Temporal characterization of psyllid colonies.

<p>Percentage of psyllids positive for the presence of <i>Candidatus</i> Liberibacter <i>solanacearum</i> (CLs) were determined by conventional PCR of a 16S rDNA specific region at irregular time points (T1 to T8). The colonies evaluated were C1, a psyllid colony with low titer of CLs, and C2 and C3, which were known to contain higher titers of CLs. The average (Avg) and the standard deviation (STDEV) for each colony were calculated and are shown above each group of time points. Y axis, percentage of positive psyllids; X axis, time points. Sample dates separated by one or more days.</p

Cyclophylin detection.

<p>A <i>Solanum sogarandinum</i> cyclophilin antibody was used for detection of cyclophylin in ZC and healthy potato plants. <b>A.</b> Western blot analysis showing cyclophilin (17.5 kDa, indicated by arrow) detection in both stem (S) and tubers (T) of healthy (H) tissues, but no detection in tubers of Zebra complex (Z) diseased plants. <b>B.</b> Coomassie brilliant blue stained gel of the protein samples used for the western blot in A, the protein concentration was calculated by Bradford assay and equal amounts of proteins (7.5 ug) were loaded. Size markers are indicated.</p

SDS-PAGE of proteins stained with Coomasie brilliant blue.

<p><b>A</b>. Profile comparison of upper stem (US) of ZC diseased plants with tubers (T) of healthy or ZC plants. <b>B</b>. Profile comparison of the root (R) and stem (S) tissue of ZC diseased (Z) and healthy (H) plants, a and b depict the differentially expressed proteins; (a) contains two proteins, cyclophilin and a putative transcription factor BTF-3 and (b) represents glycoprotein-like product. Approximately 7.5 ug of protein per lane were loaded and run in a 15% polyacryalmide gel, size markers in kDa are shown on the edges.</p

Protein content of healthy and ZC affected plants.

*<p>Bradford assays were used to determine the protein concentration of healthy and ZC affected stem tissue. The plant samples were measured in three replicates.</p>**<p>A statistical analysis one-way ANOVA using SPSS 14.0 and Tukey mean comparison with P = 0.01 showed that the ZC diseased plants have a significant increase of protein content per gram of tissue compared to healthy potato plants. The potato plants were sampled in the field and were of different varieties (FL1867, Norkotah or Norgold).</p

Assessment of CLs in individual psyllids.

*<p>Percentage of psyllids positive for CLs are represented in this table.</p

Relative expression of specific gene amplicons by RT-qPCR.

<p>Columns represent the different microbial titers in the potato psyllid vector and bars denote average values of three technical repeats for individual psyllids normalized to psyllid 28S rDNA. C1 and C2 correspond to the psyllid colonies with low- and high-CLs abundance, respectively. Notice that the values for the <i>Ca</i>. Liberibacter <i>solanacearum</i> in colony 1 (C1) are extremely low and therefore are shown on a 1/1000 fold scale compare to the other graphs. The average and SDEV for each group of samples were calculated and shown in boxes above their respective graphs. A statistical analysis one-way ANOVA using SPSS 14.0 and Tukey mean comparison with P = 0.01 showed that there are significant statistical differences in the CLs titers of C1 and C2 psyllid colonies, but no differences between titers of <i>Candidatus</i> Carsonella <i>ruddi</i> or <i>Wolbachia sp</i>.</p

Percentage of C1 psyllids with detectable levels of CLs.

*<p>Transmission studies were performed with the low-CLs psyllid colony. A conventional and nested PCR was used to assess CLs percentage in sampled population at 5 and 12 weeks of plant exposure to psyllids.</p

Lugol staining of potato stems.

<p>Potato stem - tops were sliced and reacted with lugol solution to determine presence of starch. Positive (+) indicates that tissue reacted with the lugol solution displaying a dark blue color, and negative (−) indicates that the tissue remained the same color.</p

Detection of starch accumulation by lugol staining.

<p><b>A</b>. Typical starch accumulation pattern in different plant tissues of healthy potato varieties. Upper portion of the stem (US), lower portion of the stem (LS). <b>B</b>. Lugol staining of the upper stem (US) of potato FL1867 chipping variety affected by ZC. H1 and H2 are the healthy controls. The results of frying test performed on tuber slices performed are shown in the lower panel.</p

Detection of patatin proteins in healthy and ZC potato plants.

<p><b>I.</b> Alkaline phosphatase western blot using a patatin-specfic antibody crossreacting with two bands; a class I patatin of 40 kDa (arrow) and a class II patatin (*), size markers in kDa. <b>II.</b> Coomasie loading control. HS represents healthy plant stem; HT, healthy tuber and ZS, ZC plant stem.</p