Plant Physiol.
(1976) 57, 486-489
Effect of Powdery Mildew Infection on Photosynthesis by Leaves
and Chloroplasts of Sugar Beets1
Received for publication October 21, 1975 and in revised form December 15, 1975
ANDREW C. MAGYAROSY, PETER SCHURMANN,2 AND BOB B. BUCHANAN
Department of Cell Physiology, University of California, Berkeley, California 94720
ABSTRACT
Chloroplasts isolated from powdery mildew-infected (E,ysiphe poly-
goni DC) sugar beet leaves (Beta vulgaris L) showed a reduction in the
rate of electron transport and in the accompanying ATP formation in
noncyclic photophosphorylation (water as electron donor, NADP as
electron acceptor) and little or no change in the rate of ATP formation in
cydic photophosphorylation catalyzed by phenazine methosulfate. The
inhibition of noncyclic photophosphorylation appeared to lead in the
parent leaves to a decreased rate of photosynthetic CO2 assimilation and
a shift in products resulting in a relative increase of amino acids. These
changes were accompanied by alterations in chloroplast ultrastructure
and by a reduction in the activity of enzymes necessay for the formation
of organic adds (phosphoenolpyruvate carboxylase and malate dehydro-
genase). These results are similar to the findings of Montalbini and
Buchanan (1974 Physiol. Plant Pathol. 4: 191-196) with chioroplasts
from rust-infected Vicia faba leaves.
Although the effects of certain etiological agents on the physi-
ological processes of host plants have been studied extensively,
investigations with obligate parasites, an important group of
plant pathogens, are scanty and in part contradictory. While it is
generally agreed that obligate parasite infections consistently
increase the rate of respiration (23, 27), conflicting reports
indicate that such infections can either decrease (1, 2, 9, 15-17,
21, 22) or increase (1, 9, 26) the rate of photosynthesis. Little is
known about the effect of obligate parasites on photosynthesis at
the chemical level.
It has recently been observed that infection by rust fungi
inhibits photochemical reactions of chloroplasts (14), perhaps by
effecting the production of a substance, functionally similar to
DCMU, which preferentially inhibits electron transport and
ATP formation by noncyclic photophosphorylation but does not
appreciably affect ATP formation by cyclic photophosphoryla-
tion. These findings raise the question whether there is a similar
inhibition of noncyclic photophosphorylation by another group
of parasites, the powdery mildew fungi, which could account for
the reduced capacity of diseased plants to produce sucrose (24).
An answer to this question seemed particularly timely in view
of the unusually high incidence of powdery mildew on sugar
beets during 1974 and 1975 throughout the United States,
particularly in California (8, 18). We, therefore, examined the
'This investigation was supported in part by Hatch and California
Statewide Critical Applied Research Funds.
20n leave from the Laboratoire de Physiologie vegetale et Biochimie,
Universite de Neuchatel, Switzerland.
486
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Copyright © 1976 American Society of Plant Biologists. All rights reserved.
effect of powdery mildew infection on certain aspects of photo-
synthesis by sugar beets, and we now report evidence that the
infection preferentially inhibits electron transport in noncyclic
photophosphorylation in chloroplasts and in so doing causes a
decrease in the rate of photosynthetic CO2 assimilation and a
shift in products. Accompanying these changes are alterations in
chloroplast ultrastructure and a reduced activity of certain en-
zymes. Preliminary accounts of this investigation have been
published (11, 12).
MATERIALS AND METHODS
Growth Conditions and Inoculation of Plants with Pathogen.
Plants (Beta vulgaris L. U.S. H1O) were grown in soil under
normal greenhouse conditions. The conidial stage of Erysiphe
polygoni DC was collected at the west side of the San Joaquin
Valley, California, and maintained on sugar beets. Leaves of 30-
day-old sugar beet plants were dusted with conidia of the patho-
gen, kept in a sealed metal refuse can for 2 days, and then placed
under normal greenhouse conditions. Leaves showing a heavy
uniform infection, indicated by profuse powdery conidia of the
fungus, were harvested about 30 days after inoculation and used
in all experiments unless otherwise stated. Leaves of younger
infected plants showed only patchy growth of the fungus and
therefore were not satisfactory for this investigation, which re-
quired uniformly infected material for cell-free preparations.
Uninoculated 60-day-old plants were used for control measure-
ments.
CO2 Fixation by Leaf Discs. Photosynthetic '4CO2 assimilation
was carried out in Warburg vessels containing duplicate leaf
discs. The reaction was stopped by placing the leaf discs for 2
min in separate scintillation vials containing boiling methanol.
One disc was counted to determine the rate of photosynthesis
(10), and the second was used for product analysis (19, 20). As
indicated, additional points were taken for rate determinations.
Cydic and Noncyclic Photophosphorylation by Isolated Chlo-
roplasts. Five to 6 g of healthy or infected leaves were deveined,
washed, and blended for a few seconds in a micromonel cup of a
Waring Blendor containing 32 ml of the following preparative
solution: 0.5 M NaCl, 0.04 M Tricine buffer (pH 8.2), and 0.01 M
Na-ascorbate. The resulting slurry was filtered through four
layers of filtering silk and centrifuged for 1 min at 3,700g. The
chloroplast pellet was resuspended in a small amount of prepara-
tive solution, and the Chl content was determined according to
Arnon (3).
Cyclic and noncyclic photophosphorylation were carried out
under a nitrogen atmosphere in Warburg vessels as previously
described (10). For cyclic photophosphorylation, the reaction
mixture (in a Warburg vessel) contained (M): Tricine buffer (pH
8.2), 0.1; MgCl2, 0.005; ADP, 0.005; 32Pi, 0.005; PMS, 5 x
10-5; and chloroplasts equivalent to 0.05 mg of Chl. Final vol-
ume was 1 ml; light intensity, 20,000 lux; gas phase, nitrogen;
Plant Physiol. Vol. 57, 1976 POWDERY MILDEW AND PHOTOSYNTHESIS
temperature, 20 C; reaction time, 5 min. Noncyclic photophos-
phorylation was also measured in spectrophotometric cuvettes,
and the concomitant changes in absorbance at 340 nm were
recorded with a modified Beckman DU monochromator
equipped with a Gilford 200 absorbance indicator (13).
AT32P formation in cyclic and noncyclic photophosphorylation
was determined as previously described (10). Radioactivity was
measured with a Geiger-Muller counter.
Enzyme Assays. For assay of phosphoenolpyruvate carboxyl-
ase and malate dehydrogenase, 2 g of leaves were ground at 5 C
with sand in a mortar containing 7 ml of a buffer solution of 40
mM Tricine (pH 8.2) and 5 mm Na-ascorbate. The slurry was
centrifuged at 48,000g for 15 min. Protein concentration of the
supernatant fraction was measured by a modified phenol method
(10) and then adjusted to 4 mg/ml. For assay of malate dehydro-
genase, the oxidation of NADH in the presence of oxaloacetate
was measured in a Cary Model 14 spectrophotometer. PEP3
carboxylase was assayed spectrophotometrically by measuring
the oxaloacetate formed from PEP and CO2 in the presence of
malate dehydrogenase and NADH (10).
Ultrastractural Changes. Leaf tissues for electronmicroscopic
examination were fixed in 2% glutaraldehyde, dehydrated, and
stained with uranyl acetate (6). Leaf sections were examined in a
Siemens Elmiskop 101 electronmicroscope.
RESULTS AND DISCUSSION
Effect of Powdery Mildew Infection on Cydic and Noncydic
Photophosphorylation. In view of the earlier evidence that obli-
gate parasites cause a decrease in the rate of electron transport in
noncyclic photophosphorylation and have little effect on cyclic
photophosphorylation (14), we examined activities of these two
processes in six preparations of chloroplasts isolated from
healthy and powdery mildew-infected leaves. The results indi-
cate that ATP formation by PMS catalyzed cyclic photophos-
phorylation was little affected by infection (204 versus 198
,umoles of ATP formed/mg of Chl-hr for healthy and infected
chloroplasts, respectively), whereas ATP formation by noncyclic
photophosphorylation with NADP as acceptor was reduced (Ta-
ble I). In general, there was a 20 to 30% reduction of ATP
formation in noncyclic photophosphorylation in chloroplasts
from infected leaves, as compared to their healthy counterparts.
Such a reduction in noncyclic photophosphorylation could result
either from a specific uncoupling or from an inhibition of elec-
tron transport from water to NADP. On the basis of the stoichi-
ometry observed (ratio of ATP formed per two electrons trans-
ferred from water to NADP), there was, as shown in Table I, no
appreciable difference in coupling between chloroplasts from
healthy and infected leaves. It appears likely that powdery mil-
dew infection reduced ATP formation selectively by inhibiting
noncyclic electron flow rather than by specifically uncoupling
photophosphorylation. Similar results were obtained by Montal-
bini and Buchanan (14) with chloroplasts from rust-infected
Vicia faba leaves.
CO2 Fixation of Healthy and Powdery Mildew-infected Leaf
Discs. In view of the reduced capacity of chloroplasts from
powdery mildew-infected leaves to catalyze noncyclic photo-
phosphorylation, the question arises whether infection affects
photosynthetic "4CO2 assimilation -a process dependent on the
products of noncyclic photophosphorylation, namely reduced
ferredoxin (or NADPH) and ATP. As shown in Figure 1, pow-
dery mildew markedly decreased the rate of "4CO2 assimilation
by leaf discs. Based on Chl content, the rates of fixation by
'Abbreviations: PEP: phosphoenolpyruvate; PMS: phenazine metho-
sulfate; 3-PGA: 3-phosphoglycerate.
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487
Table I. Effect of Powdery Mildew Infection on Noncyclic
Photophosphorylation by Isolated Sugar Beet Chloroplasts
The reaction mixture (in a cuvette of 2-mm light path) contained (M):
Tricine buffer (pH 8.2), 0.1; MgCl2, 0.005; ADP, 0.005; Pi, 0.005;
NADP, 0.005; spinach ferredoxin, 1 x 1O-5; and chloroplasts equivalent
to 0.05 mg of Chl. Final volume, 1 ml; light intensity (red light, A > 600
nm), 2.4 x 104 ergs/cm2 *sec; gas phase, nitrogen; temperature, 20 C.
Source of Chloroplasts ATP Formed NADPH, Formed Ratio P.2e-
( wmoleslmg Chilhr)
Healthy leaves 109 121 0.90
Infected leaves 80 64 1.25
qb
t3
!zt
minutes
FIG. 1. Effect of powdery mildew infection on the rate of photosyn-
thetic 14CO2 assimilation by leaf discs. Duplicate discs were floated
stomata side up in a Warburg vessel containing 1.5 ml of H20. The side-
arm contained 0.1 ml of NaHCO3 solution (10 ,umoles; 5 x 106 cpm/
Mmole). After 5-min preillumination, gaseous CO2 was liberated by
injecting 0.2 ml of 9 N H2SO4 into the side-arm. Light intensity, 20,000
lux; temperature, 20 C.
uninfected leaf discs were as high as 153 Lmoles of CO2 fixed/
mg Chl-hr, whereas those of infected leaf discs were consistently
about half of that value. It is noteworthy that repeated analyses
of healthy and infected leaf discs revealed the absence, on an
area basis, of a significant difference in Chl content.
Products of CO2 Assimilation. An indication that the reduc-
tion in the rate of "4CO2 assimilation induced by the pathogen
was due to a reduced capacity for noncyclic photophosphoryla-
tion came from product analyses that revealed a striking differ-
ence between healthy and infected leaves. Compared with their
healthy counterparts, after 5-min photosynthesis, infected leaves
showed increased amounts of "4CO2 fixed into amino acids and
decreased amounts of "4CO2 fixed into 3-PGA, sucrose, and
other sugars (Table II). There were respective increases of 6-fold
and 3-fold in the labeling of alanine and glutamate + aspartate,
compared to decreases of 50% and 25% in 3-PGA and sucrose.
A similar shift to amino acids was reported for squash leaves
infected with squash mosaic virus (10) and in Chinese cabbage
leaves infected with turnip yellow mosaic virus (4). In the case of
squash, a change in photophosphorylation did not appear to be
involved.
There was a slight reduction in organic acids formed by dis-
eased leaves (Table II). Such a reduction may be due to
the reduced activity of PEP carboxylase and malate dehydrogen-
488 MAGYAROSY, SCHURMANN, AND BUCHANAN Plant Physiol. Vol. 57, 1976

ase that was observed in this investigation in cell-free extracts
from powdery mildew-infected leaves. Malate dehydrogenase
and PEP carboxylase activities in leaf extracts were 37% and
50%, respectively, of the activities in extracts from healthy
leaves. The reduction of enzyme activity in extracts from pow-
dery mildew-infected leaves is to be compared with a previous
report of an increase in activity of PEP carboxylase in extracts
from rust-infected leaves (25).
Changes in Ultrastructure of Chloroplasts from Powdery Mil-
dew-infected Sugar Beet Leaves. Because change in chloroplast
ultrastructure could accompany a change in chloroplast activity
in diseased leaves, we examined by electron microscopy the
chloroplasts of powdery mildew-infected sugar beet leaves. This
examination revealed that, although the inner and outer chloro-
plast membranes appeared to be unaffected, infection by the
parasite caused a marked change in the morphological organiza-
Table II. Effect of Powdery Mildew Infection on Photosynthetic
Products Formed by Sugar Beet Leaf Discs
Products were analyzed after 5-min photosynthesis under conditions
in Fig. 1 by the thin layer electrophoresis-chromatography technique
used earlier for algal cells and spinach chloroplasts (19, 20). Total cpm
for the healthy and infected samples were 258,500 and 267,100, respec-
tively. Counts were determined in a scintillation counter.
Product
1. 3-Phosphoglyceric acid
2. Sugar mono- and di-phosphates
3. Phosphoenolpyruvate, pyruvate
tion of the stroma and grana regions of chloroplasts (Fig. 2). The
stacking of thylakoids was disturbed, starch granules did not
appear to be formed in the stroma, and large osmiophilic glob-
ules ("lipid material") were abundant. It is not possible at
present to relate these structural aberrations to the observed
inhibition of noncyclic photophosphorylation or to other bio-
chemical changes induced by infection. It may be noted that a
similar disturbance in thylakoid stacking was observed with rust-
infected leaves (5, 7).
CONCLUSIONS
The present results provide evidence that powdery mildew (on
sugar beets) resembles rust (on Vicia faba) in effecting a prefer-
ential inhibition of noncyclic photophosphorylation by chloro-
plasts of host leaves. The inhibition in both cases stems from a
diminution in electron flow from water to NADP and, in the case
of powdery mildew, leads to an inhibition of photosynthetic CO2
assimilation and a shift in products that can account for the
reported reduced capability of infected plants to form sucrose.
These changes are accompanied by aberrations in chloroplast
ultrastructure and by a decrease in the activity of enzymes
leading to the production of organic acids. It is possible that the
inhibitory effect on noncyclic photophosphorylation by powdery
mildew-infected chloroplasts, as was earlier proposed for rust
infection, is the result of the production of a substance that is
of Total
14
CO, Fixed
functionally similar to the herbicide DCMU.
Healthy leaves Infected leaves Acknowledgments-The authors gratefully acknowledge the assistance of V. Breazeale in the
preparation of thin sections for electron microscopy and the valuable advice of K. Esau of the
18 9 University of California, Santa Barbara, concerning the preparation of electron micrographs. The
advice and guidance of J. E. Duffus of the United States Agricultural Research Station, Salinas,
22 23 Calif., throughout this investigation is greatly appreciated.
2 1

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