Techniques for Distinguishing Strains of Xanthomonas campestris pv. Campestris (Xcc) for Seed Testing

1. The focus here is on best techniques for distinguishing strains of the same species.

2. Introduction: • Xanthomonas campestris pv. Campestris (Xcc) causes black rot of crucifers. • Mode of disease transmission: seed-borne • Other X.c. pathovars are seed-borne too. • What is the best way to test seed stocks for strains of Xcc versus other Xc pathovars that may not cause as severe crop damage? • Specifically, how does BioLog, FAME and pathogenicity testing compare with repPCR? • What is the strain diversity in Tanzania where black rot of cabbage is a concern?

3. Methods • Isolation of putative Xc • Confirmation • Phenotypic tests/stains • ELISA • Pathogenicity bioassays • Comparison of Strain Methods • BioLog • FAME • repPCR • Cluster Analysis

4. Indirect Enzyme Linked Immuno-Sorbance Assay (ELISA) • Cell suspension of known density is added to 96-well titer plate and dried to stick cells to plastic of wells. • Remaining exposed plastic surface of wells blocked with milk proteins. (Most proteins will bind to plastic by hydrophobic interactions; antibodies are proteins.) • Primary monoclonal antibody (made by mouse B-lymphocytes) targets a Xcc cell surface antigen ; then excess rinsed. Y Y Y Y Y Y Y Y

5. 4)Secondary antibody targets mouse antibody; Alkaline phosphatase (AP) is covalently bound; excess rinsed away. 5) React with p-nitrophenol phosphate (PNPP) as substrate for AP; p-nitrophenol product is yellow. 6) Color intensity read in a “plate reader” and is equated to amount of antigen (Xcc cells in this case). Controls and standard known amounts of antigen are reacted at the same time/plate as samples. Y Y Y Y * * * * * * * * Y Y Y Y Y Y Y Y * * Y Y Y Y Y Y Y Y Y Y Y Y Y Y

6. Polymerase Chain Reaction (PCR): Specific target sequences of DNA (e.g., gene for cloning) found in very low levels in a sample can be amplified to billions of copies to use in further manipulation (e.g., gene cloning, DNA fingerprinting, genetic screening). • Repeated cycles of replication for a specific DNA sequence will exponentially increase copies on only that DNA sequence. N=2n • Each cycle has 3 major steps: • Denaturing DNA from helix to single strands. • Annealing primers; one specific to each end of the target DNA sequence. • Extension of new DNA strand by a heat tolerant DNA Polymerase (from a thermophilic bacterium)

8. Repetitive Element PCR(repPCR) • PCR amplification of spacer fragments lying between repetitive elements of the genome by use of one or two outwardly-directed primers at high stringency. • ERIC PCR (enterobacterial repetitive intergenic consensus); not used here. • BOX PCR (154 bp BOX element) • REP PCR (repetitive extragenic palindrome)

9. Results: • 111 of 127 pathogenic but symptoms varied. • 45 of 104 FAME tested were identified as Xcc. • FAME dendrogram found two main clusters. • BioLog identified 41 of 89 only to genus • BioLog got 42 to a Xcc pathovar type • BioLog clustered into two groups. • Two repPCR protocols were reproducible. • 7 BOX groups; one dominant (B1, ~70%) • B1 REP-PCR gave 5 groups; mostly R2 or R1.

11. Conclusions: • Most isolates caused “true” black rot symptoms, versus “blight”. • Reference strain B147 not Xcc! • FAME-MIS worked well to species, but was limited in identifying pathovars. • BioLog was OK to species but not pathovar. • repPCR fingerprints linked to geographic regions in Tanzania, Africa. • No strong relationship between rep-PCR, FAME and pathogenicity for Xcc. • Unique strains exist in Tanzania. • Local diversity matters in assessing plant resistance.