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This is to certify that the
dissertation entitled

EFFECTS OF POWDERY MILDEW ON CARBON ASSIMILATION
OF POTTED CHARDONNAY GRAPEVINES.

presented by

WILLIAM ROGERS NAIL IV

has been accepted towards fulfillment
of the requirements for the

 

 

 

 

Ph.D. degree in Horticulture
Major Wessor’s Signature
30 5,7044. 2003
Date

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6/01 c'JCIRC/DateDuo.p65-p.15

EFFECTS OF POWDERY MILDEW ON CARBON ASSIMILATION OF POTTED
CHARDONNAY GRAPEVINES.

By

William Rogers Nail IV

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Horticulture

2003

ABSTRACT

EFFECTS OF POWDERY MILDEW ON CARBON ASSIMILATION OF POTTED
CHARDONNAY GRAPEVINES.

By

William Rogers Nail IV

Potted Chardonnay (Vitis vinifera L.) grapevines were inoculated with conidial
suspensions of powdery mildew of grape (Uncinula necator (Schw.) Burr.) (GPM), and
the effects of GPM infection were studied over two seasons. In Season 1, grapevines
infected with GPM had reduced C02 assimilation rates (A) compared to noninfected
vines. Vines inoculated prior to bloom (Early) showed declines in A throughout the
growing season and had reduced fresh and dry weight at the end of the season compared
to other treatments. Plants inoculated after the 5mm berry stage (Late) showed
subsequent declines in A, with no significant reduction in fresh or dry matter compared to
control vines. Leaves on both infected treatments senesced earlier than those of control
vines. Reductions in A were correlated with reductions in stomatal conductivity (gs) and
transpiration (E), and increased internal C02 concentration (C1). The effects were more
pronounced in Season 2. Plants not destructively harvested in Season I were grown a
second season in a greenhouse and had no GPM infection. Destructively harvested and
partitioned plants after Season 2 that had been infected with GPM in season 1 showed
reduced fresh and dry weights, shoot lengths, and estimated leaf area compared to control
plants. The amounts of the reductions were related to the length of infection time in
Season 1. Leaves of infected and noninfected plants were studied for the effects of
varying light (PAR) and C02 concentrations. Infection by GPM reduced carboxylation

efficiency (k), A, gs, and C; under ambient C02, Amax at >900ppm C02, stomatal

limitations to A (lg), and photochemical efiiciency (cp), while having no effect on the C02
compensation point (F) or the light compensation point (cp). Infection by GPM had no

effect on chlorophyll fluorescence (F V/F m).

DEDICATION

To all the artists, especially musicians, who have provided perspective for my scientific
endeavors. Thanks!

iv

ACKNOWLEDGMENTS

Thanks to all the people at Michigan State who have made my time here so rewarding:
Stan Howell, for his mentorship and friendship; George Bird, Jim Flore, Annemiek
Schilder, and Bob Schutzki, for their help as committee members; and Gerry Adams, who
was originally a committee member and who taught a great course in mycology. I
appreciate the help and friendship of all the people who have been in the Viticulture and
Enology program here: Dan, Leah, Jon, Marcel, Kasey, Melissa, Todd, David, and,
especially, Jen and Hongying, with whom I spent so much time in the office and in the
field; and Erik Brasher and Stephanie Jones, who helped me so much with my field
experiments. Other friends, many of whom have gone on to bigger and better things,
include Dave and Sandy, Sarah and Mark, Andy and Carmen, Russell, and Erik Runkle.
Thanks to Adriana, Costanza, Marlene, Leo, and Rita, for their help and advice with my
research, and Paul, Adam, and Mike, who spent time with us while far away from their

homes in New ZeaIand. And special thanks to Carol, for her love and support.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................ viii
KEY TO SYMBOLS AND ABBREVIATIONS .............................................. xiii
LITERATURE REVIEW ............................................................................ 1
CHAPTER 1

EFFECTS OF POWDERY MILDEW INFECTION ON CARBON
ASSIMILATION 0F POTTED CHARDONNAY (Vitis Vinifera L.)
GRAPEVINES ....................................................................................... 12

CHAPTER 2

EFFECTS OF TIMING OF POWDERY MILDEW INFECTION ON CARBON
ASSIMILATION AND SUBSEQUENT SEASONAL GROWTH OF POTTED
CHARDONNAY (Vitis vinifera L.) GRAPEVINES ........................................... 44

CHAPTER 3
EFFECTS OF POWDERY MILDEW OF GRAPE ON THE CARBON
ASSIMILATION MECHANISMS OF POTTED CHARDONNAY

(Vitis vinifera L.) GRAPEVINES ................................................................. 75
APPENDIX ........................................................................................... 93

vi

LIST OF TABLES

CHAPTER 3

Table l. The effect of powdery mildew of grape infection on CO2 compensation point
(P), carboxylation efficiency (k), C02 assimilation rate (A 360), stomatal conductance
(gs360), and internal C02 concentration (C360) under ambient C02, A...ax at >900 ppm C02,
stomatal limitations to A (lg), photochemical efficiency ((p), and light compensation point
(cp) on most recently fully expanded grape leaves on potted Chardonnay grapevines. . . .83

APPENDIX

Table 1. Effects of powdery mildew of grape (GPM) infection on single leaf
photosynthesis (Pn), stomatal conductance (gs), internal C02 concentration (C ,-), and
transpiration (E) of potted Chardonnay grapevines at four phenophases in Year 1.
Grapevines were either infected or not infected with GPM ................................... 94

Table 2. Effects of powdery mildew of grape (GPM) infection on single leaf
photosynthesis (Pn), stomatal conductance (gs), internal C02 concentration (C,), and
transpiration (E) of potted Chardonnay grapevines at four phenophases in Year 2.
Grapevines were either infected or not infected with GPM ................................... 95

Table 3. Effects of powdery mildew of grape (GPM) infection on single leaf
photosynthesis (Pn), stomatal conductance (gs), internal C02 concentration (C;), and
transpiration (E) of potted Chardonnay grapevines at four phenophases in Season 1.
Grapevines were inoculated with Uncinula necator prior to bloom (Early), after the 5mm
berry stage (Late), or not inoculated (Control) .................................................. 96

Table 4. Effects of powdery mildew of grape (GPM) infection on fresh and dry weights
of potted Chardonnay grapevines. Grapevines were inoculated with Uncinula necator
prior to bloom (Early), afier the 5mm berry stage (Late), or not inoculated (Control). ...97

Table 5. Effects of powdery mildew of grape (GPM) infection on fresh and dry weights

of potted Chardonnay grapevines in Season 2. Grapevines were inoculated with Uncinula
necator prior to bloom (Early(2°)), after the 5mm berry stage (Late(2°)), or not inoculated
(Control(2°)) in Season 1, and were not infected with GPM in Season 2 .................... 98

vii

LIST OF FIGURES

CHAPTER 1

Figure. 1. Whole vine photosynthesis chambers for measuring carbon assimilation on
potted Chardonnay grapevines,, showing Mylar chambers, blowers with ductwork, and
CIRAS-2 infrared gas analyzer (after Miller et a1. 1996) ...................................... 21

Figure. 2. Impact of powdery mildew of grape (GPM) disease severity on single leaf
photosynthesis (Pn) rates of greenhouse-grown Chardonnay grapevines. Week 1
measurements (A) were conducted 23 days after inoculation with Uncinula necator, and
every 14 days thereafter (B and C) ............................................................... 24

Figure 3. Effect of powdery mildew of grape infection on single leaf C02 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator twice
during the growing season ......................................................................... 25

Figure 4. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (gs) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (FEL) (A), and the original, initial F EL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator twice
during the growing season ......................................................................... 26

Figure 5. Effect of powdery mildew of grape infection on single leaf transpiration (E)
on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator twice
during the growing season ......................................................................... 27

Figure 6. Impact of powdery mildew of grape (GPM) infection on whole vine C02
assimilation (A) of potted Chardonnay grapevines at midseason (£1200 growing degree
days, base 50°F) (A and B) ........................................................................ 28

Figure 7. Effect of powdery mildew of grape (GPM) infection on whole-vine C02
assimilation (A) on potted Chardonnay grapevines as related to stages of vine growth
phenology, growing degree days (GDD) (base 50°F ), and days afier inoculation
(infection days) with a conidial suspension of Uncinula necator ............................. 30

viii

Figure 8. Effect of powdery mildew of grape infection on single leaf C02 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season ......................................................................... 31

Figure 9. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (g) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season ......................................................................... 32

Figure 10. Effect of powdery mildew of grape infection on single leaf internal C02
concentration (C,) on potted Chardonnay grapevines on the most recent fully expanded
leaf at time of measurement (F EL) (A), and the original, initial FEL from the first series
of measurements (ORF EL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season ......................................................................... 33

Figure 11. Effect of powdery mildew of grape infection on single leaf transpiration (E)
on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season ......................................................................... 34

Figure 12. Effect of powdery mildew of grape (GPM) infection on whole-vine C02
assimilation (A) on potted Chardonnay grapevines at harvest (22170 growing degree

days, base 50°F) ..................................................................................... 35
Figure 13. Effect of powdery mildew of grape infection on season-long C02
assimilation and carbon partitioning of potted Chardonnay grapevines ..................... 36
Figure 14. Effect of powdery mildew of grape infection on season-long C02
assimilation and carbon partitioning of potted Chardonnay grapevines ..................... 37
CHAPTER 2

Figure 1. Effect of powdery mildew of grape infection on single leaf C02 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORF EL) (B) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season ......................... 54

ix

Figure 2. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (gs) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season ......................... 55

Figure 3. Effect of powdery mildew of grape infection on single leaf internal C02
concentration (C,-) on potted Chardonnay grapevines on the most recent fully expanded
leaf at time of measurement (FEL) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season ......................... 56

Figure 4. Effect of powdery mildew of grape infection on single leaf transpiration (E) on
potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology, growing _
degree days (GDD) (base 50°F ), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season ......................... 57

Figure 5. Effect of powdery mildew of grape infection on CO2 assimilation and carbon
partitioning of potted Chardonnay grapevines. “Early” plants were inoculated with
Uncinula necator seven days pre-bloom; and “Late” vine were inoculated three days after
the 5mm berry stage. Data were combined from three sequential destructive harvests...58

Figure 6. Effect of powdery mildew of grape infection on CO2 assimilation and carbon
partitioning of potted Chardonnay grapevines. “Early” plants were inoculated with
Uncinula necator seven days pre-bloom; and “Late” vine were inoculated three days after
the 5mm berry stage. Data were combined from three sequential destructive harvests...59

Figure 7. Fresh and dry leaf weights of plants infected at two inoculation times with
Uncinula necator. “Early” plants were inoculated seven days pre-bloom; “Late” vine
were inoculated three days after the 5mm berry stage .......................................... 60

Figure 8. Effect of powdery mildew of grape infection on single leaf C02 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (F EL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) status at different stages of vine growth phenology and
growing degree days (GDD) (base 50°F). “Early(2°)” plants were inoculated with
Uncinula necator seven days pre-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season. . .63

Figure 9. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (g,) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) status at different stages of vine growth phenology and
growing degree days (GDD) (base 50°F). “Early(2°)” plants were inoculated with
Uncinula necator seven days pre-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season. . .64

Figure 10. Effect of powdery mildew of grape infection on single leaf internal C02
concentration (C,-) on potted Chardonnay grapevines on the most recent firlly expanded
leaf at time of measurement (F EL) at different stages of vine growth phenology and
growing degree days (GDD) (base 50°F ). “Early(2°)” plants were inoculated with
Uncinula necator seven days pre-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season. . .65

Figure 11. Effects of powdery mildew of grape on shoot length of potted Chardonnay
grapevines during the growing season following infection at different stages of vine
growth phenology, and growing degree days (GDD) (base 50°F). “Early(2°)” plants
were inoculated seven days pre-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season. . .66

Figure 12. Impact of the previous season’s infection by powdery mildew of grape on
fresh weight of component plant parts of potted Chardonnay grapevines. “Early(2°)”
plants were inoculated with Uncinula necator seven days prior to bloom, and “Late(2°)”
plants were inoculated three days after the 5mm berry stage in Season 1 ................... 67

Figure 13. Impact of the previous season’s infection by powdery mildew of grape on
biomass of component plant parts of potted Chardonnay grapevines. “Early(2°)” plants
were inoculated with Uncinula necator seven days prior to bloom, and “Late(2°)” plants
were inoculated three days after the 5mm berry stage in Season 1 ........................... 68

Figure 14. Effects of powdery mildew of grape on subsequent season accumulated fresh
weight (A) and biomass (B) of potted Chardonnay grapevines. “Early(2°)” plants were
inoculated with Uncinula necator seven days prior to bloom, and “Latc(2°)” plants were
inoculated three days after the 5mm berry stage in Season 1 ................................. 69

CHAPTER 3

Figure 1. C02 (A) and light (B) response curves of single leaves of potted Chardonnay
grapevines infected and not infected with powdery mildew of grape ........................ 84

Figure 2. Relationships between single leaf C02 assimilation (A) and stomatal
conductance (gs) (A), and single leaf A and internal C02 concentration (C,-) (B) in leaves
of potth Chardonnay grapevines infected and not infected with powdery mildew of

grape ................................................................................................... 85

xi

Figure 3. Chlorophyll fluorescence on leaves of potted Chardonnay grapevines infected
and not infected with powdery mildew of grape (GPM). Fluorescence is expressed as the
ratio between variable fluorescence (F..) and maximum fluorescence (Fm) ................. 86

xii

A360

Amax

C1360
CP

FEL

F v
GDD
GPM

85360

1%
ORFEL
PAR

Pn

Key to Symbols and Abbreviations

C02 assimilation

C02 assimilation at ambient C02

C02 assimilation at 2900 ppm C02
Internal C02 concentration

Internal C02 concentration at ambient C02
Light compensation point

Transpiration

The most recently fully expanded leaf on a shoot at time of measurement
Maximum fluorescence

Variable fluorescence

Growing degree days

Powdery mildew of grape

Stomatal conductance

Stomatal conductance at ambient C02
Carboxylation efficiency

Stomatal limitations to C02 assimilation
Original most recently fully expanded leaf at bloom (=FEL at bloom)
Photosynthetically active radiation

Net photosynthesis

C02 compensation point

Photochemical efficiency

xiii

Literature Review

The grapevine is one of the oldest cultivated crops in human history. Culture of
the grapevine probably originated in Asia Minor (Winkler et al. 1974) which is also the
presumed origin of Vitis vinifera (L.), the most widely cultivated grape species in the
world (ibid.). Other Vitis species flourish in many other parts of the world as wild and/or
cultivated species, especially in the Americas (Hedrick 1908; Munson 1909; Perold
1927). Human movement has resulted in the spreading of grapevine species all over the
world. Most of the spread of grapevine species has been to introduce V. vinifera into
non-native regions, although small amounts of American species were imported to
Europe in the nineteenth century as museum specimens (Mullins et al. 1992) or by
horticultural hobbyists (Pearson and Gadoury 1992). These importations resulted in
widespread epidemics of disease and arthropod infestation, as V. vinifera species were
susceptible to damage by many organisms to which native American species were
resistant. The most famous of these is phylloxera (Daktulospharia vitifoliae F itch), a
root-feeding arthropod, which almost caused the destruction of European viticulture in
the mid-nineteenth century. Powdery mildew of grape (GPM), caused by the fungus
Uncinula necator (Schw.) Bum, was also presumably introduced into Europe at this time.
The fimgus was first described in North America in 1834 by Schweinitz, and its
anamorph was first described in England as Oidium tuckeri in 1847 (Pearson and
Gadoury 1992). By 1850, GPM had caused crop losses in most of the major grape-
growing regions of Europe (Bulit and Lafon 1978), and is today the most widespread and
destructive disease of grapevines worldwide (Pearson and Gadoury 1992). It is also the

most widespread pest problem in California vineyards (Sall and Teviotdale 1981).

Uncinula necator is an obligate parasite that can infect all green tissues of the
grapevine (Bulit and Lafon 1978; Sall and Teviotdale 1981). There are two sources of
inoculum. The most common source is conidia produced on the surface of infected
tissues (Pearson and Gadoury 1992). These conidia can be produced throughout the
growing season, and are responsible for the “powdery” appearance of infected tissues.
Ascospores produced in cleistothecia form the other source of inoculum (Pearson and
Gadoury 1987, 1992; Pearson and Goheen 1988). These sexual spores are generally
released early in the growing season. The fungus can overwinter as cleistothecia and/or
by perennation as mycelia in dormant buds (Pearson and Gartel 1985; Pearson and
Goheen 1988; Sall and Wryzinski 1982; Ypema and Gubler 2000). Infected shoots
arising from the latter are commonly called “flag shoots”.

Powdery mildew of grape has long been known to result in inferior fi'uit quality
(Gadoury et al. 2001; Ough and Berg 1979; Pool et al. 1984). Early season fi'uit infection
may result in decreased fruit set, and may cause berry splitting and tissue scarring
(Chellemi and Marois 1992). Infected fruit is unsuitable for fresh market use and may be
unsuitable for the production of high quality wine (Ough and Berg 1979; Pool et al.
1984). Infection of fruit by U. necator may also predispose berries to secondary infection
by Botrytis cinerea Pers. and spoilage microorganisms (Ficke et a1. 2002).

Grapevine species and cultivars differ in their susceptibility to GPM. The disease
is believed to be native to North America, as that is where it was first described, and most
native American grapevine species are relatively resistant, while Eurasiatic species such
as V. vinifera, V. betulifolia Diels & Gilg., V. pubescens Schltdl., V. davidii (Carr.)Foex.,

and V. piasezkii Maxim. are highly susceptible (Pearson and Gadoury 1992). Cultivars

within a species may also show differences in susceptibility (Doster and Schnathorst
1985; Pearson and Gadoury 1992).

Grapevine berries (F icke et al. 2002; Gadoury et al. 2001) and leaves (Doster and
Schnathorst 1985) have demonstrated ontogenic resistance to GPM infection, although
rachises have a more protracted period of susceptibility (Gadoury et al. 2001). Therefore
it is possible that infections later in the season would be less severe. Berries of V.
vinifera cultivars showed resistance to infection three weeks after bloom (Ficke et al.
2002), while Concord berries became mostly resistant to infection within two weeks after
fi'uit set (Gadoury et al. 2001). The youngest leaves on individual shoots showed
increased conidial germination rates compared to leaves two and four nodes proximal to
the youngest leaf.

Powdery mildews and gas exchange in plants

Powdery mildews constitute a diverse group of ascomycotal fungi. All are genus-
specific obligate parasites of their host plants, and may affect plant growth by reducing
photosynthesis (Pn), increasing respiration and/or transpiration, with subsequent growth
impairment and reduced yields (Agrios 1997). There is relatively little scientific
literature quantifying the specific effects of powdery mildew infection on carbon
assimilation in plants. Powdery mildews have been shown to reduce net C02
assimilation (A) in apple (Ellis et al. 1981), pecan (Gottwald and Wood 1984), barley
(Hibberd et al. 1996; Holloway et al. 1992; Williams and Ayers 1981), pepper
(Shtienberg 1992), Prunus spp. (Layne and Flore 1995), sour cherry (Layne and Flore
1992), winter wheat (Rabbinge et al. 1985; Shtienberg 1992), pea (Ayers 1981) and sugar

beet (Magyarosy et al. 1976), as well as grape (Lakso 1982; Shtienberg 1992). Studies of

specific effects of powdery mildews on host plant A showed that powdery mildew of
barley (Blumeria (syn. Erysiphe) gramim’s D.C. ex Merat f.sp. hordei Marchal) resulted
in decreases in chlorophyll content after four days of infection and loss of electron
transport activity, with no loss of electron carrier concentration in remaining chlorophyll
(Holloway et al. 1992). Powdery mildew of sugar beet (Erysiphe polygoni DC) inhibited
electron transport in noncyclic proteins, accompanied by alterations in chloroplast
ultrastructure and reduction of enzyme activity (Magyrarosy et al. 1976). Carboxylation
resistance increased in winter wheat infected by powdery mildew (Blumeria (syn.
Erysiphe) graminis D.C. ex Merat f.sp. tritia), with consequent negative effects on
stomatal resistance, boundary layer resistance, and transport resistance (Rabbinge et al.
1 985 .)
Powdery mildew of grape and gas exchange

Grapevine leaves infected with GPM have shown declines in net Pn (Lakso et al.
1982). Infected vines have demonstrated negative growth patterns, compared to
noninfected vines, consistent with reduction in Pn, both in the susceptible hybrid variety
Rosette (Seibel 1000) (Pool et al. 1984), and the relatively resistant variety Concord
(Gadoury and Seem 2001). Inhibition of Pn can be detrimental to plant health, as 290%
of plant dry matter is derived from C fixed through Pn (Flore and Lakso 1989).

Reduction in functional leaf area, whether from physical damage (lacerations due
to wind, rain, hail, etc.), arthrOpod predation, infection by pathogens, or deliberate leaf
removal as a cultural practice, may negatively affect plant carbon assimilation. Such
reductions Operate by simply reducing the photosynthetically active leaf area of a plant,

and do not alter any specific biochemical pathways as, for instance, herbicide-induced A

reduction might cause. Experiments attempting to approximate arthropod damage on a
single-leaf or whole-plant basis by removing portions of leaves, usually with a paper
punch, have been largely successful in mimicking A reduction caused by predation
(Boucher et al. 1987; Layne and Flore 1992; Poston et al. 1976), although care must be
taken to ensure that hole punching position with respect to the midrib be consistent with
typical arthropod feeding behavior (Layne and Flore 1992; Poston et al. 1976).

Many plants have demonstrated photosynthetic compensation for loss of
fimctional leaf area. Photosynthetic compensation has been demonstrated for apple
(Flore and Irwin 1983; Hall and F erree 1976), bean (von Caemmerer and F arquhar 1984),
Iucerne (Hodgkinson 1974), mulberry (Satoh et al. 1977), and soybean (Proctor et al.
1982), as well as grape (Boucher et al. 1987; Candolfi-Vasconcelos and Koblet 1991;
Hofacker I978; lntrieri et al. 1997; Petrie et al. 2000). Photosynthetic compensation has
also been demonstrated in the case of powdery mildew infection of pea (Ayers 1981).
Therefore, reductions in functional leaf area may not reflect actual reductions in total
plant A. The proposed mechanism for photosynthetic compensation is through feedback
inhibition caused by carbohydrate buildup in vines which are not source-limited (Layne
and Flore 1995; Petrie et al. 2000), implying that leaves of non-source-limited plants
typically operate at less than their optimum photosynthetic rate (Edson et al. 1993; Edson
et al. 1995; Petrie et al. 2000). It is also possible that grapevines may compensate for

reductions in functional leaf area by the production of new leaves, especially on lateral

shoots (Koblet et al. 1994).

Estimating photosynthesis

Photosynthesis has most commonly been estimated by measuring gas exchange
parameters on a section of an individual leaf. Advances in technology have made
measurement of whole-plant Pn more practical (Garcia et al. 1990; lntrieri et al. 1998;
Miller et al 1996; Pefia and Tarara 2002; Poni et al. 1997; Wiinsche and Palmer 1997).
Measurement of Pn of individual leaves may not be an accurate measure of whole-vine
Pn (Edson et al. 1995; Miller et al. 1996). Edson et al. (1995) found that Pn on the most
recently fully expanded leaf on a shoot was more highly correlated with whole-vine Pn
than measurements taken at other leaf positions; however, the relationship was quite
variable (r2=0.59, p=0.003). Single leaf Pn was correlated with whole vine Pn early in
the season in another experiment, but the relationship was weaker later in the season as
the canopy density increased (Miller et a1. 1996).

The experiments conducted to determine the effects of GPM on A in potted
Chardonnay grapevines are described in three chapters. The experiments in the first
chapter were designed to test the hypotheses that foliar infection by GPM inhibits single
leaf and whole plant A, and that photosynthetic compensation for reduction in A may
occur. The experiments in the second chapter were designed to test the hypotheses that
grapevines vary in susceptibility to GPM infection at different phenophases, that effects
of GPM infection may be cumulative over a growing season, and that reductions in A as a
result of GPM infection may have consequences in subsequent growing seasons. The
experiments in the third chapter were designed to determine the mechanisms by with

GPM might affect A in grapevines.

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10

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ll

Chapter 1
Effects of Powdery Mildew Infection on Carbon Assimilation of Potted Chardonnay
(Vitis vinifera L.) Grapevines

ABSTRACT
Potted Chardonnay (Vitis vinifera L.) grapevines were inoculated with conidial
suspensions of powdery mildew of grape (Uncinula necator (Schw.) Burr.) (GPM), and
the effects of GPM on infection on CO2 assimilation (A) were studied over two seasons.
Vines infected with GPM had reduced single leaf and whole vine A compared to
noninfected plants in both years. Reductions in A were correlated with reductions in
stomatal conductivity (g,) and transpiration (E), and increased internal C02 concentration
(C,). The effects were more pronounced in Season 2. Leaves on infected vines senesced
earlier than those on noninfected vines. Infected vines had reduced fresh and dry weights
at the end of the season compared to noninfected vines.
Introduction

Biotic and abiotic stresses on plants frequently result in reductions in plant growth
and productivity. Knowledge of specific plant physiological responses to stress, and
combinations of stresses, is becoming increasingly important as integrated crop
management systems are being developed and improved. Many interactions between
plants and biotic stress factors are incompletely understood.

Foliar injury caused by biotic and abiotic factors can reduce the ability of a plant
to assimilate C02. Powdery mildews, which are species-specific foliar fungal pathogens,
have been associated with reductions in photosynthesis (Pn) and transpiration (E) in a

variety of crops, including barley (Williams and Ayers 1981), apple (Ellis et al. 1981),

12

and grape (Lakso et al. 1982, Shtienberg 1992). Powdery mildew of grape, caused by
Uncinula necator (Schw.) Burr. (GPM) is the most widespread and destructive disease of
grapevines worldwide (Pearson and Gadoury 1992), and has long been known to result in
inferior fruit quality (Gadoury et al. 2001a; Ough and Berg 1979; Pool et al. 1984).
Early-season fruit infection may result in decreased fruit set, and may cause berry
splitting and tissue scarring (Chellemi and Marois 1992). Infected fruit is unsuitable for
fresh market use and may be unsuitable for the production of high quality wine (Ough
and Berg 1979; Pool et al. 1984).

GPM infections have been associated with reduced vine size (as determined by
cane pruning weights) and yield in susceptible varieties (Pool et al. 1984), or only with
vine size in relatively resistant varieties (Gadoury et al. 2001b). Infections have also
been demonstrated to cause reductions in C metabolism, but not E, of individual leaves of
susceptible grape species (Lakso et al. 1982). Reduction of net C02 assimilation (A)
caused by GPM infection may be caused by a reduction in photosynthetically active leaf
area, although Shtienberg (1992) found that visual assessments of foliar pathogen damage
frequently underestimate a foliar pathogen’s effect on gas exchange. Lakso et al. (1982)
found that leaf necrosis associated with GPM infection was primarily associated with
palisade layer destruction in infected grape leaves.

Reductions in C02 assimilation have been associated with delayed ripening and/or
decreased yields. Many plants have demonstrated photosynthetic compensation for
losses in functional leaf area (Boucher et al. 1987; lntrieri et al. 1997; Layne and Flore
1992; Poston et al. 1976; Proctor et al. 1982; van Caemmerer and F arquhar 1984).

Defoliation experiments have sometimes been used to mimic functional leaf area

13

reduction caused by biotic stresses. Results from several studies indicate that grapevines
can compensate photosynthetically for some degree of leaf area loss (Candolfi-
Vasconcelos and Koblet 1991; Hoflicker 1978; lntrieri et al. 1997), although, in another
experiment, removal of entire leaves of grapevines (Vitis vinifera cv. Pinot noir) did not
result in increased A in remaining leaves (Candolfi-Vasconcelos et al. 1994).

Grapevine species and cultivars have demonstrated variable susceptibility to GPM
(Doster and Schnathorst 1985). Only members of the Vitaceae are susceptible to GPM
(Pearson and Goheen 1988); however, this includes almost all of the economically
important grapes in the world. The fungus is presumably native to North America (ibid.);
consequently, V. vinifera L. species are relatively susceptible, while native American
species, especially V. Iabruscana Bail., are considered relatively resistant, although they
can also be negatively affected by GPM infection (Gadoury et al. 2001 a; Gadoury et al.
2001b). There is also a large degree of within-species variability in susceptibility to
GPM (Gut et al. 2002).

The goal of these experiments was to evaluate the effects of GPM on grapevine C
status using single leaf and whole plant gas exchange measurements and its influence on
seasonal C sequestration and partitioning.

Materials and Methods

Plant material. Experiment I . Two-year-old dormant grapevines (V. vim'fera cv.
Chardonnay, Dijon clone 96 grafted to C. 3309 rootstock) were planted in 19L pots in a
pasteurized medium of 45% sand, 45% loam, and 10% sand, and grown and maintained
in a greenhouse on the campus of Michigan State University, East Lansing, MI, USA,

during the spring of 2001. Minimum and maximum temperatures were maintained at

14

23°C and 32°C, respectively. Plants were thinned to two shoots per vine and defruited at
bloom. Vines were watered regularly and fertilized at bloom and monthly thereafier with
a soluble fertilizer at a rate of 0.38g N, 0.17g P, and 0.32g K per pot (Peter’s 20-20-20).

Experiment 2. Two-year-old dormant grapevines (V. vinifera cv. Chardonnay,
Dijon clone 96 grafted to 03309 rootstock) were planted in 19L pots in a medium of
70% loam, 20% sand, and 10% peat, and grown and maintained on a gravel pad outdoors
at the Horticultural Teaching and Research Center, Michigan State University, East
Lansing, MI, USA during the 2001 and 2002 growing seasons. Plants were thinned
shortly after full bud burst to three shoots per vine. Vines were watered regularly and
fertilized monthly with Peter’s 20-20-20 solution as above. Plants were largely fruitless;
the fruit on a few plants, not used in the experiment, were retained to determine
phenological stages during the growing season. Fruit was removed from all treatment
plants prior to bloom. Laterals were removed as they appeared throughout the growing
season. Two applications of Sevin (l-naphthyl N-methylcarbamate (carbaryl), Aventis,
Bridgewater, NJ) liquid were made as needed to control Japanese beetle (Popillia
japonica Newman) infestations. All applications were made at least seven days prior to
gas exchange measurements.

Experimental design and treatments. Experiment 1. Eighteen plants were
arranged in a completely randomized design and inoculated with a conidial suspension of

U. necator (produced by soaking infected leaves of Marechal Foch (Kuhlmann 188-2)

grapevines for 810mm and agitating to dislodge conidia) when three leaves had

appeared on most shoots. Each plant constituted an individual experimental unit.

15

Experiment 2. Plants were blocked according to fresh weight of the dormant,
unpotted vines and arranged in a completely randomized block designs as follows: each
block contained vines of similar initial fresh weight, and all phenological stages based on
fruit development were determined based on observations of the fi'uited, non-
experimental vines:

Year 1: Plants were arranged in six blocks, with seven subsamples per treatment
randomly arranged within each block to allow for three sequential destructive harvests,
each consisting of one plant per treatment per block. Four plants per treatment per block
were not destructively harvested at the end of the season, and were retained for another
experiment. Treatments were assigned randomly within blocks and were:

1. Plants inoculated with a conidial suspension of U. necator in distilled water as
described above just prior to bloom (as determined from the non-treatment, fi'uited vines),
using a hand sprayer and sprayed to runoff. This treatment was designated “Early”.

2. Plants were sprayed with myclobutanil (a-butyl-a-(4-chlorophenyl)-lH-1,2,4,
triazole-l-propanenitrile (NOVA), Rohm and Haas, Philadelphia, PA) at bloom and
inoculated with a conidial suspension of U. necator as above between the 5mm berry
stage and 1200 growing degree days (GDD) (base 50°F ), which was 35 days after Early
inoculation. This treatment was designated “Late”.

3. Plants were protected fiom GPM infection with myclobutanil at bloom,
between 5mm berry size and 1200GDD, and at veraison. This treatment was designated
as “Control”.

Year 2: Plants were arranged in 32 blocks with one vine of each treatment per

block. Treatments were identical to those of Year 1, although there was very little

16

inoculum available for imposing the Early treatment; Early plants were reinoculated
along with the Late plants, and a single Late inoculation was assumed for analysis
purposes.

Plants sprayed with myclobutanil were separated from inoculated plants by 21 0m
for 48h to help eliminate the potential effects of drift and/or volatiles from affecting
inoculated plants in both years.

Gas exchange measurements. Single leaf measurements were conducted using a
portable infrared gas analyzer (IRGA) (CIRAS-2, PP Systems, Amesbury, MA) fitted
with a leaf cuvette with light source (PLC6, ibid.). Measurements were taken between
900 and 1500hr at 1000 PAR and 27°C (i3°C).

Experiment I . Single leaf measurements were taken on the most recent fully
expanded leaf (FEL) on each shoot beginning 23 days post-inoculation, by which time
symptoms of GPM were evident on many leaves, and thereafter at two-week intervals for
the next 28 days. Prior to each leaf measurement, the leaf to be measured was evaluated
for GPM disease severity, expressed as the percentage of the leaf area with visible GPM
symptoms. Each leaf was measured twice, in case there was significant variability within
the leaf, and the results were averaged.

Experiment 2. Single leaf measurements.

Year 1. Single leaf measurements were conducted at bloom, the 5mm berry stage,

midseason(”~"12006DD), and 817 days post-veraison. At bloom, a representative shoot

was selected on each plant, and the most recent FEL on that shoot was measured and
marked. Subsequent measurements were conducted on the same, original, leaf (ORFEL),

and also on the current most F EL on the same shoot. GPM infection severity was

17

determined on each leaf prior to each gas exchange measurement, and expressed as the
percentage of the leaf area which showed GPM symptoms. 0n leaves having 220% PM
infection, two measurements per leaf were taken and the values averaged, in case
infection caused significant variances across the leaf surface.

Year 2. Leaves were selected as in Year I. ‘ Single leaf measurements were

conducted at bloom, 5mm berry stage, midseason (~1200GDD base 50°C), veraison,

and harvest. Only one measurement was taken on each leaf, as data from Year 1 showed
no significant differences between taking one or two measurements on infected leaves, as
determined by analysis of variance.

Whole vine measurements. Whole vine gas exchange measurements were
conducted using an open gas exchange system as described by Miller et al. (1996).
Mylar M-30 film (polyethylene terephthalate, polyvinylidene chloride coated; DuPont,
Wilmington, DE) was formed into a cylinder with a 4.0cm interior diameter (i.d.) piece of
polyvinylchloride (PVC) pipe at the top, and attached to a wooden base with elastic
(“bungee”) cord. The wooden base had holes drilled into it to allow for the grape trunk
(3.8cm diameter) and air inlet (4.0cm) to help minimize the effects of soil and root
respiration on gas exchange measurements. The 3.8cm hole was further insulated with
small strips of foam weather-strip material. Air was supplied using a small shaded pole
blower fan (model 4C004, Dayton, Inc, Dayton, OH). The fan was attached to a section
of 10.2cm i.d., 2.7m section of PVC pipe. The outlet end consisted of reduction and
angled couplings just before the chamber inlet (Figure l). A small piece of tape was
loosely placed over the inlet to diffuse airflow entering the chamber. Airflow and

temperature were measured with a thermal anemometer (Tri-Sense model 37000-60,

18

Cole-Farmer, Chicago, IL). Airflow was measured through a hole drilled midway on the
inlet pipe; measurements were taken at incremental depths of 2.5cm, and averaging the
readings. Volume of air was calculated from the averaged flow measurements by the

formula:

2
V=0.51Wl
10

 

]— 0.1 (Miller et al. 1996)

where V=volume of air in Us, r= radius of the air supply cylinder in cm, and l=the linear
flow rate in m/s.

C02 measurements were performed using the CIRAS-2 unit as an IRGA only;
inlet air was sampled first through a z1.3m section of flexible tubing, then the air at the
outlet of the chamber. Three pairs of measurements were made, and the average of the
values was used for calculating Pn. Whole vine C02 assimilation was calculated by the

formula:

 

PnOmoI/vine/s)= [($5.31 flngfisZZL/ :n ‘3”) (ibid).

Temperature inside and outside the chamber was measured prior to each series of
Pn measurements, and airflow was adjusted to maintain the temperature difference inside
the chamber to within 2°C of ambient temperature; if the airflow required adjustment to
reduce the temperature difference, the chamber was allowed to reequilibrate prior to
taking Pn measurements. Prior to enclosing plants in the Mylar chambers, GPM severity
was visually determined and expressed as the percentage of plant leaf area showing
disease symptoms. Measurements were taken on cloudless days between 900 and 1500h

to help ensure uniformity of plant light interception.

l9

Leaf area per vine was estimated by measuring shoot lengths on each measured
vine. Shoot length in grapevines is correlated with leaf area (Miller et al. 1996). The
relationship between shoot length and leaf area was determined by destructively
harvesting 30 non-treatment vines between midseason and veraison and measuring shoot
length and actual leaf area as determined by a belt-driven leaf area meter (LI-COR Model
LI-3000, Ll-3050ASH, LI-COR Inc., Lincoln, NE), and was determined to be
(y=12.01x°-°7“, r2=0.916).

Destructive harvests. Year 1. After the completion of each series of gas
exchange measurements, one plant from each treatment per block was selected at random
and destructively harvested. Plants were cut into component plant parts (roots, trunk,
shoots, and leaves), and fresh weights were measured. These plant parts were dried in a
forced-air drying oven at 45°C for 22 weeks, and dry weights were measured. Fresh
shoot lengths were also measured.

Year 2. Plants from 24 blocks were harvested after veraison; shoot lengths and
fresh and dry weights were measured as in Year 1.

Statistical analysis. Statistical analysis was performed using SAS statistical
software (version 8.2; SAS Institute Inc., Cary, NC). ANOVA mean separation was
performed using Fisher’s protected LSD. Regression p-values were obtained using linear

regression.

20

 

Figure. 1. Whole vine photosynthesis chambers for measuring carbon assimilation on
potted Chardonnay grapevines, showing Mylar chambers, blowers with ductwork, and
CIRAS-2 infrared gas analyzer (after Miller et al. 1996).

21

Results

Experiment I . Carbon assimilation was negatively correlated with disease
severity at all three dates of measurement as determined by regression (Figure 2). The
relationship was linear in Week 1, becoming more curvilinear in Weeks 3 and 5. Using
combined data from all three series of measurements, there was little decrease in A from
0-20% GPM severity, and little apparent decrease in A with increasing GPM severity
over 250%, which was confirmed by analysis of variance of the combined data (not
shown).

Experiment 2. Year 1. GPM inoculation resulted in decreased single leaf A
compared to control plants in Year 1 (Figure 3). A declined throughout the growing
season on both F EL and ORFEL, but the effect was greater on the ORFEL. Differences
between infected and noninfected F EL were significant at all measurement times, while
differences between ORFEL were not significant after the midseason (pre-veraison)
period. Stomatal conductance (gs) also declined over time (Figure 4), but no significant
trends were apparent. Differences in E were evident on ORFEL at the 5mm berry stage
and post-veraison (Figure 5), in a pattern similar to that of gs. There were no significant
effects of infection on intemal C02 concentration (C,) in the experiment (data not shown).

There was no effect of GPM infection on whole vine A at the 5mm berry stage.
At midseason, whole vine A decreased with increasing GPM severity (Figure 6). There
were no statistically significant differences in fresh or dry weights at any individual
destructive harvest date, probably due to the small sample size. Data from all three

destructive harvests were combined; only leaf weights were significantly different among

22

treatments, due to a high degree of senescence after veraison on infected vines (data not

shown).

23

 

 

 

 

 

 

 

 

 

 

 

 

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3 Would
14~ 1
0
12 y = 0.0014112 - 0.10221: + 9.9903?-

 

R’ - 0.6314
“0.0001

 

 

 

 

 

 

 

 

 

4
e . 0

2 s 0
0 . f v . w . . r a

o 10 20 30 40 so so 70 so 90

Disease Severity (56 GPM)
‘3 Week 5
14 -
’2 1, ' y - 0.00an - 0.1050: +10.4as
10 o R2 a 0.0107
p<o.0001

 

Pn szlaec)
b O
O O
1i

 

 

 

 

' \
e
o . - , ' . i
l, 20 40 00 so 100
2 . -- ,- .- W. -H---_,-__ .__— -e. . ~ -
Dbeaseaeverlty MGPII)

 

 

 

 

Figure 2. Impact of powdery mildew of grape (GPM) disease severity on single leaf
photosynthesis (Pn) rates of greenhouse-grown Chardonnay grapevines. Week 1
measurements (A) were conducted 23 days after inoculation with Uncinula necator, and
every 14 days thereafter (B and C).

24

 

A FEL Year 1

Ir 1 1.91m

p=o.0004 'p=0.0044 p=0.o4'37‘ " ' p=0.0064

 

 

 

 

 

Inoculation

 

_e

 

A tumouin'lsec)

1470 1600

 

 

Pre-xeraison Post Veraison
“I. Phenology

 

 

B ORFEL Year 1

05610206 ’ #63215

A almoum'mc)

 

850 1100 1470 1600

Bloom 5mm Pro-sermon Post Venison
GOD. Phenology

 

 

 

Figure 3. Effect of powdery mildew of grape infection on single leaf CO2 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (F EL) (A), and the original, initial FEL from the first series of
measurements (ORF EL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator twice
during the growing season.

25

 

A FEL, Year 1

350Inoculation .. .. .
p=0.1261 p=0.5115 p=0.2247 p=0.0552

 

Inoculation

l

I

 

 

g. (mmollm'laec)

 

1470 1600

 

Pro-veraison Post Veraison

 

 

 

B ORFEL Year 1

Inoculation

#03501" W Ea‘ooos' ' p=0.5035' ' p=0’fo1aahm

 

 

 

 

 

g. (mmollm'leec)

 

1600

 

 

Poet Veraison

Gm. Phonology

 

 

 

Figure 4. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (g) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (FEL) (A), and the original, initial F EL fiorn the first series of
measurements (ORFEL) (B) at different stages of vine grth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator twice
during the growing season.

26

 

A FEL Year 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

hocuation

p=0.09m p=0.4425 p=0.2113 p=0.0552
.3
§ ......“
E IICernu
15.
MI

PostVeraison

B caravan
.3
g ; Ilnteeted
E . lConttol
g .
In

 

 

 

 

 

 

 

650 1160 1470

 

 

 

 

 

Bloom 5mm Pro-veraison
GOD, Phenology

 

 

 

Figure 5. Effect of powdery mildew of grape infection on single leaf transpiration (E) on
potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (F EL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator twice

during the growing season.

27

 

A Whole Vlne A at lldseason. Year 1

y- 55-05% 0.0500“ 0.1120

”.0000

A unm‘bee)

 

0 10 20 30 40 50 00
Mae eeverly ($0! leafaree)

 

 

B Whole Vlne A at lldseason. Year 1

A menu's»)

 

 

 

 

Figure 6. Impact of powdery mildew of grape (GPM) infection on whole vine C02
assimilation (A) of potted Chardonnay grapevines at midseason (~1200 growing degree
days, base 50°F) (A and B).

28

Year 2. No infection resulted fiom the pre-bloom inoculation of U. necator;
therefore, most leaves became infected at the same time, after being inoculated after the
5mm berry stage. No effects of GPM infection were detected at mid-season (16 days
after inoculation) on single leaf measurements, although leaves had begun showing
symptoms. Whole vine A was negatively affected by infection; infected vines had
significantly lower A than noninfected vines (Figure 7), although the correlation between
disease severity and A was not significant at the p=5% level (p=0.0766, r2=0.1319). By
veraison, all single leaf parameters were affected by GPM infection on both FEL and
ORFEL (Figures 8-11). A, g,, and E were reduced, while C. was increased on infected
vines. These relationships also existed at harvest. There was no statistically significant
difference in whole vine A between infected and noninfected vinesat veraison at the
p55% level (p=0.0890), nor correlation between disease severity and A (p=0.0863,
r2=0.0693). At harvest, there were differences between infected and noninfected vines,
and there was a significant correlation between disease severity and A (Figures 7, 12).

Both total fresh weight and dry weight (biomass) were affected by GPM infection
(Figures 13-14). All plants infected with GPM had reduced root, shoot, leaf, and total
fresh and dry weights compared to control plants. Trunk weights were not affected by
GPM. Most of the differences in carbon partitioning among plant parts were due to much
greater leaf senescence on infected plants; infected plants had an average of 37 leaves,

while noninfected plants had an average of 54 leaves (p<0.0001).

29

 

Whole Vlne Carbon Aeelmllatlon. Year 2

p=0.0026 p=0.0642 p=0.0097
a

 

 

fl Infected
I Noninfeaed

A almovm'iuc)

   
  

 

 

 

 

 

1250 (16) 1850 (44) 2170 (66)

     

Mdseason Veraison
GD, Phonology. (Idectlon days)

 

Harvest

 

 

 

Figure 7. Effect of powdery mildew of grape (GPM) infection on whole-vine C02
assimilation (A) on potted Chardonnay grapevines as related to stages of vine grth
phenology, growing degree days (GDD) (base 50°F), and days after inoculation
(infection days) with a conidial suspension of Uncinula necator.

30

 

 

 

 

 

 

 

 

A FEL Year!
p=0.0998 p=0.9747 p=0.1153 p<0.0001 p<0.0001
Inoculation a
12 —
10 -
ch 8
3
S 6-
<
4 4
2 .
o _
B ORFEL Year2
14_ - . . , . ...... . ..... . , ..
p=0.0998 p=0.9824 p=0.1017 p<0.0001 p<0.0001
12
Inoculation

 

 

 

 

 

coo, Phenology

 

 

 

Figure 8. Effect of powdery mildew of grape infection on single leaf C02 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (F EL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F ). Vines were inoculated with Uncinula necator once
during the growing season.

31

 

 

A FEL Year 2

30.0001"

 

 

 

g. (mmollm‘leee)

 

 

 

 

ORFEL Year 2

p=o.9759" ' [#01120 W’Hpéoooo1 p<0.ooo1

 

Inoculation

 

 

 

g. (mmollm‘laec)

 

 

 

 

 

Figure 9. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (g,) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season.

32

 

A FEL Year 2

‘ wti£0liiit§zi " "Si-303005 V " "#07211?” W””"o=0.'0017'"m" “I,"iléoooosm;

Inoculation

 

 

 

GOD. Phonology

 

 

3 carat. Year2

p=0.0853 p=0.5652 p=0.5252 7 Hp<010001 7 I 1';’p=o.0005

 

 

 

Midseason

coo. Phenolouy

 

 

 

 

 

Figure 10. Effect of powdery mildew of grape infection on single leaf internal C02
concentration (C,) on potted Chardonnay grapevines on the most recent fully expanded
leaf at time of measurement (FEL) (A), and the original, initial FEL from the first series
of measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season.

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A FELYearZ
p=0.0145 p=0.7285 p=0.4335 p<0.0001 p<0.0001
a l
5 4
A ‘ "‘
g
3 3 Illnbcted
E ICOI'III'OI
.5.
In 21
14
o ..
B ORFELYearZ
p=0.014s p=0.7285 p=0.5320 p<0.0001 p=0.0109
8
.3
g : Ilnbcted
E IControl
E
In

 

 

 

GOD, Phenology

 

 

 

Figure 11. Effect of powdery mildew of grape infection on single leaf transpiration (E)
on potted Chardonnay grapevines on the most recent firlly expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology and growing
degree days (GDD) (base 50°F). Vines were inoculated with Uncinula necator once
during the growing season.

34

 

10

Whole Vlne Carbon Admllatlon at Harveu

 

 

 

 

y I -0.0493x + 6.9993
1? 8 0.4556

 

P'.0003

 

 

 

 

A (nanotm'leee)
0|

 

 

 

 

 

 

 

 

weaee eeverlty (new leaf area)

\ O .
. e e 0
e
O\
O Q 9
e
e
0
e
o 4 a . . . . . e .
0 10 20 30 40 50 60 70 80 90

100

 

Figure 12. Effect of powdery mildew of grape (GPM) infection on whole-vine C02
assimilation (A) on potted Chardonnay grapevines at harvest (~2170 growing degree

days, base 50°F).

35

 

Fresh Weight Infected, Year 2

Leaf
1 0%

 

161.89 Total

Fresh Weight Nonlnfected, Year 2

Leaf
1 8%

  

Shoot
1 0%

Tnlnk
1 8%

229.69 Total

Figure 13. Effect of powdery mildew of grape infection on season-long C02
assimilation and carbon partitioning of potted Chardonnay grapevines.

36

 

Biomass Infected, Year 2

Leaf
Shoot 6%
12% »

    

Root
57%

80.39 Total

Biomass Nonlnfected, Year 2

Leaf

Root
57%

 

102.09 Total

Figure 14. Effect of powdery mildew of grape infection on season-long C02
assimilation and carbon partitioning of potted Chardonnay grapevines.

37

F
'.

 

Discussion

Infection with GPM negatively affected carbon assimilation whenever it was
present in these experiments. The degree of damage as a consequence of disease severity
was extremely variable at low (320%) GPM severity, among different single leaf
measurements. Consequently, evidence of photosynthetic compensation for GPM
infection was variable and inconclusive. Results fi'om Experiment 1 suggest that infected

leaves may tolerate up to 20% disease severity. Results from Experiment 2 sometimes

 

showed tolerances to GPM between 0 and 20%, but at other times showed that any GPM
infection may be detrimental to single leaf A. This was most apparent in the Year 1
single leaf assessment of A at bloom, when there was a significant decrease in A on
inoculated leaves in the absence of GPM symptoms. At other times, there were no
measured leaves with infection rates between 0 and 20%, so no valid inferences could be
made on these degrees of disease severity. An approximate damage threshold level of
20% leaf damage is consistent with the estimated compensation levels for other perennial
fruit crops (Flore and Irwin 1983; Layne and Flore 1992). Whole vine results suggest
that grapevines may photosynthetically compensate on a whole plant level up to
approximately a 20% infection level. If single leaf compensation does not occur,
grapevines may compensate for GPM foliar damage through their ability to continue to
produce new leaves and/or lateral shoots throughout the growing season.

The effect of GPM infection on g,, C,-, and E was much more pronounced in Year
2. There are no obvious climatological explanations for this. Grapes grown in Year I

were potted later in the season (mid-June, vs. mid-May in Year 2), but both growing

38

seasons were fairly typical for mid-Michigan, and temperatures were never high or low
enough to inhibit gas exchange until late in the season.

The effects of GPM on E in Year 2 indicated that water use efficiency was not
compromised in leaves infected with GPM, since infected vines also had reduced E rates.
These data are in agreement with Shtienberg (1992), who found decreases in E associated
with GPM infection, but in contrast to the findings of Lakso et al. (1982), who found no
reduction in E of grape leaves infected with GPM. However, there was a great degree of
variability in their study on the susceptible V. vinifera cultivar White Riesling (r=0.11),
whose susceptibility to GPM is similar to that of Chardonnay (Gut et al. 2002), while
there was relatively little variability in the resistant variety Concord. Such variability in
crops infected with powdery mildews has been widely reported by Shtienberg (1992).
There was no perceptible pattern to the E differences observed in Year 1.

Plants showing reductions in A also showed reduced gs, indicating that stomatal
conductance is a barrier to carbon assimilation. This disagrees with Clearwater’s
findings on ‘Riesling’, in which the carbon assimilation mechanism had relatively little
association with decreases in A associated with GPM (Clearwater et al. 2002). The
association between reduced A and g, was particularly strong after GPM caused declines
in A in Year 2 (r2=0.8655, p<0.0001), indicating a strong stomatal limitation to A in
infected plants. Infected plants showing reduced A also had greater C, in Year 2, further
indicating that the carbon assimilation mechanism was compromised by GPM.

The reductions in A due to GPM infection resulted in decreased seasonal biomass
accumulation. This may have implications for firture seasons, as the amount of perennial

wood on grapevines has been associated with subsequent vine growth, yield, and link

39

quality (Koblet et al. 1994). This association was primarily concerned with above-
ground wood (trunks), which, in the potted vine study, was the only plant part whose
biomass was not significantly affected by GPM infection. This is probably due to the
architecture of the potted vines, which have much less relative trunk area and weight than
do mature, field-grown vines. Since all woody tissues of the plant contribute to
carbohydrate storage during the dormant season, it is probable that the reduced root
biomass as a consequence of GPM infection would also result in similar negative effects.
Reduction in woody tissue biomass would also explain the gradual vine size declines on
vines infected with GPM as reported by Gadoury et al. (2001a) and Pool et al. (1984).
These data suggest that GPM reduces A in infected grapevines, and that infected
plants may be able to compensate for disease severities 320%. The observed reductions
in A were probably a consequence of reductions in stomatal functions. Reductions in
biomass accumulation as a result of decreased A in infected plants may have negative
implications for future plant growth and development. Further research is needed to
more accurately quantify photosynthetic compensation, so that damage thresholds can be

established for vineyard management.

40

I. . .1 AZ"'A‘T~

 

Literature Cited

Boucher, T.J., D.G. Pfeiffer, J .A. Barden, and J .M. Williams. 1987. Effects of simulated
insect injury on net photosynthesis of potted grapevines. HortScience 22:927-928.

Candolfi-Vasconcelos, M.C. and W. Koblet. 1991. Influence of partial defoliation on
gas exchange parameters and chlorophyll content of field-grown grapevines.
Mechanisms and limitations of the compensation capacity. Vitis 30:129-141.

Candolfi-Vasconcelos, M.C., W. Koblet, G.S. Howell, and W. Zweifel. 1994. Influence
of defoliation, rootstock, training system, and leaf position on gas exchange of Pinot noir
grapevines. Am. J. Enol. Vitic. 45:173-180.

Chellemi, D0. and J .J . Marois. 1992. Influence of leaf removal, fungicide applications,
and fruit maturity on incidence and severity of grape powdery mildew. Am. J. Enol.
Vitic. 43:53-57.

Clearwater, L., G.S. Howell, and M. Trought. 2002. Impact of powdery mildew
infection and fungicide application on grapevine photosynthesis. Abstr. ASEV 53rd
Annu. Meet. Am. J. Enol. Vitic. 53:26.

Doster, MA. and WC. Schnathorst. 1985. Comparative susceptibility of various

grapevine cultivars to the powdery mildew fungus Uncinular necator. Am. J. Enol.
Vitic. 36:101-104.

Ellis, M.A., D.C. Ferree, and DE. Spring. 1981. Photosynthesis, transpiration, and
carbohydrate content of apple leaves infected by Podosphaera Ieucotricha.
Phytopathology 71 :392-395.

Flore, J.A. and C. Irwin. 1983. The influence of defoliation and leaf injury on leaf
photosynthetic rate, diffusive resistance, and whole tree dry matter accumulation in apple.
Abstr. HortScience 18:72.

Gadoury, D.M., R.C. Seem, R.C. Pearson, W.F. Wilcox. 2001a. Effects of powdery
mildew on vine growth, yield, and quality of Concord grapes. Plant Dis. 85:137-140.

Gadoury, D.M., R. C. Seem, A. Ficke, and W.W. Wilcox. 2001b. The epidemiology of
powdery mildew on Concord grapes. Phytopathology 91 :948-955.

Gut, L.J., R. lsaacs, J .C. Wise, A.L. Jones, A.M.C. Schilder, B. Zandstra, and E. Hanson.
2002. 2003 Michigan Fruit Management Guide. Michigan State University Extension,
East Lansing, MI.

Hofacker, W. 1978. Investigations on the photosynthesis of vines. Influence of
defoliation, topping, girdling and removal of the grapes. Vitis 17:10-22.

41

lntrieri, C., S. Poni, B. Rebucci, and E. Magnanini. 1997. Effects of canopy
manipulations on whole-vine photosynthesis: Results fi'om pot and field experiments.
Vitis 36:167-173.

Koblet, W., M. C. Candolfi-Vasconcelos, W. Zweifel, and G.S. Howell. 1994. Influence
of leaf removal, rootstock, and training system on yield and fruit composition of Pinot
noir grapevines. Am. J. Enol. Vitic. 45:181-187.

Lakso, A.M., C. Pratt, R.C. Pearson, R.M. Pool, and M.J. Welser. 1982. Photosynthesis,
transpiration, and water use efficiency of mature grape leaves infected with Uncinula
necator (powdery mildew). Phytopathology 72:232-236.

Layne, DR, and J .A. Flore. 1992. Photosynthetic compensation to partial leaf area
reduction in sour cherry. J. Amer. Soc. Hort. Sci. 117:279-286.

Miller, D.P., G.S. Howell, and J .A. Flore. 1996. A whole-plant, open, gas-exchange
system for measuring net photosynthesis of potted woody plants. HortScience 31 :944-
946.

Ough, CS. and H.W. Berg. 1979. Powdery mildew sensory effect on wine. Am. J.
Enol. Vitic. 30:321.

Pearson, R.C. and A.C. Goheen. 1988. Compendium of Grape Diseases, APS Press St.
Paul, MN.

Pearson, R.C. and Gadoury, D.M. 1992. Grape powdery mildew. In Plant Diseases of
International Importance. Vol. III, Diseases of Fruit Crops. J. Kumar, HS. Chaube, U.S.
Singh, and AN. Mukhopadhyay (Eds.), pp. 129-146. Prentice Hall, Englewood Cliffs,
NJ.

Pool. R.M., R.C. Pearson, M.J. Welser, A.N. Lakso, and R.C. Seem. 1984. Influence of
powdery mildew on yield and growth of Rosette grapevines. Plant Dis. 68:590-593.

Poston, F.L., L.P. Pedigo, R.B. Pearce, and RB. Hammond. 1976. Effects of artificial
and insect defoliation on soybean net photosynthesis. J. Econ. Entomol. 69:109-112.

Proctor, J .T.A., J .M. Bodnar, W.J. Blackburn, and R. L. Wilson. 1982. Analysis of the
effects of the spotted tentifonn leaf miner (Phyllonorycter blancardella) on the
photosynthetic characteristics of apple leaves. Can. J. Bot. 60:2734-2740.

Shtienberg, D. 1992. Effects of foliar diseases on gas exchange processes: A
comparative study. Phytopathology 82:760-765.

von Caemmerer, S. and GD. Farquhar. 1984. Effects of partial defoliation, changes of

irradiance during growth, short-term water stress and grth at enhanced p(C02) on the
photoSynthetic capacity of leaves of Phaseolus vulgaris L. Planta 160:320-329.

42

Williams, G.M., and P.G. Ayers. 1981. Effects of powdery mildew and water stress on
CO2 exchange in uninfected leaves of barley. Plant Physiol. 68:527-530.

 

43

 

Chapter 2
Effects of Timing of' Powdery Mildew Infection on Carbon Assimilation and

Subsequent Seasonal Growth of Potted Chardonnay (Vitis vinifera L.) Grapevines
ABSTRACT
Potted Chardonnay (Vitis vinifera L.) grapevines were inoculated with conidial
suspensions of powdery mildew of grape (Uncinula necator (Schw.) Burr.) (GPM) just .
prior to bloom (Early), just after the 5mm berry stage (Late), or not inoculated and treated g
with myclobutanil fungicide (Control) and the effects of timing of GPM infection were

studied over two seasons. Early vines had reduced C02 assimilation rates (A) than Late

 

and Control vines until the pro-veraison period, when Late vines also showed reductions
in A. Early vines had reduced fresh and dry weights compared to other treatments.
Leaves on both Early and Late vines senesced earlier than those on Control vines.
During the following growing season, shoot lengths and fresh and dry weights were
negatively correlated with the length of infection time in Season 1.
Introduction

Factors that interfere with carbon assimilation in grapevines may have different
effects based on the phenological stage of the plant when interferences occur. Powdery
mildew of grape (Uncinula necator (Schw.) Burr.) (GPM) is a pathogen of all green
tissues of the grapevine, and inhibits carbon assimilation (A) when leaves are infected
(Lakso et al. 1982). Reductions in A near bloom may cause increased competition for
carbohydrates within a grapevine, and may result in decreased fruit set (Smithyman et al.
1998). Between bloom and veraison, grapevines are usually operating at less than

optimal photosynthetic capacity, and the fruit is a relatively weak carbohydrate sink,

although sink strength increases over time (Edson et al. 1995). Between veraison and
harvest, the fruit is the predominant sink of the plant.

Grape species and cultivars differ in their overall susceptibility to GPM (Doster
and Schnathorst 1985); only Vitaceae genera are susceptible (Pearson and Goheen 1988),
although this family includes almost all of the economically important grapes in the
world. Vitis vinifera L. species are particularly susceptible (Pearson and Gadoury 1992). I
Grape berries also have demonstrated the development of ontogenic resistance to GPM as

they mature (Gadoury and Seem 1995; Gadoury et al. 2001a), becoming virtually

 

resistant by the 5 to 7mm berry stage (Gadoury and Seem 1995). In contrast, rachises

TW"
’0

remain susceptible throughout the season in the relatively resistant V. labruscana variety
Concord (Gadoury et al. 2001a). Grape leaves have also demonstrated ontogenic
resistance to GPM infection with increasing age (Doster and Schnathorst 1985; Sall and
Teviotdale 1981), generally becoming resistant to new infections after two months (Sall
and Teviotdale 1981). The mechanism for this resistance is not known. However,
germinated conidia develop hyphae on susceptible leaves, but not on resistant leaves
(Doster and Schnathorst 1985).

Grapevines remain somewhat susceptible to GPM infections throughout the
season in spite of the resistance of individual leaves, as new leaves are continuously
being produced, especially prior to veraison. Pool et al. (1984) found that early-season
applications of protectant fungicides controlled GPM on fruit as well as regular, periodic
sprays throughout the growing season, while leaves remained susceptible to infection all

season, showing no definite patterns of variable susceptibility as a consequence of vine

45

phenology. The effects of timing of the infection of GPM on grape leaves have not been
examined with regard to whole plant behavior.

Inhibition of A resulting from GPM infection may affect plant growth in
subsequent growing seasons (Gadoury et al. 2001b; Pool et al. 1984). Infection of GPM
on the relatively susceptible interspecific hybrid variety Rosette (Seibel 1000) resulted in
lower cane pruning weights and yield during the seasons following infection compared to
noninfected plants, as well as reduced fi'uit quality due to higher acidity (Pool et al.

1984). Cane maturity (as determined by the number of mature nodes on hardened canes) '!

 

and winter hardiness of canes were also reduced on infected plants compared to

If

noninfected plants. Bud fertility in the absence of severely cold winter temperatures was
lower in vines infected the previous season. GPM infection of the relatively resistant
variety Concord showed similar effects in years following infection, except that, even
though fewer buds matured, those that did mature were not less cold-hardy, and yield was
not reduced in the following growing season (Gadoury et al. 2001b).

These experiments examine the effects of the timing of GPM infection on carbon
assimilation of potted Chardonnay grapevines during the growing season, and subsequent
effects on vine performance in the following season.

Materials and Methods

Plant material. Season 1. Two-year-old dormant grapevines (V. vinifera cv.
Chardonnay, Dijon clone 96 grafted to 3309 rootstock) were planted in 19L pots in a
medium of 60% loam, 25% sand, and 15% peat and grown and maintained on a gravel
pad outdoors at the Horticultural Teaching and Research Center, Michigan State

University, East Lansing, MI, USA during the 2001 growing season. Plants were thinned

46

shortly after full bud burst to three shoots per vine. Vines were watered regularly and
fertilized monthly with a soluble fertilizer at a rate of 0.38g N, 0.17g P, and 0.32 g K per
pot (Peter’s 20-20-20). Plants were largely fruitless; the fi'uit on a few plants, not used in
the experiment, was retained to determine phenological stages during the growing season.
Fruit was removed from all treatment plants prior to bloom. Laterals were removed as
they appeared throughout the growing season. Two applications of Sevin (l-naphthyl N-
methylcarbamate (carbaryl), Aventis, Bridgewater, NJ) liquid were made as needed to
control Japanese beetle (Popilliajaponica Newman) infestations. All applications were
made at least seven days prior to gas exchange measurements.

Season 2. Plants not destructively harvested in Season 1 were left outdoors for
two months during dormancy, then pruned to two nodes per cane and moved into an
environmentally controlled greenhouse (high and low temperatures 34°C and 20°C,
respectively). Plants were thinned to three shoots per plant at bloom and fertilized
monthly as in Season 1. Laterals were removed as they appeared.

Experimental design. Season 1 . Plants were blocked according to the fresh
weights of the dormant, unpotted vines and arranged a completely randomized block
design with six blocks, and seven subsamples per treatment randomly arranged within
each block to allow for three sequential destructive harvests, each consisting of one plant
per treatment per block. Four plants per treatment per block were not destructively
harvested at the end of the season, and were retained for Season 2. Treatments were
assigned randomly within blocks and were:

1. Plants inoculated with a conidial suspension of U. necator (produced by

soaking infected leaves of Marechal Foch (Kuhlmann 188-2) grapevines for 8 10min and

47

 

agitating to dislodge conidia) just prior to bloom (as determined from the non-treatment,
fruited vines), using a hand sprayer and sprayed to runoff. This treatment was designated
“Early”.

2. Plants were sprayed with myclobutanil (a-butyl-a-(4-chlorophenyl)-1H-1,2,4,
triazole-l -propanenitrile (NOVA), Rohm and Haas, Philadelphia, PA) at bloom and
inoculated with a conidial suspension of U. necator as above between the 5mm berry
stage and 1200 growing degree days (GDD) (base 50°F), which was 35 days after Early
inoculation. This treatment was designated “Late”.

3. Plants were protected from GPM infection with myclobutanil at bloom,
between 5mm berry size and 1200GDD, and at veraison. This treatment was designated
as “Control”. Plants sprayed with myclobutanil were separated from inoculated plants by
=10m for 48h to help eliminate the potential effects of drift and/or volatiles from
affecting inoculated plants.

Season 2. Plants were arranged in a randomized complete block design, keeping
the same blocking arrangement as in Season 1, with four subsamples per block. No
plants were inoculated with U. necator during the growing season.

Fruitfulness measurements. Season 2. F lorets were counted on all shoots on all
vines in two randomly selected blocks prior to bloom. There were typically two clusters
per shoot; third clusters were removed from those shoots on which they occurred. Apical
and basal clusters were evaluated separately. Two weeks after fruit set, set berries were
counted. Fruit set was calculated as the ratio of the number of set berries to the number

of florets on each cluster, and expressed as a percentage.

48

 

 

Gas exchange measurements. Single leaf measurements were conducted using a
portable infiared gas analyzer (IRGA) (CIRAS-2, PP Systems, Amesbury, MA) fitted
with a leaf cuvette with light source (PLC6, ibid.). Measurements were taken between
900 and 1500hr at 1000 PAR and 27°C (i3°C). GPM infection was determined on each
leaf prior to each gas exchange measurement, and expressed as the percentage of the leaf
area which showed GPM symptoms. 0n leaves having 220% GPM infection, two *
measurements per leaf were taken and the values averaged, in case infection caused I

significant variances across the leaf surface.

 

Whole vine gas exchange measurements were conducted using an open gas 3,
exchange system as described by Miller et al. (1996). Mylar M-30 film (polyethylene
terephthalate, polyvinylidene chloride coated; DuPont, Wilmington, DE) was formed into
a cylinder with a 4.00m interior (i.d.) diameter piece of polyvinylchloride (PVC) pipe at
the top, and attached to a wooden base with elastic (“bungee”) cord. The wooden base
had holes drilled into it to allow for the grape trunk (3.80m diameter) and air inlet
(4.00m) to help eliminate the effects of soil and root respiration on gas exchange
measurements. The 3.8cm hole was further insulated with small strips of foam weather-
strip material. Air was supplied using a small shaded pole blower fan (model 4C004,
Dayton, Inc, Dayton, OH). The fan was attached to a section of 10.2cm i.d., 2.7m-long
section of PVC pipe. The outlet end consisted of reduction and angled couplings just
before the chamber inlet. A small piece of tape was loosely placed over the inlet to
diffuse airflow entering the chamber. Airflow and temperature were measured with a
thermal anemometer (Tri-Sense model 37000-60, Cole-Parmer, Chicago, IL), Airflow

was measured through a hole drilled midway on the inlet pipe; measurements were taken

49

at incremental depths of 2.5cm, and averaging the readings. Volume of air was

calculated from the averaged flow measurements by the formula:

2
V=0.51 ”1
10

 

]-0.1 (Miller et al. 1996)

where V=volume of air in Us, r= radius of the air supply cylinder in cm, and l=the linear
flow rate in m/s.

C02 measurements were performed using the CIRAS-2 unit as an IRGA only; ‘
inlet air was sampled first through a 21.3m section of flexible tubing, then the air at the

outlet of the chamber. Three pairs of measurements were made, and the average of the

 

values was used for calculating Pn. Whole vine C02 assimilation was calculated by the

formula:

 

, _ ((AC02)/,uL)((flow)L/ min) . .
PanoI/vme/s)—( (29.2/1L/ranol)(60s/min) ] (rbrd).

Temperature inside and outside the chamber was measured prior to each series of
Pn measurements, and airflow was adjusted to maintain the temperature difference inside
the chamber to within 2°C of ambient temperature; if the airflow required adjustment to
reduce the temperature difference, the chamber was allowed to reequilibrate prior to
taking Pn measurements. Prior to enclosing plants in the Mylar chambers, GPM severity
was visually determined and expressed as the percentage of plant leaf area showing
disease Symptoms.

Leaf area per vine was estimated by measuring shoot lengths on each measured
vine. Shoot length in grapevines has been correlated with leaf area (Miller et al. 1996).

The relationship between shoot length and leaf area was determined by destructively

50

harvesting 30 non-treatment vines between midseason and veraison and measuring shoot
length and actual leaf area as determined by a belt-driven leaf area meter (LI-COR Model
LI-3000, LI-3050ASH, LI-COR Inc., Lincoln, NE), and was determined to be
(y=12.o1x°-°744, r2=0.9l6).

Season 1. Single leaf measurements were conducted at bloom, 5mm berry stage,

pro-veraison (~1200GDD base 50°F ), and post-veraison. At bloom, a representative I

I“.-

shoot was selected on each plant, and the most recent fully expanded leaf (FEL) on that

shoot was measured and marked. Subsequent measurements were conducted on the

 

same, original, leaf (ORFEL), and also on the current most FEL on the same shoot.
Whole vine measurements were conducted at the 5mm berry size and pre-veraison stages.
Season 2. Leaves were selected as in Year 1. Single leaf measurements were

conducted at bloom, 5mm berry stage, midseason (81200 GDD, base 50°F), veraison,

and harvest. Since plants were not inoculated with U. necator, any treatment effects
measured were from infection in Season 1. Treatments were designated “Early(2°)”,
“Late(2°)”, and “Control(2°)” according to their treatments in Season 1.

Destructive harvest and shoot length measurements. Season 1 . After the
completion of the series of gas exchange measurements at the 5mm berry stage, pre-, and
post-veraison, one plant from each treatment per block was selected at random and
destructively harvested. Plants were cut into component plant parts (roots, trunk, shoots,
and leaves), and fresh weights were measured. These plant parts were dried in a forced-

air drying oven at 45°C for 22 weeks, and dry weights were determined.

51

Season 2. Shoot lengths were measured prior to each series of single leaf gas
exchange measurements. Plants were destructively harvested after harvest, and fresh and
dry weights were determined as in Season 1.

Statistical analysis. Statistical analysis was performed using SAS statistical
software (version 8.2; SAS Institute Inc., Cary, NC). ANOVA mean separation was
performed using Fisher’s protected LSD. Regression p-values were obtained using linear

regression.

r-.. m _-'....o§r.rr'

Results

Season 1 . Early inoculated plants showed reductions in A seven days after

 

inoculation compared to noninoculated plants, although GPM symptoms were not present
(Figure 1). Early FEL continued to have lower A than noninfected FEL throughout the
season. F EL of Late plants showed reductions in single leaf A 21 days after inoculation,
at which point A was statistically the same as for Early FEL. A declined throughout the
season in all treatments. A of infected FEL was lower than that of noninfected F EL
throughout the growing season; by post-veraison, A on infected FEL was approximately
half that of Control FEL.

Only Early ORF EL showed effects of GPM infection at the 5mm berry stage, as
Late plants had not yet been inoculated. After pro-veraison, there was no difference
between treatments on ORFEL. By the post-veraison period, OFREL were practically
nonfunctional, and many infected leaves had senesced.

Differences in stomatal conductance (gs) among treatments were only apparent on
F EL at post-veraison, when Early and Late F EL had reduced gs compared to Control

plants (Figure 2). Reductions in gs occurred on ORFEL of infected leaves at the 5mm

52

berry stage; there were also significant differences between treatments at pre- and post-
veraison; although Late ORFEL gs was slightly higher than other treatments, no trends
were apparent. Differences in internal CO2 concentration (C,-) were only apparent on
FEL at post-veraison, where Early and Late plants had slightly higher C,- than Control
plants (Figure 3). Transpiration (E) was only affected on FEL at post-veraison, when it
was reduced in infected leaves. Early ORF EL showed reduced E at the 5mm berry stage L
and, to a lesser extent, at pro-veraison (Figure 4). There were no differences between . *

treatments in whole vine A at either the 5mm berry stage or pre-veraison. There were

 

also no significant differences between treatments on fresh weights and component
biomass when evaluating each harvest date separately, presumably due to the small
sample size. Data from the three destructive harvests were combined to make a
composite sample for the growing season. Early infected plants had lower fresh and dry
root, shoot, and total plant weights than Late and Control plants, which were not
statistically different (Figures 5-6). Total plant fresh and dry weights of Early plants
were 86% and 81% of maximum, respectively, while Late and Control plants differed by
only 1% and were statistically identical. Early and Late plants had much greater leaf
senescence during the post-veraison period compared to Control plants (Figure 7). Fresh
and dry weights of individual leaves were statistically identical (data not shown); the

relative lack of leaves on infected plants is responsible for the large differences.

53

 

FEL Year 1

a. oII ”cum“

.1 .a .o
.‘ .O
O O

—l
p
o

A (pmomn'looc)
.0 8
O O

.0
o

   

650 (7, 0) 1160 (33. 0) 1470 (56, 21) 1660 (79. 44)
Bloom 5mm Ple-leraisen Poet-wraison
GOD (Infection daya "any" and "Late". noecttvely), phenology

 

 

ORFEL Year 1

p=0.0096

A lpmoum’looc)

1100 (33. 0) 1470 (so. 21) 1000 (79. 44)

 

5mm ' pm son " ' balm
GOD (Infection daye “Earty” and ”Lab". nqredlvely). phenology

 

 

 

Figure 1. Effect of powdery mildew of grape infection on single leaf C02 assimilation
(A) on potted Chardonnay grapevines on the most recent firlly expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season.

54

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
   
   

 

 

 

A FEL Year 1
”p=oeae7 .. . . .. . -.. ”p=02564“ _..... .. .. $00051 ..
Inoculation
l
I Early
I Late
uControl
a
650 (7. 0) 1160 (33. O) 1470 (56. 21) 1660 (79, 44)
Bloom 5mm Pro-veraison Post-veraison
GOD (Infection days. ”Early“ and "l.ate". nqrecflvely), phenology
B ORFEL Year 1
I Early
I Late
. DContml
l
ab 3 b
1160 (33. 0) 1470 (56. 21) 1660 (79. 44)
5mm Pre-wraison Poet-\ereiaon
ODD (Infection day: “Early” and ”Lab”. remectlveiy). phenology

 

 

 

Figure 2. Effect of powdery mildew of grape infection on single leaf stomatal
conductance (g,) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (F EL) (A), and the original, initial F EL fiom the first series of
measurements (ORF EL) (B) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season.

55

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

FEL Year 1
30°inoculation
l p=0.3573 p=0.4004 'noculafim p=0.2623 a p=0.0445
.7 a
250 1 I b
200 -— __
A I Early
E 150 -~ _— — a Late
6 nControl
100 lfl #fl _ s
50 -— s—
0 ,.
650 (7, 0) 1160 (33, 0) l 1470 (56. 21) 1660 (79, 44)
Bloom 5mm ‘ Pre-veraison Post-veraison
6130 (Infection days. “Early" and "Late". nqrec‘flveiy). phenology

 

Figure 3. Effect of powdery mildew of grape infection on single leaf internal C02
concentration (C,-) on potted Chardonnay grapevines on the most recent fully expanded
leaf at time of measurement (FEL) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season.

56

 

 

 

 

 

 

 

    
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1160 (33, 0)

 

 

5mm

 

 

 

 

 

1470 (so. 21)

 

 

PW

1660 (79, 44)

Post-\eraieon

GOD (Infection days, ”Early“ and “Late", reqeetlvely). phenology

     
    

 

 

 

‘DControl

 

FEL Year 1
Fluctuation
7 L .. . .. - -. . _ ..----- . 2.-.-.. -.___.._.. . _-..- ..--.- __.__._..._.__M -...-.2.-. _- .. _......_-.. _ --., ,. _--.,
I p=0.2112 p=0.6134 p=0.2109 p=0.0170 l
l
6 < ...... i
5 . Inoculation
a; 4 - l I Early
3 a Late
2 3 _1 0001111!”
In a 1
2 « '
1 .. ._i
0 ‘ ., .
650 (7. 0) 1160 (33. 0) 1470 (56. 21) 1660 (79. 44)
Bloom 5mm Pre-veraison Poet-veraison
GOD (Infection days. “Early“ and “Lab". respectively). phenology
B ORFEL Year 1
. .. . $00005 .... . ,. """9-4'010‘406 ., .. . #005132 ._ ,. .
hoculatlon -
,% ......
3 . a l I Late
E
E.
In

 

Figure 4. Effect of powdery mildew of grape infection on single leaf transpiration (E) on
potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) at different stages of vine growth phenology, growing
degree days (GDD) (base 50°F), and days from inoculation (infection days). Vines were
inoculated with Uncinula necator twice during the growing season.

57

 

 

 

 

 

Early Fresh Weight Season 1

 

101.09 Total

Late Flesh Weight Season 1

 

100.79 TotI

Control Fresh Weight Season 1

 

104.29 Total

Figure 5. Effect of powdery mildew of grape infection on CO2 assimilation and carbon
partitioning of potted Chardonnay grapevines. “Early” plants were inoculated with
Uncinula necator seven days pro-bloom; and “Late” vine were inoculated three days after
the 5mm berry stage. Data were combined from three sequential destructive harvests.

58

. tam."

Early Dry Weight Season 1

 

59.99 Total

Late Dry Weight Season 1

 

10x
73. 79 Tom

Control Dry Weight Season 1

 

1011 2110
74.29 Total

Figure 6. Effect of powdery mildew of grape infection on CO2 assimilation and carbon
partitioning of potted Chardonnay grapevines. “Early” plants were inoculated with
Uncinula necator seven days pre-bloom; and “Late” vine were inoculated three days after
the 5mm berry stage. Data were combined from three sequential destructive harvests.

59

 

Post-Harvest Leaf Weights, Season-1

25

p=0.0045 p=0.0m7

 

 

 

 

 

 

I Early

 

I Late
DControl

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

Fresh Loolwagm Dry Looiwagm

 

 

 

Figure 7. Fresh and dry leaf weights of plants infected at two inoculation times with
Uncinula necator. “Early” plants were inoculated seven days pro-bloom; “Late” vine

were inoculated three days after the 5mm berry stage.

60

Season 2. There were no differences in number of florets, number of set berries,
or percentage fruit set among treatments (data not shown). No GPM was observed on
any leaves throughout the growing season, so any treatment effects were only ascribed to
infection in Year 1. Gas exchange parameters were not affected by the previous year’s
infection by GPM at bloom. At the 5mm berry stage, there were differences in single leaf
A on both FEL and ORFEL (Figure 8). A was negatively correlated with the previous
season’s infection duration on F EL, and ORFEL leaves infected the previous season had
reduced A compared to Control(2°) plants. Between the 5mm berry stage and midseason,
an outbreak of mites occurred on most plants; early signs of damage were on leaves
above eye level, which was also the general area of the FEL. By the time the degree of
damage was assessed and treatment for mites was applied, significant foliar damage had
occurred, and no further differences were observed between treatments, presumably as a
consequence of mite damage. The distribution of mite damage was consistent over
individual blocks, so no treatment should have been affected more than any other. The
only other differences between treatments were a reduction in g, on Early(2°) plants at
midseason compared to Late(2°) and Control(2°) treatments (Figure 9), and elevated C,- in
treatments infected the previous year compared to Control(2°) plants (Figure 10). No

significant differences in E were observed during the growing season.

Shoot lengths were highly correlated with treatment effects in Season 1
throughout the season (Figure 11). Shoot lengths did not change much after bloom; there
was some apical meristem necrosis, presumably due to excessive handling when taking
measurements and moving plants into position for gas exchange measurements. It is also

presumed that the relatively heavy fruit crop (Figures 12-13) in Season 2 served as a

61

 

 

strong sink, redirecting resources to the cluster at the expense of shoot growth. Most
plants had two clusters per shoot, and there were no treatment effects on fruit set, berries

per cluster, berry weight, or total cluster weight at harvest (data not shown).

Plant fresh weight and biomass were affected by GPM infection in the previous
season (Figures 12-14). Root, shoot, leaf, and total plant flesh and dry weights were
negatively impacted by the previous season’s infection, while trunk and fruit weights I .

were not affected.

 

62

 

A FEL Season 2; Vines Only Infected Season 1

p=0.0537 p=0.0011 mltedamege p=0.0040 p=0.5245 p=0.6396
a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

935 1255 1555
5mm Midseeson Veraison
ODD. Phenoiogy
B ORFEL Season 2; Vines Only Infected Season 1
14 ‘ 9:005:37 p=0.0000 p=0.0565 p=0.2243 p=0.8754

mite dantege
" l

 

 

 

B 581M?)
I LIN?)
D Control(z‘)

trim; ‘zrrxr .2:

 

 

 

 

 

 

   

5mm Midseeson
one. Phenology

 

 

 

Figure 8. Effect of powdery mildew of grape infection on single leaf CO2 assimilation
(A) on potted Chardonnay grapevines on the most recent fully expanded leaf at time of
measurement (FEL) (A), and the original, initial FEL from the first series of
measurements (ORFEL) (B) status at different stages of vine growth phenology and
growing degree days (GDD) (base 50°F ). “Early(2°)” plants were inoculated with
Uncinula necator seven days pre-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season.

63

 

A FEL Season 2; Vines Only Infected Season 1

p=0.9356 p=0.014rla "‘5‘" “Fiwowizm 950.723017'”... 9:01.702-

 

 

g. (mmollrn'leec)

 

 

 

 

1255

 

Midseason
GDD. Phenology

 

I Early(2')

 

 

B ORFEL Season 2; Vines Only Infected Season 1

' 9:090'00 ’ i ” 9:02:72 m,.,‘.,,,,.,‘,;";eo;om"""'some "#07004

 

 

g. (mmollm'laec))

 

 

 

 

1255

 

Midseason
GDD, Phenology

 

 

 

 

Figure 9. Effect of powdery mildew of grape infection on single leaf stomatal

conductance (gs) on potted Chardonnay grapevines on the most recent fully expanded leaf
at time of measurement (F EL) (A), and the original, initial F EL from the first series of
measurements (ORF EL) (B) status at different stages of vine growth phenology and
growing degree days (GDD) (base 50°F). “Early(2°)” plants were inoculated with
Uncinula necator seven days pro-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season.

64

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEL Season 2; Vines Only Infected Season 1

p=0.9303 =0.0358 p=0.6560 p=0.8569 p=0.8182 l

250

a mite damage

245

E 240
6 235 __
230 . " _s
i
225 - 9.4

220 -
575 935 1255 1555 1955
Bloom 5mm Mdseason Veraison Harvest
“Philology

 

 

I Eany(2°)
I Lath’)
0 Control(2°)

 

 

 

 

Figure 10. Effect of powdery mildew of grape infection on single leaf internal CO2
concentration (C 2‘) on potted Chardonnay grapevines on the most recent fully expanded
leaf at time of measurement (F EL) at different stages of vine growth phenology and
growing degree days (GDD) (base 50°F). “Early(2°)” plants were inoculated with

Uncinula necator seven days pro-bloom in the previous growing season; “Late(2°)” vine

were inoculated three days after the 5mm berry stage in the previous growing season.

65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Season 2; Vines Only infected In Season 1
350 -
p=0.0030 p=0.0059 p=0.0119 p=0.0113
a a a a
r—* ,_. A
300 b ab b
9. b
E c o o b
.5, 250 fi. , ——-
g 200 fi‘ —— - A 2*
3;...9 L L _
E
E
g 100 -~ —« tfl
l-
50 — s A
0 _
575 935 1255 1555
Bloom 5mm Midseason Veraison
GDD. Phenology

 

 

 

 

T

n Early(Z')
a Late(2')
n Control(2°)

 

Figure 11. Effects of powdery mildew of grape on sheet length of potted Chardonnay
grapevines during the growing season following infection at different stages of vine
growth phenology, and growing degree days (GDD) (base 50°F). “Early(2°)” plants
were inoculated seven days pre-bloom in the previous growing season; “Late(2°)” vine
were inoculated three days after the 5mm berry stage in the previous growing season.

66

 

Fresh Weight Season 2, Early(2°)

 

Lee!
14%

431.09 Total

Figure 12. Impact of the previous season’s infection by powdery mildew of grape on
fresh weight of component plant parts of potted Chardonnay grapevines. “Early(2°)”
plants were inoculated with Uncinula necator seven days prior to bloom, and “Late(2°)
plants were inoculated three days after the 5mm berry stage in Season 1.

99

67

Dry Weight Season 2, Early(2°)

 

173.99 Toto

Dry Weight Season 2, Control(2°)

 

“95 Sheet
991

190.29 Total

Figure 13. Impact of the previous season’s infection by powdery mildew of grape on
biomass of component plant parts of potted Chardonnay grapevines. “Early(2°)” plants
were inoculated with Uncinula necator seven days prior to bloom, and “Late(2°)” plants
were inoculated three days after the 5mm berry stage in Season 1.

68

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A Season 2; Vines Only Infected In Season 1
9m _..--... . _ . . . .-- . .- ---. ---L--2 _-.,_.--.-. ..-. ...-E2“... ._- --,_- ...-.-. 2H_-____-_--.-. . . _. . __ _..
p=0.0057 p=0.2822 p=0.0016 p=0.0380 p=0.5000 p=0.0515
800 H_
700 . l—-1
. 000 5f; _-
S
5 500 ~ IEarty(2’)
i ILate(2°)
E 400 3... ~ DControI(2‘)
300 -I
a
200 I b ‘b .“ —-
100 « _-
0 "" I. 7
Root Total
Plant Part
3 Season 2; Vines Only Infected In Season 1
p=0.0072 p=0.4113 p=0.0002 p=0.0452 p=0. 1993 p=0.0004
a Eeliy(2')
ILetetZ')
DControl(2')

 

 

 

 

 

 

 

 

 

 

Plant Part

 

 

Figure 14. Effects of powdery mildew of grape on subsequent season accumulated fresh
weight (A) and biomass (B) of potted Chardonnay grapevines. “Early(2°)” plants were
inoculated with Uncinula necator seven days prior to bloom, and “Late(2°)” plants were
inoculated three days after the 5mm berry stage in Season 1.

69

 

Discussion

Grapevine leaves infected with GPM at any time consistently showed reduced A
compared to controls, with the exception of older leaves from midseason on. The
negative effect of leaf aging on Pn has been described by Kriedmann et al. (1970) and
Poni et al. (1994) on vines which, like those in this experiment, were not sink-limited.
The lack of differences in A older leaves between infected and noninfected vines may be
due to the equilibrating effects of leaf aging. The consistent negative effect of GPM on
the FELs and leaves up to £30 days older indicates that GPM can compromise the carbon
assimilation capacity of grapevines at any time during the growing season, and implies
that the impact may be cumulative over time. This is reflected in the destructive harvest
data, as Early plants had reduced average total season fresh weights and biomass
compared to Late and Control plants. Even though Late plants had reduced A from
midseason on, their total fresh weights and biomass were not different from Control
plants. Plants infected with GPM continued to develop disease symptoms on newer
leaves until late in the season, when temperatures were no longer favorable for conidial
germination.

The premature senescence of leaves in Season 1 may have strongly influenced the
reduced vine grth in Season 2. The ability to accumulate carbon during the period
after harvest is extremely important for subsequent seasonal growth, especially in cooler
climates, where there is typically a relatively small period of time between harvest (prior
to which the ripening fruit is the strongest sink on the plant for photosynthates), and the
onset of either temperatures too cold for carbon assimilation to occur, or leaf senescence

due to cold weather. Therefore, anything that will inhibit the ability of a plant to

70

accumulate carbon during this period has the potential to predispose the plant to

suboptimal growth in future seasons. Loss of substantial leaf area, even if leaves were
performing optimally, is likely to result in decreased carbon accumulation. This effect
may be exacerbated by the reduced A resulting from GPM infection during this period.

The effects of the previous season’s GPM infections were evident in Season 2.
The influence of the previous season’s GPM infections were inconclusive for single leaf
gas exchange parameters; while plants infected in Season I began to show differences in
A at the 5mm berry stage consistent with GPM treatment in the prior season, no other
significant trends were observed over the growing season. It is possible that the mite
damage obscured Season 1 treatment effects, as the data at 5mm berry size is highly
correlated with Season 1 treatments, and differences between treatments were not
apparent after the presence of significant mite damage.

Plants infected with GPM in Season 1 showed no signs of compensation for
suboptimal Season 1 A in Season 2. GPM treatments that reduced A in Season 1 showed
diminished growth in Season 2 in relation to their duration of GPM infection in Season 1.
Early(2°) plants consistently had the lowest shoot length, fresh weight, and dry weight
(biomass) accumulation of all treatments. Late(2°) plants, which had the same biomass
accumulation as Control plants in Season 1, also had significantly reduced shoot length,
fresh weight, and biomass accumulation than Control(2°) plants. These data are in
agreement with those of field-grown grapevines which have shown reduced growth in
season’s following foliar infection by GPM (Pool et al. 1984). Carbon partitioning was

not significantly influenced by Season 1 treatments, only total carbon accumulation.

71

The reduction in Season 2 shoot length in plants infected with GPM in Season 1
corresponded to a reduction in leaf area, as determined by regression in Season 1
(y=12.01x°'9m, r2=0.916). The reduction in leaf area would account for the reduced
biomass at the end of Season 2 for both Early(2°) and Late (2°) plants.

These data suggest that inhibition of A caused by GPM infection can result in both
within-season and long-term degradation in vine health. This may apply to other foliar
pathogens that inhibit A in grapevines. The earlier the onset of infection in the growing
season, the greater the plant’s ability to assimilate C02 is compromised, indicating the
need to control the disease early in the growing season. This corresponds to the initial
period of infection, whether from cleistothecia] ascospores or from conidia. Later
infections than those in this study may not result in such significant reductions in season-
long biomass accumulation. The determination of the latest phenophase at which GPM

ceases to affect leaf senescence deserves firrther study.

72

Literature Cited

Doster, MA. and WC. Schnathorst. 1985. Comparative susceptibility of various

grapevine cultivars to the powdery mildew fungus Uncinular necator. Am. J. Enol.
Vitic. 362101-104.

Edson, C.E., G.S. Howell, and J .A. Flore. 1995. Influence of crop load on
photosynthesis and dry matter partitioning of Seyvel grapevines. III. Seasonal changes
in dry matter partitioning, vine morphology, yield, and fruit composition. Am. J. Enol.
Vitic. 46:478-485.

Gadoury, D.M. and R. C. Seem. 1995. Development of ontogenic resistance to powdery
mildew (Uncinula necator) in fruit of Concord grapevines. Phytopathology 85:1149.

Gadoury, D.M., R. C. Seem, A. Ficke, and W.W. Wilcox. 2001a. The epidemiology of
powdery mildew on Concord grapes. Phytopathology 91:948-955.

Gadoury, D.M., R.C. Seem, R.C. Pearson, W.F. Wilcox. 2001b. Effects of powdery
mildew on vine growth, yield, and quality of Concord grapes. Plant Dis. 85:137-140.

Kriedemann, RE. 1968. Photosynthesis in vine leaves as a function of light intensity,
temperature, and leaf age. Vitis 7:213-220.

Lakso, A.M., C. Pratt, R.C. Pearson, R.M. Pool, and M.J. Welser. 1982. Photosynthesis,
transpiration, and water use efficiency of mature grape leaves infected with Uncinula
necator (powdery mildew). Phytopathology 72:232-236.

Miller, D.P., G.S. Howell, and J .A. Flore. 1996. A whole-plant, open, gas-exchange
system for measuring net photosynthesis of potted woody plants. HortScience 31 :944-
946.

Pearson, R.C. and D.M. Gadoury. I992. Grape powdery mildew. In Plant Diseases of
International Importance. Vol. III, Diseases of Fruit Crops. J. Kumar, H.S. Chaube, U.S.
Singh, and AN. Mukhopadhyay (Eds.), pp. 129-146. Prentice Hall, Englewood Cliffs,
NJ.

Pearson, R.C. and A.C. Goheen. 1988. Compendium of Grape Diseases, APS Press St.
Paul, MN.

Petrie, P.R., M.C.T. Trought, and G.S. Howell. 2000. Influence of leaf ageing, leaf area
and crop load on photosynthesis, stomatal conductance and senescence of grapevine
(Vitis vinifera L. cv. Pinot noir) leaves. Vitis 39:31-36.

Pool, R.M., R.C. Pearson, M.J. Welser, A.N. Lakso, and R.C. Seem. 1984. Influence of
powdery mildew on yield and growth of Rosette grapevines. Plant Dis. 68:590-593.

73

Poni, S., C. lntrieri, and O. Silvestroni. 1994. Interactions of leaf age, fruiting, and

exogenous cytokinins in Sangiovese grapevines under non-irrigated conditions. 1. Gas
exchange. Am. J. Enol. Vitic. 45:71-78.

Sall, MA. and BL. Teviotdale. 1981. Powdery mildew. In Grape Pest Management.
D. Glaherty, F. Jensen, A. Kasimatis, H. Kido, and W. Moller (Eds), pp. 46-50.
Agricultural Sciences Publications, University of California, Berkeley, CA.

Smithyman, R.P., G.S. Howell, and DP. Miller. 1998. The use of competition for
carbohydrates among vegetative and reproductive sinks to reduce fruit set and botrytis
bunch rot in Seyval blanc grapevines. Am. J. Enol. Vitic. 49:163-170.

Williams, LE. 1995. Grape. In Carbon Partitioning and Source-Sink Interactions in

Plants. M.A. Madore and W.J. Lucas (Eds.), pp. 851-881. Am. Soc. Plant Physiol.,
Rockville, MA.

74

Chapter 3
Effects of Powdery Mildew of Grape on the Carbon Assimilation Mechanisms of
Potted Chardonnay (Vitis vinifera L.) Grapevines
ABSTRACT
Potted Chardonnay (Vitis vinifera L.) grapevines were inoculated with conidial
suspensions of powdery mildew of grape (Uncinula necator (Schw.) Burr.) (GPM).
Leaves of infected and noninfected plants were studied for the effects of varying light
(PAR) and CO2 concentrations on factors affecting carbon assimilation. Infection by
GPM reduced carboxylation efficiency (11:), net CO2 assimilation rate (A), stomatal
conductance (gs), and internal C02 concentration (C,-) under ambient CO2, Amax at >900
ppm CO2, stomatal limitations to A (18), and photochemical efficiency (1p), while having
no effect on the CO2 compensation point (P) or the light compensation point (cp).
Infection by GPM had no effect on chlorophyll fluorescence (F v/F m).
Introduction
Plant responses to foliar biotic and abiotic stresses may vary with the nature of the
stress. Net C02 assimilation (A) by foliage is a critical factor influencing plant
productivity, since 290% of plant dry matter is derived from C fixed through
photosynthesis (Pn) (Flore and Lakso 1989). Therefore, factors that inhibit assimilation
through photosynthesis may be detrimental to productivity.
Photosynthesis in plants can be limited by biotic stresses in a variety of ways.

Johnson (1987) divided the seven categories of pest effects on plants as described by
Boote et al. (1983) into two groups: a) those whose major effects are on solar radiation

interception (tissue consumers, leaf senescence accelerators, stand reducers, and light

75

stealers), and b) those whose major effects are on relative use efficiency (photosynthetic
rate reducers, assimilate sappers, and turgor reducers). Damage to the photosynthetic
apparatus may occur by more than one of these effects; reductions in A caused by the
effects of most foliar pathogens on photosynthetic activity result from a decrease in the
photosynthesizing leaf area and/or its reduced efficiency (Goodman et al. 1986;
Shtienberg 1992; Yarwood 1967).

Response patterns affecting reductions in Pn and transpiration (E) have been
related to the general type of trophic relationships involved (Shtienberg I992); powdery
mildews tended to have more similar response patterns as compared to other foliar
pathogens, for example. Infections of powdery mildew of barley (BIumeria (syn.
Erysiphe) graminis D.C. ex Merat f.sp. hordei Marchal) resulted in both decreases in
chlorophyll after four days of infection and loss of electron transport activity, with no
loss of electron carrier concentration in remaining chlorophyll (Holloway et al. 1992).
Infections of powdery mildew of sugar beet (Erysiphe polygoni DC) inhibited electron
transport in noncyclic proteins, accompanied by alterations in chloroplast ultrastructure
and reduction of enzyme activity (Magyrarosy et el. 1976). Carboxylation resistance
increased in winter wheat infected by powdery mildew of wheat (BIumeria (syn.
Erysiphe) graminis D.C. ex Merat f.sp. tritici), with consequent negative effects on
stomatal resistance, boundary layer resistance, and transport resistance (Rabbinge et al.
1985). A was negatively affected by powdery mildew infection in all three studies.
There does not appear to be a relationship between decreases in A and E among
pathosystems; rather, E has been shown to increase, decrease, or stay the same in

response to foliar pathogens, including powdery mildews (Shtienberg 1992).

76

Grape leaves infected with powdery mildew of grape (Uncinula necator (Schw.)
Burr.) (GPM) have demonstrated reduced photosynthetic rates compared to uninfected
leaves (Lakso et al. 1982), due to destruction of palisade cells by the fungus. E was not
affected; consequently, water use efficiency was less in infected leaves. Field
experiments have demonstrated negative effects of GPM on grapevine health during the
season of infection, including decreased fruit quality (Gadoury et al. 2001; Ough and
Berg 1979; Pool et al. 1984) and fruit set (Chellemi and Marois 1992). Multiseasonal
effects include reduced vine size (as determined by cane pruning weights) and yield in
susceptible varieties (Pool et al. 1984), or only with vine size in relatively resistant
varieties (Gadoury et al. 2001).

Defoliation experiments have been conducted on grapevines for a variety of
reasons, including manipulation of fruit set, modifying the fruit microclimate, and to
simulate pest damage. Grapevine responses to defoliation by removing whole leaves
frequently include increased A by the remaining leaves (Hofticker 1978; Candolfi-
Vasconcelos and Koblet 1990; Candolfi-Vasconcelos and Koblet 1991; lntrieri et al.
1997), although Candolfi-Vasconcelos et al. (1994) found no increase in photosynthetic
rate in the remaining leaves. Punching holes in the leaves of other crop species have
been used to simulate the effects of damage by phytophagous arthropods (Boucher et al.
1987; Flore and Irwin 1983; Poston et al. 1976). Stacey (1983) found that leaf removal
on tomato plants largely approximated pest damage. Defoliation experiments have been
inconsistent in apprOxirnating damaged caused by foliar pathogens, as visual estimates of
infection do not always adequately indicate the effects of a pathogen on photosynthetic

and transpirational activities (Shtienberg 1992).

77

Measurements of chlorophyll fluorescence have also been employed to determine
the health of photosynthetic mechanisms in plants (Buwalda and Noga 1994; Krause and
Weis 1991), and have been correlated with end-product inhibition of leaf A due to
damage to photosystem 11 (P811) (Layne and Flore 1993). Depending on the nature of
pathogen-induced foliar damage, damaged leaves may exhibit less potential maximal
photochemical efficiency than uninfected leaves.

These experiments were designed to determine the physiological effects of GPM
infection on individual grape leaves regarding gas exchange and chlorophyll
fluorescence.

Materials and Methods

Plant material. Two-year-old dormant grapevines (V. vinifera L. cv.
Chardonnay, Dijon clone 96, grafted to 3309 rootstock) were planted in 19L pots in a
medium of 50% loam, 40% sand, and 10% peat. The plants were grown and maintained
on a gravel pad outdoors at the Horticultural Teaching and Research Center, Michigan
State University, East Lansing, MI, USA during the 2002 growing season. Plants were
thinned shortly after full bud burst to three shoots per vine. Vines were watered regularly
and fertilized monthly with a soluble fertilizer at a rate of 0.38g N, 0.17g P, and 0.32 g K
per pot (Peter’s 20-20-20). Plants were largely fruitless; a few plants which did have fruit
were retained to determine phenological stages during the growing season. Flower
clusters were removed from all treatment plants prior to bloom. Laterals were removed
as they appeared throughout the growing season. Two applications of Sevin(1-naphthyl

N-methylcarbamate (carbaryl), Aventis, Bridgewater, NJ) liquid were made as needed to

78

control Japanese beetle (Popilliajaponica Newman) infestations. All chemical
applications were made at least seven days prior to gas exchange measurements.
Experimental design. Plants were blocked according to the fresh weight of the
dormant, unpotted vines and arranged in a randomized complete block design with 32
blocks. Treatments were assigned randomly within blocks and were:
(1) Plants inoculated with a conidial suspension of GPM in distilled water
(produced by soaking infected leaves of Marechal Foch (Kuhlmann 188-2) grapevines for

6510 minutes and agitating to dislodge conidia) between the 5mm berry (as determined

from the non-treatment fi'uited vines) and 1200 growing degree days (GDD) (base 50°F )
stages using a hand sprayer and sprayed to runoff. This treatment was designated
“Infected”.

(2) Plants were sprayed with myclobutanil (u-butyl-a-(4-chlorophenyl)-lH-l,2,4,
triazole-l-propanenitrile (NOVA), Rohm and Haas, Philadelphia, PA) at bloom and

between the 5mm berry stage and midseason (81200GDD). This treatment was

designated “Noninfected”.

Plants sprayed with myclobutanil were separated from inoculated plants by 210m
for 48b to help eliminate the potential effects of drift and/or volatiles from affecting
inoculated plants.

Ten plants from each treatment were selected for gas exchange responses to
varying CO2 concentrations and photosynthetically active radiation (PAR) level
measurements by the following criteria: The most recent fully expanded leaves on the
longest shoot on each plant were examined just prior to veraison; leaf health was

evaluated based on visual ratings of disease severity, expressed as a percentage of the leaf

79

surface with visible GPM infection. The most recent fully expanded leaves from each of
the 10 blocks which had both the healthiest Noninfected leaves and an obviously
Infected, but otherwise undamaged (by insects, wind laceration, etc.) leaf, were selected
for gas exchange measurements. Disease severity on Infected leaves ranged from 50-
90% infected leaf area.

Gas exchange measurements. Gas exchange measurements were conducted
using a portable infrared gas analyzer (IRGA) (CIRAS-2, PP Systems, Amesbury, MA)
fitted with a leaf cuvette with light source (PLC6, ibid.). Effects of C02 concentration
were determined by gradually increasing C02 from 0 to 200ppm at SOppm increments,
and from 200 to 1000ppm at 100ppm increments at photosynthetically active radiation
(PAR)=1500, allowing the IRGA to equilibrate between each measurement using the
onboard computer (Fujitsu PenCentra 130, Fujitsu PC Corporation, Santa Clara, CA) and
software (version 1.0, PP Systems, Amesbury, MA). Responses to changes in PAR were
taken immediately afterward, using the same equipment and software, by reducing PAR
from 2000 to 200 in 200PAR increments, and from 200 to 0 in 50PAR increments.
Measurements were taken between 0900 and 1500hr at 26°C (i=2°C). Plants were
measured within each block according to their random placement to help alleviate the
effects of natural diurnal variances in A (Downton et al. 1987).The data were analyzed by
applying the Marquardt-Levenberg algorithm for nonlinear regression analysis for curve
fitting (Marquardt 1963; Layne and Flore 1992, 1995).

Parameters calculated fi'om plant responses of A to variable PAR (light response
curves) were: the light compensation point (op), extrapolated from the data where A=0,

and quantum yield (0), as determined by the slopes of the linear portion of the curve.

80

Parameters calculated from plant responses of A to variable internal CO2 concentration
(C,-) were the CO2 compensation point (P), extrapolated from the data where A=0;
carboxylation efficiency (k), as determined by the slopes of the linear portion of the
curve; stomatal limitation to A (13,), calculated according to the differential method of
Jones (1985); and Am”, the maximum A value at saturating CO2. A, g,, and C,- at ambient
C02 concentrations and saturating light conditions were also measured (A 300, gm, and
C2360, respectively).

Single leaf measurements were also performed on the most recent firlly expanded
leaf of the longest shoot on all plants in the plot over a period of two days to determine
relationships, if any, between A and g8 and C, at PAR=1000 and CO2=375ppm.

Chlorophyll fluorescence measurements

Three blocks were randomly selected for chlorophyll fluorescence measurements.
The longest shoot on each plant, also used for gas exchange measurements, was selected
and each leaf evaluated for disease severity, expressed as the percentage of leaf area with
visible PM symptoms. A clip with a sliding window to admit or exclude light was
attached to each leaf, and the leaf section was allowed to dark acclimate for 230 min.
Chlorophyll fluorescence was measured with a Hansatech Plant Efficiency Analyzer
(model PEA, Hansatech Instruments, Norfolk, England). Fluorescence was expressed as
the ratio of variable fluorescence (F9) to the maximum fluorescence (F I“) (F v/F m).

Statistical analysis. Statistical analysis was performed using SAS statistical
software (version 8.2; SAS Institute Inc., Cary, NC). AN OVA mean separation was
performed using Fisher’s protected LSD. Curve fitting was performed using SigmaPlot

software (version 8.01; SPSS Ltd., Chicago, IL).

81

Results

While A and g5 were negatively affected by GPM infection under ambient CO2
and saturating light conditions, there was no negative effect of GPM on C,- (Table 1).
Values for A 300 and 85360 on Infected plants were 38% and 36% of those of Noninfected
plants. k and Am, were also negatively affected by GPM infection (37% and 47%,
respectively, on Infected compared to Noninfected plants). There were no significant
differences in 1' between treatments. lg was higher in Infected plants compared to
Noninfected plants. There was no decline in A at high CO2 levels.

Single leaf measurements showed a strong relationship between A and g5 on both
Infected and Noninfected plants (Figure 2), although the linear relationships between A
and g, were different for the two treatments. There was a general negative correlation
between A and C, in Infected plants; the relationship between A and C,~ in Noninfected
plants was not significant at the pSOJO level, but was generally positive.

Infected plants showed reduced (52%) (0 compared to Noninfected plants. There
were no differences in cp between treatments. There were also no significant differences
in chlorophyll fluorescence between treatments (Figure 3) or between different levels of

disease severity.

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of potted Chardonnay grapevines infected and not infected with powdery mildew of

grape.

85

 

 

 

 

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Figure 3. Chlorophyll fluorescence on leaves of potted Chardonnay grapevines infected
and not infected with powdery mildew of grape (GPM). Fluorescence is expressed as the
ratio between variable fluorescence (Fv) and maximum fluorescence (Fm).
Discussion

GPM infection compromised the carbon assimilation mechanism of grape leaves
at several levels. The reduced k values for Infected plants indicate that the carboxylation
reactions, on a leaf area basis, were negatively affected by GPM infection. Similarly,
reductions in (p in Infected plants indicate a reduction in overall quantum efficiency on a
leaf area basis. These data are consistent with those of Lakso et al. (1982), who found
that GPM damaged the photosynthetic apparatus of grape leaves by causing death of
palisade cells. The lack of differences in chlorophyll fluorescence between treatments
indicates that there was no significant effect of GPM infection on the specific PSII
thylakoid reactions, and that the reduction of A as a consequence of GPM infection was

not due to disruptions of specific biochemical pathways, but rather to relatively large-

86

scale destruction of entire cells. GPM fungi do not actually invade palisade cells, only
epidermal cells (Pearson and Goheen 1988). However, the death of adjacent palisade
cells has been consistently noted (Lakso et al. 1982; Doster and Schnathorst 1985),
presumably due to a hypersensitive response similar to that observed on fruit (Seem, RC
2000, personal communication), and the results of this experiment are consistent with
photosynthetic losses as a consequence of palisade cell destruction.

The positive association between g8 and A in leaves of both Infected and
Noninfected plants indicates a strong mechanistic relationship between the two, and that
the correlation of gs on A is stronger in leaves of Infected plants than in leaves of
Noninfected plants. This stronger relationship is reflected in the negative relationship
between A and C,- in Infected leaves. The relationship between A and C,- was much
weaker, but positive, in Noninfected leaves. The correlation between increased gS and A
is similar to that observed in defoliation experiments on grapevines, when remaining
leaves demonstrated photosynthetic compensation for reduced leaf area (Hoflicker 1978,
Candolfi-Vasconcelos and Koblet 1991, Petrie et 211.2000). However, in this experiment,
any possible photosynthetic compensation was apparently overridden by the negative
effects of the high levels of GPM infection, as A levels on leaves of Infected plants were
consistently lower than those of leaves of Noninfected plants. The lack of compensation
was also evident in the reduced k and (p of infected plants; previous studies of
photosynthetic compensation for reduction in leaf area on sour cherry showed that k and,
to a lesser extent, (p increased afier partial (20%) defoliation (Layne and Flore 1992).

Disease severity in this experiment was much higher than 20%.

87

Increased lg in Infected leaves also shows stomatal influences on A, and implies
that the stronger positive relationship between g5 and A on Infected leaves might be
partially alleviated by increased stomatal resistance. The lack of decrease in A at
saturating PAR for either Infected or Noninfected plants indicates that ribulose-1,5-
bisphosphate (RuBP) regeneration capacity is not affected by GPM infection.

Photosynthetic responses of plants in response to infection by foliar pathogens
vary with the nature of the infection (Shtienberg 1992). Results from this experiment are
consistent with those to be expected from necrosis of palisade cells, with which GPM has
been associated (Lakso et al. 1982), but not by interfering with specific metabolic C02
assimilation pathways. The reduction in carboxylation efficiency was similar to that
observed in winter wheat infected with powdery mildew (Rabbinge et al. 1985). The
reduced electron transport in response to powdery mildew of barley (Holloway et al.
1992), attributed to the destruction of chloroplasts and not inhibition of metabolic
pathways, also resembled the results of this study. Powdery mildew of sugar beets did
alter metabolic pathways by reducing enzyme activity (Magyarosy et al. 1976), indicating
that the mechanisms of inhibition of the photosynthetic apparatus vary with the obligate
pathogen and/or host plant reaction.

Results from these experiments suggest that GPM inhibits single leaf A in
grapevines by quantitatively interfering with the carbon assimilation apparatus of
individual leaves. These reductions in A are caused mostly by disruptions of stomatal
and photochemical fimctions. Cultural practices designed to reduce GPM infection of
berries in vineyards may have both short— and long-term health benefits for grapevines as

a result of a lack of GPM-induced reduction of A in foliage. Additional research should

88

address the impact of lower levels of GPM on the photosynthetic apparatus of individual

leaves and whole vines.

89

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Boote, K.J., J .W. Jones, J .W. Mishoe, and RD. Berger. 1983. Coupling pest to crop
growth simulators to predict yield reductions. Phytopathology 73:1581-1587.

Boucher, T.J., D. G. Pfeiffer, J .A. Barden, and J .M. Williams. 1987. Effects of

simulated insect injury on net photosynthesis of potted grapevines. HortScience 22:927-
928.

Buwalda, J .G. and G. Noga. 1994. Intra-plant differences in leaf chlorophyll
fluorescence parameters in perennial fruiting plants. New Zealand J. Crop Hort. Sci.
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Candolfi-Vasconcelos, M.C. and W. Koblet. 1990. Yield, fruit quality, bud fertility and
starch reserves of the wood as a function of leaf removal in Vitis vinifera- Evidence of
compensation and stress recovering. Vitis 29:199-221.

Candolfi-Vasconcelos, M.C. and W. Koblet. 1991. Influence of partial defoliation on
gas exchange parameters and chlorophyll content of field-grown grapevines.
Mechanisms and limitations of the compensation capacity. Vitis 30:129-141.

Candolfi-Vasconcelos, M.C., W. Koblet, G.S. Howell, and W. Zweifel. 1994. Influence
of defoliation, rootstock, training system, and leaf position on gas exchange of Pinot noir
grapevines. Am. J. Enol. Vitic. 45:173-180.

Chellemi, DO. and J .J . Marois. 1992. Influence of leaf removal, fungicide applications,
and fi'uit maturity on incidence and severity of grape powdery mildew. Am. J. Enol.
Vitic. 43:53-57.

Dorster, MA. and W.C. Schnathorst. 1985. Comparative susceptibility of various
grapevine cultivars to the powdery mildew fungus Uncinula necator. Am. J. Enol. Vitic.
36:101.

Downton, W.J.S., W.J.R. Grant, and B.R. Loveys. 1987. Diurnal changes in the
photosynthesis of field-grown grape vines. New Phytol. 105:71-80.

Flore, J .A. and C. Irwin. 1983. The influence of defoliation and leaf injury on leaf
photosynthetic rate, diffusive resistance, and whole tree dry matter accumulation in apple.
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Flore, J .A. and AN. Lakso. 1989. Environmental and physiological regulation of
photosynthesis in fruit crops. In Horticultural Reviews Vol. II. J. Janick (Ed.), pp. 111-
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Gadoury, D.M., R.C. Seem, R.C. Pearson, W.F. Wilcox. 2001. Effects of powdery
mildew on vine growth, yield, and quality of Concord grapes. Plant Dis. 85: 137-140.

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Goodman, R.N., Z. Kiraly, and KR. Wood. 1986. Photosynthesis. In The Biochemistry
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92

APPENDIX

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