Detection Of Clavibacter Michiganensis Research Articles

Detection of Clavibacter michiganensis subsp. michiganensis Assisted by Micro-Raman Spectroscopy under Laboratory Conditions.

Clavibacter michiganensis subsp. michiganesis (Cmm) is a quarantine-worthy pest in México. The implementation and validation of new technologies is necessary to reduce the time for bacterial detection in laboratory conditions and Raman spectroscopy is an ambitious technology that has all of the features needed to characterize and identify bacteria. Under controlled conditions a contagion process was induced with Cmm, the disease epidemiology was monitored. Micro-Raman spectroscopy (532 nm λ laser) technique was evaluated its performance at assisting on Cmm detection through its characteristic Raman spectrum fingerprint. Our experiment was conducted with tomato plants in a completely randomized block experimental design (13 plants × 4 rows). The Cmm infection was confirmed by 16S rDNA and plants showed symptoms from 48 to 72 h after inoculation, the evolution of the incidence and severity on plant population varied over time and it kept an aggregated spatial pattern. The contagion process reached 79% just 24 days after the epidemic was induced. Micro-Raman spectroscopy proved its speed, efficiency and usefulness as a non-destructive method for the preliminary detection of Cmm. Carotenoid specific bands with wavelengths at 1146 and 1510 cm−1 were the distinguishable markers. Chemometric analyses showed the best performance by the implementation of PCA-LDA supervised classification algorithms applied over Raman spectrum data with 100% of performance in metrics of classifiers (sensitivity, specificity, accuracy, negative and positive predictive value) that allowed us to differentiate Cmm from other endophytic bacteria (Bacillus and Pantoea). The unsupervised KMeans algorithm showed good performance (100, 96, 98, 91 y 100%, respectively).

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (108 CFU mL-1) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 103 to 107 CFU mL-1. The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.

Detection of Clavibacter michiganensis subsp. michiganensis in tomato and chili seeds and farming area of Sinaloa, Mexico

Detection of Clavibacter michiganensis subsp. michiganensis in tomato and chili seeds and farming area of Sinaloa, Mexico

Enrichment of Clavibacter michiganensis subsp. michiganensis in tomato seed extracts

An enrichment procedure was developed to improve detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato seed extracts. In the final procedure, seeds were soaked overnight in a buffer at 4°C and homogenised. Seed extracts were then filtered, supplemented with antibiotics and incubated for three days at 25°C. In seed extracts spiked with known amounts of Cmm suspensions, a 0- to 10,000-fold increase of Cmm populations was found in the incubation buffer supplemented with but not without the selective compounds, dependent on the seed lot. The procedure also enhanced enrichment of Cmm in seeds artificially infected with low or high numbers of a GFP-tagged strain of Cmm. The presence of the fluorescent bacteria in these internally-infected seeds was confirmed with epifluorescence stereo- and confocal laser scanning microscopy. Eight naturally-infected seed lots stored for three years were used for further evaluation of the procedure. Cmm was detected in seven out of eight lots by TaqMan, although Cmm was only detected by spread-plating in four lots. Enrichment resulted in an increase in Cmm population densities in two of these four seed lots.

Utility of a loop-mediated amplification assay for detection of Clavibacter michiganensis subsp. michiganensis in seeds and plant tissues

A loop-mediated amplification (LAMP) assay developed previously to specifically identify Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato, was compared with a commercial lateral flow immunoassay, three PCR assays, and culturing on agar medium for efficacy in detecting the pathogen in pepper and tomato tissues and tomato seeds. The LAMP assay was as effective in identifying C. m. michiganensis-positive tomato seed and tissue samples as the Cmm ImmunoStrip®. The LAMP assay was performed using a handheld real-time fluorescence-monitoring device (Smart-DARTTM) and the iQTM5 Multicolor Real-Time PCR Detection System to compare both systems. Both devices produced equivalent results, thus demonstrating the potential of this LAMP detection system for field detection. A side-by-side comparison of the C. m. michiganensis-LAMP with the Cmm ImmunoStrip® assay, two conventional PCR assays, using Cmm5/6 and PSA8/R primer sets, and one quantitative PCR assay (primers RZ_ptssk 10/11, probe RZ_ptssk12), showed that the C. m. michiganensis-LAMP/Smart-DARTTM assay can provide real-time portable in-field test results comparable to accepted standards. These results, combined with the specificity established previously, suggest that this LAMP assay can be an alternative to current diagnostic tests designed to detect C. m. michiganensis. This LAMP assay could be seamlessly integrated into standardized testing regimens, such as those of the European and Mediterranean Plant Protection Organization (EPPO), International Seed Federation (ISF), and commercial seed companies, as the definitive C. m. michiganensis confirmatory test, following isolation of colonies by plating.

Comparison of specificity and sensitivity of immunochemical and molecular techniques for determination of Clavibacter michiganensis subsp. michiganensis

Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 degrees C for Dreier's and 10 s at 69 degrees C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 10(5) CFU/mL and 10(3) CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10(3) CFU/mL with our designed primers and 104 CFU/mL with Dreier's primer pair.

A multiplex PCR method for detection of Clavibacter michiganensis subsp. michiganensis with co-amplification of its host DNA

A multiplex PCR assay system was developed for the detection of Clavibacter michiganensis subsp. michiganensis (Cmm), which combined two tests in one reaction mixture. Cmm-specific primers PSA-4/PSA-R and Solanum lycopersicum-specific primers NS-7-F/NS- 8-R (internal PCR control primer) were combined in one PCR reaction mixture with Cmm and plant DNA as template. The primer sets could amplify the target product successfully. Different combinations and concentrations of primers and annealing temperatures were tested, respectively. The detection level of the optimized multiplex PCR assay was up to 5×102 cfu·mL−1. To verify the applicability of this system, it was employed to detect Cmm in tomato seeds and plantlet samples. Seeds mixed with Cmm and diseased plantlets were detected successfully. The multiplex PCR system will avoid false-negative results and provide a reliable method for the detection of Cmm.

Detection of Clavibacter michiganensis subsp. sepedonicus in daughter tubers of volunteer potato plants

Daughter tubers of volunteer potatoes were tested for their ability to maintain <I>Clavibacter michiganensis</I> subsp. <I>sepedonicus (Cms)</I>. In different areas of the CR, volunteer potatoes were searched for in crops grown in rotation with potatoes and where one or two years before <I>Cms</i> had been detected and identified in samples of harvested seed or commercial potatoes using the test scheme in accordance to EC Directive 93/85/EEC. During May and June of 2005 and 2006, emerging or emerged plants of volunteer potatoes were collected at nine locations of Bohemia and transplanted to the experimental field in the Diagnostic Service Laboratory at Šluknov-Kunratice in Northern Bohemia. The daughter tubers of these plants were harvested and stored at 6°C for 1 month and then at 22°C for 3 months for multiplication of <I>Cms</i> cells. Samples of the daughter tubers were divided into 215 partial samples and tested for the occurrence of <I>Cms</i> at five terms which differed in length of storage time. The DAS ELISA test was used to detect <I>Cms</i> in the tuber samples. <I>Cms</i> was detected in eight of the nine potato volunteer tuber samples from different locations. The presence of <I>Cms</i> in positively tested tuber samples was confirmed using a pathogenicity test on eggplants (<I>Solanum melongena</I>). The optimal time for the detection of the pathogen in the harvested daughter tubers was between 4 and 10 weeks of storage at 22°C.

Maritime Regional Meeting, 2006 The Canadian Phytopathological Society

Muscodor albus Volatiles control toxigenic fungi in media under controlled atmosphere in storage conditions. P.G. Braun, M. Vailati, R.K. Prange, and E.W.E. Bevis. Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Mian Street, Kentville, NS B4N 1J5, Canada. Evaluation of products for the control or suppression of common scab [Streptomyces scabies] in ‘Shepody’ potatoes. R. Coffin, C. Goyer, W. Hardy, M. Webber, B. Drummond, L. Johnson, and S. Watts. Cavendish Farms, Kensington, PE C1N 5J5, Canada; (C.G.) Potato Research Centre, Agriculture and Agri-Food Canada,P.O. Box 20280 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada; and (S.W.) Engage Agro Corporation, 848 Gordon Street, Guelph, ON N1G 1Y7, Canada. Evaluation of products for prevention of rot in stored potato tubers exposed to Phytophthora infastans inoculum R. Coffin, W. Hardy, B. Drummond, L. Johnson, S. Watts, M. McQuillan, and D. Bell. Cavendish Farms, Kesington, PE C1N 5J5, Canada; (S.W.) Engage Agro Corporation, 848 Gordon Street, Guelph, ON N1G 1Y7, Canada(M.M) Agricultural Storage Management Inc., 16 Donald Drive, Charlottetown, PE C1A 1Z5, Canada; and (D.B) United Agri Products, 75 Tweedside Road, Harvey, Your Country, NB E6K 2G9, Canada. A novel technique for inoculation of field and horticultural lettuce crops with Sclerotinia spp. B.D. Gossen and M.R. McDonald. Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada; and (M.R.M.) Department of Plant Agriculture, University of Guelph,Guelph ON N1G 2W1 Canada. Valdensinia leaf spot[Valdensia heterodoxa] of commercial lowbush blueberry in Atlantic Canada: an emerging new threat. P.D. Hildebrand and W.E. Renderos. Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Main Street, Kentville, NS B4N 1J5, Canada. Trimming the carrot canopy to reduce the incidence of sclerotinia [Sclerotinia sclerotiorum] R.D. Peters and K.R. Sanderson. Crops and Liverstock Research Centre,Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada. An artificial positive control for use in the real-time PCR detection of Clavibacter michiganensis subsp. sepedonicus in potato. D.S. Smith, J.Gourley, and S.H. De Boer. Canadian food inspection Agency, Charlottetown Labratory, 93 Mount Edward Road, Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada. Molecular detection of Clavibacter michiganensis subsp, insidiosus causal agent of bacterial wilt of alfalfa L.J. War, J. Gourley, and S.H. De Boer. Canadian Food Inspection Agency, Charlottetoen, PE C1A 5T1, Canada.

Parameters for specific detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers by multiplexed PCR-ELISA

A multiplex PCR-ELISA protocol for detection ofClavibacter michiganensis subsp.sepedonicus (Cms) was developed that is based on primers for amplification of three single-copy, unique DNA sequences, Cms50, Cms72, and Cms85. The three sequences were simultaneously amplified from the genomes of all 42 strains of Cms that were tested including variant mucoid forms, but not from strains representing five related subspecies, andRathayibacter rathayi andRhodococcus faciens. The lowest limit of detection by gel electrophoresis was estimated to be approximately 300 CFU per mL when cells were spiked into potato core fluid, but sensitivity increases approximately 10-fold using PCR-ELISA. Inclusion of a sea anemone DNA fragment engineered so it could be amplified from the Cms72 primer set provided the simultaneous signal that the system functioned properly when any sample was free of the pathogen. The addition of hydrolyzed casein to the reaction mix was demonstrated to markedly reduce or eliminate inhibition of PCR by plant cell components or contaminants. Multiplex PCR-ELISA detection of Cms was determined to be verifiable for analysis of both stems and tubers based on the amplification of multiple sites in its genome, it provides absolute specificity, and it was more sensitive than detection based on gel electrophoresis of PCR products and serological approaches.

PCR detection ofclavibacter michiganensis subsp.sepedonicus- infected tuber samples in a plate capture assay

The speed and sensitivity of PCR-based assays allow shorter turnaround times for the detection of pathogens for which culture and serological methods are difficult or unavailable. PCR was performed with primer sets Cms50 and Cms72, designed previously by Millset al. (1997) through subtractive hybridization to detectClavibacter michiganensis subspeciessepedonicus (Cms). In bacterial suspensions, fewer than three cells/10 ul reaction were detected after PCR amplicons were hybridized with specific DIG-labeled DNA probes in an enzyme-linked oligonucleosorbent assay (ELOSA). In naturally infected tuber samples representing three cultivars of potato, the diagnostic sensitivity of PCR/ ELOSA was 96%, while the specificity exceeded 99%. PCR/ELOSA detectedCms in infected tuber samples with equal sensitivity regardless of colony morphology, potato cultivar, or primer sets.

Abstracts, Alberta Regional Meeting, The Canadian Phytopathological Society, 1999

Fludioxonil (Maxim®) provides effective control of fusarium dry rot of potatoes (Fusarium spp.). P.S. Bains, H. Bennypaul, L.M. Kawchuk, and J.D. Holley. Crop Diversification Centre North, Alberta Agriculture, Food and Rural Development, R.R. 6, Edmonton, AB T5B 4K3, Canada; (L.M.K.) Agriculture and Agri-Food Canada, Research Centre, Box 3000, Lethbridge, AB T1J 4B1, Canada; (J.D.H.) Crop Diversification Centre South, Alberta Agriculture, Food and Rural Development, SS4, Brooks, AB T1R 1E6, Canada. Virulence and molecular diversity in isolates of Botrytis cinerea recovered from greenhouse tomato in Alberta. J.T. Calpas, M.N. Konschuh, C.C. Toews, and J.P. Tewari. Crop Diversification Centre South, Alberta Agriculture, Food and Rural Development, SS4, Brooks, AB T1R 0K8, Canada; and (J.P.T.) Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada. Report of aster yellows phytoplasma in poker statice and Queen Anne’s lace in Alberta. K.F. Chang, S.F. Hwang, L.M. Kawchuk, G.D. Turnbull, and R.J. Howard. Crop Diversification Centre South, Alberta Agriculture, Food and Rural Development, SS4, Brooks, AB T1R 1E6, Canada; (S.F.H., G.D.T.) Alberta Research Council, P.O. Bag 4000, Vegreville, AB T9C 1T4, Canada; and (L.M.K.) Agriculture and Agri-Food Canada, Research Centre, Box 3000, Lethbridge, AB T1J 4B1, Canada. Molecular differentiation of three Tilletia species: T. indica, T. walkeri, and T. barclayana. T. Despins, A. Laroche, D.A. Gaudet, L. Knorr, and L. Lintott. Agriculture and Agri-Food Canada, Research Centre, Box 3000, Lethbridge, AB T1J 4B1, Canada. Screening of lentil cultivars for resistance to fusarium root rot. S.F. Hwang, B.D. Gossen, G.D. Turnbull, K.F. Chang, and R.J. Howard. Alberta Research Council, P.O. Bag 4000, Vegreville, AB T9C 1T4, Canada; (B.D.G.) Agriculture and Agri-Food canada, Saskatoon Research Centre, Saskatoon, SK S7N 0X2, Canada; and (K.F.C., R.J.H.), Crop Diversification Centre South, Alberta Agriculture, Food and Rural Development, SS4, Brooks, AB T1R 1E6, Canada. The first report of fusarium wilt of canola in Alberta, 1999. R.M. Lange, L.M. Harrison, M. Hall, K. MacDonald, and P.D. Kharbanda. Alberta Research Council, P.O. Bag 4000, Vegreville, AB T9C 1T4, Canada; (L.M.H.) Alberta Agriculture, Food and Rural Development, Box 29, Beaverlodge, AB T0H 0C0, Canada; (M.H.) Alberta Agriculture, Food and Rural Development, Box 40, Fort Vermilion, AB T0H 1N0, Canada; and (K.M.) Alberta Agriculture, Food and Rural Development, Box 519, Vegreville, AB T9C 1R6, Canada. Symptomless infection of barley seed by Rhynchosporium secalis. H.K. Lee, J.P. Tewari, and T.K. Turkington. Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; (T.K.T.) Agriculture and Agri-Food Canada, Lacombe Research Center, 6000 C & E Trail, Lacombe, AB T4L 1W1, Canada. Polymerase chain reaction detection of Clavibacter michiganensis subsp.sepedonicus. L. Lintott, S. Eldridge, and D. Fujimoto. Agriculture and Agri-food Canada, Research Centre, Box 3000, Lethbridge, AB T1J 4B1, Canada. Association of aster yellows and witches’-broom phytoplasmas with several plant hosts in Alberta. A.-H. Khadhair, J.P. Tewari, R.J. Howard, and B. Choban. Alberta Research Council, Vegreville, AB T9C 1T4, Canada; (J.P.T.) Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; (R.J.H.) Crop Diversification Centre South, Brooks, AB T1R 1E6, Canada; and (B.C.) Crop Diversification Centre North, Edmonton, AB T5B 4K3, Canada. First report of the Dutch elm disease from Alberta, Canada. J.P. Tewari, J. Feddes-Calpas, and S. Brezden. Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada; and (J. F.-C.) Crop Diversification Centre South, SS4, Brooks, Alberta T1R 1E6, Canada.

Detection of Clavibacter michiganensis subsp. sepedonicus by DNA amplification

Control of bacterial ring rot ultimately depends on the accurate and sensitive detection of C. michiganensis subsp. sepedonicus in infected potato tissues and tubers. Polymerase chain reaction (PCR) based detection of C. michiganensis subsp. sepedonicus appears to have the potential to circumvent many of the problems currently associated with detection of this phytopathogen. PCR reactions using primers specific to C. michiganensis subsp. sepedonicus and genomic DNAs from related strains, including phytopathogens, did not produce any amplification products. C. michiganensis subsp. sepedonicus was detectable from a mixture of potato and bacterial DNA by amplification of a DNA sequence specific to C. michiganensis subsp. sepedonicus. Detection by DNA amplification allowed direct processing of plant tissue samples, and circumvented the need for prior isolation of the suspected phytopathogen.

Detection of Clavibacter michiganensis ssp. michiganensis in tomato seeds by immunofluorescence microscopy and dilution plating

A method for detectingClavibacter michiganensis ssp.michiganensis in tomato seeds was evaluated. The method is based on rapid screening of tomato seed lots using indirect immunofluorescence staining (IF), followed by dilution plating of IF positive seed lots. Different polyclonal antisera, prepared againstC. michiganensis ssp.michiganensis were tested for their specificity using IF. All strains ofC. michiganensis ssp.michiganensis tested reacted with the polyclonal antisera. Two of nine saprophytic isolates from tomato seeds were positive with the antisera as well as with the control normal serum, but cells of these isolates were distinct in shape from cells ofC. michiganensis ssp.michiganensis.

Detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers grown from seed pieces with various levels of inoculum

An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was used to monitor potato stems of three cultivars for the presence of the ring rot pathogen,Clavibacter michiganensis subsp.sepedonicus, during the growing season and in progeny tubers after harvest. The highest ELISA values were obtained with the highest concentration of bacteria used to inoculate seed pieces in all cultivars tested. Low ELISA values were obtained for stems and progeny tubers selected from plants grown from seed inoculated at lower bacterial concentrations. Estimates of bacterial densities in stems and progeny tubers by immunofluorescence indicated that low ELISA values were most probably caused by low bacterial numbers. It is suggested that the sensitivity of ELISA for detecting the ring rot pathogen in potato stems and progeny tubers is a function of the concentration of bacteria in individual seed pieces.

A comparative study in five laboratories on detection ofClavibacter michiganensis subsp.sepedonicus in potato stems and tubers

Potato stems and tubers grown in the field from seed tubers inoculated withClavibacter michiganensis subsp.sepedonicus, which causes bacterial ring rot, were tested by indirect, sandwich enzyme-linked immunosorbent assay (ELISA) in five laboratories. Correlation between values for each experimental treatment from the five laboratories was greater (r=0.86) than correlation between values for individual samples (r=0.71). When three or more laboratories obtained ELISA values of ≥0.200 for a sample, that sample was presumed to be positive. Conversely, when three or more laboratories obtained ELISA values <0.200, the consensus determination was regarded as negative. The percentage of stem and tuber samples that were in agreement with the consensus ELISA determination varied from 65.5 to 96.7%. Indirect immunofluorescence tests, conducted on the same samples in two laboratories, were consistent with 83.4–91.9% of the consensus ELISA determinations. Presence or absence ofC.m. sepedonicus was confirmed in some samples by an eggplant bioassay and direct isolatiion of the bacterium. The ELISA procedure was well suited for screening large numbers of samples and this study confirms it to be a promising procedure in routine indexing of seed potatoes forC.m. sepedonicus.

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 108 colony forming units (cfu) per ml.