Download Photosynthetic and leaf water potential responses

Tree Physiology 28, 1703–1711
© 2008 Heron Publishing—Victoria, Canada
Photosynthetic and leaf water potential responses of Alnus glutinosa
saplings to stem-base inoculaton with Phytophthora alni subsp. alni
CHRISTIAN CLEMENZ,1 FRANK FLEISCHMANN,1 KARL-HEINZ HÄBERLE,2 RAINER
MATYSSEK2 and WOLFGANG OßWALD1,3
1
Phytopathology of Woody Plants, Technische Universität München, Am Hochanger 13, 85354 Freising, Germany
2
Ecophysiology of Plants, Technische Universität München, Am Hochanger 13, 85354 Freising, Germany
3
Corresponding author ([email protected])
Received April 7, 2008; accepted July 7, 2008; published online September 2, 2008
Summary Three-year-old Alnus glutinosa (L.) Gaertn. (alder) saplings were single or double inoculated at the stem base
with Phytophthora alni subsp. alni Brasier & S.A. Kirk under
natural climatic conditions. Lesion formation on the bark
showed a biphasic pattern of development, with extension occurring at a moderate rate in spring, and more rapidly during
late summer. However, large variability was encountered in
pathogen development within the population of infected
saplings, ranging from high susceptibility to almost complete
resistance. Infection resulted in severe growth retardation, and
death within two years of inoculation in 75% of the saplings.
During disease development, rates of transpiration and CO2
uptake were significantly reduced. Consequently, minimum
leaf water potentials were less negative in infected saplings
than in control saplings. Surviving saplings matched control
trees in photosynthetic capacity, transpiration rate and water
potential during the second year of infection. Leaf starch concentration of infected saplings was significantly higher than in
control saplings, possibly indicating that the destruction of
bark tissue by the pathogen impaired phloem transport from
leaves to roots.
Keywords: photosynthesis, starch, stem inoculation, twig water potential.
Introduction
Phytophthora is a genus of soil-borne pathogens to which
many herbaceous and woody plants are susceptible (Erwin and
Ribeiro 1996, Oßwald et al. 2004). Recently, new aggressive
strains of Phytophthora ramorum Werres, De Cock & Man in’t
Veld have been reported to cause death of bark-infected adult
Lithocarpus densiflorus (Hook. & Arn.) Rehder trees and
trees of several American Quercus species (Werres et al. 2001,
Rizzo et al. 2002). Earlier, Gibbs (1995) drew attention to unusual mortality of Alnus glutinosa (L.) Gaertn. (alder) in
southern Britain. Dying trees were mainly found on river
banks and were characterized by leaf loss, crown dieback and
cankers, mostly spreading from roots to the trunk (Jung et al.
2000). Brasier et al. (1995) showed the disease to be caused by
a hitherto unknown Phytophthora species similar to
P. cambivora (Petri) Buisman, and thought to be a hybrid of
P. cambivora and an unknown taxon similar to P. fragariae
Hickman (Brasier et al. 1999). This pathogen was described
by Brasier et al. (2004) as Phytophthora alni sp. nov., and its
naturally occurring variants were classified in three subspecies: P. alni subsp. alni Brasier & S.A. Kirk, P. alni subsp.
uniformis and P. alni subsp. multiformis. Phytophthora alni is
found on Alnus glutinosa and A. incana (L.) Moench
throughout Europe (Hartmann 1995, Cech 1997, Werres 1998,
Streito et al. 2002, Jung and Blaschke 2004). Besides alder,
wild Prunus avium L. is susceptible to Phytophthora alni
(Santini et al. 2006). Recently, the genetic relationship between the different Phytophthora alni subspecies was elucidated by Ioos et al. (2006). According to their nuclear and mitochondrial DNA analyses, P. alni subsp. alni is a hybrid of
P. alni subsp. multiformis and P. alni subsp. uniformis. Recently, in Hungary, France and Belgium, specific primers from
randomly amplified polymorphic DNA fragments have been
developed (De Merlier et al. 2005, Ioos et al. 2005, Bakonyi et
al. 2006) to detect and discriminate among the subspecies of
P. alni in various substrates (e.g., river water, necrotic alder
bark and baiting leaves).
Phytophthora alni mainly infects fine roots or adventitious
roots, from which it extends to the trunk where it destroys
phloem and cambial tissues (Jung and Blaschke 2001). Infected plants, as in the case of plants infected by other Phytophthora pathogens, show typical wilt symptoms on aboveground organs before they die (Oßwald et al. 2004).
Sterne et al. (1978) investigated the influence of P. cinnamomi Rands on 8- to 10-year-old avocado trees under field
conditions. They found that transpiration of healthy trees
reached a mean maximum in early afternoon (at 1400 h) of
1.78 µg cm – 2 s – 1 in full sun, whereas transpiration of diseased
trees peaked between 1000 and 1200 h at a rate of only
0.2–0.5 µg cm – 2 s – 1, which hardly varied with solar irradi-
1704
CLEMENZ, FLEISCHMANN, HÄBERLE, MATYSSEK AND OßWALD
ance. They found that predawn and minimum leaf water potentials were more negative in infected trees than in control
trees and suggested that fine root destruction by the pathogen
limits water uptake, thereby inducing water stress. Ploetz and
Schaffer (1989) investigated the effects of the same host–parasite system on net CO2 assimilation and found significant reductions in rates of assimilation and transpiration, and in
stomatal conductance soon after infection. Similar effects of
different Phytophthora species on beech saplings (Fagus
sylvatica L.) were observed by Fleischmann et al. (2002). Subsequently, Fleischmann et al. (2005) demonstrated that CO2
assimilation and stomatal conductance of F. sylvatica decreased in response to the root pathogen Phytophthora
citricola Swada before declines in leaf water potential and the
development of wilt symptoms were observed.
Effects of Phytophthora pathogens on the water relations of
woody plants may be complex, depending on the extent of
phloem and xylem destruction. It was reported recently that,
besides invading phloem tissue, Phytophthora pathogens occur in the adjacent xylem where they reduce sap flux and sapwood specific conductivity (Brown and Brasier 2007, Parke et
al. 2007). Lowered conductivity may be accompanied by reduced water potential (e.g., Sterne et al. 1978). In this study,
we investigated the Alnus glutinosa–P. alni system with a
view to understanding how infection at the root collar causes a
reduction in leaf water potential and photosynthetic capacity.
Materials and methods
Field sampling, isolation and characterization of
Phytophthora alni
Phytophthora species were isolated from the margins of typical lesions at the stem base of infected Alnus glutinosa trees in
the field near Freising (Bavaria, southern Germany) according
to the method of Jung et al. (2000). The isolates were characterized by morphological features. The oogonia of all isolates
showed moderate ornamentation and two-celled amphygynous antheridia as described for P. alni subsp. alni (Brasier
et al. 2004). Total DNA was extracted from fresh mycelium of
all isolates using the Plant DNeasy Minikit (Qiagen, Hilden,
Germany). The ITS1, 5.8S and the ITS2 regions of the rDNA
were amplified by PCR with the primers ITS4 and ITS6 according to Brasier et al. (1999). After restriction digestion of
the amplified polynucleotides with the enzymes AluI, AciI,
EcoO109I, MspI and TaqI, nucleotides were separated on
NuSieve agarose gels. Elution profiles were identical for all
P. alni isolates and were comparable with those of the reference P. alni subsp. alni isolate ALN 377 (data not shown). One
isolate (ALN 403) was chosen for the inoculation experiments.
2.5 cm, respectively). All saplings were raised from seeds (Bavarian pre-alpine provenance, origin Landsberg am Lech, Germany). Trees were grown in 30-l containers in a substrate consisting of equal amounts of calcareous sand, vermiculite, sterile peat and sandy loam from an agricultural area. The sandy
loam used in the experiment was free of Phytophthora pathogens proven by baiting experiments of representative samples
(data not shown).
Saplings were inoculated at the root collar in April 2004 before buds flushed. Each experimental set of plants consisted of
six inoculated and six control saplings. Six saplings were each
inoculated at the stem base either on the north side (single
stem inoculation) or at the stem base on both the north and the
south side (double stem inoculation). Mycelial plugs of a
14-day-old culture of P. alni subsp. alni (ALN 403) grown on
carrot agar (CA) were inserted into holes in the bark to the
depth of the cambium, which were made with a sterile cork
borer. Sterile CA plugs were implanted into the bark of six
control trees for each inoculation variant. All inoculation
wounds were sealed with Parafilm, covered with moist gauze
and wrapped with aluminum foil. Lesion extension was assessed 12 times from April to October 2004 by measuring the
vertical and circumferential extent of the lesion visible at the
bark surface. Bark necrosis was most clearly visible after the
stem was moistened with sterile water. Phytophthora alni
subsp. alni was successfully re-isolated from bark shortly after
the saplings had died in 2004 and 2005.
The stem surface temperature of two inoculated saplings
was recorded at the stem base close to the inoculation site with
Thermor 1 NTC (UMS, Munich) sensors. Saplings were watered each evening with equal amounts of tap water throughout
the experiment, but they were not fertilized.
Measurements of leaf photosynthesis, transpiration and twig
water potential
Rates of net photosynthesis and transpiration of upper-crown
leaves of each sapling were measured at least twice per month
from May to mid August 2004 with a CO2 /H2O diffusion
porometer (LI-6400, Li-Cor). Only healthy, completely green
leaves were chosen for investigations. Gas exchange measurements were conducted between 1000 and 1500 h (CET) at a
saturating photosynthetic photon flux of 1500 µmol m – 2 s – 1,
with a leaf temperature of 20–25 °C and relative humidity in
the porometer chamber of 35 to 45%. During measurements,
the CO2 concentration of the incoming air was held at 360 ppm
with a gas-mixing system. Twig xylem water potential was
measured before 0400 h (predawn) and at 1400–1500 h (minimum potential) on July 31, 2004 with a Scholander pressure
chamber (Eijkelkamp, Giesbeek, The Netherlands) in parallel
with the ninth set of photosynthetic measurements.
Extraction and quantification of starch from alder leaves
Inoculation experiments
Single and double stem inoculation experiments were conducted at the same time under natural climatic conditions on
24 healthy 3-year-old Alnus glutinosa container-grown
saplings (with a mean height and diameter of 1.7 m and
In June and August 2004, five healthy leaves per sapling (adjacent to leaves selected for gas exchange measurements) were
collected and pooled. Starch was assessed in 20 mg of
freeze-dried and ground leaf material per tree as glucose
equivalents after enzymatic degradation of starch to glucose.
TREE PHYSIOLOGY VOLUME 28, 2008
ALNUS GLUTINOSA INFECTED WITH PHYTOPHTHORA ALNI
Soluble sugars were subsequently extracted three times with
hot water (60 °C, once with 1.0 ml, twice with 0.5 ml). After
centrifugation for 5 min at 10,000 g, the supernatant was removed. The remaining pellet was air-dried, resuspended in
1 ml of heat-stable amylase (500 U ml –1; Sigma, Germany)
and incubated for 30 min at 60 °C in a water bath. The suspension was centrifuged for 5 min at 10,000 g and 400 µl of the
supernatant was mixed with 300 µl amyloglucosidase (3 U
ml –1; Sigma) and incubated in a water bath at 37 °C overnight.
After a final centrifugation step (5 min at 10,000 g), the
supernatant was filtered (0.45 µm nylon) and stored at –25 °C
until analyzed. The glucose content of 20 µl of supernatant was
quantified by high performance liquid chromatography
(HPLC) with an Aminex HPX-87N column (Biorad, Germany) at 60 °C in combination with a refractive index detector
(RI-Monitor 1755, Biorad); 20 mM H2SO4 was used as the
mobile phase at a flow rate of 0.6 ml min –1.
Statistical analysis
Normal (i.e., Gaussian) distribution of datasets was verified by
the Kolmogorow-Smirnow test. Whenever necessary, data
were transformed (log or arc-sin transformation) to achieve
normal distribution. Comparison between treatments was performed with a t-test for independent samples. Depending on
the signficance of differences in the variances of compared
datasets (assessed by the F-test), means were compared by the
Welch’s test. Otherwise the Student’s t-test was used.
1705
portion of circumference that was necrotic.
The progress of girdling in the three saplings remaining
alive after two years is shown in Figure 2b. Two surviving saplings (Nos. 3 and 6) that received a single inoculation showed
a biphasic pattern of girdling in 2004, which was followed by a
smooth decrease from August 2004 to autumn 2005, with final
values for the two saplings of 8 and 31%. In comparison, the
surviving sapling that received a double inoculation (No. 5)
showed girdling values ranging between 90 and 95% throughout the whole vegetation period in 2004. However, from spring
to autumn 2005, the girdling values dropped sharply and
reached a final value comparable with those for the surviving
saplings in the single inoculation treatment. All saplings that
died showed girdling values between 95 and 100% in 2005
(data not shown).
One of six double inoculated saplings died in August 2004,
whereas four more died during leaf flush in 2005. Following a
single inoculation, three saplings died during bud break in
2005 and an additional sapling died one month later.
Where the bark of dead saplings was peeled close to the
stem lesions, the outer xylem tissue was slightly reddish in
Results
Vertical extension of Phytophthora alni subsp. alni caused
lesions in alder stem tissue
There was high heterogeneity in lesion development following
single and double stem inoculation with P. alni subsp. alni
(Figure 1). The responsiveness of individual alder saplings
ranged from high susceptibility to almost complete resistance.
The mean lesion growth rate (April to October) of the highly
susceptible saplings was 1.6 and 1.7 mm day –1 for the single
and the double inoculation treatment, respectively.
Figure 2a shows the amount of girdling (horizontal lesion
spread) was dependent on the time of single and double inoculation in 2004. Double inoculation of the pathogen at sites on
opposite sides of the stem caused more severe girdling than
single inoculation. Maximum girdling occurred about 40 days
after inoculation.
The time course of girdling in the single inoculation experiment exhibited a biphasic pattern with rapid inital expansion,
followed by a decrease from mid May to mid June and a second phase of expansion starting in July and reaching mean values of about 80% of stem circumference in August. Early
summer stagnation was not caused by the current stem surface
temperature, which from May to September 2004 remained
within the range of 5–28 °C, which is known to allow growth
of P. alni subsp. alni (Brasier et al. 2004). The calculated decrease in girdling from mid May to mid June is explained by
growth of uninfected parts of the stem, which reduced the pro-
Figure 1. Vertical lesion extension in the cortical tissue of Alnus
glutinosa saplings after (a) single and (b) double inoculation with
Phytophthora alni subsp. alni at the stem base. Values in (b) are given
as the sum of the lesion extensions on the opposite sides of the stem
divided by two. The identities of saplings still alive at the end of the
2005 growing season are underlined. Abbreviation: dpi = days post
inoculation.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
1706
CLEMENZ, FLEISCHMANN, HÄBERLE, MATYSSEK AND OßWALD
Figure 2. Comparison of cortical tissue destruction of Alnus glutinosa saplings by
Phytophthora alni subsp. alni. (a) Mean
girdling values at the stem base after single (䊊) and double (䊉) inoculation with
P. alni subsp. alni in 2004. (b) Progression
of girdling in saplings that survived to the
end of the 2005 vegetation season (single
inoculation: 䉭, No. 3; and 䊐, No. 6; and
double inoculation: 䊏, No. 5). Abbreviation: dpi = days post inoculation. Asterisks
show significant differences between single and double inoculated saplings at *P <
0.05; **P < 0.01 according to a t-test of
unpaired samples.
color, Sapling mortality was preceded was by shedding of
chlorotic and necrotic leaves.
Gas exchange and starch levels of leaves
Net CO2 uptake and transpiration rates of leaves were measured in single and double inoculated saplings in 2004 and
2005. In 2004, rates of net CO2 uptake and transpiration were
reduced in leaves of inoculated saplings compared with the
controls (Figures 3a, 3c, 4a and 4c). Significant differences for
both parameters were found in double inoculated saplings
from June to August 2004, whereas single inoculated saplings
differed in net CO2 uptake and transpiration only in late summer. However, saplings that survived the inoculation with
P. alni subsp. alni in 2004 did not differ from controls in rates
of net CO2 uptake and transpiration during the following
growing season (Figures 3b, 3d, 4b and 4d). The inhibitory effects of the pathogen on photosynthesis and transpiration were
more pronounced in 2004 in double inoculated saplings than
in single inoculated saplings, and were significant in June and
July (data not shown). The water-use efficiency (WUE) of leaf
gas exchange hardly differed between inoculation treatments
and controls in either year (Figure 5).
An inverse linear regression was found when comparing the
pooled girdling values of single and double inoculated saplings with the corresponding mean net assimilation rates in
2004 (Figure 6).
With the exception of the surviving double inoculated sapling (No. 5), twig water potentials reflected leaf gas exchange
rates, with the lowest water potentials being observed in the
surviving single innoculated saplings (Nos. 3 and 6), which
did not differ from the controls (Figure 7a), whereas minimum
leaf water potentials of saplings that eventually died remained
high (i.e., were significantly less negative than control values),
particularly in double inoculated saplings (Figure 7b).
Predawn water potentials of single and double inoculated saplings, however, did not differ significantly from values for control saplings (Figure 7).
Although inoculaton caused a sharp reduction in photosynthesis, leaf starch concentration was greater than in control
saplings (Table 1). This may indicate reduced or interrupted
phloem transport of assimilates in the inoculated saplings, the
assimilates that would otherwise have been exported being
stored in leaves.
About 7 weeks after inoculation, some leaves became
chlorotic before becoming necrotic and being abscised. This
development intensified as the season advanced (data not
shown). Nevertheless, some green leaves were present in all
living tree crowns throughout the growing season and were
thus available for physiological measurements. At the end of
the 2004 growing season, the annual increment in radial stem
growth and shoot length were significantly less in inoculated
saplings than in controls (Table 1), particularly in the double
inoculation treatment. The effect of the double inoculation on
these growth parameters was significantly greater than that of
the single inoculation.
Discussion
The stem inoculation experiments were carried out to gain detailed insight into the interaction between A. glutinosa and the
highly aggressive pathogen P. alni subsp. alni over a 2-year period. Rates of net photosynthesis and transpiration of inocu-
TREE PHYSIOLOGY VOLUME 28, 2008
ALNUS GLUTINOSA INFECTED WITH PHYTOPHTHORA ALNI
1707
Figure 3. Rates of photosynthesis (Amax)
of (a, b) single (䊉) and (c, d) double (䊏)
inoculated Alnus glutinosa saplings compared with control saplings (䊊, 䊐) in
2004 and 2005. Values for controls in
2004 and 2005 and for inoculated saplings
in 2004 are means of six replicates ±
SE. In 2005, (b) two single inoculated
saplings and (d) one double inoculated
sapling were measured. Asterisks show
significant differences between control
and Phytophthora alni subsp. alni inoculated saplings at *P < 0.05; **P < 0.01;
and ***P < 0.001 according to a t-test of
unpaired samples.
Figure 4. Transpiration rates of (a, b) single (䊉) and (c, d) double (䊏) inoculated
Alnus glutinosa saplings compared with
control saplings (䊊, 䊐) in 2004 and 2005.
Values for controls in 2004 and 2005 and
for inoculated saplings in 2004 are means
of six replicates ± SE. In 2005, (b) two
single inoculated saplings and (d) one
double inoculated sapling were measured.
Asterisks show significant differences between control and Phytophthora alni
subsp. alni inoculated saplings at *P <
0.05; **P < 0.01; and ***P < 0.001 according to Student’s t-test of unpaired
samples.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
1708
CLEMENZ, FLEISCHMANN, HÄBERLE, MATYSSEK AND OßWALD
Figure 5. Water-use efficiency (WUE) of
leaves of (a, b) single (䊉) and (c, d) double (䊏) inoculated Alnus glutinosa saplings compared with control saplings (䊊,
䊐) in 2004 and 2005. Values for controls
in 2004 and 2005 and for inoculated saplings in 2004 are means of six replicates ±
SE. In 2005, (b) two single inoculated saplings and (d) one double inoculated sapling were measured. Asterisks show
significant differences between control
and Phytophthora alni subsp. alni inoculated saplings at *P < 0.05; **P < 0.01;
and ***P < 0.001 according to a t-test of
unpaired samples.
lated alders were significantly reduced. Stomatal closure was
consistent with less negative afternoon twig water potentials in
the inoculated saplings than in the controls. The difference
was most pronounced in saplings that were double inoculated.
Hence, our working hypothesis that inoculation of Alnus
glutinosa with P. alni inhibits photosynthesis by impeding sap
flow and thus lowering leaf water potentials was not corroborated. Furthermore, the hydraulic conductance of the xylem
was apparently unchanged, as may be inferred from the ratio
of transpiration rate versus water potential (Schulze and Hall
1982). This ratio did not differ significantly between inocu-
Figure 6. Regression analysis of girdling values versus net CO2 uptake rates (Amax) of single (䊉) and double (䊊) inoculated Alnus
glutinosa saplings in 2004. Values are replicate means ± SE.
lated and control trees (data not shown), apparently indicating
unchanged whole-tree hydraulic conductivity. Thus, if P. alni
invaded the xylem as well as the stem cortex, it must have done
so only to a minor extent, or without affecting xylem conductivity. Rather, the water potential of inoculated trees was increased (i.e., became less negative), and therefore, the observed stomatal closure was not a result of reduced xylem conductivity due to infection. Phytophthora pathogens can invade
and grow in xylem tissue as was recently shown by Parke et al.
(2007) and Brown and Brasier (2007). Confirming that there
was adequate whole-tree hydraulic functionality in the infected trees, we observed no difference in predawn leaf water
potential between inoculated and control saplings. Thus both
water-absorbing fine roots as well as the xylem appear to have
been functioning adequately (Schulze and Hall 1982).
Leaf stomatal closure was more likely, therefore, related to
leaf starch accumulation, which can down-regulate photosynthesis (Stitt and Schulze 1994), than to impaired uptake and
conduction of water. The accumulation of leaf starch was apparently a result of reduced translocation of assimilates to the
root because of the local pathogen-induced destruction of
phloem tissue (Krapp et al. 1991, Araya et al. 2006). This conclusion is corroborated by the finding that net CO2 uptake rates
decreased with the extent of phloem tissue destruction (girdling) at the stem base (see Figures 6 and 7). Thus, the
photosynthetic decline in the inoculated trees was likely a result of end product inhibition. Given such a scenario, stomatal
closure reflected the usual tight linkage between stomatal conductance and CO2 assimilation rate (Schulze and Hall 1982).
Such a relationship is typically reflected in an unchanged CO2
concentration of the mesophyll intercellular space (Ci ) and
constant WUE (Schulze and Hall 1982). Similar WUE (and
TREE PHYSIOLOGY VOLUME 28, 2008
ALNUS GLUTINOSA INFECTED WITH PHYTOPHTHORA ALNI
1709
Figure 7. Twig water potentials of control
Alnus glutinosa saplings (white bars) after
single (1 × P. alni) or double (2 × P. alni)
inoculation with Phytophthora alni subsp.
alni (gray bars) at the end of July 2004.
Hatching indicates predawn water potentials and open bars indicates minimum
water potentials during early afternoon.Values are means of six replicates ±
SE.
hence, Ci; not shown) prevailed at most sampling dates in control and inoculated saplings (Figure 5).
Hence, the gas exchange responses indicated a systemic
effect of the local pathogen infection at the stem base. Krapp
and Stitt (1995) reported that from Day 4 onward, after cold-
Table 1. Annual increments in shoot length (cm) and stem diameter
(cm), and leaf starch concentration of Alnus glutinosa saplings after
single and double stem inoculation with Phytophthora alni subsp. alni
in 2004. Inoculation was performed in the spring and measurements
were made at the end of the growing season. Values are means of six
replicates ± SE. The change in increment due to inoculation is given
as a percentage of the increment in control saplings (representing
100%). Means for inoculated saplings followed by different letters
differ significantly (P < 0.05).
Shoot length
Single inoculation
Control
P
Change (%)
40.0 ± 9.9 a
77.9 ± 6.0
< 0.05
–49
Double inoculation
27.4 ± 5.3 a
Control
73.9 ± 6.9
P
< 0.01
Change (%)
–63
Stem
diameter
1.6 ± 0.4 a
2.5 ± 0.1
n.s.
–36
Leaf starch
(mg g –1 )
59.5 ± 11.6 a
39.9 ± 16.8
n.s.
49
0.8 ± 0.1 b
87.7 ± 10.2 b
2.5 ± 0.1
35.8 ± 9.3
< 0.001
< 0.01
–67
145
girdling of the petioles of spinach leaves, photosynthesis decreased in association with a decline in maximum Rubisco activity and a down-regulation of the rbcS gene (small subunit of
Rubisco). In parallel, a two- to fivefold accumulation of sucrose, glucose, fructose and starch was recorded in girdled
leaves compared with ungirdled leaves. When the cold-girdle
was removed on Day 5, photosynthesis and transcripts for rbcS
gradually recovered. The coordinated interplay of photosynthesis and transpiration in leaves of inoculated alder is indicated by the WUE measurements, which showed little variation between inoculated and control plants at each sampling
date (Figure 5).
As we observed in the case of P. alni infection of Alnus
glutinosa, Robin et al. (2001) and Maurel et al. (2004) showed
that P. cinnamomi infection of Quercus ilex L. (holm oak) or
Q. suber L. (cork oak) or P. cinnamomi infection of Castanea
sativa Mill. reduced stomatal conductance and raised leaf water potential. Ploetz and Schaffer (1989) investigated the effect
of P. cinnamomi on net CO2 assimilation of avocado (Persea
americana Mill.) plants in soil infestation experiments. Significant reductions in photosynthesis, transpiration and stomatal
conductance were measured 3 days after infection, and these
parameters declined toward zero within 1 week. In contrast to
our findings, Sterne et al. (1978) reported that leaf xylem water potential was more negative in avocado trees infected with
P. cinnamomi than in healthy trees. The authors concluded that
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
1710
CLEMENZ, FLEISCHMANN, HÄBERLE, MATYSSEK AND OßWALD
root infection with P. cinnamomi so impaired water transport
between roots and leaves that mechanisms normally regulating leaf water potential (e.g., stomatal closure) were inadequate to prevent the development of leaf water stress.
A large reduction in root hydraulic conductivity was measured by Dawson and Weste (1984) in roots of Eucalyptus
sieberi L. Johnson infested with P. cinnamomi. In parallel to
reduced water flow and leaf conductance, leaf xylem water potential decreased (i.e., became more negative) with increasing
infection. Similar results were reported by Fleischmann et al.
(2005) for Fagus sylvatica saplings after root infection with
zoospores of P. citricola. The same authors measured a sharp
decrease in water potential of infected saplings relative to that
of control saplings shortly before leaves of infected plants
witled.
The discrepancy between our findings and those of Sterne et
al. (1978), Dawson and Weste (1984) and Fleischmann et al.
(2005) reflects differences in the tissue primarily damaged by
the pathogen. In the case of A. glutinosa inoculation with
P. alni, damage began at the stem base, whereas P. cinnamomi
and P. citricola primarily infested the root system of their host
plants, thereby impairing water uptake and water flow. Hence,
the effect of the pathogen in the other studies was primarily on
the water conducting system, which induced water stress,
which was counteracted, in part, by stomatal closure. The
stomatal response, in turn, limited photosynthesis. In our
study, however, the primary effect of the pathogen was on the
cortical tissue, including phloem near the stem base, which
disrupted carbon translocation from leaves to roots. The reduction in photosynthesis in A. glutinosa by P. alni subsp. alni
had a marked effect on growth. In the first year, annual shoot
increment was reduced by 50 to 67%.
The effects of P. alni subsp. alni infection that we observed
on photosynthesis in A. glutinosa were systemic, which might
be explained by toxic metabolites released by the pathogen
(Heiser et al. 1998, Oßwald et al. 2004) or by hormonal imbalances of leaves caused after stem infection. Cahill et al. (1986)
reported changes in the cytokinin concentrations of xylem
exudates of eucalypt seedlings following infection with
P. cinnamomi. They found significant reductions in the
zeatin-type and isopentyladenine-type cytokinins in the xylem
exudates of susceptible Eucalyptus marginata Donn ex Sm.
The authors suggested that failure of cytokinin transport from
the root system to leaves is responsible for the symptoms of
P. cinnamomi infection observed in infected susceptible Eucalyptus plants. Specific small Phytophthora proteins, so-called
elicitins, which are formed by Phytophthora species during
proliferation in host tissue and released into adjacent tissue,
have been discussed as possible systemic signals (Brummer et
al. 2002, Oßwald et al. 2000). However, recent results by
Fleischmann et al. (2005) and Maurel et al. (2004) show that
these small proteins are neither involved in signalling nor in
systemic symptom development. Electrical signals are known
to be of importance in many systemic plant interactions, including effects on photosynthesis and stomatal regulation, as
demonstrated in woody plants (Koziolek et al. 2004, Lautner
et al. 2005). However, the physiological significance of elec-
trical signaling under field conditions during host–pathogen
interactions remains to be evaluated.
Acknowledgments
We thank Dr. T. Jung (Bavarian State Institute of Forestry) for the
P. alni subsp. alni reference isolate (ALN 377). The investigations
were funded by Deutsche Forschungsge-meinschaft (DFG) Os 86-10.
References
Araya, T., K. Noguchi and I. Terashima. 2006. Effects of carbohydrate accumulation on photosynthesis differ between sink and
source leaves of Phaseolus vulgaris L. Plant Cell Physiol. 47:
644–652.
Bakonyi, J., Z.A. Nagy and T. Ersek. 2006. PCR-based DNA markers
for identifying hybrids within Phytophthora alni. J. Phytopathol.
154:168–177.
Brasier, C.M., J. Rose and J.N. Gibbs. 1995. An unusual Phytophthora associated with widespread alder mortality in Britain. Plant
Pathol. 44:999–1007.
Brasier, C.M., D.E.L. Cooke and J.M. Duncan. 1999. Origin of a new
Phytophthora pathogen through interspecific hybridization. Proc.
Natl. Acad. Sci. USA 96:5878–5883.
Brasier, C.M., S.A. Kirk, J. Delcan, D.E.L. Cooke, T. Jung and
W.A. Man in’t Veld. 2004. Phytophthora alni sp. nov. and its variants: designation of emerging heteroploid hybrid pathogens
spreading on Alnus trees. Mycol. Res. 108:1172–1184.
Brown, A.V. and C.M. Brasier. 2007. Colonization of tree xylem by
Phytophthora ramorum, P. kernoviae and other Phytophthora species. Plant Pathol. 56:227–241.
Brummer, M., M. Arend, J. Fromm, A. Schlenzig and W.F. Oßwald.
2002. Ultrastructural changes and immunocytochemical localization of the elicitin quercinin in Quercus robur L. roots infected with
Phytophthora quercina. Physiol. Mol. Plant Pathol. 61:109–120.
Cahill, D.M., G.M. Weste and B.R. Grant. 1986. Changes in
cytokinin concentrations in xylem extrudate following infection of
Eucalyptus marginata Donn ex Sm with Phytophthora cinnamomi
Rands. Plant Physiol. 81:1103–1109.
Cech, T.L. 1997. Phytophthora–Krankheit der Erle in Österreich.
Forstschutz Aktuell. 19/20:14–6.
Dawson, P. and G. Weste. 1984. Impact of root infection by Phytophthora cinnamomi on water relations of two Eucalyptus species that
differ in susceptibility. Phytopathology 74:486–490.
De Merlier, D., A. Chandelier, N. Debruxelles, M. Noldus, F. Laurent,
E. Dufays, H. Claessens and M. Cavelier. 2005. Characterization
of alder Phytophthora isolates from wallonia and development of
SCAR primers for their specific detection. J. Phytopathol. 153:
99–107.
Erwin, D.C. and O.K. Ribeiro. 1996. Phytophthora diseases worldwide. Am. Phytopathol. Soc., St. Paul, MN, 592 p.
Fleischmann, F., D. Schneider, R. Matyssek and W.F. Oßwald. 2002.
Investigations on net CO2 assimilation, transpiration and root
growth of Fagus sylvatica infested with four different Phytophthora species. Plant Biol. 4:144–152.
Fleischmann, F., J. Koehl, R. Portz, A.B. Beltrame and W. Oßwald.
2005. Physiological change of Fagus sylvatica seedlings infected
with Phytophthora citricola and the contribution of its elicitin
“Citricolin” to pathogenesis. Plant Biol. 7:650–658.
Gibbs, J.N. 1995. Phytophthora root disease of alder in Britain. EPPO
Bull. 25:661–664.
TREE PHYSIOLOGY VOLUME 28, 2008
ALNUS GLUTINOSA INFECTED WITH PHYTOPHTHORA ALNI
Hartmann, G. 1995. Wurzelfäule der Schwarzerle (Alnus glutinosa)–eine bisher unbekannte Pilzkrankheit durch Phytophthora
cambivora. Forst Holz 50:555–557.
Heiser, I., W. Oßwald and E.F. Elstner. 1998. The formation of reactive oxygen species by fungal and bacterial phytotoxins. Plant
Physiol. Biochem. 36:703–713.
Ioos, R., C. Husson, A. Andrieux and P. Frey. 2005. SCAR-based
PCR primers to detect the hybrid pathogen Phytophthora alni and
its subspecies causing alder disease in Europe. Eur. J. Plant Pathol.
112:323–335.
Ioos, R., A. Andrieux, B. Marcais and P. Frey. 2006. Genetic characterization of the natural hybrid species Phytophthora alni as inferred from nuclear and mitochondrial DNA analyses. Fungal
Genet. Biol. 43:511–529.
Jung, T. and M. Blaschke. 2001. Phytophthora–Wurzelhalsfäulen der
Erlen (Phytophthora collar rot of alders). LWF leaflet No. 6,
Freising, Germany, Bavarian State Institute of Forestry (LWF),
LWF Merkblatt, 2 p.
Jung, T. and M. Blaschke. 2004. Phytophthora root and collar rot of
alders in Bavaria: distribution, modes of spread and possible management strategies. Plant Pathol. 53:197–208.
Jung, T., A. Schlenzig, M. Blaschke, B. Adolf and W. Oßwald. 2000.
Erlensterben durch Phytophthora–Droht Bayerns Erlen eine
Epidemie? LWF-aktuell 24:22–25.
Koziolek, C., T.E.E. Grams, U. Schreiber, R. Matyssek and J. Fromm.
2004. Transient knockout of photosynthesis mediated by electrical
signals. New Phytol. 161:715–722.
Krapp, A. and M. Stitt. 1995. An evaluation of direct and indirect
mechanisms for the sink-regulation of photosynthesis in spinach:
changes in gas-exchange, carbohydrates, metabolites, enzyme-activities and steady-state transcript levels after cold-girdling source
leaves. Planta 195:313–323.
Krapp, A., W.P. Quick and M. Stitt. 1991. Ribulose-1,5-bisphosphate
carboxylase-oxygenase, other Calvin-cycle enzymes, and chlorophyll decrease when glucose is supplied to mature spinach leaves
via the transpiration stream. Planta 186:58–69.
Lautner, S., T.E.E. Grams, R. Matyssek and J. Fromm. 2005. Characteristics of electrical signals in poplar and responses in photosynthesis. Plant Physiol. 138:2200–2209.
Maurel, M., C. Robin, T. Simonneau, D. Loustau, E. Dreyer and
M.L. Desprez-Loustau. 2004. Stomatal conductance and root-toshoot signalling in chestnut saplings exposed to Phytophthora
cinnamomi or partial soil drying. Funct. Plant Biol. 31:41–51.
Oßwald, W., M. Brummer, A. Schlenzig, J. Koehl, T. Jung, I. Heiser
and R. Matyssek. 2000. Investigations on photosynthesis of oak
seedlings infected with Phytophthora quercina and characterisation of the P. quercina toxin quercinin. In Proc. 1st Intl. Meeting on
Phytophthoras in Forests and Wildland Ecosystems. Eds.
E.M. Hanson and W. Suttin. Oregon State University, Corvallis,
OR, pp 66–70.
1711
Oßwald, W., J. Koehl, I. Heiser, J. Nechwatal and F. Fleischmann.
2004. New insights in the genus Phytophthora and current diseases
these pathogens cause in their ecosystems. In Progress in Botany.
Eds. K. Esser, U. Lüttge, W. Beyschlag and J. Murata. SpringerVerlag, Berlin, pp 436–466.
Parke, J.L., E. Oh, E. Voelker, E.M. Hansen, G. Buckles and
B. Lachenbruch. 2007. Phytophthora ramorum colonizes Tan oak
xylem and is associated with reduced stem water transport. Phytopathology 97:1558–1567.
Ploetz, R.C. and B. Schaffer. 1989. Effects of flooding and Phytophthora root rot on net gas exchange and growth of avocado.
Phytopathology 79:204–208.
Rizzo, D.M., M. Garbelotto, J.M. Davidson, G.W. Slaughter and
S.T. Koike. 2002. Phytophthora ramorum as the cause of extensive
mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant Dis. 86:205–214.
Robin, C., G. Capron and M.L. Desprez-Loustau. 2001. Root infection by Phytophthora cinnamomi in seedlings of three oak species.
Plant Pathol. 50:708–716.
Santini, A., F. Biancalani, G.P. Barzanti and P. Capretti. 2006. Pathogenicity of four Phytophthora species on wild cherry and Italian alder seedlings. J. Phytopathol. 154:163–167.
Schulze, E.D. and A.E. Hall. 1982. Stomatal responses, water loss
and CO2 assimilation rates of plants in contrasting environments.
In Encyclopedia of Plant Physiology. New Series, Vol. 12B,
Physiol. Plant Ecol. II. Eds. O.L. Lange, P.S. Nobel, C.B. Osmond
and H. Ziegler. Springer-Verlag, Berlin, pp 182–230.
Sterne, R.E., M.R. Kaufmann and G.A. Zentmyer. 1978. Effect of
Phytophthora root rot on water relations of avocado: interpretation
with a water transport model. Phytopathology 68:595–602.
Stitt, M. and E.D. Schulze. 1994. Plant growth, storage, and resource
allocation: from flux control in a metabolic chain to the
whole-plant level. In Flux Control in Biological Systems. Ed.
E.D. Schulze. Academic Press, San Diego, pp 57–118.
Streito, J.C., P. Legrand, F. Tabary and G.J. De Villartay. 2002. Phytophthora disease of alder (Alnus glutinosa) in France: investigations between 1995 and 1999. For. Pathol. 32:179–191.
Werres, S. 1998. Erlensterben. Allgemeine Forstzeitschrift/Der Wald.
10:548–549.
Werres, S., R. Marwitz, W. Veld, A. De Cock, P.J.M. Bonants, M. De
Weerdt, K. Themann, E. Ilieva and R.P. Baayen. 2001. Phytophthora ramorum sp. nov., a new pathogen on Rhododendron and Viburnum. Mycol. Res. 105:1155–1165.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com