Detection of the plant pathogenic bacterium Xanthomonas campestris pv. Campestris in seed extracts of Brassica sp. Applying fluorescent antibodies and flow cytometry.
BACKGROUND
Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted plant pathogenic bacterium that causes black rot of crucifers. Seed lots and plants are screened for contamination with this pathogen using plating or serological assays. These methods, however, are time consuming and not very sensitive, respectively. Therefore, flow cytometry (FCM) was evaluated as a tool for the rapid detection and quantification of Xcc cells labeled with a mixture of specific fluorescein isothicyanate (FITC)-monoclonal antibodies (mAb) in pure culture, in mixed cultures of Xcc with either the common saprophyte Pseudomonas fluorescens (Psf) or a nonpathogenic X. campestris isolate (Xc), and in crude seed extracts.


METHODS
The mAb 18G12, conjugated with FITC, was tested at dilutions of 1:50, 1:100, 1:200, and 1:400. For mixed suspensions of Xcc and Psf, mAb 18G12 was used at a dilution of 1:100. The combination of mAbs 18G12, 2F4, and 20H6, all conjugated with FITC, was used at a dilution of 1:100 for the detection and quantification of Xcc cells in mixed suspensions containing Xcc and Xc and in crude seed extracts. The analyses were performed with a Coulter EPICS XL-MCL flow cytometer, at low flow rate during 2 min.


RESULTS
Using FCM, Xcc cells labeled with FITC-conjugated mAbs (18G12, 2F4, and 20H6) were detected and quantified rapidly at low numbers, i.e., 10(3) colony-forming units per milliliter in pure and in mixed cultures with Psf. The presence of the nonpathogenic Xc in the seed extracts did not interfere with the FCM results. Xcc cells were distinguished from the cells of other organisms and from small particles present in the seed extract based on the high-intensity fluorescence of the labeled cells.


CONCLUSIONS
The application of FCM in combination with FITC-conjugated mAbs appears to be a promising technique for the detection and quantification of Xcc cells in seed extracts of crucifers.
Chitarra
L G
LG
Business Unit Plant Development and Reproduction, Plant Research International B.V., Wageningen, The Netherlands.
Langerak
C J
CJ
Bergervoet
J H W
JH
van den Bulk
R W
RW
D000911:Antibodies, Monoclonal / Q000276:immunology; D001937:Brassica / Q000737:chemistry / Q000382:microbiology*; D004306:Dose-Response Relationship, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique / Q000379:methods*; D010935:Plant Diseases / Q000382:microbiology*; D010936:Plant Extracts / Q000032:analysis; D011551:Pseudomonas fluorescens / Q000276:immunology / Q000302:isolation & purification / Q000472:pathogenicity; D012639:Seeds / Q000382:microbiology*; D013194:Staining and Labeling; D016959:Xanthomonas campestris / Q000276:immunology / Q000302:isolation & purification* / Q000472:pathogenicity
D000911:Antibodies, Monoclonal; D010936:Plant Extracts; D016650:Fluorescein-5-isothiocyanate