Legends
Fig. 1. Symptoms of phytoplasma infected apple, pear and peach trees. (a) The apple proliferation (AP) induced by ‘Candidatus Phytoplasma mali’ led to typical disease symptoms like witches’ broom, enlarged and highly serrated stipules. The leaf size ratio (length width-1) and the midrib ratio (diameter midrib width-1) did not show any effects.(b) The leaves of ‘Candidatus Phytoplasma pyri’ affected trees inducing pear decline (PD) were characterized by premature foliar reddening and a significant decrease of the leaf size ratio but not by an impact on the midrib. (c) The leaves of ‘CandidatusPhytoplasma prunorum’ infected trees inducing European stone fruit yellows (ESFY) showed chlorosis, and a significant increase of the leaf size and midrib ratio. Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from mixed effect models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.
Fig. 2. Analysis of morphological leaf parameters in healthy and phytoplasma infected apple trees. (a) The infection of apple trees with a virulent classified ‘Candidatus Phytoplasma mali’ accession (3/6) was investigated with the morphology of the leaf main vein in the midrib. (b) The morphological analysis consisted of the vascular bundle area, the xylem area, the phloem area, the sieve element area, the xylem/phloem ratio and the sieve element/phloem ratio and showed a significant decrease for nearly all studied parameters in AP infected trees but not for the sieve element/phloem ratio. Box-whisker plots with median as lines and jittered raw values as closed circles (corresponding to each measurement). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from mixed effect models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.
Fig. 3. Analysis of morphological leaf parameters in healthy and phytoplasma infected pear trees. (a) The infection of a pear tree with ‘Candidatus Phytoplasma pyri’ inducing pear decline (PD) was studied by the morphology of the leaf main vein.(b) The morphological analysis consisted of the vascular bundle area, the xylem area, the phloem area, the sieve element area, the xylem/phloem ratio and the sieve element/phloem ratio and showed no significant changes. Box-whisker plots with median as lines and jittered raw values as closed circles (corresponding to each measurement). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from mixed effect models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.
Fig. 4. Analysis of morphological leaf parameters in healthy and phytoplasma infected peach trees. (a) The infection of a peach tree with ‘Candidatus Phytoplasma prunorum’ inducing European stone fruit yellows (ESFY) was studied by the morphology of the leaf main vein. (b) The morphological analysis consisted of the vascular bundle area, the xylem area, the phloem area, the sieve element area, the xylem/phloem ratio and the sieve element/phloem ratio and showed significant decreases for the sieve element areas and ratios of sieve element to the phloem. Box-whisker plots with median as lines and jittered raw values as closed circles (corresponding to each measurement). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from mixed effect models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.
Fig. 5. Analysis of translocation in phloem sieve elements of healthy and phytoplasma infected apple, pear and peach trees. The translocation was examined with the determination of the velocity of the phloem mass flow (cm h-1) using fluorescence and with the calculation of volumetric flow rates (cm³ h-1) in mean single sieve elements. Both parameters were individually determined for (a) apple, (b) pear and (c) peach trees. Apple trees were infected with ‘Candidatus Phytoplasma mali’ inducing apple proliferation (AP). Pear trees were infected with ‘Candidatus Phytoplasma pyri’ causing pear decline (PD) and peach trees were infected with ‘Candidatus Phytoplasma prunorum’ inducing European stone fruit yellows (ESFY). Infected apple trees (AP) showed a significant decrease of phloem mass flow velocity and volumetric flow rates in contrast to infected pear trees (PD) where a significant rise was observed. In infected peach trees (ESFY) the phloem mass flow velocity was not affected but the volumetric flow rate decreased significantly. Box-whisker plots with median as lines and jittered raw values as closed circles (corresponding to each measurement). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from mixed effect models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.
Fig. 6. Analysis of callose deposition in the leaf phloem tissue of healthy and phytoplasma infected apple, pear and peach trees. At cross sections of the leaf mid rip, the callose deposition in sieve elements was stained with aniline blue and detected via fluorescence microscopy (see panels on the left side). The callose fluorescence was quantified after subtracting auto-fluorescence (see panels on the right side). (a) In apple trees, an infection with the virulent accession (3/6) inducing apple proliferation (AP) did not show any differences in the callose deposition in comparison to healthy plants.(b+c) The phytoplasma infection of pear trees (PD) and peach trees (ESFY) induced a significant (p<0.05) increase of callose deposition in sieve elements. Box-whisker plots with median as lines and jittered raw values as closed circles (corresponding to each measurement). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from generalized least square models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.
Fig. 7. The phytohormone balance in healthy and phytoplasma infected apple, pear and peach trees. The concentrations (ng gFM-1) of several phytohormones – salicylic acid (SA), jasmonic acid-iso leucine (JA-Ile), jasmonic acid (JA) and abscisic acid (ABA) – were measured in the leaves of healthy and phytoplasma infected (a) apple, (b) pear and(c) peach. For apple, a virulent accession (3/6) was considered inducing apple proliferation (AP). Pear trees showed pear decline (PD) and peach trees showed the European stone fruit yellows (ESFY). Box-whisker plots with median as lines and jittered raw values as closed circles (corresponding to each measurement). Boxes represent the interquartile range (IQR) and whiskers extend to 1.5*IQR. Bars represent the 95% confidence intervals with the estimated marginal means obtained from linear models as dots (both back transformed to the response scale). Letters indicate statistical differences between EMMs of groups at the 0.05 significance level.