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.