Neighbor-Joining Tree based on the concatenated sequences of four housekeeping genes of several <i>Xanthomonas</i>.

<p>The sequences of the housekeeping genes <i>atpD</i>, <i>dnaK</i>, <i>efp</i> and <i>gyrB</i> were concatenated and used to infer the MLST profile of <i>X. euvesicatoria</i> and <i>X. vesicatoria</i> strains used in this study, which are highlighted in yellow. The Neighbor-Joining tree was derived from the TN93+G+I model and a bootstrap analysis of 1000 replicates.</p

Dot blot validation of selected probes.

<p>Nine probes were evaluated with total DNA from a collection of BSX, consisting of 19 <i>Xeu</i>, five <i>Xv</i>, three <i>Xg</i> and two <i>Xp</i> strains. Probability values, obtained with a customized MATLAB algorithm for the automatic data analysis, are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037836#pone-0037836-t003" target="_blank">Table 3</a>.</p

PCR validation.

<p>The selected primer-pairs were tested for efficiency using eight different <i>Xeu</i> strains. For each assay, three different annealing temperatures were tested: 57°C, 59°C and 61°C.</p

Detection of BSX in infected plant material using an inverted dot blot platform.

<p>Crude bacterial suspensions, obtained from tomato and pepper plants leaves after one and two weeks of infection with <i>Xeu</i> 905, were used as templates for PCR enrichment using the markers' primer pairs. PCR products corresponding to each plant were labeled with Digoxigenin and used as probes. Purified DNA from <i>Xeu</i> 905 was used as positive control. Negative controls consisted of tomato plants infected with <i>Pst</i> DC3000 for 2 weeks and uninfected plants. The raw ChemiDoc captures and processed images, using the automatic image analysis algorithm, are shown.</p

Detection of BSX in infected plant material using a duplex PCR (markers XV7 and XV11).

<p>Tomato and pepper plants inoculated with <i>Xeu</i> 905, <i>Xv</i> 919, <i>Xg</i> 962 and <i>Xp</i> 4321were processed after one and two weeks to obtain crude bacterial suspensions used as PCR templates. Plants inoculated with <i>Pst</i> DC3000 were used as controls. M – DNA marker (GeneRuler DNA Ladder Mix); Ø-Duplex PCR using distilled water as template; C- healthy tomato and pepper plants.</p

List of bacterial strains used in this study.

*<p>LMG-Belgian Co-Ordinated collections of micro-organisms, Gent, Belgium; CPBF-Colecção Portuguesa de Bactérias Fitopatogénicas, Lisboa, Portugal; NCPPB-National Collection of Plant Pathogenic Bacteria, York, United Kingdom.</p>a<p>- Bacterial spot-causing xanthomonads (BSX);</p>b<p>- Pathovar reference strain;</p>c<p>- Type strain; NM- Not mentioned.</p