Immunofluorescence microscopy and dilution-plating for the detection of Xanthomonas campestris pv. campestris in crucifer seeds : methods to determine seed health and seed infection

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 not in IF and EIA. The other monoclonal antibodies (MCA 20H6, 2F4, 18G12, and 1OC5) did not react with all strains of X. c. pv. campestris and did react with some other xanthomonads, such as X. c. pv. vesicatoria and amoraciae, in IF, EIA and DBI. It was concluded that some polyclonal antisera and monoclonal antibodies may be used for identification of (a group of) strains of X. c. pv. campestris. The question, whether monoclonal antibodies, as compared to a polyclonal antiserum, may give rise to false- negative or false-positive results when testing seed lots, is dealt with in chapter 5. IF with one polyclonal antiserumand monoclonal antibodies was used for direct detection of X. c. pv. campestris in seed washings and identification of colonies. For direct detection of X. c. pv. campestris in seed washings, it was found that IF-results (cell counts) depend in part on the seed lot tested, the extraction method and antibodies used. This confirmed the interactions found in plating assays. The polyclonal antiserum tested (PCA 94) did not always result in detection of more fluorescent cells in IF. On the contrary, for some seed lots and extraction methods the opposite was found; monoclonal antibodies sometimes detected more fluorescent cells than polyclonal antisera. When using two extraction methods (5 min and 2.5 h shaking at room temperature) for one seed lot the risk of false-negative results in IF could be minimized (chapter 5 and 6). When monoclonal antibodies were used for identification of colonies, it was found that some X. c. pv. campestris colonies did not react with antibodies MCA 16B5, MCA 17C12, and to a lesser extent with MCA 1OC5, MCA 2F4, and MCA 18G12. Saprophytic isolates of one seed lot cross-reacted with MCA 17C12 and to a lesser extent with MCA 2F4, 18G12 and PCA 94. On the basis of this study MCA 20H6 was considered to be the most suited antibody for detection and identification of X. c. pv. campestris .Chapter 6 shows that the correlation between IF and dilution-plating was similar for one monoclonal antibody (MCA 20H6) and one polyclonal antiserum tested (PCA 94). With increasing cell numbers in IF the chance of isolating X. c. pv. campestris also increased. With IF generally much more seed lots were found positive than with dilution-plating. It was shown that the correlation between IF and dilution-plating depended in part on the volume of seed extract examined. When examining relatively large volumes (e.g. 50 μl), the sensitivity of IF will be enhanced and the risk of false-negative reactions in IF as compared to dilution-plating will be decreased. On the other hand the correlation with dilution-plating decreased. It was concluded that IF generally gives a good prediction of 'health' of a seed lot and that dilution-plating generally gives a good prediction of 'disease'. Chapter 7 summarizes the problems with serological techniques for seed-borne bacteria and gives possible solutions to be worked out in future.To minimize the risk of false-negative results, it is advisable to use at least two plating media (e.g. NSCAA and CS20ABN) in dilution-plating and two extraction methods (5 min and 2.5 h shaking at room temperature) in both IF and dilution-plating. For IF, it is advisable to screen either with different monoclonal antibodies separately, with a mixture of monoclonal antibodies (e.g. MCA 20H6, MCA 2F4 and MCA 18G12), or both a monoclonal antibody (MCA 20H6 or 2F4) and a high quality polyclonal antiserurn (PCA 94).More research is needed, however, to assess the significance of interference by saprophytes and antibiotic substances, released by the seed or saprophytes, with the detection assay and the survival of the pathogen in the seed