodenal mucosal and reduced inflammatory infiltration. ELISA revealed that XSLJZT reduced the content of inflammatory factors (VCAM-1, IL-6, TNF-α, and ICAM-1) and histamine. In addition, WB and IFC uncovered that the protein levels of ZO-1 and β-catenin were up-regulated and MC/Tryptase/PAR-2 signaling pathway was inhibited by XSLJZT. ConclusionXSLJZT significantly improved the integrity of duodenal mucosa and decreased the inflammation in FD rats through the inhibition of MC/Tryptase/PAR-2 signaling pathway response.

Establishment of pancreatic cancer cell lines with endoscopic ultrasound-guided biopsy via conditionally reprogrammed cell culture.

Recent studies have identified the mutational landscape of pancreatic cancer and suggested tumor‐specific subtypes. However, the major hurdle against personalized treatment is the difficulty to obtain sufficient cancer tissues from most inoperable cases. We investigated whether patient‐derived conditionally reprogrammed cells (CRCs) can be constructed using a small piece of tumor tissue using endoscopic ultrasound (EUS)‐guided fine needle biopsy (FNB). Thirty patients with pancreatic solid mass (mean size, 34.6 mm) were enrolled prospectively. Among 22 patients who were diagnosed with pancreatic ductal adenocarcinoma, we established patient‐derived pancreatic cancer cell lines from eight patients (36.4%). Immunofluorescence colony staining for CRCs showed that the cytoplasm of cancer cells was clearly stained with anti‐cytokeratin 19 monoclonal antibody. In the soft agar colony formation assay, CRCs formed colonies compared with the negative control by day 15. In vivo, implanted CRCs showed tumor engraftment and hematoxylin and eosin staining showed pancreatic cancer ductal structure. All established CRCs showed a KRAS mutation. In conclusion, we established patient‐derived pancreatic cancer cell lines with a small tumor tissue obtained by EUS‐FNB. With in vitro drug sensitivity and genomic studies, established patient‐derived cell lines can be used in identification of new targets for diagnosis and treatment of pancreatic cancer.

Anaerobic disinfestation of tare soils contaminated with Ralstonia solanacearum biovar 2 and Globodera pallida

Tare soil is soil attached to harvested products like potato tubers. Tare soil becomes a considerable waste stream after storage, washing and processing of harvested products. There is a high risk on contamination of tare soils with (quarantine) phytopathogens, because of import of harvested products from different regions of the world. Disinfestation is necessary to make tare soils applicable for agricultural production. Anaerobic (non-chemical) soil disinfestation (ASD) and inundation are suitable methods to disinfest tare soils. Two different soils (marine loam and glacial sand) were either treated with five (2011) or four (2012) treatments and these were: (i) no treatment (control), (ii) freshly cut grass, (iii) Herbie 7025, (iv) inundation (five cm water on top of the soil surface), and (v) combination of Herbie 7025 and inundation (2011 only). Containers with treated and untreated soils were inoculated with two quarantine phytopathogens, Ralstonia solanacearum biovar 2 and Globodera pallida. After soil inoculation, the containers were airtight closed and only opened for destructive sampling after 84 days. Then, soils were analysed for the presence of R. solanacearum biovar 2, using immunofluorescence colony staining, and for the presence of viable G. pallida eggs, determined by the number of juveniles hatched from eggs and lured to potato root exudate. Strong (> 99.4 %) declines of both pathogens in treated versus the appropriate control soils were observed. Repetition of the experiment revealed the same pattern and therefore it was concluded that ASD and inundation, as separate treatments, have promise for remediation of tare soils from contaminating quarantine pathogens.

Development of immunofluorescence colony staining (IFC) for detection of Xanthomonas campestris pv. vesicatoria and Clavibacter michiganensis subsp. michiganensis in tomato seeds

Immunofluorescence colony-staining (IFC) is based on sample pour plating in combination with immunofluorescence staining for recognition of the target colony. IFC was optimised for detecting Xanthomonas campestris pv. vesicatoria (Xcv) and Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato seed lots. Optimum incubation periods for colony growth were 2 d for Xcv and 3 d for Cmm. For both targets, the fluorescence intensity of the target colonies was positively correlated with the incubation temperature in the range tested and the period of colony growth. In general a lower brilliance was observed in IFC preparations of pure cultures than when seed extracts was added. The maximum density of bacterial colonies in the pour plates for both targets was about a thousand times higher per square cm as for surface plating. In comparison with IF (immunofluorescent cell staining) and isolation, IFC was 10 times more sensitive than IF. In samples with a target to saprophyte ratio higher than 1/100, 10 to 100 times higher numbers of positive colonies of the target were found in IFC using 16 mm diameter wells in miniaturized routine system, than by dilution plating on trypticase soy agar (TSA) and selective media. The recovery of Xcv in pour plates at various density of the target was still 46-68% for samples with a high microbial background (105 or more colonies per cm). For the slow growing Clavibacter, recovery was reduced to ca. 12-17% for samples with a high microbial background. A series of 23 commercial seed lots were tested for Xcv with IF and IFC. Using IF seven samples were positive based on IF-positive cells in the preparation. In IFC no typical fluorescent positive Xcv colonies were observed. From the weak cross-reacting colonies, viable cells were collected for pure culturing. Verification confirmed that the colonies of the weakly cross-reacting species belonged to other species

Fate of Clavibacter michiganensis ssp. sepedonicus, the Causal Organism of Bacterial Ring Rot of Potato, in Weeds and Field Crops

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inoculation. About 6–8 weeks after inoculation, Cms could be detected in most crops except onion and sugar beet, in larger numbers in stems (105–106 cells/g of tissue) than in roots (≤103 cells/g of tissue) in immunofluorescence cell-staining (IF). Cms was successfully re-isolated only from IF-positive stem samples of maize, bush bean, broad bean, rape and pea, but not from roots. Twelve solanaceous weeds and 13 other weeds, most commonly found in potato fields in Europe, were tested in IF as hosts of Cms by stem and root inoculations. Only in Solanum rostratum, a weed present in northern America, Cms persisted in high numbers (108 cells/g tissue) in stems and leaves, where it caused symptoms. In the other solanaceous weeds, Cms persisted at low numbers (approximately 105 cells/g of tissue) in stems but less so in roots. The bacteria could be frequently re-isolated from stem but not from root tissues. In 2 consecutive years, plants from 14 different weed species were collected from Cms-contaminated potato field plots and tested for the presence of Cms by dilution plating or immunofluorescence colony-staining (IFC), and by AmpliDet RNA, a nucleic acid-based amplification method. Cms was detected in roots but not in stems of Elymus repens plants growing through rotten potato tubers, and in some Viola arvensis and Stellaria media plants, where they were detected both in stems and roots, but more frequently by AmpliDet RNA than by IFC.

Pig slurry reduces the survival of Ralstonia solanacearum biovar 2 in soil.

The effect of added pig slurry and solarization on the survival of Ralstonia solanacearum biovar 2 strain 1609 in soil was analysed in soil microcosms and field plots. In addition, the invasion of potato plants by R. solanacearum and the development of disease symptoms were determined, as measures of induced disease suppressiveness. In untreated soil, R. solanacearum showed slow population declines in both microcosms and the field from, initially, 10(6-)10(7) to 10(3)-10(4) CFU.(g dry soil)(-1) in about 9 weeks. The suppressiveness assays of these untreated soils after this period revealed that most of the plants that were used developed wilting symptoms and (or) contained the pathogen in their lower stem parts, as shown by immunofluorescence colony staining and PCR. The addition of pig slurry resulted in a significantly lower population size of R. solanacearum as well as reduced numbers of infected and (or) diseased plants in the soil suppressiveness tests. On the other hand, solarization of soil also decreased R. solanacearum survival but did not enhance soil suppressiveness as measured by development of disease symptoms and (or) plant invasion after 9 weeks. Combined soil solarization and pig slurry addition showed an additive effect of both treatments. Healthy-looking plants, primarily from soils treated with pig slurry and solarization, incidentally revealed the latent presence of R. solanacearum in the lower stem parts. The mechanism behind the enhanced population declines and disease suppressiveness induced by pig slurry is unclear but shifts in community profiles were clearly discernible by PCR - denaturing gradient gel electrophoresis 9 weeks after pig slurry addition in the field experiment, indicating induced changes in the bacterial community structure.

Production of antifungal compounds by Lysobacter enzymogenes isolate 3.1T8 under different conditions in relation to its efficacy as a biocontrol agent of Pythium aphanidermatum in cucumber

Lysobacter enzymogenes strain 3.1T8 is a potential biocontrol agent of Pythium aphanidermatum (Edson) Fitzp. in cucumber ( Cucumis sativa L.). It showed in vitro antagonistic activity, hemolytic activity, and the production of a surface active compound, which decreased in media of increasing strength. One or more low molecular compounds, present in culture filtrates of 3.1T8, caused rapid immobilization of zoospores of P. aphanidermatum and inhibited cyst germination. Bioassays for suppression of root and crown rot were performed with 2-week-old cucumber plants in nutrient solution. Complete disease suppression occurred after addition of a suspension of unwashed cells grown on nutrient-poor R2A, or the filtrate of this suspension, 1 day before inoculation with zoospores. Application of washed cells caused slight suppression in one of two bioassays only. No disease suppression occurred when bacterial inoculum grown on 1/10 TSBA or in 1/10 TSB was used. In two bioassays on cucumber plants grown for 4–5 weeks in 18-L containers, application of R2A-grown cells of 3.1T8 did not cause disease suppression. Density of 3.1T8, monitored using immunofluorescence colony staining, decreased at least 100-fold during these experiments to approximately log 3–4 cfu ml −1 of nutrient solution and log 5–6 cfu g −1 roots. Overall, the findings indicated a strong influence of nutritional resources on production of antifungal compounds by strain 3.1T8. The lack of disease suppression in hydroculture containers suggested that in situ production of the compounds was insufficient or absent, although the bacteria persisted in the nutrient solution and on the roots of cucumber.

Effects of compost addition and simulated solarisation on the fate of Ralstonia solanacearum biovar 2 and indigenous bacteria in soil

Abstract The effects of compost addition and simulated solarisation of soil on the survival of Ralstonia solanacearum biovar 2 strain 1609, as well as on the structure of indigenous soil bacterial communities, were analysed. In addition, effects on the invasion of susceptible test plants by strain 1609 were assessed. In untreated soil in microcosms and the field, strain 1609 showed slow progressive declines, from 10(6)-10(7) to roughly 10(4)-10(5) CFU per g dry soil in around 60 days. When these soils were used in suppressiveness tests, a majority of plants developed symptoms of wilting and revealed the presence of the pathogen in their lower stem parts, as evidenced by immunofluorescence colony staining (IFC) and polymerase chain reaction (PCR). Solarisation of unamended soil did not drastically affect R. solanacearum survival or plant invasiveness. However, the addition of household compost resulted in enhanced R. solanacearum population decline rates, as well as reduced numbers of diseased plants in suppressiveness tests. Combined solarisation and compost addition yielded differential results between microcosms and the field. Some healthy-looking plants, primarily from soils treated with compost, revealed the latent presence of strain 1609 in the lower stem parts. The eubacterial and beta-subgroup proteobacterial communities in the differentially treated soil microcosms were rather stable, as evidenced by analysis of PCR-denaturing gradient gel electrophoresis (DGGE) generated molecular profiles. However, compost amendment clearly induced changes in these communities, which were detectable until the end of the experiment; two major bands, affiliated with Variovorax paradoxus and Aquaspirillum psychrophylum, were associated with the compost amendment. The decrease in abundance of R. solanacearum in the compost-amended soils was confirmed by the DGGE profiles.

Effects of compost addition and simulated solarisation on the fate of Ralstonia solanacearum biovar 2 and indigenous bacteria in soil

The effects of compost addition and simulated solarisation of soil on the survival of Ralstonia solanacearum biovar 2 strain 1609, as well as on the structure of indigenous soil bacterial communities, were analysed. In addition, effects on the invasion of susceptible test plants by strain 1609 were assessed. In untreated soil in microcosms and the field, strain 1609 showed slow progressive declines, from 10 6–10 7 to roughly 10 4–10 5 CFU per g dry soil in around 60 days. When these soils were used in suppressiveness tests, a majority of plants developed symptoms of wilting and revealed the presence of the pathogen in their lower stem parts, as evidenced by immunofluorescence colony staining (IFC) and polymerase chain reaction (PCR). Solarisation of unamended soil did not drastically affect R. solanacearum survival or plant invasiveness. However, the addition of household compost resulted in enhanced R. solanacearum population decline rates, as well as reduced numbers of diseased plants in suppressiveness tests. Combined solarisation and compost addition yielded differential results between microcosms and the field. Some healthy-looking plants, primarily from soils treated with compost, revealed the latent presence of strain 1609 in the lower stem parts. The eubacterial and β-subgroup proteobacterial communities in the differentially treated soil microcosms were rather stable, as evidenced by analysis of PCR-denaturing gradient gel electrophoresis (DGGE) generated molecular profiles. However, compost amendment clearly induced changes in these communities, which were detectable until the end of the experiment; two major bands, affiliated with Variovorax paradoxus and Aquaspirillum psychrophylum, were associated with the compost amendment. The decrease in abundance of R. solanacearum in the compost-amended soils was confirmed by the DGGE profiles.

Detection of Pseudomonas syringae pv. aptata in Irrigation Water Retention Basins by Immunofluorescence Colony-staining

Bacterial blight of cantaloupe (Cucumis melo) caused by Pseudomonas syringae pv. aptata was first observed in south-western France and has since spread to all cantaloupe-growing areas of this country. Use of pesticides registered for this disease has proved ineffective and no commercial cultivars of cantaloupe are resistant to this blight. To develop control strategies for this disease, the principal sources of inoculum were investigated. Among the different sources of inoculum studied, we report the isolation of P. syringae pv. aptata from irrigation water retention basins in south-western France using the immunofluorescence colony-staining (IFC) method. In this study, the pathogen was detected at a low concentration (12 and 70cful−1) in two different retention basins. These results suggest that P. syringae pv. aptata can survive in water used to irrigate cantaloupe crops and could be a source of inoculum for epidemics of bacterial blight. To develop control strategies for this bacterial disease, the importance of water retention basins as sources of inoculum for bacterial blight of cantaloupe needs to be evaluated relative to other potential sources such as seeds, plants from nurseries and plant debris in the soil.

Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato

An indirect immunoflouorescence colony staining method was developed for the detection of important seed-borne bacterial pathogens of tomato. The method involves the use of specific antiserum for initial binding of target bacteria and visualization of positive colonies with a commercially available secondary antiserum conjugated with FITC and observed under a fluorescence microscope. The indirect method is especially suitable for laboratories, seed companies, and quarantine stations which have no facilities for conjugation of primary antiserum. It is more economical and overcomes the problems generally encountered with variable conjugate quality in new batches of conjugates prepared from the same stock of primary antiserum. The assay is easy to perform and results can be easily assessed by visual scoring or image analyser. Results are available in 4–5 days as compared to 30–45 days in traditional methods. The resulting bacterial culture can be tested by PCR or host infectivity and a culture can be stored for future reference. Used in combination with highly specific antibodies (commercially available monoclonal and recombinant antibodies) it can be used as a very sensitive detection tool and has application potential in localization studies as well. Choosing the right secondary conjugate is however necessary to get best results in the assay.

Survival of Ralstonia solanacearum Biovar 2, the Causative Agent of Potato Brown Rot, in Field and Microcosm Soils in Temperate Climates

ABSTRACT After outbreaks of potato brown rot in three different fields in the Netherlands, the fate of the brown rot pathogen, Ralstonia solanacearum biovar 2, was monitored in soil by immunofluorescence colony staining (IFC) supported by R. solanacearum division-2 specific polymerase chain reaction. In selected areas of all fields, the R. solanacearum population densities were initially on the order 10(4) to 10(6) per g of topsoil. These population densities then declined progressively over time. In two fields, however, the pathogen persisted for periods of 10 to 12 months. The survival of a selected R. solanacearum biovar 2 isolate, strain 1609, in three soils, a loamy sand and two different silt loam soils, was further studied in soil microcosm experiments. The effects of temperature and soil moisture content were assessed. At 12 or 15 and 20 degrees C, a gradual decline of the population densities was observed in all three soils, from the established 10(5) to 10(6) CFU g(-1) of dry soil to significantly reduced levels, occasionally bordering the limit of detection (10(2) CFU g(-1)of dry soil), in periods of approximately 90 to 210 days. Soil type affected the rate of population decline at 20 degrees C, with the greatest decline occurring in loamy sand soil. In all three soils, the survival of IFC-detectable R. solanacearum 1609 cells at 4 degrees C was severely impaired, reflected in an accelerated decline of CFU counts, to undetectable numbers. Moreover, indications were found for the occurrence of viable but nonculturable strain 1609 cells in the loamy sand as well as in one silt loam soil under these conditions. In addition, a single freezing-thawing cycle caused a significant additional reduction of the culturable R. solanacearum 1609 populations in the three soils, though detectable populations remained. Moderate soil moisture fluctuations of approximately pF 2 did not affect the survival of R. solanacearum 1609 in soil. Severe drought, however, drastically reduced the populations of strain 1609 CFU in all three soils.

Blackleg risk potential of seed potatoes determined by quantification of tuber contamination by the causal agent and Erwinia carotovora subsp. atroseptica: a critical review*

Blackleg is a seed‐borne disease of potato caused by the soft rot bacterium Erwinia carotovora subsp. atroseptica in temperate regions. Although most seed stocks are extensively contaminated, blackleg incidence is related to seed contamination level, the threshold level for disease development being about 103 cells of E. c. atroseptica per tuber. Disease control relies primarily on the production and use of‘clean’seed potatoes. This is better achieved by planting seed potatoes with a low contamination level than by certification based on blackleg inspection and rogueing. Testing seed‐potato stocks for E. c. atroseptica involves four steps, which have not all been fully evaluated. First, sampling to reflect variation in tuber contamination level. Second, tuber tissue is prepared for testing. Third, determination of numbers of E. c. atroseptica, which can be carried out using a selective diagnostic medium (CVP), immunofluorescence colony staining after immuno‐capture of the target bacteria, enzyme‐linked immunosorbent assay (ELISA) applied to tuber extract after enrichment and a quantitative polymerase chain reaction (PCR) assay. Although all methods have the necessary sensitivity level, they suffer from certain important drawbacks, including reliability, specificity and ease of use. Last, interpretation of contamination results in terms of blackleg risk assessment. These steps are discussed critically as a basis for future studies.

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers.

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.

Immunofluorescence Colony-staining (IFC) for Detection and Quantification of Ralstonia (Pseudomonas) solanacearum Biovar 2 (Race 3) in Soil and Verification of Positive Results by PCR and Dilution Plating

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of sucrose and the antibiotics cycloheximide and crystal violet to the non-selective trypticase soy broth agar resulted in increased colony size and staining intensity of R. solanacearum in IFC. Verification of IFC-results by picking cells from IFC-positive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with ‘spiked’ and naturally contaminated soils respectively. Several other bacterial species which cross-reacted with polyclonal antibodies in IFC also grew on SMSA and were difficult to distinguish from R. solanacearum, thereby necessitating confirmation of the results. Rapid verification of IFC-positive results directly by PCR-amplification with primers D2/B specific to division 2 of R. solanacearum had a success rate of 86% and 96% with ‘spiked’ and naturally contaminated soil samples, respectively. Primers D2/B reacted with all R. solanacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to detect consistently ca. 100 cfu g−1 of soil, a detection level similar to that found with direct plating on SMSA, but less laboriously, whereas detection level with a bioassay on tomato plants was only 104−105 cfu g−1 of soil.

Immunofluorescence colony staining and selective enrichment in liquid medium for studying the population dynamics of Ralstonia solanacearum (race 3) in soil1

Survival of Ralstonia solanacearum (race 3) in different soil types in The Netherlands is currently under investigation to determine its significance in the epidemiology of brown rot. For detection of the brown‐rot pathogen, immunofluorescence colony staining (IFC) and selective enrichment in liquid media were optimized and evaluated. In IFC, pour‐plating of extracts from a clay soil and a loamy sand in the semi‐selective SMSA medium resulted in relatively low recoveries. Using non‐selective trypticase soy agar (10% TSA) medium, colonies of target bacteria added to extracts, particularly the sandy loam, remained very small and were weakly stained. Increased colony size and improved staining quality were found after addition of sucrose and crystal violet to 10% TSA. IFC detected 103‐104 cfug‐1 of dry soil. Detection of R. solanacearum by dilution plating on selective SMSA agar was complicated by the growth of many saprophytic bacteria which was confused with that of R. solanacearum. For detection of populations lower than 103 cfu g‐1 of dry soil, selective enrichment in SMSA broth containing the same nutrients as SMSA agar medium was evaluated. After enrichment, R. solanacearum was detected by serial dilution of the sample and immunofluorescence cell staining. EFC was less suitable here, due to the high densities of non‐target bacteria in the samples. In clay soil, initial populations of 1–10 cfu g‐1 of dry soil were enlarged within 48h to higher than 108 cfu g‐1. In sandy soil, enrichment occurred only when initial populations exceeded 100 cfu g ‐1 of dry soil. IFC was used to study the survival of R. solanacearum in sandy soil from the north‐east of The Netherlands (Drenthe). The pathogen survived at 15°C in soil for at least 92 days, but was not detectable at 4 or 20°C after 50–92 days. When the soil was frozen for 24 h at ‐8°C, the pathogen could not be detected by IFC. In soil from three different locations in The Netherlands from which brown rot‐infected potatoes were harvested, the pathogen was not detected by IFC, nor by bioassay in tomato plants. Cross‐reacting bacteria were detected in IFC preparations at a low frequency. They were weakly stained and could be distinguished from target bacteria in control preparations. Reisolated bacteria from these weakly stained colonies did not grow on SMSA and were negative in PCR amplifications, based on primers OLI‐1 and Y‐2.

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.

Detection of soft rot Erwinia spp. on seed potatoes: conductimetry in comparison with dilution plating, PCR and serological assays

Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 104 colony forming units (cfu) ml-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 105 cfu ml-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 104 cfu ml-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 104 cfu ml-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection.

Polymerase chain reaction for verification of fluorescent colonies ofErwinia chrysanthemiandPseudomonas putidaWCS358 in immunofluorescence colony staining

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw. chrysanthemi and non-target colonies from potato peel extracts, the identity of 90% of 113 target colonies was confirmed. Using primers directed against sequences of the ferric-pseudobactin receptor gene pupA of Pseudomonas putida WCS358, the identity of 96% of 22 target colonies was confirmed in IFC-stained samples with pure cultures. In pour plates with mixtures of Ps. putida WCS358 and non-target bacteria from compost extracts, the identity of 59% of 108 fluorescent colonies was confirmed by PCR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.

Immunofluorescence colony‐staining (IFC) as a concept for bacterial detection in quality testing of plant materials and ecological research 1

Immunofluorescence colony‐staining (IFC), as a concept in bacterial detection, has developed during the past decade, providing several new options for the study of culturable plant‐associated bacteria: (1) in situ detection of target colony‐forming units on or in plant parts, such as on roots or in stem sections; (2) selective isolation of cross‐reacting bacteria for research on the improvement of the reliability of serological detection systems; (3) distinction between culturable and non‐culturable cells; (4) simultaneous detection of different target bacteria using different fluorescent markers and a dual fluorochrome‐filter system. IFC‐positive colonies can be directly identified by isolation or PCR, which makes it easier to recognize false positive results. The use of IFC for the selective isolation of cross‐reacting bacteria forms a major tool for the further improvement of the specificity of IFC and serological assays.