摘要：

Black rot is one of the most threatening diseases of crucifers. The causal agent of this disease is the bacterium Xanthomonas campestris pv. campestris . The bacterium attacks all cultivated brassicas, radishes and numerous weeds, and is able to survive on plant debris in the soil. The primary source of inoculum is often infected seed. The most important ways to control black rot are the use of resistant cultivars and the use of 'healthy' seed. To obtain healthy seed, chemical or physical seed treatments may be used. These seed treatments may, however, seriously damage the seed quality (germination), may cause phytotoxicity or may not sufficiently eliminate the black rot pathogen from the seed (chapter 2). To check the seed health, sensitive and specific methods are needed to detect X. c. pv. campestris in the seed. Several methods for detecting X. c. pv. campestris are summarized in chapter 2. The most commonly used assays are plating assays, in which seed washings are plated onto isolation media, such as BSCAA (basal starch cycloheximide agar with nitrofurantoin and vancomycin), CS20ABN (a starch medium with bacitracin, neomycin and cycloheximide), FS (a medium with starch trimethoprim, cephalexin, cycloheximide, methyl green), NSCA (nutrient starch cycloheximide agar), NSCAA (NSCA with nitrofurantoin and vancomycin), and SMA-medium (starch-methionine agar with cephalexin and nitrofurantoin). Serological assays such as enzyme immuno-assays, Ouchterlony double diffusion, agglutination and immunofluorescence microscopy (IF) can be used for identification of pure cultures of X. c. pv. campestris . Cross-reactions with other pathovars of X. campestpis have, however, been reported. So far, IF using polyclonal antisera was the only serological technique employed for detecting X. c. pv. campestris in seed washings.The aim of this study was to analyse important characteristics of IF and plating assays, and to improve their use for identification and detection of X. c. pv. campestris in crucifer seeds. Methods for the detection and identification of X. c . pv. campestris are reviewed in chapter 2. In chapter 3 some aspects of plating assays for isolation of Xanthomonas campestris pv. campestris from crucifer seeds are discussed. Little differences were found between results obtained with NSCA, NSCAA and FS medium. It was, however, noted that the performance of the media often depends on the seed lot and extraction method used. Therefore, probably other methods such as immunofluorescence microscopy (IF) are needed to confirm presence of X. c. pv. campestris with higher certainty. With the extraction methods 2.5 h shaking and 1.5 h soaking, more colonyforming units were recovered from some seed lots than with the standard 5 min shaking of seed lots. However, prolonged extraction did not result in finding more seed lots infected. However, the use of two methods for extracting X. c. pv. campestris from crucifer seed, will enhance the chance of isolating the pathogen from the seed (chapter 3 and 6).In chapter 4 the specificity of polyclonal antisera and monoclonal antibodies for identification of X. c. pv. campestris is discussed. Polyclonal antisera reacted in IF with all strains of X. c. pv. campestris and other xanthomonads (e.g. X. c. pv. vesicatoria and amoraciae ) at low dilution (1:100). Non-xanthomonads also reacted with 2 out of 3 polyclonal antisera at this dilution. At higher dilutions (1:900), however, most crossreactions with non-xanthomonads disappeared as well as reactions with some strains of X. c. pv. campestris and other pathovars. Six monoclonal antibodies (MCA 17C12, MCA 16B5, MCA 20H6, MCA 2F4, MCA 18G12, MCA 10C5), produced against X. c. pv. campestris were tested in immunoblotting (IB), an enzyme immunoassay (EIA), dot-blot immunoassay (DBI) and IF. The monoclonal antibodies reacted with the lipopolysaccharide (MCA 20H6, 2F4, 18G12, and IOC5) or membrane proteins (MCA 17C12 and 16B5) of X. c. pv. campestris in IB Two monoclonal antibodies (MCA 17C12 and 16B5) reacted with all xanthomonads tested in DBI, but no

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