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

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Introduction
Introduction: strains of the same species.

  • 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?


Methods
Methods strains of the same species.

  • Isolation of putative Xc

  • Confirmation

    • Phenotypic tests/stains

    • ELISA

    • Pathogenicity bioassays

  • Comparison of Strain Methods

    • BioLog

    • FAME

    • repPCR

  • Cluster Analysis


Indirect enzyme linked immuno sorbance assay elisa
Indirect Enzyme Linked strains of the same species.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.

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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.

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Polymerase chain reaction pcr
Polymerase Chain Reaction (PCR): phosphatase (AP) is covalently bound; excess rinsed away.

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)


Repetitive element pcr reppcr
Repetitive Element PCR phosphatase (AP) is covalently bound; excess rinsed away.(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)


Results
Results: phosphatase (AP) is covalently bound; excess rinsed away.

  • 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.


Conclusions
Conclusions: phosphatase (AP) is covalently bound; excess rinsed away.

  • 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.


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