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

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.