Figure 1.
Detection of starch accumulation by lugol staining.
A. 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. 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.
Table 1.
Protein content of healthy and ZC affected plants.
Figure 2.
SDS-PAGE of proteins stained with Coomasie brilliant blue.
A. Profile comparison of upper stem (US) of ZC diseased plants with tubers (T) of healthy or ZC plants. 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.
Figure 3.
A Solanum sogarandinum cyclophilin antibody was used for detection of cyclophylin in ZC and healthy potato plants. A. 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. 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.
Figure 4.
Detection of patatin proteins in healthy and ZC potato plants.
I. 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. II. Coomasie loading control. HS represents healthy plant stem; HT, healthy tuber and ZS, ZC plant stem.
Figure 5.
Analysis of total phenolic compounds in ZC potato stems.
A. Total phenolic compounds were analyzed using the Folin-Ciocalteu method. B. Results of lugol staining on the stems of the tissues used for phenolic content assays. C. Results of frying test performed on tuber slices of the same plants samples used in B. H, healthy stem and chips, ZC, ZC diseased.
Figure 6.
Temporal characterization of psyllid colonies.
Percentage of psyllids positive for the presence of Candidatus Liberibacter solanacearum (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.
Figure 7.
Relative expression of specific gene amplicons by RT-qPCR.
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 Ca. Liberibacter solanacearum 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 Candidatus Carsonella ruddi or Wolbachia sp.
Table 2.
Percentage of C1 psyllids with detectable levels of CLs.
Figure 8.
Symptoms of potato plants exposed to C1 psyllids with a low–CLs content.
Differences between caged plants with or without psyllids containing low amounts of CLs. A shows a caged control potato plant, non-exposed to psyllids and B, C and D are potato plants exposed to low-CLs psyllids. Symptoms observed vary from curling up of new leaves (B), Psyllid yellows-like symptoms (C) and formation of aerial tubers (red arrow, D).
Figure 9.
Nested PCR screening of plants exposed to low-CLs psyllids.
Nested PCR for the amplification of a 580 bp 16S rDNA amplicon was conducted in all DNA samples for Treatment I and II. Plant tissue was collected at 5 and 12 weeks from the initiation of the experiment. IA – IH and IIA-IIF, DNA samples of plants exposed to psyllids; samples II and IIG, DNA samples of control plants non-exposed to psyllids. Molecular markers are indicated in kilo bases (Kb).
Table 3.
Lugol staining of potato stems.
Table 4.
Assessment of CLs in individual psyllids.
Figure 10.
Disease progression on potato plants after psyllid exposure.
A. Atlantic plants exposed for 2.5 weeks to low-CLs psyllids, no drastic symptoms were observed, and plants appear normal at the end of experiment. B. Atlantic plants exposed for 9 weeks with the low-CLs psyllid colony, disease progression is visible by leaf edges curling up proceeded by a strong psyllid yellows like symptoms. C. Atlantic plants exposed 2.5 weeks to high-CLs psyllids. At the time of insecticide treatment the plants have a normal appearance, but 2 weeks later the symptoms develop, the plant collapses and dies 3 weeks later. Plants caged without psyllids are labeled as control, and the time points shown (2.5, 5 and 9 weeks) are in weeks after psyllid inoculation.