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Molecular detection and quantification of Xanthomonas arboricola pv. juglandis, the causal agent of walnut blight

James Adaskaveg: Department of Microbiology and Plant Pathology, University of California

<div>Walnut blight, caused by <em>Xanthomonas arboricola</em> pv. <em>juglandis</em> (<em>Xaj</em>), is a serious disease worldwide. Identification of the bacterium is limited to using morphological and biochemical methods that are time- and labor-intensive. Identification of <em>Xaj</em> on isolation plates is often difficult because multiple bacteria of similar appearance to <em>Xaj </em>are often recovered from walnut tissues (<em>Arthrobacter, Curtobacterium, Frigoribacterium, Microbacterium </em>spp.). Using a whole genome sequence of an <em>Xaj</em> strain in GenBank, we identified a gene conserved in <em>Xaj</em> that was not shared with closely related <em>X. arboricola </em>pvs. <em>pruni </em>and <em>corylina</em>. Sequence alignment using BLASTN resulted in exclusive matches with <em>Xaj </em>strains. A TaqMan-based probe was designed for the gene sequence. In qPCR, 9 <em>Xaj</em> strains that were genetically diverse based on Rep-PCR resulted in a positive signal, whereas 15 strains of other genera listed above that were morphologically similar to <em>Xaj</em> did not amplify. A standard curve that was constructed using walnut bud tissue spiked with 10-fold dilutions of <em>Xaj</em> resulted in a detection threshold of 10 cells/µl. The standard curve had an r<sup>2 </sup>value of 0.999. Quantification of <em>Xaj</em> in walnut buds as determined by qPCR Ct values corresponded with the number of <em>Xaj</em> cells recovered after isolation. These results indicate that the TaqMan probe specifically and sensitively detected <em>Xaj</em> in walnut tissue and can be used to identify and quantify the pathogen rapidly and efficiently.</div>