Development of a qPCR assay for Xf in planta
A qPCR assay was designed to detect and quantify Xf from grapevine tissue. The highly conserved target gene, EFTu was chosen. Figure 1A shows a multiple sequence alignment of EFTu across the target region. Included are two Xf strains, 9a5c and Temecula, and three sequences from closely related Xanthomonas species. This region contains 100% sequence identity between these Xf strains and extensive base pair mismatching with closely related Xanthomonas species across both the forward and reverse primers, and the probe (Fig. 1A, shading). When total genomic DNA from Xf (Temecula) and the Xanthomonas species are used as templates for traditional PCR, utilizing the primer pair described above, no amplification of the Xanthomonas isolates occurred (Fig. 1B).
Three methods of isolating DNA from combinations of plant tissue and known concentrations of Xf were compared with dilutions of Xf genomic DNA isolated from culture in order to determine the most robust procedure (Fig. 2). The assay was extremely reliable, resulting in r2 values from 0.970 to 0.998 regardless of the DNA isolation method used. The variability that was present increased with decreasing concentration as would be expected. The cycle thresholds for any given concentration of Xf cells were greater for all of the preparation methods than for Xf DNA from culture. More simply, in all cases, the presence of plant tissue resulted in a decrease in the sensitivity of the assay. The Qiagen Plant extraction method was chosen because of its low background and similar behaviour across the full range of Xf concentrations (Fig. 2, closed triangles). The assay was also very sensitive. Theoretically the assay was able to detect plant extract. Table 1 shows the cycle thresholds obtained for each individual run, and the mean cycle thresholds and standard deviation of these values across all runs. With standard deviations that are less than a third of a cycle, the standard curve was highly reproducible.
Relationship between Xf population and leaf-scorch symptoms
Xf-inoculated greenhouse-grown grapevines began exhibiting symptoms at 60 DAI. Xf was detected in 94% of leaves, but only 51% of the individual leaf punch/petiole samples. Bacterial populations in leaf discs were found ranging anywhere from as little as 102 to as much as 109 cells g–1 tissue. Xf populations across leaf lamina were relatively homogenous in some leaves (Fig. 3E, Q) and extremely variable in others (Fig. 3J, N). Two of the leaves analysed were leaves whose petioles were inoculated originally. Clearly these leaves harboured much greater populations of bacteria across the entirety of the lamina (Fig. 3P, Q). The relationship between bacterial population and the severity of leaf-scorch symptoms was also highly variable. For example, some of the leaves that exhibited the most severe symptoms had almost no detectable Xf (Fig. 3O and R), while other less symptomatic leaves harboured higher concentrations (Fig. 3C, F).
Populations were averaged across each leaf and these averages were compared with leaf-scorch symptoms. When all leaves were considered there was a weak but positive relationship between the average Xf population and symptom severity (Fig. 4A, r2=0.21, P=0.056). When examined more closely this relationship was established almost entirely by the high average populations in the two inoculated leaves (Fig. 4A, circled). If these leaves were not considered, regression analysis resulted in an insignificant r2=0.015 (P=0.073). The average population across all leaves increased significantly between 76 DAI and 91 DAI (Fig. 4B, P increased almost 10-fold from 2.9 to 3.7 log10 cells g–1 in 15 d.
Field-grown Chardonnay grapevines were identified that exhibited all of the hallmarks of Xf infection, including shrivelled fruit, leaf-scorch symptoms, petiole matchsticks, and green islands (Fig. 5, white arrows). Whole plants were designated as exhibiting either severe or moderate symptoms as defined by the overall extent of scorched and abscised leaves (Fig. 5A severe, B moderate), and an equivalent number of leaves exhibiting the spectrum of symptom severities was harvested from each plant (Fig. 5C, D). Bacterial concentrations were determined for individual leaves in a combined tissue sample that included all five primary veins and a 1-cm portion of the petiole. In these field samples, Xf was detected in only 30% of leaves, a much lower percentage than was found in greenhouse experiments. Bacterial populations were more uniform among samples, ranging from 104 to 106 cells g–1 tissue (Fig. 5C, D), than among leaves in the greenhouse experiments. Comparing symptom severity and Xf concentration in individual leaves, regression analysis resulted in an r2=0.09, demonstrating that similar to greenhouse experiments there was no significant relationship (P=0.115). However, when whole plants are considered, plants exhibiting severe symptoms had a higher percentage of Xf-positive leaves (Fig. 5E). Forty-seven percent of leaves tested positive for the presence of Xf in plants exhibiting severe symptoms, while only 13% of leaves tested from plants with moderate symptoms tested positive. In addition, average Xf populations across all leaves were greater in the plants exhibiting severe symptoms (P