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This is to certify that the
thesis entitled

“DOWNY MILDEW: HOST SPECIALIZATION
AND EFFECTS ON
PHOTOSYNTHESIS AND CARBON
PARTITIONING IN ‘NIAGARA’ GRAPEVINES"

presented by

BRIAN LEE LEHMAN

has been accepted towards fulﬁllment
of the requirements for the

 

 

 

 

 

MASTER OF degree in PLANT PATHOLOGY
SCIENCE
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erssor’s Signature
5 / 9 / o 5"
Date

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2/05 c:/ClRC/DateDue.indd-p.15

DOWNY MILDEW: HOST SPECIALIZATION AND EFFECTS ON
PHOTOSYNTHESIS AND CARBON PARTITIONING IN ‘NIAGARA’
GRAPEVINES
By

Brian Lee Lehman

- A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Plant Pathology

2005

ABSTRACT
DOWNY MILDEW: HOST SPECIALIZATION AND EFFECTS ON
PHOTOSYNTHESIS AND CARBON PARTITIONING 1N ‘NIAGARA’
GRAPEVINES
By

Brian Lee Lehman

Michigan is the fourth largest producer of juice grapes (Vitis Iabrusca) in the
United States, averaging more than 400,000 tons annually. Downy mildew (Plasmopara
viticola) is an important pathogen Of juice grapes in Michigan and throughout the grape-
growing regions of the United States. Downy mildew can damage leaves and fruit
Clusters, resulting in losses in fruit yield and quality. The objectives of this research were
to: 1) study the effect of downy mildew on photosynthesis of ‘Niagara’ leaves, 2) study
the effect of infection on dry weight and carbon partitioning in ‘Niagara’ vines and, 3)
evaluate the ability of P. viticola to cross-infect different grape cultivars and Species.

In ﬁeld studies, ‘Niagara’ leaves infected with downy mildew showed a reduction in total
carbon assimilation with increasing disease severity. On potted ‘Niagara’ vines,
inoculated leaves showed a Signiﬁcant reduction in photochemical efﬁciency,
carboxylation efﬁciency, maximum rate of photosynthesis, and the Stomatal limitation to
photosynthesis before symptoms appeared. In ﬁeld studies, downy mildew reduced total
net dry weight in vines and affected the rate of carbon translocation, but had no effect on
carbon partitioning.’Cross infection studies carried out with isolates of P. viticola from
different host species and isolates in Michigan showed that ‘Niagara’ leaves were

resistant to infection by isolates of P. viticola obtained from several other host Species.

DEDICATION

This thesis is dedicated to my wife Marcie for her continuous love and support.

iii

ACKNOWLEDGMENTS

I would like to thank my advisor Dr. Annemiek Schilder for her friendship,
advice, and support. I would also like to acknowledge my guidance committee members,
Dr. Ray Hammerschmidt and Dr. James Flore for their advice and support and for giving
me conﬁdence throughout my graduate research.

I would like to give Special thanks to Adriana Nikoloudi and Costanza Zavalloni
for taking time away from their own research to help me with ﬁeld work and analyzing
data.

I greatly appreciate the help, advice, and friendship of Jerri Gillett, Wendy Mann,
Phillip Wharton, Siva Sabaratnam, Jan Byme, Roger and Jennifer Sysak, April Sanders,
Steve Jordan, Amanda Gevens, Janette Jacobs, Paolo Sabbatini, Ryan Bounds, Jackie
Smith, Angela Tenney, William Kirk, Tim Maret, Ben Munn, Khawlah Munshi, Brent
Hoag, Cliff Zehr, Pete Callow, Susan Butterworth, and Gerry Skeltis.

My Sincere gratitude goes to my wife Marcie for her understanding, support, and

encouragement.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. ix

LIST OF FIGURES .......................................................................................................... xiii
CHAPTER 1

LITERATURE REVIEW .................................................................................................... l

The Grapevine ........................................................................................................ .1

History ......................................................................................................... 1

Grape production ......................................................................................... 2

Grapevine physiology ................................................................................. 4

Carbon assimilation ......................................................................... 5

Effect of stomata and leaf age on photosynthesis ........................... 7

Effect of light on photosynthesis ..................................................... 7

Source-Sink relationships and carbon partitioning .......................... 8

Downy Mildew of the Grapevine .......................................................................... l 1

Classiﬁcation ............................................................................................. 1 1

History ....................................................................................................... 12

Host range ................................................................................................ l3

Symptoms .................................................................................................. l4

Morphology ............................................................................................... 17

Disease cycle and epidemiology ............................................................... 19

Primary infection ........................................................................... l9

Sporulation .................................................................................... 22

Survival of Sporangia .................................................................... 23

Overwintering ................................................................................ 24

Distribution and economic importance ..................................................... 24

Disease management ................................................................................. 25

Fungicides ..................................................................................... 26

Biological control .......................................................................... 28

Forecasting .................................................................................... 28

Rationale and objectives of the research ................................................... 29

Literature cited .......................................................................................... 30

CHAPTER 2
THE EFFECTS OF DOWNY MILDEW (Plasmopara viticola) INFECTION ON

PHOTOSYNTHESIS OF ‘NIAGARA’ GRAPEVINE LEAVES .................................... 36

Introduction ........................................................................................................... 36

Estimating photosynthesis and gas exchange parameters ......................... 36

Effects of biotrophic pathogens on photosynthesis ................................... 40

Effects of biotrophic pathogens on respiration ........................................ 43

Effects of biotrophic pathogens on chlorophyll ﬂuorescence ................... 44

Pathogen effects on grapevine photosynthesis .......................................... 44

Effects of Plasmopara viticola on photosynthesis of grapevines ............. 45

Rationale and objectives ............................................................................ 46

Materials and methods .......................................................................................... 46
Assimilation rates of leaves of downy mildew-infected ‘Niagara’ vines

in the ﬁeld .................................................................................................. 46

Data analysis ................................................................................. 47

Leaf photosynthesis in potted vines as infection progresses ..................... 47

Plant material ................................................................................. 47

Inoculation ..................................................................................... 48

Measurements ................................................................................ 48

Data analysis ................................................................................. 49

Results ................................................................................................................... 50
Assimilation rates of leaves of downy mildew-infected ‘Niagara’ vines

in the ﬁeld .................................................................................................. 50

Leaf photosynthesis in potted vines as infection progresses ..................... 54

Discussion ............................................................................................................. 68

Literature cited ...................................................................................................... 74

vi

CHAPTER 3
THE EFFECT OF DOWN Y MILDEW INFECTION ON CARBON PARTITIONING

AND BIOMASS ACCUMULATION IN ‘NIAGARA’ VINES ...................................... 79

Introduction ....................................................................................................... 79

Pathogen effects on carbon allocation ....................................................... 79

Effects of biotrophic pathogens on carbon allocation ............................... 80

Carbon isotope composition in plant tissues ............................................. 81

Isotopes and carbon partitioning in grapevines ........................................ 83

Rationale and objectives ............................................................................ 84

Materials and Methods .......................................................................................... 85
Effect of downy mildew on plant dry weight and dry weight

partitioning ................................................................................................ 85

Inoculation ..................................................................................... 85

Statistical analysis ......................................................................... 86

'3C translocation and dry weight accumulation in infected vines ............. 86

Inoculation ..................................................................................... 87

Administration of 13CO; to plants (pulsing) .................................. 87

Sampling for I3C ............................................................................ 88

Dry weight analysis ....................................................................... 88

Stable isotope analysis .................................................................. 89

Data analysis ................................................................................. 89

Statistical analysis ......................................................................... 91

Results ................................................................................................................... 91
Effect of downy mildew on plant dry weight and dry weight

partitioning ................................................................................................ 9]

3 C translocation in infected vines ............................................................ 92

Discussion ........................................................................................................... 103

Dry weight accumulation and allocation ................................................. 103

'3C distribution ........................................................................................ 104

Other implications ................................................................................... 105

Conclusion ............................................................................................... 106

Literature cited .................................................................................................... 107

vii

CHAPTER 4
VARIATION AMONG Plasmopara viticola ISOLATES FROM DIFFERENT HOST

SPECIES ......................................................................................................................... 111
Introduction ......................................................................................................... l 1 1

Host range of P. viticola .......................................................................... 112

Host resistance to P. viticola ................................................................... 112

Growth of P. viticola on resistant cultivars ............................................ 113

Molecular studies on P. viticola .............................................................. 114

Rationale and objectives .......................................................................... 116

Materials and methods ........................................................................................ 117

Plant material and isolate collection ........................................................ 117

Inoculum preparation .............................................................................. 117

Leaf disk preparation ............................................................................... 118

Inoculation ............................................................................................... 118

Data analysis ........................................................................................... 119

Results ................................................................................................................. 121
Discussion ........................................................................................................... 129
Literature cited .................................................................................................... 133
APPENDIX A ................................................................................................................. 137
APPENDIX B ................................................................................................................. 148

viii

LIST OF TABLES

 

 

CHAPTER 2
Table 2.1. Photosynthetic parameters affected in ‘Niagara’ grapevine leaves inoculated
with Plasmopara viticola in 2003 ..................................................................................... 67
CHAPTER 4
Table 4.1. Plasmopara viticola isolates used in cross-infection studies on different host
Species and cultivars in 2003 and 2004. .......................................................................... 120
Table 4.2. Analysis of variance of isolate-host combinations used in 2003 .................. 124
Table 4.3. Analysis of variance of isolate-host combinations used in 2004. ................. 125

Table 4.4. Analysis of variance of isolate-host combinations used in 2003 and 2004... 126

APPENDIX A

Table A.l. The effect of downy mildew disease development in ‘Niagara’ grapevine
leaves on chlorophyll fluorescence, photosynthetic efﬁciency (0), Aamb, and the light
compensation point determined by ﬂuorescence measurements and light response curves
in 2003 ............................................................................................................................. 138

Table A.2. The effect of downy mildew disease development in ‘Niagara’ grapevine
leaves on carboxylation efﬁciency (k), C02 compensation point (P), internal C02
concentration, Am”, stomatal conductance (gs), and stomatal limitation (lg (in) determined
by C02 response curves in 2003 ..................................................................................... 139

Table A.3. The effect of downy mildew disease development in ‘Niagara’ grapevine
leaves on chlorophyll fluorescence, photosynthetic efﬁciency (0), Aamb, and the light
compensation point determined by fluorescence measurements and light response curves
in 2004 ............................................................................................................................. 140

Table A.4. The effect of downy mildew disease development in ‘Niagara’ grapevine
leaves on carboxylation efﬁciency (k), C02 compensation point (P), internal C02
concentration, Am”, stomatal conductance (gs), and stomatal limitation (1200) determined
by C02 response curves in 2004 ..................................................................................... 141

Table A.5. Repeated measures analysis of variance of fluorescence in leaves of ‘Niagara’
grapevines infected with Plasmopara viticola compared to healthy leaves in 2003 ...... 142

ix

Table A.6. Repeated measures analysis of variance of the assimilation rate at ambient
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2003 ..................................................................................................... 142

Table A.7. Repeated measures analysis of variance of the light compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003. ................................................................................................................. 142

Table A.8. Repeated measures analysis of variance of the photosynthetic efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003. ................................................................................................................. 142

Table A.9. Repeated measures analysis of variance of the assimilation rate at maximum
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2003 ..................................................................................................... 143

Table A.10. Repeated measures analysis of variance of the stomatal conductance in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003 ....................................................................................... 143

Table A.11. Repeated measures analysis of variance of the CO2 compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003 .................................................................................................................. 143

Table A.12. Repeated measures analysis of variance of the carboxylation efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003 .................................................................................................................. 143

Table A.13. Repeated measures analysis of variance of the intercellular CO2
concentration in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2003 ................................................................................ 144

Table A.14. Repeated measures analysis of variance of the stomatal limitation to
photosynthesis in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2003 ................................................................................ 144

Table A.15. Repeated measures analysis of variance of ﬂuorescence in leaves of
‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy leaves in
2004 ................................................................................................................................. 144

Table A.16. Repeated measures analysis of variance of the assimilation rate at ambient
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2004 ..................................................................................................... 144

Table A.17. Repeated measures analysis of variance of the light compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004 .................................................................................................................. 145

Table A.18. Repeated measures analysis of variance of the photosynthetic efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004 .................................................................................................................. 145

Table A.19. Repeated measures analysis of variance of the dark respiration in leaves of

‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy leaves in
2004 ................................................................................................................................. 145

Table A.20. Repeated measures analysis of variance of the assimilation rate at maximum
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2004 ..................................................................................................... 145

Table A.21. Repeated measures analysis of variance of the Stomatal conductance in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004 .................................................................................................................. 146

Table A.22. Repeated measures analysis of variance of the C02 compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004 ................................................................................................................. 146

Table A.23. Repeated measures analysis of variance of the carboxylation efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004 .................................................................................................................. 146

Table A.24. Repeated measures analysis of variance of the intercellular CO2
concentration in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2004 ................................................................................ 146
Table A.25. Repeated measures analysis of variance of the stomatal limitation to
photosynthesis in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2004 ................................................................................ 147
APPENDIX B
Table 8.1. Percent of pulsed l3C02 taken up by ‘Niagara’ grapevines in 2004 ............. 149
Table B.2. Analysis of variance of total dry weight of ‘Niagara’ grapevines infected with

Plasmopara viticola at the 5-mm berry Stage and bunch closure compared to healthy
vines in 2003 ................................................................................................................... 150

xi

Table B.3. Analysis of variance of dry weight of individual organs of ‘Niagara’
grapevines infected with Plasmopara viticola at the 5-mm berry stage and bunch closure
compared to healthy vines in 2003 .................................................................................. 150

Table B.4. Repeated measures analysis of variance of '3 C content in leaves of three-year-
old potted ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves at 1, 24, 48, and 168 h in 2004 ............................................................................. 150

Table B.5. Repeated measures analysis of variance of '3 C content in roots of three-year—
old potted ‘Niagara’ grapevines infected with Plasmopara viticola compared to roots of
healthy vines at l, 24, 48, and 168 h in 2004 .................................................................. 150

Table B.6. Repeated measures analysis of variance of '3 C content in lesions of leaves of
three-year-old potted ‘Niagara’ grapevines infected with Plasmopara viticola compared
to healthy leaves at 1, 24, 48, and 168 h in 2004 ............................................................ 151

xii

LIST OF FIGURES
(Images in this thesis are presented in color)

 

CHAPTER 1
Figure 1.1. Total grapevine acreage in Michigan from 1980 to 2004 ................................ 3
Figure 1.2. Total yield of ‘Niagara’ and ‘Concord’ juice grapes produced in Michigan
from 1992 to 2003 ............................................................................................................... 4
Figure 1.3. The transfer of energy during photosynthesis .................................................. 6
Figure 1.4. Signs and symptoms caused by Plasmopara viticola .................................... 16
Figure 1.5. Morphological structures of Plasmopara viticola ......................................... 18
Figure 1.6. Disease cycle of Plasmopara viticola. ........................................................... 20

CHAPTER 2

 

Figure 2.1. A representation of a light response curve showing the relationship between
the carbon assimilation rate and light intensity ................................................................. 38

Figure 2.2. A representation of a C02 response curve showing the relationship between
the carbon assimilation rate and the intercellular C02 concentration ............................... 39

Figure 2.3. Relationship between the carbon assimilation rate and increasing disease
severity in ﬁeld-grown Vitis labrusca ‘Niagara’ vines in 2002 (A), 2003 (B), and 2004
(C) measured in an 18-mm diameter cuvette of a CIRAS I infrared gas analyzer ........... 52

Figure 2.4. Relationship between carbon assimilation rate and intercellular C02
concentration (A) and stomatal conductance (B) in healthy and downy mildew-infected
leaves of ﬁeld-grown Vitis labrusca ‘Niagara’ vines measured with a CIRAS I infrared
gas analyzer in Lawton, MI in 2003 .................................................................................. 53

Figure 2.5. The effect of downy mildew disease development on chlorophyll
ﬂuorescence measured as the ratio of F VIF m in leaves of two-year-old potted ‘Niagara’
grapevines in 2003 (A) and 2004 (B) ................................................................................ 56

Figure 2.6. Effect of downy mildew disease development on the light compensation
point in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B) .. 57

xiii

Figure 2.7. The effect of downy mildew disease development on the photosynthetic
efﬁciency in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004
(B) ...................................................................................................................................... 58

Figure 2.8. The effect of downy mildew disease development on the C02 compensation
point in leaves of two—year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B) .. 59

Figure 2.9. Effect of downy mildew disease development on the carboxylation efﬁciency
in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B) ........... 60

Figure 2.10. Effect of downy mildew disease development on the rate of photosynthesis
at ambient CO2 levels in leaves of two-year—old potted ‘Niagara’ grapevines in 2003 (A)
and 2004 (B) ...................................................................................................................... 61

Figure 2.11. Effect of downy mildew disease development on the assimilation rate at
increased C02 concentrations (1400 umol mol") in leaves of two-year-old potted
‘Niagara’ grapevines in 2003 (A) and 2004 (B) ............................................................... 62

Figure 2.12. Effect of downy mildew disease development on the stomatal conductance
in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B) ........... 63

Figure 2.13. Effect of downy mildew disease development on the stomatal limitation to
photosynthesis (lg (in) in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A)
and 2004 (B) ...................................................................................................................... 64

Figure 2.14. Effect of downy mildew disease development on the intercellular C02
concentration in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004

(B) ...................................................................................................................................... 65

Figure 2.15. Effect of downy mildew disease development on dark respiration in leaves

of two-year-old potted ‘Niagara’ grapevines in 2004 ....................................................... 66
CHAPTER 3

 

Figure 3.1. Total dry weight of two-year-old potted ‘Niagara’ grapevines inoculated with
Plasmopara viticola at the 5-mm berry Stage and bunch closure compared to non-
inoculated vines in 2003 .................................................................................................... 93

Figure 3.2. Total dry weight of three-year-old potted ‘Niagara’ grapevines inoculated
with Plasmopara viticola at bunch closure compared to non-inoculated vines in 2004... 94

Figure 3.3. Percent of total dry weight in leaves, roots, and canes of two-year-old potted

‘Niagara’ grapevines inoculated with Plasmopara viticola at the 5-mm berry stage and
bunch closure compared to non-inoculated vines in 2003 ................................................ 95

xiv

Figure 3.4. Percent of the total dry weight in leaves, roots, new canes, and woody parts
(including canes and stem) of three-year—old potted ‘Niagara’ grapevines infected with
Plasmopara viticola compared to non-inoculated vines in 2004 ...................................... 96

Figure 3.5. The relationship between leaf dry weight and disease severity of two-year-old
potted ‘Niagara’ grapevines infected with Plasmopara viticola in 2003 .......................... 97

Figure 3.6. The relationship between total plant dry weight and disease severity of two-
year-old potted ‘Niagara’ grapevines infected with Plasmopara viticola in 2003 ........... 98

Figure 3.7. Percent of the total '3 C sampled relative to the natural abundance in leaves,
roots, new shoots, and woody (including canes and Stem) parts of three-year-old potted
‘Niagara’ grapevines infected with Plasmopara viticola compared to non-inoculated
vines in 2004 .......................... 99

Figure 3.8. Percent of total labeled l3C sampled relative to the natural abundance in non-
symptomatic downy mildew-infected leaves compared to healthy leaves of 3-year-old
potted ‘Niagara’ grapevines in 2004 ............................................................................... 100

Figure 3.9. Percent of total '3 C sampled relative to the natural abundance in the roots of
downy mildew-infected vines compared to the roots of healthy three-year-old potted
‘Niagara’ grapevines in 2004 .......................................................................................... 101

Figure 3.10. Percent of total '3 C sampled relative to the natural abundance of '3 C in the

lesions of leaves of three-year-old ‘Niagara’ grapevines infected with Plasmopara

viticola in 2004 ................................................................................................................ 102
CHAPTER 4

Figure 4.1. Sporulation of Plasmopara viticola isolates from different hosts on leaf disks
of various grape cultivars and species in 2003 ................................................................ 122

Figure 4.2 Sporulation of Plasmopara viticola isolates from different hosts on leaf disks
of various grape cultivars and species in 2004 ................................................................ 123

Figure 4.3. Sporulation of Plasmopara viticola isolates inoculated onto the same host
from which they were collected in 2004 ......................................................................... 127

APPENDIX B

Figure B.1. Equation used to calculate percent uptake of l3C02 from Ba'3C02 in
‘Niagara grapevines in 2004. 149

................................................................................

 

XV

Chapter 1

Literature review

The gramvine
History

Written records of the European grape (Vitis vimfera L.) date back ﬁve or Six
thousand years making it one of the longest known and earliest reported cultivated fruits,
but cultivation in North America did not happen extensively until the late 1700’s
(Anderson 1956, Hedrick 1945, Mullins et al. 1992). When several large companies
failed at growing European varieties due to susceptibility to diseases in the New World,
the colonists turned to the native grapevine species.

Most of the grapes in eastern North America were bred from two native species,
Vitis labrusca L. and V. rotundifolia Michx. (Anderson 1956). Vitis labrusca was initially
the grape the American viticulturist employed for wine and table grape production. Being
native to North America, it is widely adapted to the climate and soils, and has furnished
more varieties of cultivated grapes than all other American species combined (Hedrick
1945). Vitis Iabrusca is somewhat cold hardy and moderately resistant to downy and
powdery mildews, but it is susceptible to black rot (Pearson and Goheen 1988). The ﬁrst
cultivated American grapevine variety was ‘Alexander’, a descendant of V. labrusca
named after John Alexander, gardener to the governor of Pennsylvania (Bush et a1.
1895).

Vitis labrusca ‘Concord’ originated from the seed of a wild grape planted in 1843

by Ephraim Wales Bull in Concord, Massachusetts. It was ﬁrst displayed in 1853 at the

25'h annual exhibition of the Massachusetts Horticultural Society (Bush et al. 1895). By
1860, it was the most popular grape used for juice production in America and still iS
today. Horace Greeley termed it “the grape for the millions” when awarding it the
Greeley prize for the best American grape (Bush et al. 1895).

In 1868, the variety Niagara was produced from the seed of a ‘Concord’ grape
fertilized by the variety Cassady by C.L. Hoag and B.W. Clark of Lockport, NY.
‘Cassady’ was a V. labrusca cross that originated in the yard of H. P. Cassady in
Philadelphia. ‘Niagara’ was described as a “true Labrusca in habit and foliage but
immensely productive” (Bush et al. 1895). Proprietors guarded the grape for many years,
not allowing its distribution by others and only furnishing vines for vineyard planting on
‘Special terms’ (Bush et al. 1895). This enabled the proprietors to market ‘Niagara’
grapes on a large scale and at premium prices. Today, ‘Niagara’ grapes are the leading
American grape used for white grape juice production in the US. ‘Niagara’ and
‘Concord’ are Similar in terms of vigor and productiveness, but ‘Niagara’ is Slightly less

cold hardy than ‘Concord’(Bush et al. 1895).

Grape production

The United States is the third largest grape producer in the world, behind Italy and
France, producing over Six million tons annually (Amanor-Boadu 2003). California is the
largest grape—producing state in the US, followed by Washington, New York, Michigan,
and Pennsylvania. Of the grapes grown in the United States, approximately 87% are
processed, while the remaining 13% are table grapes. Approximately 60% of all
processed grapes is made into wine, 30% into raisins, 9% into juice, and less than 1% is

canned (Amanor-Boadu 2003).

Michigan is the fourth largest producer of grapes in the US, currently producing
over 90,000 tons annually (NASS 2004). Of the grapes grown in Michigan approximately
90% (by weight) are juice grapes (‘Concord’ and ‘Niagara’), with the largest portion
being ‘Concord’. Figure 1.1 Shows the total number of acres used for grape production
Since 1980 in Michigan. Michigan was the fourth largest producer of ‘Concord’ grapes by
weight and the largest producer of ‘Niagara’ grapes in 2003. Figure 1.2 shows trends in

the production of ‘Concord’ and ‘Niagara’ grapes in Michigan since 1992.

16000

 

14000 .

12000 ‘

10000“

8000-

Acreage

6000 4

4000-

2000 "

 

 

 

O l I I T I I

1980 1 985 1 990 1995 2000 2005

Year

Figure 1.1. Total grapevine acreage in Michigan from 1980 to 2004 (National
Agricultural Statistics Service 1980-2004).

 

70000

 

+ 'Concord'
60000 . -----O 'Niagara'

 

 

 

50000‘

400004

30000-

Grape production (tons)

20000 a

10000 -

 

 

 

 

0 I T I I I I 1 I I 1 r I

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Year

Figure 1.2. Total yield of ‘Niagara’ and ‘Concord’ juice grapes produced in Michigan
from 1992 to 2003 (NASS, 1992-2003). Low yield was likely due to poor pollination and
adverse weather conditions during ﬂowering in 2001 and an early spring frost in 2002.
Grapevine physiology

The grapevine belongs to the family Vitaceae, a group of angiosperms containing
14 genera of both woody and herbaceous perennials characterized by the occurrence of
tendrils arising opposite from a leaf (Mullins er al. 1992, Pearson and Goheen 1988). The
grapevine is a woody perennial, and the woody parts of the vine contain the stores of
carbohydrates and minerals needed for growth in the early parts of the next growing
season. Grapevine Shoots usually produce from one to three ﬂower clusters. The number

is dependent upon the variety and conditions of the previous season under which the

dormant bud developed (Hellman 2003).

Carbon assimilation

Carbohydrates are produced in plants through the process of photosynthesis which
utilizes solar energy for the reduction of carbon dioxide from the atmosphere, as follows:

light

C02 + 2H20——> (CH20) + 02 + H2O

Solar energy is converted to chemical energy by the light-harvesting antenna
complexes (groups of pigment molecules that absorb solar energy) and photosystems I
and 11 located in the chloroplasts. Electrons provided by the Splitting of water molecules
in Photosystem 11 provide energy in the form of ATP (adenosine triphosphate) and
NADPH (nicotinamide adenine dinucleotide phosphate) needed for the assimilation of
C02 (Figure 1.3). The chloroplasts also contain high concentrations of ribulose l, 5-
bisphosphate carboxylase-oxygenase (Rubisco), the enzyme that catalyzes the reaction of
ribulose 1, 5-bisphosphate and C02 to produce 3-phosphoglycerate, which is
subsequently reduced to carbohydrates. These reactions are termed the photosynthetic
carbon reduction (PCR) cycle or Calvin cycle and utilize the ATP and NADPH captured
in the light reactions. The enzyme Rubisco has a dual function as a carboxylase as well
as an oxygenase enzyme. Carbon dioxide and oxygen compete for binding Sites at
Rubisco, so the concentration of these molecules has a direct effect on carbon ﬁxation.
The oxygenase reaction (termed photorespiration) results in the uptake of 02 and release
of C02 resulting in a decrease in net photosynthesis (Lawlor 2001). The biological
function of the oxygenase reaction is not known, but one possible explanation is that it
helps to dissipate excess ATP under high light intensities and low intercellular C02

concentrations (Taiz and Zeiger 2002).

light
ﬂuorescence

heat
‘ e'\ g l

Photosystem l ATP

   

Reaction ‘ ADP -

centers k,
Synthesis of sugars,
starch, amino acids, 4‘ glyceraldehyde
and fatty acrds 3-phosphate

Figure 1.3. The transfer of energy during photosynthesis. Light is captured by pigments
in the antenna complexes of the photosystems. Energy in the form of electrons is used for
the production of NADPH and ATP. Energy from NADPH and ATP is needed for the
regeneration of ribulose l, 5-bisphosphate (RuBP) in the Calvin cycle. The enzyme
Rubisco uses RuBP and CO2 as substrates for synthesis of sugars, Starch, amino acids,
and fatty acids.

Photosynthetic intermediates produced in the PCR cycle are used to produce
starch in the chloroplasts and sucrose in the cytoplasm of cells. Sucrose is the main
carbohydrate translocated throughout the grapevine. If the rate of sucrose synthesis
exceeds the removal or transport out of the cytoplasm, photosynthates are diverted into
starch synthesis (Mullins et al. 1992). These reserves are utilized in the absence of newly
formed photosynthates, mainly at night and early in the growing season. The amount of

carbon assimilated and partitioned to woody parts of the vine varies with vine age,

genotype, and time of year (Mullins er al. 1992).

Effect of Stomatﬂnd leainge on photosynthesis

Grape leaves are hypostomatal, meaning that the majority of the stomata are
located on the lower leaf surface (Mullins er al. 1992). Diffusion of carbon dioxide
through the cuticle is minimal. As a consequence, the size of the stomatal opening can
directly affect the diffusion of carbon dioxide and consequently, the rate of
photosynthesis in the leaf. Several factors can affect stomatal function including light,
C02 concentration, vapor pressure differential, water deﬁcits, and pollutants (Mullins er
al. 1992). The photosynthetic apparatus of young grape leaves is not fully developed and,
therefore, photosynthates from other parts of the vine must be imported to support their
growth (Kriedman 1968, Mullins et al. 1992). Photosynthesis in mature grapes leaves
generally reaches a maximum when the leaves are fully expanded and may maintain this
rate for extended periods before decreasing towards senescence (Kriedman 1968).
Developing berries are photosynthetically active, but their contribution to the carbon

budget of the berry is probably insigniﬁcant (Mullins et al. 1992).

Effect of light on photosynthesis

Solar radiation provides the energy needed for photosynthesis to occur.
Approximately 90% of solar radiation striking a leaf is absorbed. Light saturation (the
point where an increase in solar radiation will not increase the rate of photosynthesis)
generally occurs at 600-700 umol rn'2 S'l PAR (photosynthetically active radiation) which
is roughly a third of full sunlight. The light compensation point (amount of light needed
for net photosynthesis to occur) of grapes is about 50 umol PAR (Mullins et al. 1992).

Some enzymes important in berry ripening are light regulated. Low light intensities can

decrease berry Size, Brix (a measure of soluble solids), and pH, and increase the titratable
acidity (measure of organic acids) in grape berries (Mullins et al. 1992).

The emission of chlorophyll ﬂuorescence, a process occurring from the
deactivation of excited pigments, is a minor process competing with photosynthesis
(Krause and Weis 1991). Excess light energy absorbed exceeds the electron carrying
capacity and the capacity of Rubisco to ﬁx C02 and is dissipated by the release of heat or
as ﬂuorescence. Most chlorophyll a ﬂuorescence is emitted by Photosystem II at normal
temperatures. Photosystem I, which is relatively stable, traps excitation energy and

dissipates excess energy as heat (Lawlor 2001).

Source-sink relationships and carbon partitioning

‘Source’ refers to plant tissue that provides and ‘Sink’ to plant tissue that
consumes photosynthetic carbon compounds (Herold 1980). Sinks are comprised of all
non-photosynthetic parts of a plant such as roots, trunks, ripening fruits, and immature
leaves. Large sinks tend to dominate the overall supply of photosynthates, while small
Sinks must obtain them from more localized supplies and storage (Wardlaw 1990). Fruits
and seeds act as large sinks and therefore, generally dominate as sinks over vegetative
growth (Wardlaw 1990).

Carbon partitioning refers to the differential distribution of photosynthates to
multiple Sinks within the plant (Taiz and Zeiger 2002). Carbon partitioning in plants can
be controlled by many factors including the supply of photosynthate, the size and number
of Sinks, vascular connections between compartments, and the potential for temporary

storage of carbohydrates in leaves (Wardlaw 1990). Individual plant cell tissues may be

spatially separated and have different functions, but they all function as a tightly
networked system which comprises the whole plant. Therefore, small changes in the
demand for assimilates produced from photosynthesis in the grapevine can have
signiﬁcant effects on the partitioning of carbon or a change in the rate of photosynthesis
or both (Wardlaw 1990).

Defoliation (a source reduction) can lead to reduced growth and yield, while the
removal or thinning of grapes (a Sink reduction) can lead to reduced rates of
photosynthesis (Candolﬁ-Vasconcelos and Koblet 1990, Petrie et al. 2000, Quereix et al.
2001). Grape berries act as a large carbon sink, and the removal or lack of fruit can affect
source-Sink relationships in vines (Coombe 1989). Since fruiting vines tend to allocate
more carbon to the fruit clusters rather than to roots and canes for storage, heavy fruiting
can result in reduced shoot growth and poor fruit production the following year (Edson
1993, Kaps and Cahoon 1989, Wardlaw 1990). Excess crop loads and reduced leaf area
on vines can also delay fruit ripening, reduce yield, and reduce total vine size and wood
maturity (Howell 2001). Reducing crop loads in ‘Seyval blanc’ grapevines increased
berry weight, Brix, and vegetative growth (Kaps and Cahoon 1989). Heavy crop loads in
‘Concord’ grapes caused greater carbohydrate partitioning to the fruit, but at the expense
of vegetative tissues (Miller and Howell 1998). High crop loads in ‘Seyval’ vines reduced
dry weight to the roots but did not affect total vine dry weight (Edson 1995b). Younger
vines typically bear little or no fruit, and if fruit develops, it is usually removed to
encourage vine growth and carbohydrate Storage for future growth.

Defoliation of vines during ripening of fruit can cause major changes in dry

matter partitioning and root growth, but this effect is strongly dependent on the timing of

the defoliation (Candolﬁ-Vasconcelos et al. 1994). Partially defoliated vines
compensated for lost leaf area by increasing leaf chlorophyll content and increasing
stomatal and mesophyll conductance in the remaining leaves to allow an increase in
photosynthetic activity (Candolﬁ-Vasconcelos 1991). A low sourcezsink ratio also causes
vines to delay senescence (Howell 2001). Two years of defoliation of V. vinifera vines
caused a signiﬁcant reduction in starch reserves in the wood. Complete recovery occurred
the second season after the removal of the stress (Candolﬁ-Vasconcelos and Koblet
1990). Retranslocation of carbon from the trunk and roots was shown to be much higher
in the defoliated vines three weeks after veraison (the onset ripening in berries)
(Candolﬁ-Vasconcelos et a1. 1994). In order to maintain the balance in the source-Sink
relationships, maximum root growth usually occurs in the Spring and fall of the year
when growth of other plant organs is less rapid (Mullins et al. 1992).

The grapevine does Show some capacity for compensation. The extent to which a
vine can compensate is highly dependent on the timing and amount of stress applied to
the vine such as very large crop loads or excessive defoliation. ‘Chardonnay’ vines with
young fruit removed Showed no difference in the partitioning of newly ﬁxed carbon as
well as the rate of sucrose and starch synthesis compared to vines with fruit (Chaumont et
al. 1994). The effects are more noticeable during the cluster ripening periods when
berries become the dominant sinks. Large vegetative and fruiting sinks have been shown
to inﬂuence photosynthetic rates of vines (Edson 1995a). The photosynthetic rates of
vines (V. vinifera) were shown to increase from veraison until harvest (Petrie et al. 2000).
The photosynthetic capacity of grapevines before veraison typically does not exceed 50%

of the total photosynthetic capacity and therefore, is not source limited (Howell 2001).

10

Understanding the balance between crop load and the photosynthetic capacity of the vine

enables growers to maximize yields without sacriﬁcing juice quality or vine health.

Downy mildew of the graggvine

Classification

The causal agent of downy mildew of grapes is Plasmopara viticola (Berk. &
Curt.) Berlese & de Toni, and belongs to a group of biotrophic pathogens (organisms
which can obtain food in nature only from living host cells) called downy mildews
(Burruano 2000). Downy mildews are fungal-like organisms belonging to the kingdom
Chromista (Stramenopila), phylum Oomycota, family Peronosporaceae (Agrios 1997,
Alexopoulos et al. 1996). The kingdom Chromista also contains brown algae and diatoms
(Agrios 1997). The phylum Oomycota is characterized by organisms containing a diploid
thallus, biﬂagellate zoospores produced in Sporangia, and cell walls containing glucans,
with small amounts of hydroxyproline and cellulose. Members of the family
Peronosporaceae have Sporangia borne on Sporangiophores of determinate growth
(Agrios 1997). In the genus Plasmopara, Sporangia germinate indirectly (via zoospores)
as opposed to Peronospora and Bremia spp., where germination is usually direct
(formation of a germ tube) (Shaw 1981).

The ﬁrst collection and identiﬁcation of the causal organism of grape downy
mildew was made in North America in 1837 by L.D. Schweinitz, who identiﬁed it as
Botrytis cana Lk. (Anderson 1956, Gregory 1915, Viennot-Bourgin 1981). In 1848, it
was renamed Botrytis viticola Berk. & Curt. by Berkeley and Curtis (Anderson 1956,

Berkeley and Curtis 1848). Anton de Bary published an extensive study on Botrytis and

11

other fungal parasites in 1863, and changed the name to Peronospora viticola de Bary
(Anderson 1956, Bary 1863). After Schroeter established the genus Plasmopara in 1886,
Berlese and de Toni (1888) renamed Peronospora viticola to Plasmopara viticola (Berk.

& Curt.) Berlese & de Toni (Gregory 1915, Lafon 1981).

History

Plasmopara viticola is believed to be native to North America. Prior to 1878, the
disease was reportedly well established throughout southern Canada and most of the
eastern and central United States (Agrios 1997, Alexopoulos et al. 1996, Anderson 1956,
Singh 2000,Viennot-Bourgin 1981). Cultivation of grapevines in America in the 1800’s
was risky due to the possibility of downy mildew outbreaks. One limitation to the
introduction of wine grapes (Vitis vinifera) from Europe was the fear of losses due to
their susceptibility to P. viticola (Viennot-Bourgin 1981).

By the late 1800’s, Europe had already suffered losses from several introduced
pathogens and pests before the introduction of downy mildew. In 1845, a few years
following the potato late blight epidemic (caused by Phytophthora infestans (Mont.) de
Bary), grape powdery mildew (Uncinula necator (Schw.) Burr.) was inadvertently
introduced into Europe from the United States (Viennot-Bourgin 1981). It Spread
throughout the grape-growing regions in Europe, causing extensive damage until the
introduction of sulfur in the 1850’s to control the spread of the disease (Viennot-Bourgin
1981). Grapevines were imported from America in the hopes of ﬁnding and breeding
plant material resistant to powdery mildew. These imports were responsible for the
introduction of grape phylloxera (Daktulosphaira vitifoliae Fitch), a root-attacking aphid,

which destroyed vineyards across Europe (Anderson 1956,Viennot-Bourgin 1981). Soon

l2

after in 1881, it was discovered that a European vine could be grafted to an American
vine rootstock making the vine resistant to phylloxera (Viennot-Bourgin 1981). There
was concern in Europe that the introduction of plant material from America could
introduce downy mildew and have devastating effects, but the need to replant the
vineyards after the phylloxera destruction was more urgent. In his bulletin, Farlow (1876)
mentioned that if the vine mildew was introduced into Europe, it would prove a repetition
of what had already happened with phylloxera due to the susceptibility of V. vinifera to
the disease. In addition, the warmer temperatures and greater moisture in areas of Europe
compared to the northeastern US, would allow infections to begin before the vine had
attained substantial growth. In August 1878, downy mildew was identiﬁed and reported
in France (Viennot-Bourgin 1981). By the early 1880’s, it had spread across Europe and
the Mediterranean, causing severe crop losses until the Bordeaux mixture (copper sulfate

and lime) was discovered and found to be effective (Lafon 1981,Viennot-Bourgin 1981).

Host range

Downy mildews are generally limited in their host range, possibly due to the close
association with the host plant with which they evolved as biotrophic pathogens (Renfro
1981). Most Plasmopara species infect broadleaf hosts, although a few have been
reported on Graminaceae such as P. oplismeni Viennot-Bourgin on basketgrass and P.
penniseti Kenneth & Kranz on pearl millet (Alexopoulos et al. 1996, Kenneth 1981,
Shaw 1981). Woody hosts of the genus Plasmopara include Vitis spp. (P. viticola), Ribes
spp. (P. ribicola J. Schrtit. Ex J .J . Davis), and Viburnum spp. (P. vibumi Peck) (Shaw

1981).

13

Numerous endogenous grape species have been identiﬁed in North America,
including Vitis aestivalis Michx. (summer grape), Vitis vulpina L. (frost grape), Vitis
riparia Michx. (riverside grape), Vitis labrusca L. (fox grape), and Vitis rotundifolia
Michx. (muscadine grape). Plasmopara viticola is known to infect all of these grape
species but virulence of the pathogen is generally low (Agrios 1997, Emmit 1992,
Viennot-Bourgin 1981). However, Farlow (1876) reported that downy mildew was very
abundant on V. aestivalis, V. Iabrusca, V. cordifolia Michx. (syn. V. vulpina), and all
American cultivated varieties, and by the middle of September most leaves had been
infected and “hang dead on the branches” (Farlow 1876). The European grape, Vitis
vinifera, is also highly susceptible. Little is known about the role of infected wild hosts in
the spread of the disease to cultivated grapes, but it appears to be minimal (Emmett 1992,
Renfro 1981). In addition to Vitis species, P. viticola also infects Parthenocissus
quinquefolia (L.) Planch., (Virginia creeper) and Parthenocissus tricuspidata (Sieb. &
Zucc.) Planch. (Boston ivy), both woody perennials in the family Vitaceae (Anderson

1956).

Symptoms

Downy mildew can infect all green parts of the vine including leaves, ﬂowers,
petioles, tendrils, and young shoots and berries (Figure 1.4). Young tissue is the most
susceptible to infection, becoming more resistant to infection at it matures (Pearson and
Goheen 1988). Mature berries and immature developing leaves are more resistant to
infection than other tissues, probably due to the presence of non-functioning stomata,
although infection of the rachis can spread into mature bem'es (Lafon 1981). Infected

young berries become discolored and develop white, felt-like sporulation. Infected berries

l4

or entire clusters may drop prematurely. Young infected Shoots are typically shorter and
thicker than normal due to hypertrophy of the cells (Anderson 1956). Infected shoots may
also curl, forming a ‘shepherd’s crook’, become white with Sporulation and eventually
die (Pearson and Goheen 1988). Leaves develop circular yellow lesions on the upper leaf
surface commonly called ‘oil spots’ due to their greasy or translucent appearance.
Sporulation occurs on the underside of the leaf and appears as a dense white, downy
growth giving rise to the common name of the pathogen (Pearson and Goheen 1988). The
infection of older leaves results in small angular lesions due to the inability of the
organism to spread across veins. Infected leaf surfaces eventually become necrotic and
leaves may drop prematurely. Symptoms typically take 1-2 days longer to appear from
the time of infection on American than on European species (V. vinifera) (Farlow 1876).
Leaf infection is the most important source of inoculum for berry infection and

overwintering oospores (Pearson and Goheen 1988).

15

 

Figure 1.4. Signs and symptoms caused by Plasmopara viticola. A) Sporulation on a
tendril of Vitis riparia, B) Sporulation and hypertrophy on a young Shoot of V. riparia, C)
Sporulation on berries of ‘Mars’ grapes, D) ‘Oil spots’ on the upper surface of a
‘Niagara’ leaf, E) Sporulation on the lower leaf surface of V. aestivalis, and F)
Sporulation along the midvein of a V. riparia leaf.

Morphology

Plasmopara viticola produces a diploid, thin-walled coenocytic (multinucleate),
nonseptate mycelium that occupies the intercellular spaces of the host (Anderson 1956,
Gregory 1915, Singh 2000). Hyphal size can vary from 1-2 to 60 um wide, which is
partially determined by the size of the intercellular spaces in which they grow. Upon
contact with the host cells, the hyphae invaginate the cells and form pear-shaped
structures called haustoria. These haustoria are the host-parasite interface through which
the exchange of nutrients between the host and pathogen occurs (Langcake and Lovell
1980). They are produced abundantly in tissues where mycelium is found, but are
generally smaller and less abundant on resistant varieties (Gregory 1915). Haustoria are
generally 4-10 um in diameter (Singh 2000).

A swollen cushion of hyphae in the substomatal Space gives rise to
Sporangiophores, which eventually emerge through the stomata or lenticels (Figure 1.5A,
B) (Anderson 1956). Sporangiophores range from 140-250 um long and are
dichotomously branched at the tips (Emmett 1992).

Sporangia are produced on branches formed at right angles to the main stem of
the sporangiophore (Figure 1.5C) (Anderson 1956, Singh 2000). Sporangia are hyaline
and range from 11-18 um in width to 15-31 urn in length, but size can vary with
environmental conditions and among hosts (Anderson 1956, Singh 2000). A sporangium
typically releases from 5-8 zoospores (Figure 1.5D, E) (Gregory 1912). Zoospores are
pear-shaped and range in size from 4-5 urn in width and 7-9 um in length (Singh 2000).
Motility is by means of two apical ﬂagella, one longer tinsel ﬂagellum with hairs and a

Shorter whiplash ﬂagellum that trails behind (Alexopoulos et al. 1996).

17

 

Figure 1.5. Morphological structures of Plasmopara viticola. A) Sporangiophores with
Sporangia on ‘Niagara’ grape leaves, B) Electron micrograph of sporangiophore and
Sporangia growing out of a stoma (Alexopoulos et al. 1996), C) Sporangiophore and
Sporangia from ‘Niagara’ grape leaves, D) Sporangium releasing zoospores, E) Empty
Sporangium and sporangium with a few zoospores inside, and F) Oospore taken from a
‘Niagara’ grape leaf.

Sexual reproduction begins by the fusion of an antheridium and an oogonium to
form a thick-walled, diploid oospore (Figure 1.5F). Oospores range from 28-40 um in
diameter. Oospores germinate to produce a sporangiophore with a single, large apical
sporangium. Sporangia produced from oospores are 25 um in width and 35-40 um in

length, and typically produce 8-20 zoospores (Singh 2000).

Disease cycle and epidemiology

Downy mildew infection and Spread is favored mainly by moisture, with
temperature being a less important factor (Lafon 1981). Even before the biology of P.
viticola was well understood, it was observed that moisture from morning dew favored
development of the disease, prompting some viticulturists to try to prevent condensation

on leaves by sheltering or covering the vines (Viennot—Bourgin 1981).

Primary infection

Primary infection is initiated by the germination of oospores in the Spring (Figure
1.6). It is believed that oospores go through a temperature-dependent dormancy period,
but it is uncertain if maturation can proceed before the dormancy is completed (Emmit
1992, Ronzon-Tran Manh and Clerjeau 1988). Gregory (1915) found that oospores would
not germinate unless they were allowed to remain on the ground until at least January or
February in the northeastern United States. Maturation is inﬂuenced primarily by winter
precipitation and soil temperature (Emmit 1992). Rainy conditions with temperatures

above 10°C favor the germination of oospores (Emmett 1992, Serra and Borgo 1995).

19

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The optimum temperature for germination is 20-22°C (Populer 1982, Ronzon-
Tran Manh Sung and Clerjeau 1988). At optimum temperatures, the formation of the
sporangium and release of zoospores is usually completed in 24 hours (Gregory 1915).
Oospores germinate to form a Single sporangiophore and primary sporangium. Moisture
is required to dissolve the cross-wall of callus connecting the sporangia to the
sporangiophores, permitting their dispersal, and for the liberation of zoospores from
sporangia (Emmett 1992, Lafon 1981). The detachment of sporangia is aided by twisting
in response to a vapor pressure differential or a decrease in relative humidity (Pinckard
1941). Primary inoculum consists of windbome Sporangia or rain-splashed sporangia or
zoospores (Emmett 1992, Gregory 1912, Singh 2000).

Release and movement of zoospores requires free water. The Sporangia are
hydrophobic which allows them to resist water and adhere to the cuticle and trichomes of
the leaf as they are dispersed. During periods of high humidity and extended periods of
leaf wetness, the sporangia become hydrophilic and begin to differentiate into zoospores
(Kortekamp et al. 1999). The optimum temperature for differentiation is 15-25°C
(Gregory 1915, Lafon 1981, Emmit 1992). The differentiation and release of zoospores is
usually complete within 30 minutes (Langcake and Lovell 1980). Zoospores are attracted
to stomata by unidentiﬁed chemical signals, which vary with the size of the stomatal
opening (Kiefer er al. 2002, Royle and Thomas 1973). Upon contact with stomata,
zoospores encyst, Shed their ﬂagella, and produce a germ tube that enters the substomatal
cavity (Emmit 1992). The rate of zoospore release, development of the germ tube, and
stomatal targeting can be affected by the absence or presence of host tissue as well as

stomatal closure (Kiefer et al. 2002). Several zoospores can encyst and produce germ

21

tubes that penetrate a Single stoma (Royle and Thomas 1973). In the substomatal cavity a
vesicle is formed which produces a Short hypha. The production of a haustorium is
initiated upon contact of the infection hypha with a host cell (Langcake and Lovell 1980).
Further hyphal development into the mesophyll and palisade cells occurs 12 to 15 hours
after the production of the initial haustorium (Emmett 1992). Additional haustoria are

produced with additional hypha] growth. Hyphal growth ceases at 30°C, but hyphae can

remain viable for more than 10 days at temperatures of 42—43°C (Singh 2000).

Sp_orulation

Under favorable conditions, sporulation occurs on the infected tissue and the
secondary infection cycle begins. The major factors affecting sporulation are relative
humidity, temperature, 1i ght/darkness, and the condition of the host tissue (Gregory 1915,
Emmit 1992). A continuous relative humidity of 95-100% is required for sporulation
(Blaeser 1979, Pearson and Goheen 1988, Emmit 1992). A dark period of at least 4 hours
followed by light is required for the production of sporangia (Rumbolz et al. 2002, Singh
2000). Continuous darkness or light produces little or no sporulation (Anderson 1956,
Yarwood 1937). White light irradiation prevents formation of sporangia and produces
abnormally shaped Sporangiophores (Brook 1979, Rumbolz et al. 2002, Yarwood 1937).

Sporulation can occur at temperatures as low as 10°C and as high as 29°C but the
optimal temperature is 18-22°C (Blaeser and Weltzien 1978, Gregory 1915, Pearson and
Goheen 1988). Production of Sporangia and Sporangiophores can be completed in as little

as 7 hours (Rumbolz et al. 2002). AS many as 20 Sporangiophores can arise through a

22

single stomatal opening (Anderson 1956, Singh 2000). Sporulation can continue from the

same infected tissue for several days under favorable conditions.

Survival of smrangia

Survival of sporangia is much shorter than oospore survival and decreases with
decreasing humidity and increasing temperature (Blaeser and Weltzien 1978, Blaeser
1979, Singh 2000). Sporangia in a vineyard showed a viability of 4 to 8 days when
Shaded by the canopy, provided temperatures did not exceed 22°C (Kast and Stark-Umau
1999, Zachos 1959). Gregory (1915) found that Sporangia cannot survive sunlight
exposure at temperatures above 30°C. Sporangia on detached leaves survived less than 48
hours at 20°C with 70% relative humidity, and less than 24 hours at 20°C with 30%
relative humidity. Viability was maintained longer when Sporangia remained attached to
Sporangiophores (Blaeser and Weltzien 1978). Older sporangia required more time to
release zoospores and showed declining viability with age (Kast and Stark—Urnau 1999).
Sporangia produced at night between 0200 and 0600 h had the highest germination rates
compared to Sporangia produced at any other time during the diurnal cycle (Srinivasan
1976). Production of sporangia can continue for several successive nights from the same
lesion under ideal conditions (Singh 2000). Secondary sporangia are wind dispersed or
water-splashed to other plant tissues where they release zoospores in free water to

continue the secondary infection cycle.

23

Overwintering

Late in the season, temperatures favor sexual reproduction, and overwintering
oospores are produced in infected host tissue. Plasmopara viticola is reported to be
heterothallic, requiring two different mating types for sexual reproduction to occur
(Wong et al. 2001). Oospores are formed in lesions on infected leaves in late summer and
fall (Figure 5.1). Oospore numbers in lesions can be as high as 250 per square mm
(Populer 1982) and are most concentrated adjacent to the leaf midrib or a major vein
(Singh 2000). Overwintering occurs mainly in the form of oospores in leaf debris on the
soil surface, however, in milder climates, P. viticola may survive as mycelium in buds
and infected Shoots (Emmit 1992, Pearson 1988, Singh 2000).

Survival of oospores is favored by moisture and their close proximity to the soil
surface with temperature being a less important factor (Lafon 1981). Oospores have been
known to survive temperatures of -26 °C for ﬁve days (Gaumann 1950). Before
germination, oospores must go through a temperature-dependent dormancy period (Singh
2000). Under controlled conditions oospores mature at alternating weekly temperatures
of -5 and 10°C. Oospores germinate within 6—10 days after shifting to 20°C, but a 4-
month minimum maturation period is required (Ronzon-Tran Manh Sung and Clerjeau

1988).

Distribution and economic importance
Downy mildew is the most destructive fungal disease of grapevines and has been
reported in 91 countries (Emmett 1992, Singh 2000). Downy mildew is particularly

destructive in parts of the world that receive frequent summer rains that maintain high

24

humidity at night and temperatures over 13°C, such as France, Germany, and South
Africa (Emmett 1992). In areas with drier climates such as Afghanistan, California,
Chile, Egypt, and Western Australia, downy mildew is of little importance (Emmett
1992, Weltzien 1981).

The pathogen infects all green tissues of the vine causing loss of photosynthetic
area and cluster infections that can lead to 100% yield loss (Emmett 1992, Singh 2000).
Early infections can lead to defoliation of vines that prevents cluster maturation resulting
in low sugar content as well as exposing fruits to sun scald (Singh 2000). Vine vigor,
grape production, and grape quality may be reduced in years following epidemics due to
depletion of carbohydrate reserves (Emmett 1992). Heavy infections may have an effect
on winter hardiness making vines more susceptible to colder temperatures. In addition,
bud burst may be Slowed and crop potential may be reduced due to a lower number of

viable buds (Emmett 1992).

Disease Management

Since the discovery of the Bordeaux mixture by Millardet in 1885, fungicides
have been used extensively to control downy mildew, although recently canopy
management practices are gaining attention (Emmett 1992, Schwinn 1981, Singh 2000).
Preventative methods such as cleaning up infected leaves can be used to help reduce
downy mildew inoculum in the vineyard, but these methods are impractical and often
only provide limited control. Vines should be Spaced and pruned to increase air

circulation and decrease humidity when practical (Mullins er al. 1992).

25

Fungicides

Application of fungicides provides the best method of control (Emmett 1992,
Schwinn 1981, Singh 2000). Several different fungicides can provide effective control of
downy mildew if applied at recommended intervals. Protectant fungicides include captan,
mancozeb, ziram, and copper fungicides. Copper-containing fungicides such as the
Bordeaux mixture provide preventative control when used with proper timing. Copper
fungicides are still used frequently for downy mildew control but are often mixed with
other fungicides to improve effectiveness and increase the spectrum of control (Schwinn
1981). Some varieties such as ‘Catawba’, ‘Merlot’, and ‘Chancellor’, are sensitive to
copper, so alternative fungicides must be employed (Wise et al. 2004). Mancozeb is a
protectant EBDC (ethylene bisdithiocarbamate) fungicide that is effective against downy
mildew as well as black rot and Phomopsis cane and leaf Spot. Even though it is primarily
a protectant, it does have some anti-sporulant activity when applied post-infection (Wong
and Wilcox 2001); however, it has a 66-day preharvest interval (Wise et al. 2004). In
addition, some juice grape processors do not allow sprays of mancozeb after bloom.
Captan is also effective for control of downy mildew and Phomopsis cane and leaf Spot
but not for black rot. While Captan currently has a 0-day preharvest interval for grapes,
its use is not allowed by some juice grape processors (Wise et al. 2004). These fungicides
are all preventative, providing control before infection occurs, and normally have little or
no effect on disease that is already established.

Ridomil Gold (mefenoxam) is a systemic fungicide that prevents hypha] growth
and the production of haustoria and provides excellent control of downy mildew (Singh

2000). Ridomil can provide complete control if applied before infection and can

26

Signiﬁcantly reduce disease severity if applied up to ﬁve days after infection (Wong and
Wilcox 2001). Post—symptom application is effective at reducing further sporulation.
Other fungicides such as copper and mancozeb are added to Ridomil as a resistance
management tool to reduce the likelihood of pathogens developing resistance, however
these additions increase the pre—harvest interval (42 days for Ridomil Gold Copper and
66 days for Ridomil Gold MZ) (Wise et al. 2004).

ProPhyt (potassium phosphite) and Aliette (fosetyl-Al) are formulations of
phosphorous acid that are considered true systemics and an alternative to mefenoxam
(Singh 2000). Application up to 12 days after infections occur can reduce the incidence
and severity of infection by reducing sporulation (Wicks et al. 1991). These fungicides
are translocated through the plant via the xylem and phloem and provide protection to
actively growing foliage and clusters for 14 to 21 days (Singh 2000).

Strobilurins are a class of protectant and curative fungicides that prevent infection
as well as provide some post-infection activity. Strobilurin fungicides include Abound
(azoxystrobin), Sovran (kresoxim—methyl), Flint (triﬂoxystrobin), and Pristine
(pyraclostrobin and boscalid). Pristine can be phytotoxic to ‘Concord’, ‘Fredonia’,
‘Worden’ and other related varieties (Wise et al. 2004). On young plants, the preventative
efﬁcacy of azoxystrobin was Shown to be similar to that of mancozeb (Singh 2000).
When applied up to ﬁve days after the start of infection, azoxystrobin can signiﬁcantly
reduce sporulation and lesion size, but has little effect on disease incidence (Wong and
Wilcox 2001). Strobilurins have a broad spectrum of activity, yet they have a single mode
of action, so management practices must be used to limit the development of fungicide

resistance (Wise 2004).

27

Biological control

F usarium proliferatum (T. Matsushima) Nirenberg is a fungus that has been
shown to act as a biocontrol agent by parasitizing the Sporangiophores of Plasmopara
viticola. Post-infection application on leaf disks reduced sporulation by more than 90%,
and weekly applications of conidial suspensions in vineyards reduced disease
development on leaves and clusters (Falk er al. 1996). Although it is not currently
registered as a fungicide, it could potentially prove effective in areas where downy
mildew is not severe or when used in conjunction with other fungicides in resistance

management programs (Falk et al. 1996).

Forecasting

Forecasting methods can be used to determine if disease severity warrants
fungicide application. These systems measure the duration of leaf wetness, relative
humidity, and temperature to identify infection periods and determine fungicide
application intervals (Agrios 1997). Since downy mildew climatic conditions and disease
outbreaks can vary from region to region, disease forecasting can be helpful in predicting
when outbreaks might occur. Several disease-forecasting systems are available for downy
mildew and many used in Europe are based on the ﬁndings of Blaeser and Weltzien
(1979) which include infection conditions, Sporulation conditions, and viability of
sporangia. Models designed to predict outbreaks in the US. may be more suited to
predict outbreaks on American vines and French hybrids (Madden and Ellis 2000, Park et
al. 1994). DMCast iS a forecasting system that also considers ontogenic resistance

(increasing resistance as tissues mature) of berries and cluster stems, the period of

28

oosporic inoculum release, the development and components of lesion productivity, and

sporangium survival (Park et al. 1994).

Rationale and objectives of the research

Plasmopara viticola has been infecting grapevines for many years in the United
States and around the world. Most research over the last century has focused on
understanding the biology of P. viticola and developing management strategies which
incorporate the use of fungicides. In order to establish damage thresholds caused by foliar
infections of P. viticola in grapevines, it is important to have an understanding of how the
pathogen affects carbon assimilation and allocation in the vine. An investigation of host
specialization in P. viticola can give a better understanding of how the pathogen spreads
between vine species and cultivars, and also improve inoculation procedures for
‘Niagara’ grapevines. This research will ultimately assist in the development of minimal
fungicide strategies and better alternate methods of control that allow maximum
grapevine productivity.

The objectives of this research were to: 1) Determine the effects of P. viticola on
photosynthesis of infected ‘Niagara’ leaves, 2) Determine the effects of infection on
biomass accumulation and carbon partitioning in ‘Niagara’ vines, and 3) Evaluate the

ability of P. viticola sporangia to cross-infect different grape cultivars and Species.

29

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34

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35

Chapter 2
The effects of downy mildew (Plasmopara viticola) infection on photosynthesis of

‘Niagara’ grapevine leaves

Introduction

Many studies have been done to determine the effect of pathogens on
physiological and biochemical changes in plants. However, little is known about the
effects on photosynthesis at the biochemical level (Scholes 1992). Studies using leaves
infected with viruses, bacteria, and fungi have shown that the rates of photosynthesis in
infected plants may decrease, remained unchanged, or in some cases may even increase
(Buchanan 1981). Blueberry leaves infected with Septoria leaf spot Showed exponential
reductions in photosynthesis as disease severity increased (Roloff et al. 2004). Potato
leaves infected by Phytophthora infestans (Mont.) de Bary showed an increase in the
photosynthetic rate in infected areas. This increase was presumably caused by the
opening of stomata allowing more efﬁcient gas exchange (Farrell 1971). Many of these
effects depend on the pathogen, host and the Speciﬁc stage of disease development

(Bassanezi et al. 2002, Shtienberg 1992).

EstimJating photosynthesis andﬁgas exchange parameters

The measurement of CO2 uptake by plants provides a direct measure of
productivity and is instantaneous and nondestructive. It can also provide information on
individual leaves or parts of leaves (Long 1985). Photosynthesis measurements on a leaf

or leaf section can accurately reﬂect changes in photosynthesis within the leaf but do not

36

necessarily reﬂect a whole-vine response, due to the variation in age and position of
leaves on the vine (Edson 1993, Kriedman 1968, Miller 1996, Mullins et al. 1992, Nobel
1985). The rate of photosynthesis can also be affected by the environmental conditions in
which it is measured, such as temperature and humidity (Scholes et al. 1994). The effects
of pathogens on photosynthesis can be evaluated by comparing the response of diseased
leaves and healthy leaves to different levels of light, CO2, temperature, or vapor pressure
changes. The analysis of chlorophyll ﬂuorescence can be used as an indicator of the
photochemical efﬁciency of photosystem II (Scholes and Rolfe 1996). The analysis of
response curves of the photosynthetic rate and absorbed light allows the changes in
quantum efﬁciency, maximum light intensity, the light compensation point, and dark
respiration to be identiﬁed (Figure 2.1) (Baker 1996, Rossing 1992). Photosynthetic
efﬁciency or yield is the number of moles of CO2 absorbed by the leaf per mole quanta of
light absorbed, giving a direct measure of the efﬁciency of light utilization by the leaf.
There is evidence that the CO2 assimilation rate of mature crop canopies is determined
more by the photosynthetic efﬁciency than by the light-saturating rate of photosynthesis
(Long 1985). Plots of the photosynthetic rate vs. intercellular CO2 concentration (A/Ci
curves) allow identiﬁcation of changes in Rubisco activity and regeneration rate of
ribulose 1, 5-bisphosphate (RuBP) (Figure 2.2) (Baker 1996, Farquhar and Sharkey
1982). A/Ci curves can also be useful in separating stomatal and mesophyll limitations to

photosynthesis (Farquhar and Sharkey 1982, Long 1985).

37

25-

 

 

Amax: maximum rate of photosynthesis
20~
Tm
'E 15-
'6 Reduction in photosynthesis relative to
:5, maximum potential represents limitations
3’ to CO2 diffusion, Rubisco activity, or
‘5 10 - .
; RuBP regeneration
0
3'9”.
'— 5- ..
g <___ Inrtral SIOpe of response curve represents
2 the quantum efﬁciency of photosynthesis
O I I I I
400 800 1200 1600

 

Photon ﬂux density (mmol rn‘2 8")
Light compensation point: zero net photosynthesis
Zero light intensity and negative C02

exchange represents dark respiration

Figure 2.1. A representation of a light response curve showing the relationship between
the carbon assimilation rate and light intensity. Variables can then be calculated from the
curve (modiﬁed from Baker 1996).

38

      

 

 

40 '1
Amax : the maxrmum rate of RuBP regeneration

__/-\ 30 " ___________________

v:

'E

'25

E 20 .

3

<1.)

‘5

l—

C

.3 10 -

r3 Initial slope represents carboxylation efﬁciency:

E the amount of active Rubisco

{I}

< O I I T I I I I I I

0 200 400 600 800 1 000 1 200 1 400 1 600
/ Intercellular C02 concentration
-10 - /

CO2 compensation point: intercellular CO2
concentration at no net photosynthesis

Figure 2.2. A representation of a C02 response curve Showing the relationship between
the carbon assimilation rate and the intercellular CO2 concentration. Variables can then
be calculated from the curve. The maximum rate of RuBP (ribulose 1, 5-bisphosphate)
regeneration is assumed equal to the rate of coupled photosynthetic electron transport
(modiﬁed from Long 1985).

39

The intercellular CO2 concentration (C;) can also provide a method for assessing
limitations to photosynthesis caused by Stomatal (resistance to CO2 movement across
stomata) and mesophyll limitations (resistance to C02 movement across cell walls, cell
membranes, cytosol, or chloroplast membranes). If a reduction in the assimilation rate is
a result of Stomatal limitation, then C, should decrease; if the reduction is caused by
mesophyll limitations, then C, should increase (Long 1985). Under normal CO2
concentrations and saturating light conditions, the rate of photosynthesis is largely
controlled by Rubisco activity. Under saturating light and saturating CO2 conditions, the
rate of photosynthesis is determined by the availability of inorganic phosphate (Pi)
(Scholes et al. 1994). The data obtained from these measurements can provide a

mechanistic understanding of how pathogens alter photosynthesis in infected plants.

Effects of biotrophic pathogens on photosynthesis

Many studies have examined the effects of infection by biotrophic pathogens,
particularly rusts and powdery mildews, on photosynthesis and respiration (Farrar 1987,
Magyarosy et al. 1976, Owera et al. 1981, Scholes and Farrar 1986, Whipps and Lewis
1981). Biotrophic pathogens typically cause a general decline in the photosynthetic rates
of plants by reducing the efﬁciency of, or decreasing functional leaf area (Bassanezi et al.
2002, Bastiaans 1991, Black 1968, Daly 1976, Livne 1964, Raggi 1978, Shtienberg
1992). The reduction in net photosynthetic rates of infected leaves can be a consequence
of increased stomatal or mesophyll resistance (Ahmad et a1. 1983, Owera et al. 1981,
Rabbinge et al. 1985, Scholes 1992). In addition, reduced rates of photosynthesis can be a

result of reduced Rubisco activity (Bassanezi et al. 2002, Bertamini et al. 2002),

40

destruction of chloroplasts (Ahmad et al. 1982, Mignucci and Boyer 1979, Sziraki et al.
1984), alterations of the cytochrome content of membranes (Magyarosy and Malkin
1978, Walters 1985), or increased respiration (Scholes 1992). Radish leaves infected with
white rust (Albugo candida (Pers.) Kuntze) Showed a reduction in photosynthesis several
days before an increase in respiration rates and the appearance of symptoms, but the
cause of the decrease was unknown (Black et al. 1968). Rust-infected beans (Bassanezi et
al. 2002), wheat (Rabbinge et al. 1985), and barley (Bassanezi et al. 2002, Owera et al.
1981) showed a decrease in the net photosynthetic rate and an increase in the internal
CO2 concentration, although significant decreases in these parameters in beans were not
seen until after symptom development. A reduction in Rubisco activity was the major
cause of the decreased photosynthetic activity of phytoplasma—infected apple leaves
(Bertamini et al. 2002) and rust-infected bean leaves (Bassanezi et al. 2002) after the
appearance of symptoms. Stomata did not signiﬁcantly limit photosynthesis in diseased
bean leaves (Bassanezi et al. 2002). There was also less damage to photosynthetic
regulation mechanisms in the remaining healthy parts of rust-infected bean leaves than in
bean leaves infected by a hemibiotrophic pathogen (a pathogen initially requiring living
host cells but eventually killing host cells and living on the remains) such as
Phaeoisariopsis griseola (Sacc.) Ferraris (angular leaf Spot) (Bassanezi et al. 2002).
Powdery mildew infecting the upper surface of barley leaves had little effect on gas
exchange, because gas exchange was for the most part through stomata of the lower leaf
surface (Aust et al. 1977). However, powdery mildew was shown to reduce
photosynthetic rates by lowering mesophyll conductance in infected leaves of sugar beet

(Gordon and Duniway 1982). Scholes (1992) reported that the reduction in

41

photosynthesis in barley leaves infected with powdery mildew was primarily due to loss
of activity or quantity of photosynthetic enzymes in the Calvin cycle (reductive pentose
phosphate [RPP] cycle). Down-regulation of the Calvin cycle was thought to be caused
by the effect of carbohydrates on gene expression encoding photosynthetic enzymes.
Electron transfer rates also decreased due to decreased demand for ATP and NADPH,
while loss of chlorophyll occurred later. Montalbini and Buchanan (1974) found that rust-
infected bean leaves had signiﬁcantly fewer electron carriers than healthy leaves, causing
a disruption of electron transport. In addition to decreased photosynthetic rates in rust-
infected beans (Bassanezi et al. 2002, Raggi 1978), barley (Aust et al. 1977) and wheat
(Bethenod et al. 2001), diseased leaves also showed an increase in respiration. Rust-
infected beans Showed an increase in the CO2 compensation point (CO2 concentration
where there is zero net photosynthesis) that was highly correlated with a decrease in
respiration (Raggi 1978). Rust-infected wheat leaves showed an increase in the
intercellular CO2 concentration (C) with increased respiration (Bethenod et al. 2001).
Increased rates of respiration of the pathogen, host or both could increase C i, causing an
apparent decrease in net photosynthesis, although the pathogen contribution to increased
respiration rates is thought to be small due to the greater biomass of plant tissue (Ayres
1981, Daly 1976, Walters 1985). Reduction in photosynthesis caused by the loss of
chlorophyll or destruction of chloroplasts has typically been evident after the appearance
of disease symptoms in soybean (Mignucci and Boyer 1979), barley (Ahmad et al. 1983),
and dry beans (Lopes and Berger 2001, Sziraki er al. 1984, Wagner and Boyle 1995).
Transient increases in photosynthetic rates have been known to occur in

uninfected areas of diseased leaves, but these increases are usually Short lived and vary

42

with disease severity and leaf age (Allen 1942, Aust et al. 1977, Daly 1976, Livne 1964).
Depending on the pathogen-host combination, the visual size of the lesion may not
always provide an accurate estimate of physiological damage to a leaf, because the area
affected can be larger than the area invaded by the pathogen (Bastiaans 1991, Giuntoli
and Orlandini 2000, Shtienberg 1992). This effect can lead to a stronger or weaker
pathogen effect on photosynthesis than what may be expected based on lesion Size.
Increased rates of photosynthesis in healthy areas of diseased leaves could contribute to
this effect.

A decrease in the export of photosynthates from source leaves caused by a strong
pathogen Sink at the site of infection can cause an accumulation of carbohydrates in the
leaf. This effect has been Shown in rust-infected beans (Zaki 1965) and radishes (Black
1968). Powdery mildew infections on cereals tend to show this effect as well (Scholes
1992). Carbohydrate accumulation at infection sites can cause a down regulation of the
Calvin cycle, thereby causing an inhibition of photosynthesis (Livne 1966, Scholes 1992,

Wagner and Boyle 1995).

Effects of biotrophic pathogens on respiration

Leaves infected with biotrophic fungi generally have greater rates of dark
respiration, and this is likely due to both the pathogen and the host (Farrar 1992, Scholes
1992). Daly (1976) attributed most of the respiration observed in a powdery mildew
infection to the host. Since rates of respiration are typically higher in infected plant
tissues, the ﬂux of CO2 into intercellular spaces from respiration raises the intercellular

CO2 concentration above that of healthy plants and thus reduces the measured net

43

photosynthesis (Farrar 1987). Increases in the intercellular CO2 concentration were
reported for some rust diseases (Owera et al. 1981), but not powdery mildew of beet

(Gordon and Duniway 1982).

Effects of biotrophic pathogens on chlorophyll ﬂuorescence

Chlorophyll ﬂuorescence can be used as a non-destructive measure of
photosynthetic activity in photosynthetic parts of plants (Chaerle and Van der Straeten
2000, Daley et al. 1989). The measurement of F vIF m (ratio of variable to maximum
ﬂuorescence) provides information about the potential photosynthetic efﬁciency of
photosystem II (Bassanezi et al. 2002, Genty et al. 1989). Rust-infected beans Showed no
Signiﬁcant reduction in F vIF m after symptoms appeared. Neither photosystem 11
efﬁciency nor chlorophyll abundance changed in powdery-mildew-infected sugar beet
leaves even in 60—day-old infections (Bassanezi et al. 2002, Magyarosy et al. 1976).
Reductions in F vIF m may be a consequence of reduced capacity of electron transport or
regeneration of ATP and NADPH, causing excess absorbed energy in photosystem II to

be dissipated as ﬂuorescence (Bassanezi et al. 2002).

Pathogen effects on grapevine photosynthesis

Few studies have focused on the impact of pathogen infection on photosynthesis
of the grapevine and knowledge is limited (Giuntoli 2000). Mature grapevine leaves
infected with powdery mildew Show a decreasing photosynthetic rate with increased
infection caused by damage to the palisade cells (Lakso et al. 1982). Powdery mildew on

grape leaves reduced the net assimilation rate, stomatal conductance, carboxylation

44

efﬁciency, and quantum efﬁciency on potted ‘Chardonnay’ grapevines (Nail and Howell
2004). Similar results were found with infection on ‘Riesling’ vines as well as an increase
in respiration in the healthy areas of infected leaves (Clearwater 2002). A decrease in
Rubisco activity was the primary mechanism causing a substantial decrease in
photosynthesis in grape leaves infected with grapevine fan leaf virus (GFLV), while
inhibition of the primary light reactions (conversion of light energy to NADPH and ATP)

was only a minor effect (Sampol et al. 2003).

Effects of Plasmopara viticola on photosynthesis of grapevines

The majority of studies on the effect of biotrophic pathogens on photosynthesis
have focused on rusts and powdery mildews, and little is known about plants infected
with downy mildews (Walters 1985). Downy mildews are known to induce many
alterations in the host physiology, but the exact mechanisms for these alterations have
only been identiﬁed in a small number of pathosystems (Giuntoli 2000, Scholes 1992).
Grapevine leaves infected with downy mildew Showed decreased rates of photosynthesis,
and when only symptomatic areas were measured the net photosynthetic rate fell below
zero (Giuntoli 2000). Healthy areas of diseased leaves did not Show a signiﬁcant
difference in their photosynthetic rates compared to healthy leaves (Giuntoli 2000). There
is no evidence to Show the mechanism of the decrease in net photosynthesis in downy-

mildew-infected grapes.

45

Rationale and objectives

Plant pathogens can cause a reduction in photosynthetic rates of plant tissues,
which in turn can potentially reduce total dry weight accumulation or adversely affect
carbon allocation in plants. Understanding the effects that pathogens have on carbon
assimilation in plants is the ﬁrst Step to predicting when pathogens will negatively affect
crop productivity.

The objectives of this study were to: 1) Determine the relationship between foliar
disease severity and the rate of photosynthesis and 2) Use gas exchange and chlorophyll
ﬂuorescence measurements to determine the sequence of changes in carbon assimilation

following inoculation of grape leaves with Plasmopara viticola.

Material and methods

Assimilation rates of leaves of downy mildew-infected ‘Niagara’ vines in the ﬁeld

 

Carbon assimilation rates were measured on Vitis labrusca ‘Niagara’ leaves in
vineyards located at the Trevor Nichols Research Complex in Fennville, MI in 2002, in a
commercial vineyard in Lawton, MI in 2003, and at the Clarksville Horticultural
Experiment Station in Clarksville, MI in 2004. All vines measured were mature and had
fruit at the time of measurement. Measurements were taken on October lst in 2002,
September 11th and 16th in 2003, and September 16th in 2004 when infections were well
established throughout the vineyard. Fully expanded leaves of approximately the same
age (ﬁrst or second fully expanded leaf) were chosen for the experiment. Measurements
of assimilation rate were taken with a CIRAS I infrared gas analyzer (PP Systems,

Amesbury, MA) equipped with a Parkinson leaf cuvette.

46

Well-developed, Sporulating downy mildew lesions on infected leaves were
enclosed in the leaf chamber of the cuvette. The location of the cuvette on the leaf was
adjusted so that areas with estimated disease severities ranging from 0 to 90% were
enclosed in the cuvette. Noninfected leaves of a Similar age in close proximity to infected
leaves were used as a control. Sunlight was used for all measurements at saturating
values of 1400 umol m'2 S'1 PAR or greater. Measurements were taken between 1000 and
1500 h. Temperatures were 22 2t 5°C. A total of 9 healthy and 9 diseased leaves were
measured in 2002, 16 healthy and 16 diseased leaves in 2003, and 12 healthy and 12
diseased leaves in 2004. Values for the assimilation rate, stomatal conductance, and

intercellular CO2 concentration were obtained for analysis.

Data analysis

The assimilation rate (A) was plotted against disease severity, Stomatal
conductance (3,), and intercellular CO2 concentrations (Ci). The data were plotted and
analyzed with nonlinear regression analysis using Sigmaplot statistical software (Systat

Software Inc., Richmond, California).

gaf photosynthesis in potted vines as infection progresses
Plant material

Two-year-old Vitis labrusca ‘Niagara’ grapevines were potted in 5 gallon pots
and maintained outdoors at the Michigan State University Plant Pathology farm, East
Lansing, MI in 2003 and the Michigan State University greenhouse courtyard in 2004.

Plants were watered regularly and fertilized every two weeks with 20-20-20 N-P-K

47

fertilizer. Any ﬂowers that developed were removed. The potting medium was a mixture
of steam-sterilized 60% loam and 40% sand. Vines were Sprayed regularly with Sevin
(carbaryl, Bayer Cropscience, Research Triangle Park, NC) to control Japanese beetle
and Nova (myclobutanil, Dow Agrosciences, Indianapolis, IN, USA) to control powdery

mildew. The experiment began on October 4 in 2003 and October 28 in 2004.

Inoculation

The ﬁrst or second fully expanded leaf was selected from each of ﬁve vines for
the experiment. For inoculum, naturally infected leaves collected from ‘Niagara’ vines in
the ﬁeld were placed in plastic bags with moistened paper towels. Bags were placed in
the dark for 12 h at 22°C to induce sporulation. Sporangia were washed into a beaker
from the leaves using a pipette and deionized water. The concentration was determined
using a hemacytometer and adjusted to 5x 104 Sporangia per ml. A suspension of
sporangia was applied to one half of the leaf using an atomizer while the other half of the
leaf was kept dry to avoid infection. The entire leaf was covered for 6 hours with a
moistened plastic bag to retain moisture. Inoculations were done before sunrise to avoid
any negative effect of light on sporangia] germination. Measurements began the day after

inoculation in 2003 and the day before inoculation in 2004.

Measurements
Gas exchange measurements were taken every 48 hours after the initial
measurement for 14 days after inoculation in 2003 and 10 days after inoculation in 2004.

Each measurement was taken on the inoculated half of the leaf, and the noninoculated

48

half was used as a control. A small Spot was marked on both Sides of the leaf with a
permanent marker so measurements could be taken in the same location. Gas exchange
measurements were taken with a CIRAS I infrared gas analyzer and a Parkinson leaf
cuvette (18-mm diameter chamber) (PP Systems, Amesbury, MA). Measurements were
taken between 1000 and 1600 h. Temperatures ranged from 24 :1: 3°C. Light values of 0,
50, 100, 200, 300, 400, 500, 700, 900, 1100, 1300, and 1500 umol m'2 S‘I PAR
(photosynthetically active radiation) and ambient CO2 concentrations were applied to the
leaves to develop the light response curves. Concentrations of 0, 100, 200, 300, 400, 500,
700, 900, 1100, 1300, and 1500 umol mol'1 of CO2 and $1400 umol m'2 S'l PAR were
applied to leaves to develop A/C, curves. The experiment was Similar in 2004 with the
exception that younger leaves were used, and only four vines were used.

Fluorescence measurements were taken with a Hansatech plant efﬁciency
analyzer (Hansatech Instruments, Norfolk, England). Infected and control areas were
measured twice (close to the same time) every 48 h and the average was used in the

analysis. Leaves were dark acclimated for 30 minutes before measurements were taken.

Data analysis

Data obtained from the light experiments in both years include the maximum
assimilation rate (Amu), light compensation point, and photosynthetic efﬁciency ((1)). In
2004, the dark respiration rate was also obtained. Data obtained from the CO2 response
curves included the maximum assimilation rate (Amax), assimilation rate at ambient CO2
(Amb), carboxylation efﬁciency (k), CO2 compensation point (P), and intercellular CO2

concentration (C). Stomatal conductance (gs) was obtained directly from the

49

measurements. The stomatal limitation ago») was calculated according to Layne (1989).
Fluorescence was expressed as the optimal yield of PSII, the ratio between variable
ﬂuorescence to maximum ﬂuorescence (F vIF m)- Data were plotted and subjected to
nonlinear regression analysis using Sigmaplot software (Systat Software Inc, Richmond,

CA) using the following equation from Layne (1989):

y=ax(l.0—b)xe('”x)

Where “y” is the net CO2 assimilation, “x” is the intercellular CO2 concentration, “a” is
the asymptotic value of the curve, “b” is the minimum value of the curve, and “c” is the
rate constant. The value of “e” is 2.7182818. Statistical analysis was done with Sigmastat

statistical software using a repeated measures analysis of variance.

Results
Assimilation rates of leaves of downy mildew-infected ‘Niagara’ vines in the ﬁeld
Downy mildew infection caused a Si gniﬁcant reduction in photosynthesis after the
appearance of symptoms. The assimilation rate of diseased leaves decreased with
increasing severity of infection. There was a linear reduction with increasing disease
severity in 2002 and a nonlinear reduction in 2003 and 2004. A 60% disease severity in
2002 and 70% severity in 2003 and 2004 produced a net assimilation rate of zero in
symptomatic infections (Figure 2.3). There was a strong negative correlation between the

assimilation rate and C; in the infected leaf (Figure 2.4A).

50

Figure 2.3. Relationship between the carbon assimilation rate and increasing disease
severity in ﬁeld-grown Vitis labrusca ‘Niagara’ vines in 2002 (A), 2003 (B), and 2004
(C) measured in an 18-mm diameter cuvette of a CIRAS I infrared gas analyzer. The zero
values for disease severity represent measurements on healthy leaves.

51

Assimilation rate (umol CO2 "12 5.1) Assimilation rate (umol CO2 m‘2 s")

Assimilation rate (umol CO2 m‘2 s“)

 

y = 0.0002x2 - 0.12x + 6.65
R2 = 0.76

 

 

 

 

 

y = 0.002x2— 0.31x + 13.03
R2 = 0.70

 

 

 

 

 

y = 0.001x2 - 0.18x2 + 7.53
R2 = 0.75

 

 

 

I
eoj 130

 

Leaf area diseased in cuvette (%)

52

 

 

 

N
U!

 

 

 

 

 

 

 

 

 

 

A
y = 0.0004x2 - 0.36x + 73.90

7" 20 "

(I)
‘1‘

EN 15 4

o

O

6 -

E 10

E},

Q)

’3 5 ‘

C

.9

E o «

.§

3

< -5 -

'10 I I I I
150 200 250 300 350 400
Intercellular CO2 concentration (umolmzs'1)
30
B

FA 25 - y = 8.57x2+ 0.014x -1.35

.f” R2 = 0.75

E 20 - .

N

o

0 15 .1

'6

E

E; 10 -

9

93

c: 5 "

.9

E

E 0 _

'6

(I)

< -5 ..

'10 I I I I
O 100 200 300 400 500

Stomatal conductance (umol CO2 mzs")

Figure 2.4. Relationship between carbon assimilation rate and intercellular CO2
concentration (A) and stomatal conductance (B) in healthy and downy mildew-infected
leaves of ﬁeld-grown Vitis labrusca ‘Niagara’ vines measured with a CIRAS I infrared
gas analyzer in Lawton, MI in 2003.

53

There was a positive correlation between the assimilation rate and Stomatal conductance

(Figure 2.43). Stomatal conductance values increased with increasing assimilation rates.

Leafyphotosynthesis in potted vines as infection progresses

In 2003, symptoms ﬁrst appeared on leaves 7 days after inoculation as slight, pale
yellow lesions which became darker and more distinct as the disease progressed.
Symptoms appeared 8 days after inoculation in 2004. Infected leaves Showed differences
from the healthy leaves in photosynthetic efﬁciency, Aamb, carboxylation efﬁciency,
intercellular CO2, and Am, in 2003 and Aamb in 2003 and 2004 before symptoms
appeared. However, chlorophyll ﬂuorescence, the light compensation point, CO2
compensation point, stomatal conductance, and stomatal limitation were not signiﬁcantly
affected until after symptoms appeared in 2003. Chlorophyll ﬂuorescence did not
decrease signiﬁcantly until eight days after inoculation in 2003 and ten days after
inoculation in 2004 (Figure 2.5A, B). In 2003, the light compensation point was variable,
becoming Signiﬁcantly different from control values 8 days after inoculation (Figure
2.6A). In 2004, the light compensation point increased slowly and became signiﬁcantly
different from the control 10 days after inoculation (Figure 2.7B). The photosynthetic
efﬁciency (0) of infected leaves decreased signiﬁcantly 6 days after inoculation in 2003
and 2 days after inoculation in 2004, although there was an increase in the control values
2 days after inoculation in 2004 (Figure 2.7A, B). The CO2 compensation point (P)
increased steadily in 2003 and became signiﬁcantly higher than control values 6 days
after inoculation (Figure 2.8A). In 2004 the CO2 compensation point became

signiﬁcantly higher 8 days after inoculation and the infected half of the leaf showed more

54

variability (Figure 2.8B). Carboxylation efficiency (k) became Signiﬁcant 4 days after
inoculation in 2003 and 2004 (Figure 2.9A, B) and decreased Sharply 4 days after
inoculation in 2003. Photosynthesis at ambient CO2 decreased sharply at 2 days after
inoculation and became signiﬁcantly lower than control values 4 days after inoculation
(Figure 2.10A). In 2004, the control values also decreased and photosynthesis in infected
areas was lower on 4, 8, and 10 days after inoculation (Figure 2.10B). Maximum
photosynthesis at increased CO2 levels (Amax) decreased Si gniﬁcantly 4 days after
inoculation in 2003 and 6 days after inoculation in 2004 (Figure 2.11A, B). Control
values in 2004 also dropped sharply 6 days after inoculation. Stomatal conductance
decreased signiﬁcantly in infected leaf areas 8 days after inoculation in 2003 and 10 days
after inoculation in 2004 (Figure 2.12A, B). Stomatal conductance also decreased in
healthy leaf areas in 2004. The stomatal limitation to photosynthesis was signiﬁcantly
lower in infected leaf areas 6, 8, and 10 days after inoculation in 2003, but was not
signiﬁcantly different from healthy leaves in 2004 (Figure 2.13A, B). The intercellular
CO2 concentration (C,) increased sharply in infected leaf areas 6 days after inoculation in
2003 compared to healthy leaf areas (ﬁgure 2.14A). In 2004, C, was only Signiﬁcantly
higher on the 8th day after inoculation (Figure 2.14B). Dark respiration was not
signiﬁcantly affected by downy mildew infection of P. viticola (Figure 2.15). Table 2.1
Shows the number of days after inoculation of leaves with P. viticola each parameter was

affected in 2003.

55

 

090
085‘

080-

l

0.75 - symptoms

F/Fm

070‘

 

+ Healthy half of leaf
-0- Infected half of leaf

0.65d

 

 

 

     

 

 

0.60 I T I I I
0 2 4 6 8 10

Days after inoculation

 

12 14 16

 

090

0.85 -

0.80 3

0.75d

FJFm

0.70 -

1

symptoms
0.65 -

 

+ Healthy half of leaf

0.60 a -O— Infected half of leaf

 

 

 

 

 
    
 

 

 

0.55

I I I I

0 2 4 6 8

Days after inoculation

10 12 14

Figure 2.5. The effect of downy mildew disease development on chlorophyll
ﬂuorescence measured as the ratio of F "/F m in leaves of two-year-old potted ‘Niagara’
grapevines in 2003 (A) and 2004 (B). Means are averages of ﬁve replications in 2003 and
four replications in 2004. Bars indicate the standard error of the mean.

56

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

500
A
:- + Healthy half of leaf
'w 400 - —O— Infected half of leaf 1-
N
E
‘2
3 300 1 symptoms
a:
c ‘—
8
x 200 -
2
c
.9
.8 100 -
0..
0 f I I r I I I
0 2 4 6 8 10 12 14 16
Days after inoculation
180
B _-
160 a
“1,, 140 .. + Healthy half of leaf
(YE —O— Infected half of leaf
3 12° ‘ symptoms >
E;
g 100 "
8
CD 80 -*
'0
§
.1: 60 "
c
9.
o 40 -
.c
0.
20 ~
0 I I I I I I

 

 

 

O 2 4 6 8 10 12

Days after inoculation

Figure 2.6. Effect of downy mildew disease development on the light compensation
point in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B).
Means are averages of ﬁve replications in 2003 and four replications in 2004. Bars
indicate the standard error of the mean.

57

 

0.06

0.05 a

0.04 a

'0

0.03 - symptoms

0.02 a , °

 

0'01 7 + Healthy half of leaf 0
—O— Infected half of leaf

Photosynthetic effucrency
(umol CO2 rn'2 s"/um0l quantum m'2 s")

 

 

 

 

 

 

0.00 I I I I j I I
0 2 4 6 8 10 12 14 16

Days after inoculation

0.040

 

0.035 ..
0.030 -
0.025 —

0.020 d

0.015 a ' symptoms
0

o
0.010 -

+ Healthy area of leaf
0005 - —O— Infected area of leaf

 

Photosynthetic effrcrency
(umol CO2 m'2 s’1/umol quantum m'2 s")

 

 

 

 

 

 

0.000

-:
cf

I I I I

O 2 4 6 8 10 12

Days after inoculation

Figure 2.7. The effect of downy mildew disease development on the photosynthetic
efﬁciency in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004
(B). Means are averages of ﬁve replications in 2003 and four replications in 2004. Bars
indicate the standard error of the mean.

58

C02 (umol moi")

co2 (umol moi")

320

 

300 -
280 -
260 -1
240 -l
220 d
200 -
180 ~
160 -1
140 -
120 -
100 -

80 a

60 -

 

40

 

+ Healthy half of leaf
—0— Infected half of leaf

   
 

 

 

 

 

  
    

1

symptoms

 

 

220

I I I I I I I

2 4 6 8 10 12 14

Days after inoculation

16

 

200 -
180 -
160 -
140 -
1204
100 -
80 4
60 -

40‘

 

 

+ Healthy half of leaf
—0— Infected half of leaf >

 

 

 

 

 

 

 

20

‘
d

I I I I

0 2 4 6 8 10

Days after inoculation

 

12

Figure 2.8. The effect of downy mildew disease development on the CO2 compensation
point in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B).
Means are averages of ﬁve replications in 2003 and four replications in 2004. Bars
indicate the standard error of the mean.

59

0J4

 

012‘-

0.08 - ’

006 - symptoms

004'- b

 

+ Healthy half of leaf
0-02 ‘ —o— Infected half of leaf . .

 

 

 

Carboxylation efficiency (umol rn'2 62 Pa")

 

 

 

0.00 T I I I r I I
0 2 4 6 8 10 12 14 16

Days after inoculation

009

 

(108-

(107-

 

006-' ~-

0.05 - ‘
symptoms

0.04 ' 0 w

(103‘

 

+ Healthy half of leaf .
0‘02 —0— lnfected half of leaf

 

 

 

Carboxylation efficiency (umol rn'2 s'2 Pa")

 

 

 

001 I u r 1 v
0 2 4 6 8 10 12

.

Days after inoculation

Figure 2.9. Effect of downy mildew disease development on the carboxylation efﬁciency
in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B). Means
are averages of ﬁve replications in 2003 and four replications in 2004. Bars indicate the
standard error of the mean.

60

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A
of” 20 -
E
N
O
0
g 15 -‘ .
3
a) + Healthy half of leaf
‘5 —O— Infected half of leaf
2 10 -1 O
.2 symptoms
3
s \l/
U) 5 -
a:
< ’ .
o . .
o I I I I I I I
0 2 4 6 8 10 12 14 16
Days after inoculation
12
B
'7‘” 10 -1 __
N O
E
ON
0 8 ‘
E
v 6 .4
9.
cu . -—
‘- o
c
.g 4 . symptoms
E
.§
0’ o
2 2 q + Healthy half of leaf .
—O— Infected half of leaf 0
0 j I T I I I

 

0 2 4 6 8 10 12

Days after inoculation

Figure 2.10. Effect of downy mildew disease development on the rate of photosynthesis
at ambient CO2 levels in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A)
and 2004 (B). Means are averages of ﬁve replications in 2003 and four replications in
2004. Bars indicate the standard error of the mean.

61

4O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A

p 35 d
'w
“3

E 30 -

N
O
2 25 -

o

E
3 20 -
9 symptoms

9 _-

c 15 '1 \l/
.9
E
E ‘0 ‘ __
'3
< 5 2 + Healthy half of leaf

—-0— Infected half of leaf
0 I I I I T I I
0 2 4 6 8 10 12 14 16
Days after inoculation
25
B

of” 20 -
E o

N symptoms
° ’
o O
'5 15 " o
E
3
2
9 10 4
r:
.9
E
._E. .
'17: 5 a
2 + Healthy half of leaf 0

—O— Infected half of leaf 0
0 I I I I r I
0 2 4 6 8 10 12

Days after inoculation

Figure 2.11. Effect of downy mildew disease development on the assimilation rate at
increased CO2 concentrations (1400 umol mol") in leaves of two-year-old potted
‘Niagara’ grapevines in 2003 (A) and 2004 (B). Means are averages of ﬁve replications
in 2003 and four replications in 2004. Bars indicate the standard error of the mean.

62

450

 

 

+ Healthy half of leaf

400 ‘ —-O— Infected half of leaf

 

 

 

350 a
300 - ‘L

250 -

200 ~
/I\ .
150 -‘

 

 

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symptoms '
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Days after inoculation
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g 120 - symptoms
:11
g 100 «
E
g 80 -
'O
S
o 60 «
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75 40 q
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Figure 2.12. Effect of downy mildew disease development on the stomatal conductance
in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004 (B). Means
are averages of five replications in 2003 and four replications in 2004. Bars indicate the
standard error of the mean.

63

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0.35 -

0.30 -

0.25 -

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+ Healthy half of leaf
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ll

symptoms

 

0.70

 

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Days after inoculation

 

0.60 -

0.50 -‘

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symptoms

 

 

 

+ Healthy half of leaf
—0— Infected half of leaf “L

 

 

 

 

0.00

 

 

I I I I

2 4 6 8 10 12

Days after inoculation

Figure 2.13. Effect of downy mildew disease development on the stomatal limitation to
photosynthesis (1:00) in leaves of two-year-old potted ‘Niagara’ grapevines in 2003 (A)
and 2004 (B). Means are averages of ﬁve replications in 2003 and four replications in
2004. Bars indicate the standard error of the mean.

64

400

380

360

340

320

300

280

260

240

Intercellular CO2 concentration (umol mol")

220

400
380
360
340
320
300
280
260
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220
200
180
160
140

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Days after inoculation
. \l/symptoms
_ + Healthy half of leaf
—0— Infected half of leaf
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.4
I I I I I I
o 2 4 6 8 1o 12

Days after inoculation

Figure 2.14. Effect of downy mildew disease development on the intercellular C02
concentration in leaves of two—year-old potted ‘Niagara’ grapevines in 2003 (A) and 2004
(B). Means are averages of ﬁve replications in 2003 and four replications in 2004. Bars
indicate the standard error of the mean.

65

 

 

 

 

 

 

 

 

 

 

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Days after inoculation

Figure 2.15. Effect of downy mildew disease development on dark respiration in leaves

of two-year—old potted ‘Niagara’ grapevines in 2004. Means are averages of four
replications. Bars indicate the standard error of the mean.

66

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67

Discussion
Downy mildew infection of grape reduced net photosynthesis in the leaves of

field-grown vines. The decrease in photosynthesis as severity increased in 2002 was more
linear than in 2003 and 2004. A similar linear decrease in photosynthesis in Vitis vinifera
infected with downy mildew was also reported by Giuntoli (2000). The differences
between the seasons could be due to the age of infection in the leaves when
measurements were taken. The nonlinear decreases in photosynthesis in 2003 and 2004
as disease severity increased could be a result of compromised healthy tissues
surrounding the symptomatic areas or “oil spots”. A similar effect was found for leaf
blast on rice (Bastiaans 1991), Septoria leaf spot on blueberry (Roloff et al. 2004), and
downy mildew on Vitis vinifera (Orlandini 1998). The effect could be the result of toxin
production, a reduced relative water potential, or accumulation of carbohydrates at the
host-pathogen interface (Bastiaans 1991, Scholes 1992). This would result in a larger
relative reduction in photosynthesis under small disease severities due to the presence of
more non-symptomatic tissue, some of which could be photosynthetically compromised.
The intercellular C02 concentration (C) of leaves had a strong negative
relationship to the rate of photosynthesis, suggesting that C decreased as disease severity
increased. Stomatal conductance increased with an increasing assimilation rate, but the
relationship was weaker. The reduction in photosynthesis could be caused in part by
reduced C02 ﬂow across stomata. Sporulation through the stomata could reduce the ﬂux
of C02 across the stomata. Sporulation was present on the leaves of all vines measured in
the ﬁeld. Closure of the stomata from the physiological effects of the pathogen in
symptomatic areas or non-symptomatic areas could also have this effect. Slight decreases

in stomatal conductance have been measured in rust-infected barley (Owera et al. I981).

68

Powdery mildew of grape also showed a decrease in stomatal conductance, but
carboxylation efﬁciency and intercellular C02 concentration decreased as well (Nail and
Howell 2004), suggesting the reduced assimilation rate may have been caused by
biochemical as well as physical mechanisms.

Carboxylation efﬁciency, Amax, and AW, were the first signiﬁcant reductions in
leaves infected with P. viticola relative to healthy leaf areas in 2003. Carboxylation
efﬁciency and Aamb are indicators of Rubisco efﬁciency suggesting the ability of Rubisco
to ﬁx C02 is compromised 4 days after the infection process begins. Rubisco activity in
powdery mildew and rust-infected plants have been attributed to a reduction of ribosomes
and rRN A in chloroplasts and RNA metabolism (Ayres 1979, Chakravorty 1982, Walters
1985). Am”, a measurement of the ability to regenerate RuBP, also decreased
signiﬁcantly 4 days after inoculation. However, if Rubisco is compromised, RuBP levels
will remain high, and the rate of photosynthesis will subsequently decrease.

Six days after inoculation, photochemical efﬁciency decreased signiﬁcantly in
infected leaves, while the C02 compensation point and intercellular C02 concentration
increased signiﬁcantly in 2003. Photosynthetic efficiency is the amount of CO2 ﬁxed
relative to the amount of light absorbed. The amount of RuBP produced in infected
leaves cannot be used by Rubisco due to decreasing Rubisco efﬁciency and therefore, the
ability to ﬁx C02 decreases while the same amount of light is being absorbed. As a result,
photosynthetic efﬁciency decreases. However, reductions in photosynthesis in the
chloroplasts of rust-infected beans were shown to be a result of reduced electron transport
(Sziraki et al. 1984). The sharp increase in the intercellular C02 concentration in infected

leaves 6 days after inoculation suggest there is a mesophyll limitation to C02 diffusion,

69

which can also be attributed to Rubisco damage. As damage to Rubisco occurs, its ability
to use C02 as a substrate becomes limited and intercellular CO2 levels increase relative to
levels in healthy leaves. The C02 compensation point can also be affected by Rubisco.
Transgenic tobacco plants with less Rubisco than control plants showed substantially
higher C02 compensation points (Lawlor 2001). The C02 compensation point can also be
affected by temperature and light, but no signiﬁcant change in the compensation point
was seen in the healthy grape leaves, suggesting that the increase in infected leaves was
caused by the pathogen. The stomatal limitation to photosynthesis (13(2)) was signiﬁcantly
lower in infected leaves at 6, 8, and 12 days after inoculation. As the mesophyll limitation
to photosynthesis (Rubisco limitation) increases in infected plants, the percent of stomatal
limitation should decrease which is further evidence that the decrease in photosynthesis
in infected leaves is a result of a mesophyll limitation.

The light compensation point and chlorophyll ﬂuorescence increased signiﬁcantly
8 days after inoculation, while the stomatal conductance decreased signiﬁcantly 8 days
after inoculation. Similar levels of the light compensation point in infected leaves before
symptoms compared to healthy leaves suggest that infected leaves have the ability to ﬁx
C02 at low light levels even with damage to Rubisco. At low light levels, the production
of ATP and NADPH and subsequently, the regeneration of RuBP are limiting. The
damage to Rubisco in infected leaves by day 8 may be enough to become the limiting
factor to photosynthesis, or possibly damage to the electron transport system has
occurred, either of which could increase the compensation point. The increase in
chlorophyll fluorescence (measured as a decrease in F vIF m) after symptoms appeared

could also be a result of damage to the electron transport system. Light energy absorbed

70

by PSII can no longer be used to produce ATP and NADPH, and a relatively larger
proportion of the excess absorbed energy is emitted as ﬂuorescence. The increase in
ﬂuorescence could also be a result of damage to PS1]. Phytoplasma infection of apple
leaves was shown to inhibit photosystem H efﬁciency and electron transport chain
activity (Bertamini et al. 2002), but the increase in ﬂuorescence after the appearance of
symptoms in downy mildew-infected leaves suggests that damage to photosystem II is
not the initial cause of the decreased rates of photosynthesis in infected leaves.

The decrease in stomata] conductance 8 days after inoculation suggests that there
was a stomatal closure or blockage, but this was not the primary cause of the reduction in
photosynthesis rates in infected leaves since there was still a greater stomatal limitation
(lg(ii)) in healthy leaves compared to infected leaves. Decreased photosynthetic rates on
powdery mildew-infected wheat, barley, and oak leaves have also been shown not to be a
result of stomatal closure or blockage (Ayres 1979).

A stomatal limitation could be imposed initially during the infection process of
Plasmopara viticola when the germ tube is formed in the substomatal cavity, but the
restriction is short lived because the primary hypha then extends into the mesophyll.
Therefore, it is not likely that this would cause a substantial reduction in the assimilation
rate. Sporulation through stomata could also cause a stomatal limitation, but this usually
occurs when lesions are well developed, and by this time in the infection process
numerous other physiological processes have been photosynthetically compromised in
the leaf.

The results in 2004 were more variable and photosynthesis was generally lower in

healthy leaves than in 2003. The lower rates of photosynthesis were due in part to

7]

younger leaf age in 2004. Also, lesions that developed did not ﬁll the entire cuvette area
in two of the leaves that were measured. The decreases in photosynthetic efﬁciency,
carboxylation efﬁciency, maximum assimilation rate at ambient C02 (Aamb), maximum
assimilation rate at 1400 umol mol'l C02 (Amax), and stomatal conductance in healthy leaf
tissue in 2004 suggest there may be some other factor affecting these parameters. The
effects could be due to temperature ﬂuctuations in the greenhouse, the amount of natural
light the plants were receiving during the experiment, or from moving the plants while
performing the experiment

The reduction in the photosynthetic rate in infected leaf tissues may be caused by
physical or biochemical limitations to C02 assimilation. The data from the response
curves suggest that the reduction in photosynthesis in infected leaves is due to a
mesophyll limitation rather than reduced photosystem II efﬁciency or a stomatal
limitation. Similar effects were found with Fusarium wilt on tomato (Nogues 2002),
phytoplasma infection on apple (Bertamini et al. 2002), powdery mildew on sugar beet
leaves (Gordon and Duniway 1982), and grapevine fan leaf virus-infected grapes
(Sampol et al. 2003). Understanding the infection process is important for ﬁnding ways
to manage diseases and preventing economic losses. Since carbon allocation in the vine is
ultimately affected by the carbon assimilation mechanisms, the reductions in
photosynthesis in vine leaves caused by downy mildew could potentially reduce the
amount of energy available for dry matter accumulation and thus affect crop yield,
quality, and vine hardiness.

In conclusion, reduced rates of photosynthesis in leaves of ‘Niagara’ vines

infected by P. viticola are likely biochemical in nature and could be a result of damage to

72

photosynthetic enzymes (such as Rubisco) and/or decreased rates of RuBP regeneration

possibly due to damage to electron transport components.

73

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77

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78

Chapter 3

The effect of downy mildew infection on carbon partitioning and biomass

accumulation in ‘Niagara’ vines

Introduction
Pathogen effects on carbon allocation

Growth of crops is usually measured by the amount of dry weight added over a
period of time and determined by destructive sampling (Long 1985). Infection by plant
pathogens often results in a change in the movement of carbon assimilates among plant
organs (de Nooij 1992). Pathogens invading leaves have access to large pools of carbon
and can cause a movement of photoassimilates into infected areas, as well as a reduction
of export to other organs. Foliar pathogens frequently increase the shoot:root ratio so
higher proportions of assimilates in infected plants are exported to the shoots (Farrar
1992, Walters 1985). In potato leaves infected with Phytophthora infestans, there was a
retention of assimilates at the infection sites (Farrell 1971). Carbohydrate concentrations
were found to be as much as ten times higher in the immediate area of rust infection on
bluegrass (Hodges and Robinson 1977). Assimilates are known to accumulate in radish
leaves infected with downy mildew (Coffey 1975, Williams 1964), but it is not known
whether they are retained by the pathogen or imported from other sites (Walters 1985).

The ﬂux of carbon into the apoplast of plants has been shown to be sufﬁcient to
support fungal growth (Ahmad et al. 1982, Farrar 1992, Kneale and Farrar 1985). The

photosynthetic capacity of one mesophyll cell of barley was shown to be enough to

79

support a colony of powdery mildew, suggesting that the carbohydrate supply was not
likely to be limiting to growth of powdery mildew (Farrar 1987). Accumulation of
carbohydrates in the leaf at the sites of infection generally appears to be at the expense of

the roots or non-infected leaves (Walters 1985).

Effects of biotrophicpathogens on carbon allocation

Biotrophic pathogens, unlike necrotrophic fungi, depend upon the host
metabolism for nutrients (Wright et al. 1995). Because of the close nutritional association
of biotrophic pathogens with their hosts, studies on the effects of carbon assimilation and
metabolism caused by the infection of these types of pathogens have been more
numerous than for other types of pathogens (Daly 1976). Biotrophic pathogens can cause
internal recycling of CO2 through repeated respiration and photosynthesis and, as a result,
there is a smaller amount of net photosynthates available for translocation (Farrar 1987).

Leaves infected by biotrophic pathogens often show a large increase in invertase
activity. Sucrose hydrolization (into glucose and fructose) requires the enzyme invertase,
but it is difﬁcult to tell whether invertase is produced by the plant or pathogen. Downy
mildew (Plasmopara viticola (Berk. & Curt.) Berlese & de Toni) infection on leaves of
grape (Vitis vimfera L.) caused a relatively small increase in invertase activity compared
to powdery mildew infection (Uncinula necator (Schwein.)) (Brem et al. 1986).

The increased accumulation of carbohydrates resulting from an increase in
invertase activity can eventually cause a down-regulation of the Calvin cycle leading to
inhibition of photosynthesis (Scholes et al. 1994). Reduced photosynthesis can reduce the

potential sucrose supply for translocation from infected leaves to other parts of the plant.

80

Brown rust (Puccinia hordei Otth.) infection has been shown to reduce carbon
translocation in barley leaves (Owera et al. 1983). Infection of grapevine leaves by
biotrophic pathogens can also have signiﬁcant effects on carbon partitioning in
grapevines. Downy mildew (P. viticola) has been shown to cause a depletion of sucrose
in grape leaves (Brem et al. 1986). ‘Concord’ grapevines infected with powdery mildew

showed reduced berry quality and wood maturity (Gadoury et al. 2001).

Carbon isotope composition in plant tissues

Two carbon isotopes occur in nature, one being the more abundant 12C, and the
other, the less abundant ”C. The isotope l3C comprises approximately 1.11% of the total
carbon (Boutton 1991, Farquhar et al. 1989). The ratio of '3C/'2C varies slightly in
natural materials due to fractionation during chemical, physical, and biological processes
(Farquhar et al. 1989). Very small differences in the carbon fractionation of materials can
be measured by mass spectrometry and can be of use to biologists (Boutton 1991).

Most of the natural isotopic variation of interest to biologists results from the
fractionation of carbon during photosynthesis (Boutton 1991, Farquhar et al. 1989).
Carbon isotopes in plant tissues are measured as the relative difference between the
isotope ratios of the sample and standard gases, and are known as the delta (5) notation.
The 5'3C values are expressed relative to a calcium carbonate standard, Pee Dee
Belemnite (PDB), a limestone fossil formation of Belemnitella americana of the
Cretaceous period (Boutton 1991). The 5'3C value is calculated as the l3CI'ZC ratio of the

sample relative to the PDB standard as follows:

81

5 13C = I (Rsample — Rstandardy Rstandard] X 1000

where 5'3 C is the parts per thousand difference in '3 C between that of the sample and the
standard, Rsamplc is the l3C/IZC mass ratio of the sample, and Rsmndam is the l3C/‘ZC mass
ratio of the Pee Dee Bee Belemite (PDB) limestone standard which has a value of
0.0112372 (Boutton 1991).

The abundance of '3 C in plant tissues is normally lower than that of atmospheric
carbon dioxide largely due to isotopic fractionation (change in the '3 C fraction) during
the photosynthetic process (Boutton 1991, Oleary 1988). In C3 plants, the enzyme
Rubisco discriminates against the reaction of 13CO2 due to small differences in chemical
and physical properties imparted by the difference in the mass of the isotopes (Farquhar
et al. 1989, Oleary 1988). Plants, including grapevines, using the conventional C3
pathway to ﬁx carbon have relatively low '3 C values because CO2 is reduced to
phosphoglycerate by the enzyme Rubisco which discriminates against l3CO2 (Boutton
1991). Conversely, C4 plants reduce CO2 to aspartic or malic acid via the enzyme PEP
carboxylase which discriminates less against l3C than Rubisco. Therefore, C3 plants
generally have lower 5'3 C values ranging from -32 to -20, whereas C4 plants have 5'3 C
values ranging from -17 to -9 (Bender 1971, Boutton 1991, Farquhar 1982). Tissue
samples of Vitis vinifera grapes were shown to have l3C values of -24.6 to -26.3
(Dimarco et al. 1977). Fractionation can also occur through limitations to diffusion of
CO2 into the leaf, allowing some aspects of stomatal control of photosynthesis to be
studied (Ehleringer 1991, Oleary 1988). Natural variation in carbon isotope abundance

can be useful in studying biochemical and physiological characteristics of photosynthesis

82

in plants under various environmental stresses such as drought stress (Ehleringer 1991,
Long 1985, Oleary 1988). Isotopic enrichment can also be used to study storage and
mobilization of carbon compounds, carbon transport between root grafts or mycorrhizal
connections, and the effects of herbicides or disease on carbon allocation (Svejcar et al.

1990).

Isotopes and carbon partitioning in grapevines

Carbon isotope labeling has been a valuable tool in studying the carbon
partitioning among sinks in plants. Plants can be enriched with '3 C or l4C through the
uptake of 13CO2 or l4CO2. Translocation of carbon from leaves has been measured by the
loss of the radioactive isotope 14C from the leaf after being pulsed with labeled MCO2
(Wardlaw 1990). Downy mildew has been shown to cause a change in assimilate
translocation in infected grape leaves using isotopic labeling (Brem et al. 1986, Owera er
al. 1983). Mature grape leaves labeled with MC have shown that assimilates can be
translocated acropetally to young leaves and shoots or basipetally to clusters and
permanent vine structures depending on their proximity to the sink (Hale 1962). Leaves
of ‘Delaware’ fed with MC exported an increasing percentage of assimilates as the
number of fruit clusters increased (Motomura 1990). A high percentage of these
assimilates came from the side of the plant in which the clusters were located. Top leaves
generally supply carbon to the apical buds and young leaves, middle leaves supply to the

stem, and basal leaves supply carbon to the stem and roots (Motomura 1990, Palit 1985).

83

Rationzﬁ and objectives

Little is known about the effect of downy mildew infection on the movement of
carbon in plants. However, grapevines infected with P. viticola do show the ability to
compensate for leaf damage by mobilization of reserves from the roots (Jermini 2003).
Stable carbon isotope (‘3 C) techniques can be used to follow the movement of carbon in
plants and determine the effect pathogens have on carbon allocation. The use of stable
carbon isotopes in agricultural research has recently become more frequent due to the
regulatory constraints and radioactive properties associated with MC labeling (Svejcar et
al. 1990). In addition, photosynthetic and metabolic processes discriminate much less
against the '3C isotope than MC (Van Norman 1952) making '3 C attractive for studying
pathogen effects on carbon allocation in plants. These studies may ultimately aid in
understanding the effects of pathogens on carbon assimilation and allocation.
Understanding the effects pathogens have on biomass accumulation and carbon allocation
in plants will aid in developing management practices that incorporate minimal fungicide
use and optimize plant growth and yield.

The objectives of this experiment were to: 1) Determine the effect of P. viticola
infection on dry weight accumulation and partitioning in ‘Niagara vines and, 2)
Determine the effect of P. viticola infection on short-term carbon movement in vines by

using stable isotope labeling.

84

Materials and Methods
Effect of downy mildew on plant dry weight and dry weight partitioning

Two-year-old bare-rooted ‘Niagara’ vines were planted in S-gallon pots in a
mixture of 80% loam and 20% sand. Plants were grown outdoors from the beginning of
July until the end of September at the Plant Pathology farm, Michigan State University,
East Lansing, MI. Vines were fertilized every two weeks with a soluble 20-20-20 N-P-K
fertilizer at a rate of 0.2 g N per vine. All vines were sprayed every two weeks at the
recommended rates with Sevin (carbaryl) (Bayer Cropscience, Research Triangle Park,
NC) to control Japanese beetle and Nova (myclobutanil) (Dow Agrosciences,
Indianapolis, IN) to control powdery mildew. Control vines were sprayed once a month
at the recommended rate with Ridomil Gold MZ (mefenoxam and mancozeb) (Syngenta

Crop Protection, Greensboro, NC) to prevent downy mildew infection.

Inoculation

Vines were arranged in a completely randomized design and inoculated with a
suspension of distilled water and P. viticola sporangia collected from infected ﬁeld-
grown ‘Niagara’ vines in Lawton MI. The suspension was adjusted to 5 x 104 sporangia
per ml using a hemacytometer. Inoculations were done between 0500 and 0700 h in the
ﬁrst week of August and the beginning of September. The undersides of the leaves were
sprayed with the suspension using an atomizer to provide uniform coverage for infection.
Vines were covered with plastic for ﬁve hours after the inoculation to retain moisture.
Two treatments and a control were used. Each treatment was replicated ﬁve times (one

vine per replicate) in a completely randomized design. One treatment was inoculated at

85

the 5-mm berry stage and the other at bunch closure. Control vines were kept free of
infection. Three separate control vines of the same age with fruit were exposed to the
same conditions and used to determine the growth stages, but were not used in the
analysis. The disease severity was estimated at harvest as a percent of the total leaf area
on the vine covered by downy mildew lesions. Abscised leaves were collected and added
to the remaining leaf samples at harvest. Vines were destructively harvested at the end of
September, seven weeks after the ﬁrst inoculation, and separated into leaves, canes, and
roots. Samples were then placed in a drying oven at 65°C for7 days and dry weights were

obtained for each tissue type.

Statistical analysis
Statistical analysis was done using an analysis of variance with Sigmastat
statistical software (Systat Software Inc, Richmond, CA). Regression analysis was

performed using Sigmaplot statistical software (Systat Software Inc, Richmond, CA).

'3 C translocation and dry weight accumulation in infected vines

Eight two-year-old, bare-rooted ‘Niagara’ vines were potted in 10—gallon pots in a
mixture of 80% loam and 20% sand in 2003. Vines were arranged in a completely
randomized design. Four vines were inoculated and four vines were used as non-
inoculated controls. Plants were allowed to grow outdoors for two summers at the Plant
Pathology Research Farm, Michigan State University, East Lansing, Michigan. Vines
were fertilized every two weeks with a soluble 20-20-20 N-P-K fertilizer at a rate of 0.2 g

N per vine. All vines were sprayed every two weeks with Sevin to control Japanese beetle

86

and Nova to control powdery mildew. Control vines were sprayed with Ridomil Gold MZ

to prevent downy mildew infection.

Inoculation

Vines were initially inoculated with P. viticola beginning in August (around
bunch closure) and inoculations continued once every week for ﬁve weeks to simulate a
natural infection process. Flowers that developed on vines were removed. Inoculations
were done by washing sporangia from infected leaves taken from ﬁeld-grown vines in
Lawton, MI. Infected leaves were incubated in the dark for 12 h at 22°C to obtain fresh
sporangia for inoculations. Sporangia were suspended in sterile, distilled water, and the
concentration was adjusted to 5 x 104 sporangia per ml using a hemacytometer. The
abaxial sides of the leaves were sprayed with the suspension using an atomizer.
Inoculations were done in the morning between 0400 and 0600 h while leaves were wet
from dew to aid in the infection process and to eliminate the need to cover leaves with

plastic bags to retain moisture.

Administration of I3C 02 to plants (pulsing)

Six weeks after the initial inoculation, all plants were exposed to CO2 labeled with
'3 C. Vines were covered with plastic bags and the opening of the bag was sealed to the
pots to create an airtight chamber around each plant. Two 6-mm holes were drilled into
each of the pots 5 cm above the soil line, and plastic tubing was installed in each hole to
allow l3CO2 to enter the chamber. l3CO2 was produced by mixing 20 ml of 85% lactic

acid (Baker, Phillipsburg, NJ, USA) with 600 mg Ba'3CO3 (Icon Isotopes, Summit, NJ,

87

USA) per plant. The reagents were mixed using 20-ml syringes. Syringes were attached
to the application tube by a two-way valve. Upon mixing, 13CO2 was released into the
chamber. Administration of l3CO2 was done between 0900 and 1200 h. Plastic chambers

were removed from plants 30 minutes after administration of 13CO2.

I Sampling for I 3C

Sampling of plant tissues was done at 1 h, 24 h, 48 h and 7 days after the
administration of 13CO2. Tissue was sampled randomly at every interval from healthy
leaves (HL), non-symptomatic tissue on diseased leaves (HDL), symptomatic (chlorotic)
leaf tissue from well-developed lesions (DL), and roots (R). New shoots (current year’s
growth) and woody tissues (stem and older canes) were sampled at 7 days only. Samples
from all tissues of plants that were not exposed to 13CO2 were taken to determine a
baseline for natural abundance of 13 C relative to the experimental vines. Harvested
samples were placed in aluminum foil packs and immediately placed in liquid nitrogen to
stop respiration. They were subsequently transferred to a freezer at -80°C for storage

prior to processing.

Dry weight analysis

The vines were destructively harvested at 7 days, and total leaf area and disease
severity were determined. Leaf area was measured with a leaf area meter to obtain total
leaf area for each vine. Disease severity was estimated on each leaf, and disease severity

on the entire vine was calculated as a ratio of total diseased area relative to total leaf area.

88

Harvested plant material was dried in a drying oven at 65°C for 7 days to obtain dry

weights from leaves, roots, shoots, and woody parts (stem and older canes) of the vines.

Stable isotope analysis

Frozen samples were removed from the freezer and placed in a drying oven at
65°C for seven days. Once dry, leaf and root samples were ground to a powder in liquid
nitrogen using a mortar and pestle. Cane samples were ground using a Wiley Mill with a
40—mesh screen. A 1 to 2-mg sample of each of the tissues was weighed with a
rnicrogram scale and placed in pressed tin capsules (Costech Analytical Technologies,
Valencia, CA) for analysis. One sample of each tissue type from each plant was taken.
Samples were sent to the UC Davis Stable Isotope Facility (University of California,
Davis, CA) and analyzed for '3 C content using a mass spectrometer (Integra automated

carbon and nitrogen gas analyzer, Sercon Ltd., Cheshire, UK).

Data analysis

The carbon isotope composition (8 '3 C) was calculated by the following formula:

6|3C = [(Rsample “ Rstandand), Rstandard] X 1000

where 6'3 C is the parts per thousand difference in 13C between that of the sample and
standard, Rsample is the l3C/IZC mass ratio of the sample, and Rsmndard is the l3C/I‘ZC mass
ratio of the Pee Dee Bee Belemite (PDB) limestone standard which has a value of

0.01 12372.

89

From this value several other values can be calculated in order to determine the
amount of '3 C in plant tissues. The absolute ratio (R) of a sample is calculated by the

formula:

R = l3c/nc = [(8301000) + 1] x Rm

Where Rpm; equals 0.01 12372. From the absolute ratio (R), the fractional abundance (F)

of ”C in plant tissue can be calculated as follows:

F=R/(R+l)

Atom % is defined as the percent of '3 C in plant tissues and is calculated as:

Atom%=Fx100

The amount of '3 C in the plant was calculated by multiplying the amount of
carbon in the sample (mg) by its corresponding fractional abundance. The fractional
abundance of '3 C in each sample was adjusted for plant size by multiplying it by the
fraction of each plant’s dry weight relative to the largest plant. The amount of '3C
attributed to labeling was determined by subtracting the '3 C present in the natural
abundance of the tissue from the total amount of '3C in each tissue sample. The amount

of '3 C in each plant organ was expressed as the ratio of excess '3 C (amount of '3 C

90

attributed to labeling) in the plant to the excess in each plant organ at the time of

sampling.

Statistical analysis

Statistical analysis using a repeated measures analysis of variance and multiple
comparisons using the Fisher’s LSD was used to separate differences in isotope
composition at sampling time. Separation of means of plant samples collected at seven
days was performed with a t-test. All statistical testing was done using Sigmastat

statistical software.

Results
Effect of downy mildew on plant dry weight and dry weight partitioniﬂz

Total estimated disease severity of the infected vines at the time of plant harvest
in 2003 averaged 22.5% for vines inoculated at the 5-mm berry stage and 16% for vines
inoculated at bunch closure. Total disease severity of the infected vines in 2004 averaged
11.4% of the total leaf area at the time of plant harvest. Whole vine estimates of disease
severity in 2003 gave an appearance of a higher overall disease severity than estimates
done on a leaf by leaf basis in 2004 due to the precision of the leaf area meter.

‘Niagara’ vines inoculated with P. viticola in 2003 accumulated less dry weight
than non-inoculated vines (Figure 3.1). Vines in 2004 did not show a significant
difference in total dry weight (Figure 3.2). Vines inoculated at the S-mm berry stage and
bunch closure did not show any signiﬁcant differences in the percent of dry weight

partitioning between the leaves, canes, or roots compared to non-inoculated vines in 2003

91

(Figure 3.3). Vines inoculated in 2004 showed a signiﬁcantly higher percent of dry
weight allocation to the new shoots of infected vines and a lower percent of dry weight
allocation to the woody parts of the vine compared to the control vines (Figure 3.4).
There was no signiﬁcant difference in percent of dry weight allocation in the leaves or
roots. The 2004 vines showed a higher leaf dry weight and lower root dry weight than
vines in 2003. The total dry weight of the canes was similar in 2003 and 2004. There was
a signiﬁcant negative relationship (P<0.05) between severity of the infection and leaf dry
weight (Figure 3.5), and between severity and total dry weight, but the latter relationship

was not as strong (Figure 3.6).

'3 C translocation in infected vines

The total enriched l3C content was slightly higher in the leaves and lower in the
roots of non-infected plants compared to the infected plants 7 d after exposure to ”CO2,
but the differences were not signiﬁcant (Figure 3.7). Total enriched 13C in the leaves of
infected vines was similar to the control vines after seven days, but the rate at which the
carbon was translocated was different. Infected leaves retained the labeled carbon longer
than the non-infected leaves. Labeled carbon accumulation in the roots showed similar,
but opposite trends (Figure 3.8). Roots of non-infected plants accumulated more labeled
BC in 24 hours than infected plants, but after 168 hours the amount of 13C in the roots
was similar (Figure 3.9). Symptomatic tissues accumulated nearly twice the amount of
13C in 24 hours as they had at the time of exposure to ”CO2, decreasing slightly after 24

hours (Figure 3.10).

92

 

250

200- b

150 -

Dry weight (g)

50-

 

 

 

5-mm stage Bunch closure Noninoculated

Time of inoculation

Figure 3.1. Total dry weight of two-year-old potted ‘Niagara’ grapevines inoculated with
Plasmopara viticola at the 5-mm berry stage and bunch closure compared to non-
inoculated vines in 2003. Each mean represents the average of ﬁve replications. Error
bars represent the standard error of the mean. Means sharing the same letter are not
signiﬁcantly different at P S 0.05.

93

500

 

400 -‘

 

300‘

200‘

Dry weight (g)

100 ‘1

 

 

 

 

 

 

 

Bunch closure Noninoculated

Time of inoculation

Figure 3.2. Total dry weight of three-year-old potted ‘Niagara’ grapevines inoculated
with Plasmopara viticola at bunch closure compared to non-inoculated vines in 2004.
Each mean represents the average of four replications. Error bars represent the standard
error of the mean. Means sharing the same letter are not signiﬁcantly different at

P S 0.05.

94

 

60

 

 

 

 

 

 

 

 

 

 

 

 

Time of inoculation
5° ‘ -
a 5-mm berry stage

,3 a a :l Bunch closure
2: I l:l Noninoculated
c 40 -
.9
s a a
_q :l:
a 30 -
E :‘r
9 it
o as:
3 20-
O

10 -

3%
0 - L1- “ T Q
leaves roots canes
Plant tissue

Figure 3.3. Percent of total dry weight in leaves, roots, and canes of two—year-old potted
‘Niagara’ grapevines inoculated with Plasmopara viticola at the 5-mm berry stage and
bunch closure compared to non-inoculated vines in 2003. Each mean represents the
average of ﬁve replications. Error bars represent the standard error of the mean. Means
sharing the same letter are not significantly different at P S 0.05.

95

50

 

 

 

 

 

 

 

 

 

 

 

 

- Healthy plant

40 [:1 Infected plant
1; a a a
o\ "E a
c :1:
.9
‘5 30 -
o
9
To a b
E
.9 20 .
a)
3
E:
D

10 'I a b

0 I I I IIﬂ

leaves roots new shoots wood
Plant tissue

Figure 3.4. Percent of the total dry weight in leaves, roots, new canes, and woody parts
(including canes and stem) of three—year—old potted ‘Niagara’ grapevines infected with
Plasmopara viticola compared to non—inoculated vines in 2004. Treatment vines were
inoculated once a week for six weeks before plant harvest. Each mean represents the
average of four replications. Error bars represent the standard error of the mean. Means
sharing the same letter are not signiﬁcantly different at P S 0.05.

96

 

 

y 2= -0.62x+50.69
60 - R = 0.50
15,-?
E
.9 40 -
m
3
2‘
U
‘26
m
.1
20 - o
0 I I I I

 

 

 

0 1 0 20 30 40

Leaf area infected (%)

Figure 3.5. The relationship between leaf dry weight and disease severity of two-year-old
potted ‘Niagara’ grapevines infected with Plasmopara viticola in 2003. Each point

represents one plant.

97

 

 

 

 

 

250
y = -2.08x+166.26
2
200 e R = 0.37

:53
E
.9
m 150 -
3
2‘
'U
E
E 100 -
Q.
79 o
C
'_ o

50 -

0 I I I I
o 10 20 30 40

Leaf area infected (%)

Figure 3.6. The relationship between total plant dry weight and disease severity of two-
year-old potted ‘Niagara’ grapevines infected with Plasmopara viticola in 2003. Each

point represents one plant.

98

70

 

 

 

 

 

 

 

 

 

 

 

 

a

60 ~ - Healthy plant

a [:1 Infected plant
a

50 - I
0
£2
8
§ 40 " a
'8
E a
a) 30
2
a:

20 -

a a
10 -
0 I I l - I '1]
leaves roots new canes wood

Plant tissue

Figure 3.7. Percent of the total '3 C sampled relative to the natural abundance in leaves,
roots, new shoots, and woody (including canes and stem) parts of three-year—old potted
‘Niagara’ grapevines infected with Plasmopara viticola compared to non-inoculated
vines in 2004. Treatment vines were inoculated once a week for six weeks before plant
harvest. Samples were taken 7 d after labeling. Each mean represents the average of four
replications. Error bars represent the standard error of the mean. Means sharing the same
letter are not significantly different at P 5 0.100.

99

 

 

 

 

 

 

 

120
+ Healthy plant
100 - —O— Infected plant
g; 80 ~
(I)
m
a 60 .
9
.9
.9
.— 40 -
20 -
0 l I I I I I I I I

 

 

0 20 40 60 80 1 00 1 20 1 40 1 60 1 80

Hours after labeling

Figure 3.8. Percent of total labeled '3 C sampled relative to the natural abundance in non-
symptomatic downy mildew-infected leaves compared to healthy leaves of 3-year-old
potted ‘Niagara’ grapevines in 2004. Samples were taken at l, 24, 48, and 168 h after
exposure to 13CO2. Each value is an average of four replications. Bars represent the

standard error of the mean.

100

60

 

50 - '-

404

 

 

30-

20-

1"’C in roots (%)

 

10-

 

 

+ Healthy plant
0 . —O— Infected plant

 

 

 

 

 

 

'10 I I I I I I I I I

0 20 4O 60 80 100 1 20 1 40 1 60 180

Hours after labeling

Figure 3.9. Percent of total '3 C sampled relative to the natural abundance in the roots of
downy mildew-infected vines compared to the roots of healthy three-year-old potted
‘Niagara’ grapevines in 2004. Samples were taken at 1, 24, 48, and 168 hours after

exposure to CO2. Each value is an average of four replications. Bars represent the
standard error of the mean.

101

 

 

 

 

13C in diseased tissue (%)
N

 

 

 

0 I I I I I I I I I

0 20 40 60 80 100 1 20 1 40 1 60 1 80

Hours after labeling

Figure 3.10. Percent of total '3 C sampled relative to the natural abundance of '3 C in the
lesions of leaves of three-year-old ‘Niagara’ grapevines infected with Plasmopara
viticola in 2004. Samples were taken at l, 24, 48, and 168 hours after exposure to I3CO2.
Each value is an average of four replications. Bars represent the standard error of the
mean.

102

Discussion
Dry weight agcumulation and allocation

In order to develop damage thresholds that can be used in integrated disease
management, it is essential to understand the effects of disease on carbon partitioning and
biomass accumulation in plants. Since biotrophic pathogens typically cause a general
decline in the photosynthetic rates of plants, there is a smaller amount of net
photosynthates available for translocation, and thus total biomass could ultimately be
reduced (Farrar 1987). Infection by P. viticola generally caused a reduction in the dry
weight of grapevines for infections lasting at least three weeks in 2003, but did not affect
dry weight in 2004. In 2004, overall dry weights were much higher and there was more
variability. This could be attributed to fewer plants. Also, vines used in 2003 were potted
in 5-gallon pots and allowed to grow for one growing season. Vines used in 2004 were
potted in 10—gallon pots and allowed to grow for two seasons which could better simulate
ﬁeld conditions.

Downy mildew infection did not affect the percent of dry weight partitioning to
different plant tissues considerably in 2003, at least not for infections lasting up to a 7-
week period. Infected vines allocated more biomas to new shoots and less to the cane and
stem (wood) in 2004. Although canes and shoots were not sampled separately in 2003,
there was more leaf biomass as well as total biomass in 2004, which could have resulted

in a larger percent of dry weight allocated to new shoots of infected vines.

103

'3c distribution

Seven days after labeling, infected vines had generally allocated more enriched
carbon to the roots and less to the leaves than the non-infected vines, but this effect may
be due to the timing of the sampling, since carbon reaching the roots in healthy plants
may have already been respired. At 24 hours, the healthy vines had allocated a larger
portion of the labeled carbon from the leaves to the roots than infected vines, suggesting
that the rate of translocation of assimilates is slowed by downy mildew infection.
Diseased bean leaves infected by rust (Uromyces phaseoli (Pers.) Wint.) also showed
reduced rates of translocation at ﬁve hours after labeling with MC (Livne 1966). Powdery
mildew infection of barley also showed transient decreases in the percentage of
assimilates translocated to the roots and subsequently, had little effect on assimilate
distribution (Walters and Ayres 1982).

White rust on radish showed an accumulation of assimilates at infection sites
(Williams 1964), a trend similar to what was seen for downy mildew on grapes although
the extent to which carbon accumulated was less for downy mildew. It is important to
remember that the disease was fully developed and some lesions had sporulated at the
time of labeling and sample collection. Downy mildew has an incubation period of
approximately seven days, and the accumulation of assimilates during that period could
be signiﬁcantly different from leaves with fully developed and/or Sporulating lesions.
Changes in photosynthesis and respiration can ultimately alter the movement of carbon
within and between diseased tissues and to non-infected plant tissues (Daly 1976). Since
there is a signiﬁcant change in the photosynthetic processes before the appearance of

downy mildew symptoms in grape leaves, it is possibly that there could be changes in

104

carbon translocation during the incubation period as well. Fungal metabolites in some
biotrophic infections increase linearly after the third day of inoculation with little or no
increase after sporulation occurs (Farrar 1987). If this is the case with P. viticola, the
accumulation of '3 C in lesions of infected grapevine leaves may have been greater before
symptoms appeared, or possibly after symptoms appeared if sporulation was prevented.
Another consideration is that it was not possible to know if a natural infection was
established in apparently healthy leaves of infected plants and symptoms had not yet
appeared during sampling, but it is unlikely that all apparently healthy leaves were
infected.

Respiration can also affect the amount of '3C in the leaves and roots of infected
and non-infected grapevines. Of the total carbon ﬁxed each day by barley plants, 25%
was translocated to the roots, and half of what was translocated was respired (Farrar
1980). Starch reserves in barley roots are relatively small compared to perennial plants,
and pathogens causing small changes in the assimilate supply could greatly affect the
availability of energy for respiration. In contrast, grapevines have large carbohydrate
reserves in the roots and trunk (Winkler 1945), and moderate downy mildew infections

may not affect the allocation of carbon substantially.

Other implications

Infected leaves eventually become necrotic and fall, allowing light to reach other
leaves in the canopy but exposing the fruit to sun scald. Defoliation was a common
occurrence on American vines in the late 1800’s before the introduction of chemical

control methods, but it did not normally have an adverse affect on fruit ripening (Farlow

105

1876). However, defoliated V. vinifera vines with ripening fruit changed their
translocation patterns by directing carbon stored in roots of the vine to the fruit (Candolﬁ-
vasconcelos et al. 1994). If carbon allocation to the roots is affected by prolonged
infection of downy mildew and too few reserves are stored in the roots, cold hardiness
and fruit set (transition of ﬂowers into young fruit) the following year could be

compromised.

Conclusion

Downy mildew infection affects the rate of carbon translocation and the total dry
weight accumulation in ‘Niagara’ grapevines. Carbon allocation and dry weight
accumulation in ‘Niagara’ vines infected with P. viticola still needs further investigation
to determine the damage thresholds of ‘Niagara’ vines infected with P. viticola.

The effects of longer periods of infection and vine defoliation need to be studied
under ﬁeld conditions to determine when control measures need to be taken in order to
prevent losses. Data also need to be obtained on the effects of the disease on carbon
allocation of fruiting vines. Finally, the effects of P. viticola infection on cold hardiness
and growth and fruit production during the next growing season should be examined.
Carbon isotope techniques can be further implemented to study the movement of
assimilates during the incubation period of the infection. The effects of disease on carbon
translocation should be extended to ﬁeld studies using fruiting vines as well. Stable
isotope techniques are not frequently employed but can serve as a powerful tool to study
many aspects of the carbon partitioning in diseased plants, and ultimately aid in the

reﬁnement of disease management practices.

106

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Wright, D. P., Baldwin, B. C., Shephard, M. C., and Scholes, J. D. 1995. Source-sink
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110

Chapter 4

Variation among Plasmopara viticola isolates from different host species

Introduction

Downy mildew pathogens are generally speciﬁc to a single host order or family, a
characteristic that may stem from co-evolution with their hosts (Crute 1981). As a result,
the movement or spread of pathogen populations may be limited by the host species.
Plasmopara viticola (Berk. & Curt.) Berlese & de Toni is a highly specialized pathogen
that has persisted on a few genera within one host family (Vitaceae). Wild plant hosts
have been an important factor in the development of downy mildew pathogens (Renfro
1981). Plasmopara viticola is commonly found on wild hosts in the US, but it does not
appear that wild hosts have a signiﬁcant role in disease epidemiology on cultivated
grapevines (Renfro 1981). Severely infected wild grapes in close proximity to cultivated
grapes do not appear to be the source of infection for the cultivated grapes (Barrett 1939,
Renfro 1981), suggesting the possibility of the existence of physiological races.
Physiological races or pathotypes have been identiﬁed in downy mildew species
parasitizing crops including sunﬂower (Vear et al. 1997), cucurbits (Lebeda and
Widrlechner 2003), lettuce (Zinkemagel 1983), pearl millet (Thakur et al. 2004), and
spinach (Irish et al. 2003), but races may also occur in downy mildews adapted to wild or
non-crop plants. Although a few studies have mentioned the variation in pathogenicity
between isolates of P. viticola on different grape cultivars (Kast 2001, Santilli 1957),

physiological races or pathotypes have not been identiﬁed.

111

Host range of P. viticola

Plasmopara viticola has been reported to cause losses in Vitis vinifera L., the
most economically important grapevine species in Europe, for more than 100 years
(Agrios 1997, Anderson 1956, Emmett 1992, Viennot-Bourgin 1981). North America has
various species of cultivated grapes including Vitis labrusca L. (fox grape), V. vinifera
(wine grape), V. rotundifolia Michx. (muscadine grape), and wild hosts including V.
riparia Michx. (riverside grape), V. aestivalis Michx. (summer grape), and
Parthenocissus quinquefolia (L.) Planch. (Virginia creeper), that can harbor the pathogen
(Pearson and Goheen 1988, Renfro 1981). Although Europe has a large pool of V.
vinifera available to P. viticola, most of the acreage of V. vinifera in the US. is located in
California where conditions are not conducive to infection by the pathogen (Gubler
1994). Plasmopara viticola is native to North America, and natural selection had
produced a balance so it could parasitize wild American grape hosts without seriously
affecting the grape’s survival while the European grape had little or no natural resistance
(Agrios 1997, Alexopoulos et al. 1996). Since fungal pathogens on crop species can
become specialized to host genotypes when the host provides a uniform environment

(Crute 1981), there is the possibility that physiological forms exist in P. viticola.

Host resistance to P. viticola

Many grape species and hybrids show at least partial resistance to P. viticola
(Barlass 1986, Dai 1995, Demaree 1937, Denzer et al. 1995, Di Gaspero and Cipriani
2002, Eibach 2000, Kast 2001, Matthews 1981, Staudt and Kassemeyer 1995). The most

likely sources of resistance are found in the American grape species, particularly V.

112

labrusca and V. aestivalis (Matthews 1981). Vitis labrusca (depending on cultivar) and
V. aestivalis are less susceptible to downy mildew than V. vinifera, and the American
species V. riparia is classiﬁed as highly resistant (Demaree 1937, Lafon 1981, Pearson
and Goheen 1988). However, infection studies using P. viticola inoculum taken from V.
vinifera cv. Muller-Thurgau showed that V. riparia was moderately resistant while V.
aestivalis showed susceptibility to infection (Staudt and Kassemeyer 1995). Studies using
P. viticola isolates collected from different hybrid varieties in Europe showed that disease
severity on leaves, especially on resistant hybrid varieties, was dependent upon the fungal
isolate being tested, suggesting that different physiological races may exist (Kast 2001).
Isolates taken from Vitis californica Benth., a wild grape species in California, were not
able to complete their life cycle on Vitis vinifera or any other species that served as hosts
outside of California (Santilli 1957). A few attempts have been made to classify P.
viticola into distinct physiological forms or pathotypes based on differences in
sporangiophore and sporangia morphology, but differences in morphology have been
shown to be highly dependent on environmental conditions, particularly humidity and

temperature (Rafaila 1968).

Growth of P. viticola on resistant cultivars

On resistant grape species and cultivars, the infection process by P. viticola is
initiated, but is terminated several days after the initiation (Kortekamp et al. 1998, Dai
1995, Langcake and Lovell 1980). The lesions on infected leaves of resistant grape
species and cultivars became necrotic soon after the infection process was terminated,

whereas on susceptible species and cultivars, the tissues did not become necrotic until

113

after sporulation occurred (Dai 1995, Kortekamp et al. 1998, Langcake and Lovell 1980).
Infection on leaves of V. riparia by isolates taken from V. vinifera caused brown necroses
around the stomata and produced sporulation only under highly favorable conditions, and
even then sporangia were few in number (Langcake and Lovell 1980). The necrosis,
which was seen 2-3 days after the infection began, was apparently triggered by a
hypersensitive response. Increased peroxidase activity and the accumulation of phenolic
compounds in the leaves of other resistant cultivars have also been reported (Dai 1995,
Kortekamp et al. 1998). Oospore formation, however, was shown to occur more readily
on species and cultivars that do not support sporangial production (Grunzel 1961, Populer
1981). Sexual reproduction by means of oospores has the potential to increase genetic
diversity through recombination, potentially yielding more virulent isolates. There is also
evidence that P. viticola is heterothallic which can increase the potential for pathogenic
specialization among isolates relative to grapevines species and cultivars (Wong et al.
2001). New isolates of P. viticola could pose a problem with control of downy mildew
over time on resistant grapevines, even though resistance of most hybrid varieties is
assumed to be polygenic (controlled by more than one gene) and generally not complete

(Eibach 2000, Kast 2001, Kortekamp et al. 1998).

Molecular studies on P. viticola

Various molecular markers have been used on plant pathogens to determine the
phylogeny of related organisms, study the diversity of populations, and follow the
movement of speciﬁc genes and genotypes (McDermott and McDonald 1993).

Techniques such as restriction fragment length polymorphisms (RFLPs), randomly

114

ampliﬁed polymorphic DNA (RAPDs), microsatellite DNA markers, and direct
sequencing of nuclear ribosomal internal transcribed spacer (ITS) regions have been used
to create a better understanding of the evolution and population dynamics of P. viticola.
Studies using large subunit ribosomal DNA sequences have shown genetic relationships
between downy mildews such as Peronospora, Bremia, and Plasmopara spp. (Goker et
al. 2003, Riethmuller et al. 2002, Voglmayr 2003, Voglmayr et al. 2004), but no studies
have focused on variation within P. viticola isolates themselves. Although there have
been an increasing number of studies on the genetic diversity of downy mildews, the
knowledge is generally limited.

Molecular studies can also be used to follow the movement and spread of
pathogens. Using microsatellite DNA markers, it was determined that at least some of the
disease establishment in an Italian vineyard was imported on either young plants or by
some type of human transport rather than wind-mediated introduction (Gobbin et al.
2003). Studies using RAPD techniques from isolates of P. viticola from the leaves of
several grapevine varieties in a single vineyard in Germany showed a high genetic
diversity among primary infections and decreasing diversity with each subsequent cycle
of infections (Stark-Umau et al. 2000). The decreasing diversity in subsequent cycles was
attributed to selection of the ﬁttest isolates to carry on the cycles. However, in a Greek
vineyard in consecutive years, it was shown that there was very low diversity among
primary infections, and that oospore germination and infection occurred throughout the
season (Rumbou and Gessler 2004). The ability of secondary sporangia to cause
infections was low, and infections occurred over short distances. The differences in

diversity of primary infections in these studies could be attributed to the environmental

115

conditions during the previous season and the amount of primary inoculum present. Since
it has been traditionally believed that secondary sporangia are largely responsible for the
temporal and spatial spread of the disease (Agrios 1997, Blaise 1996, Gobbin et al. 2003,
Pearson and Goheen 1988), ﬁndings in these studies could change theories on the disease
cycle and epidemiology of P. viticola, and could have implications for disease

management strategies.

Rationale and objectives

Most of the studies on P. viticola in the past century have focused on
understanding the biology of the pathogen and developing management strategies to
control the disease. Not much attention has been given to the possibility that
physiological races exist in P. viticola or if the taxa contains more than one species. If
races or other species of P. viticola exist, it could affect the way the disease is managed
on different grape species and cultivars as well as in different geographical areas.

The objectives of this study were to: 1) Determine if downy mildew isolates
collected from different grape cultivars were able to infect other grape cultivars, and 2)
Determine if P. viticola isolates collected from wild host species were able to infect

leaves of cultivated grapevines.

116

Materials and Methods
Plant material and isolate collection

Healthy, fully expanded leaves of ﬁeld-grown Vitis labrusca ‘Niagara’ and
‘Delaware’, interspeciﬁc hybrids ‘Seyval’ and ‘Vi gnoles’, and wild vines of Vitis riparia,
V. aestivalis, V. vinifera, and Parthenocissus quinquefolia (used as a positive control
when P. quinquefolia inoculum was used) were collected in July in 2003 and July
through September in 2004. Healthy leaves used for inoculation were collected at the
Clarksville Horticultural Station in Clarksville, MI and a greenhouse courtyard of
Michigan State University in East Lansing, MI. Healthy leaves of ﬁeld-grown vines were
chosen from untreated vineyards or untreated vines in experimental plots to avoid leaves
containing fungicide residues. Isolates of P. viticola were collected from leaves and fruit
of wild (V. riparia, V. aestivalis, and P. quinquefolia), potted (V. labrusca ‘Niagara’ and
‘Delaware’) and ﬁeld-grown (V. interspeciﬁc hybrid ‘Vignoles’ and ‘Seyval’) vines in
Lawton, Clarksville, Onondaga, East Lansing, and Jackson, MI. The isolates collected

and the date and location of collection are shown in Table 4.1.

Inoculum preparation

Infected leaves, shoots, and clusters were collected from the ﬁeld and any
sporulation was removed by rinsing with deionized water. Infected tissues were then
placed in plastic bags with moistened paper towels, sealed, and incubated overnight in the
dark at 22°C to induce sporulation. The following morning the sporangia were washed

from Sporulating tissues with sterile, deionized water into a 20—ml vial to create a

117

suspension of inoculum. The suspension was adjusted to a concentration of 5 x 104

sporangia per ml using a hemacytometer.

Leaf disk preparation

Healthy leaf disks measuring\2.5 cm in diameter were taken from a leaf of each of
the six vine species/cultivars and placed abaxial side up in a 150 mm Petri dish. Another
Petri dish was prepared with six more leaf disks that were left uninoculated to serve as a
control. Leaf disks that served as the inoculated and the control were taken from the same
area of the same leaf to conﬁrm that an infection did not already exist in the leaf. The
abaxial surfaces of the leaves were wetted with sterile, deionized water using an atomizer
before inoculation. Leaf disks in one Petri dish were inoculated with a sporangial
suspension, while sterile, deionized water was added to leaf disks of the control. Each

Petri dish (inoculated and control) was replicated ﬁve times (a total of ten Petri dishes).

Inoculation

A ZOO-pl volume of each prepared sporangial suspension was applied to each leaf
disk using a pipette. The same amount of sterile water was applied to the control leaf
disks. Sterile ﬁlter paper was wetted and applied to the inside of the lid of each Petri dish
to maintain humidity. The plates were sealed with paraﬁlm and incubated under
ﬂuorescent light at 21°C for 24 hours with a 12-hour light and 12-hour dark period. In the
2004 experiments, the leaf disks were removed after the incubation period and washed

with sterile water to remove any remaining sporangia from the inoculation to avoid

118

counting them during the analysis. Leaf disks were patted dry with sterile towels and the
plates were rescaled with paraﬁlm and incubated for 7 additional days at 21°C.

After a total of 8 days of incubation, the leaf disks were examined under a
dissecting microscope to see if any sporulation had occurred. The leaf disks were then
removed from the Petri dish, and the sporangia were removed by drawing them into a
pipette using 60 ul of sterile, deionized water. The total number of sporangia produced
per leaf disk was determined using a hemacytometer. The average of two counts (one on
each side of hemacytometer) per leaf disk was used in the analysis. If sporulation was
seen under the dissecting microscope and the hemacytometer counts were zero, it was
given a count of one to indicate that there were sporangia on the leaf disks. Experiments
with isolates not collected in 2003 were repeated twice in 2004. Isolates taken from wild

hosts were also included in 2004.

Data analysis

The analysis of the isolate-host combinations was conducted using a two-factor
analysis of variance (ANOVA) in both years. In addition, results from isolates used in
both seasons were analyzed together using a three-factor analysis of variance using year
as a factor. Statistical analysis was done using Sigmastat statistical software (Systat

Software Inc., Richmond, California, USA).

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Results

In 2003, four different isolates of downy mildew were used to inoculate vines.
There was substantial variation in the number of sporangia produced on inoculated leaf
disks for each host-isolate combination (Figure 4.1). The effect of the host was
statistically signiﬁcant (P<0.001), while the effect of the isolate was not (P<O.526).
However, there was a signiﬁcant host-isolate interaction (P<0.001) (Table 4.2).
Generally, each host-pathogen combination resulted in an infection that was capable of
producing sporulation. The highest production of sporangia on ‘Niagara’ leaves was
produced by the Ni-03 isolate, and it was the only isolate that caused significant
sporulation on ‘Niagara’ leaves in 2003. In addition, the Ni-O3 isolate was not effective at
producing sporulation on Vitis riparia, but isolates Ta—03, Se-O3, and Ma-O3 all did.
Sporangium production on ‘Delaware’ was generally low in 2003 with all isolates. All

isolates produced some sporulation on ‘Seyval’ and ‘Vignoles’.

121

 

 

 

 

 

 
 
  

 

 

   

 

15000 . Inoculated leaves
x [:1 V. Iabrusca'Niagara'
E 14000 . m V. Iabrusca'Delaware'
E - V. ripan‘a
8 12000 _ V. aestivalis
C [ZZZ V. hybrid 'Seyval'
g s\\\\V v. hybrid 'Vignoles'
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Figure 4.1. Sporulation of Plasmopara viticola isolates from different hosts on leaf disks
of various grape cultivars and species in 2003. Isolate origin: Ni—03 (‘Niagara’ leaves in
Lawton, MI), Ta—03 (unidentiﬁed table grape clusters, Clarksville, MI), Se-03 (‘Seyval’
leaves, Clarksville, MI), and Ma-O3 (‘Mars’ table grape leaves, Onondaga, MI). Values
are mean sporangium counts from ﬁve leaf disks. Error bars represent the standard error
of the mean.

122

 

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% .
=6 [:1 V. labrusca 'Niagara‘
“a 30000 ~ . m V. labrusca 'Delaware'
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5 20000 -
5
o.
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Ni-03 Ri-O4 Ae-04 Se-03 Ta-03
Isolate

Figure 4.2 Sporulation of Plasmopara viticola isolates from different hosts on leaf disks
of various grape cultivars and species in 2004. Isolate origin: Ni-04 (‘Niagara’ leaves in
Lawton, MI), Ri-04 (Vitis riparia leaves, East Lansing, MI), Ae-04 (V. aestivalis leaves,
Lawton, MI), Se-04 (‘Seyval’ leaves, Clarksville, MI), and Ta-04 (unidentiﬁed table
grape clusters, Clarksville, MI). Values are mean sporangium counts from ﬁve leaf disks.
Error bars represent the standard error of the mean.

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126

35000

 

 

 

 

 

 

 

 

 

 

 

 

x 30000 ‘ Inoculated leaves
(I)
‘6 [:1 V. Iabrusca'Niagara'
'8 _ - V. riparia
% 25000 m V. aestivalis
8. 7///// V. hybrid 'Seyval'
as 20000 _ m P. quinquefolia
'6)
C
S
8 15000 -
(I)
‘6
83 10000 -
.o
E
3
Z 5000 -
0 .
Ni-04 Ri-04 Ae-04 Se-04 Pa-04
Isolate

Figure 4.3. Sporulation of Plasmopara viticola isolates inoculated onto the same host
from which they were collected in 2004. Isolate origin: Ni—04 (‘Niagara’ leaves in
Lawton, MI), Ri-04 (Vitis riparia leaves, East Lansing, MI), Ae-04 (V. aestivalis leaves,
Lawton, MI), Se-04 (‘Seyval’ leaves, Clarksville, MI), and Pa-04 (Parthenocissus
quinquefolia leaves, Lawton, MI), Values are mean sporangia counts from ﬁve leaf disks.
Error bars represent the standard error of the mean.

127

In 2004, the Ni-04 isolates produced more sporangia on Vitis labrusca ‘Niagara’
vines than any other host-isolate combination (Figure 4.2), while producing little
sporulation on V. riparia, V. aestivalis, V. hybrid ‘Seyval’ and V. hybrid ‘Vignoles’.
Sporulation on V. labrusca ‘Delaware’ with the N i-04 isolate was substantially higher
than with Ni-03 in 2003. Isolates Ri-04, Se-04, and Ta-04 produced little sporulation on
‘Niagara’ leaves and Ae-04 produced none at all. The other isolates generally produced
more sporulation on leaf disks other than ‘Niagara’. Isolates Ni-04, Ae-04, and Se-04 all
produced more sporangia on the leaves of the species or cultivar from which they were
collected than on other species and cultivars. Isolates Ta—O3, Se-03, Ta—04, and Se-04
produced similar results on all leaf disks. There was a signiﬁcant difference among the
isolates (P<0.001) in 2004, but not among the hosts (Table 4.3). There was also a
signiﬁcant host-isolate interaction (P<0.001) in 2004. Analysis of the data from both
seasons showed a signiﬁcant difference between years (P=0.029) and a signiﬁcant host x
isolate x year interaction (P<0.001) (Table 4.4). The N i-04 produced over four times the
number of sporangia in 2004 than Ni-03 did in 2003. Sporangia collected from
Parthenocissus quinquefolia did not cause sporulation or visible infection on any host
except for on leaves of P. quinquefolia, on which it sporulated readily. Figure 4.3 shows
the number of sporangia produced on each leaf disk by isolates collected from the same
host species or cultivar. Inoculum from Ge-04 did not cause any sporulation or sign of
infection on any host in either replication, but a leaf of V. vinifera could not be obtained

for a positive control.

128

Discussion

Data obtained in 2003 and 2004 suggest that P. viticola isolates collected from V.
labrusca ‘Niagara’ vines (Ni-O3, Ni-04) are more efﬁcient at infecting and causing
sporulation on ‘Niagara’ leaves than on any other host tested, with the exception of
‘Seyval’ in 2003. In addition, all other isolates were not very successful at causing
sporulation on ‘Niagara’ vines, suggesting that the Ni-03 and Ni-04 isolates may be
adapted speciﬁcally to infect ‘Niagara’ vines or possibly other cultivars of the species V.
labrusca. These results are similar to those of Kast (2001), who found variation in the
ability of P. viticola isolates collected in Germany and Switzerland to infect the hybrids
‘Johanniter’, ‘Regent’ and ‘Trollinger’. The signiﬁcant host-isolate interaction in both
seasons may be due in large part to the ability of the Ni-03 and Ni-04 to produce large
quantities of sporangia on ‘Niagara’ leaves consistently but not on other grape species.
Isolates Ta-03, Se-03, and Ma-03 all showed the ability to produce infection and
sporulation on the wild and interspeciﬁc hybrid leaves somewhat consistently, suggesting
they may be more similar to each other than the Ni-03 and Ni-04 isolates. Interestingly,
isolates obtained from other wild host species (Ri—04, Ae-O4) produced little or no
sporulation on any ‘Niagara’ leaves, but were able to infect the host from which they
were collected (V. riparia and V. aestivalis). The isolates Se-04 and Ta-O4 were able to
infect the wild hosts as well, suggesting they may be able to overcome resistance factors
in these grapevines but not in the Labrusca vines. Isolates taken from the unidentiﬁed
grape and ‘Seyval’ (Ta-O3, Ta-04, Se-03, Se-04) were collected from the same location
and produced similar results, so it is possible that they were the same isolate or closely

related. Inoculum taken from P. quinquefolia seems to be very speciﬁc to the leaves of P.

129

quinquefolia, suggesting physiological races or species of P. viticola may exist, but little
has been reported about downy mildew infection of P. quinquefolia, and infection is
generally considered to be caused by P. viticola (Renfro 1981).

The signiﬁcant interaction between host and isolate in both seasons indicates that
the quantity of sporangia produced on a speciﬁc host greatly depends on the isolate being
used for the inoculation. The signiﬁcant interaction between isolate, host, and year
suggests that the variability in the number of sporangia produced could also be affected
by conditions that isolates and hosts are exposed to during a particular season,
particularly temperature and moisture. Isolates from Romania showed high
morphological variability, particularly in sporangia, which was shown to be influenced
by temperature and humidity (Rafaila 1968). Favorable conditions in 2004 could have
allowed isolate Ni-04 to produce more sporulation on leaf disks of V. labrusca than
isolate Ni-03 in 2003. The same conditions could be true for the host, V. labrusca, being
less resistant to certain isolates during a particular season. Leaf age can also strongly
inﬂuence susceptibility (Emmett 1992, Pearson and Goheen 1988, Rafaila 1968). Even
though leaves of approximately the same age were chosen for the experiment, there was
inevitably some variability in susceptibility of individual leaves. Although healthy leaves
were chosen from untreated vineyards, they could have also contained fungicide residues
that drifted from adjacent vineyards or plots. Washing the leaf disks after inoculations in
2004 may have provided a more realistic count of sporangia produced by the infection,
although the sporangia counts were generally higher in 2004 when the leaf disks were
washed, and it is doubtful that this had much of an effect on total counts of sporangia

between 2003 and 2004.

130

There is other evidence that could possibly support the idea of physiological races
of ‘Niagara’ isolates. Downy mildew infection is not typically seen on cultivated V.
labrusca vines until July or August in Michigan. Farlow (1876) reported that the fungus
did not usually appear in New England on American species and cultivars until August
and was very common from the middle of August until frost. Other reports in the late
1800’s stated that downy mildew did not appear in the eastern United States until late
summer and fall (Bush at al. 1895), similar to what is typically seen on V. labrusca in
Michigan today (personal observation). In contrast, substantial downy mildew infections
were seen in late June on table grape clusters and V. riparia in 2004, two weeks before
the ﬁrst infection was seen on leaves of ‘Niagara’ vines. The difference could be due to
susceptibility of the vine at different times throughout the growing season, or that host-
speciﬁc sporangia were released earlier or later in the season from germinating oospores.

Immature berries are reported to be highly susceptible to downy mildew infection
(Pearson and Goheen 1988). Inoculations with P. viticola on ‘Niagara’ in New York
showed that ﬂowers remained susceptible until several days after bloom (Kennelly 2002).
Inoculations done on ‘Niagara’ and ‘Delaware’ in the early 1900’s showed that P.
viticola attacked the leaves, stems, tendrils, and fruit on American varieties (Gregory
1912). However, infections of flowers and clusters of ‘Niagara’ were not commonly
observed under ﬁeld conditions in the northeastern U.S. (Farlow 1876). In his study on
the biology of the pathogen, Farlow ( 1876) stated that in New England, “Peronospora
viticola infection was limited to the leaves and stems of American species and cultivars,
and does not attack the fruit”, suggesting that berry infection was rare or non-existent on

ﬁeld vines in the late 1800’s. This conﬂicting evidence may be a result of the young

13]

berries of some American species or cultivars being resistant during the period of oospore
germination, or that there are isolates infecting berries of certain cultivars that do not
have the ability to infect the berries of others. The lack of resistance factors in V. vinifera
in Europe may have limited the need for the pathogen to carry speciﬁc virulence factors
that enable it to overcome resistance found in its native range.

Knowledge of host-pathogen interactions can aid in analyzing and predicting
disease pressure and epidemics. If different physiological races do exist, there is the need
to identify them and determine how frequently they can be found, where they are found,
and how they change with different selection pressures (Crute 1981). An increased
understanding of host speciﬁcity of P. viticola isolates and a better understanding of
individual host resistance and tolerance could ultimately lead to alternative and more
efﬁcient methods of disease control. Future studies should focus on the relationship
between the genetic diversity of P. viticola isolates and the pathogenicity on different

host species over a wide range of environmental conditions and geographic areas.

132

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136

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141

Table A5. Repeated measures analysis of variance of ﬂuorescence in leaves of ‘Niagara’
grapevines infected with Plasmopara viticola compared to healthy leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 0.0756 0.0126 26.25 <0.001
Treatment 1 0.0529 0.0529 3 1 .32 0.005
Day x treatment 6 0.0518 0.0086 21.10 <0.001
Residual 24 0.0098 0.0004

Total 69 0.2 1 10 0.0031

 

Table A.6. Repeated measures analysis of variance of the assimilation rate at ambient
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2003. ‘

 

Source of Variation DF SS MS F P
Day 6 684.55 1 14.09 7.87 <0.001
Treatment 1 2206.81 2206.81 247.89 <0.001
Day x treatment 6 574.37 95.73 6.94 <0.001
Residual 24 331.20 13.80

Total 69 4204.40 60.93

 

Table A.7. Repeated measures analysis of variance of the light compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 107690.14 17948.36 6.31 <0.001
Treatment 1 1 12135.58 1 12135.58 14.673 0.02
Day x treatment 6 78134.65 13022.44 6.42 <0.001
Residual 18 36542.19 2030.12

Total 63 533103.61 8461.96

 

Table A.8. Repeated measures analysis of variance of the photosynthetic efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 0.0048 0.0008 14.64 <0.001
Treatment 1 0.01 10 0.01 10 347.10 <0.001
Day x treatment 6 0.0037 0.0006 14.37 <0.001
Residual 24 0.0010 0.000]

Total 69 0.0200 0.0003

 

142

Table A.9. Repeated measures analysis of variance of the assimilation rate at maximum
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 1721.88 286.98 14.70 <0.001
Treatment 1 4577.35 4577.35 124.00 <0.001
Day x treatment 6 1131.78 188.63 20.86 <0.001
Residual 22 198.96 9.04

Total 67 8909.31 132.98

 

Table A.10. Repeated measures analysis of variance of the stomatal conductance in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 31230.68 5205.1 1 0.44 0.847
Treatment 1 180369.48 180369.48 15.98 0.016
Day x treatment 6 150749.72 25124.96 5.54 0.001
Residual 24 108839.29 4534.97

Total 69 907623.96 13153.97

 

Table A.11. Repeated measures analysis of variance of the C02 compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 44271.07 7378.51 2.56 0.046
Treatment 1 1551 15.63 1551 15.63 25.77 0.007
Day x treatment 6 68580.45 11430.08 6.06 <0.001
Residual 23 43383.53 1886.24

Total 68 427452.50 6286.07

 

Table A.12. Repeated measures analysis of variance of the carboxylation efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2003.

 

Source of Variation DF SS MS F - P
Day 6 0.0207 0.0035 6.34 <0.001
Treatment 1 0.0686 0.0686 72.05 0.001
Day x treatment 6 0.0148 0.002 8.93 <0.001
Residual 24 0.0067 0.0003

Total 69 0. 1290 0.0019

 

143

Table A.13. Repeated measures analysis of variance of the intercellular C02
concentration in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 48061.23 8010.21 6.30 <0.001
Treatment 1 85424.50 85424.50 26.07 0.007
Day x treatment 6 32025.71 5337.62 9.94 <0.001
Residual 24 12883.99 536.83

Total 69 231815.59 3359.65

 

Table A.14. Repeated measures analysis of variance of the stomatal limitation to
photosynthesis in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2003.

 

Source of Variation DF SS MS F P
Day 6 0.252 0.0420 4.3 1 0.004
Treatment 1 0.074 0.0738 6.63 0.061
Day x treatment 6 0.056 0.0094 1.52 0.216
Residual 23 0. 142 0.0062

Total 68 0.835 0.0123

 

Table A.15. Repeated measures analysis of variance of ﬂuorescence in leaves of
‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy leaves in
2004.

 

Source of Variation DF SS MS F P
Day 6 0.0491 0.0082 5.44 0.002
Treatment 1 0.0487 0.0487 20.86 0.020
Day x treatment 6 0.0722 0.0120 13.48 <0.001
Residual 18 0.0161 0.0009

Total 55 0.2280 0.0042

 

Table A.16. Repeated measures analysis of variance of the assimilation rate at ambient
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 149.77 29.95 4.54 0.010
Treatment 1 85.97 85.97 10.53 0.048
Day x treatment 5 52.06 10.41 6.89 0.002
Residual 15 22.66 1.51

Total 47 476.22 10.13

 

144

Table A.17. Repeated measures analysis of variance of the light compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 11585.15 2317.03 2.14 0.116
Treatment 1 5152.83 5152.83 5.24 0.106
Day x treatment 5 9750.74 1950.15 1.20 0.137
Residual 15 14640.55 976.04

Total 47 69727.48 1483.56

 

Table A.18. Repeated measures analysis of variance of the photosynthetic efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 0.0007 0.0001 2.30 0.097
Treatment 1 0.0013 0.0013 125.69 0.002
Day x treatment 5 0.0002 0.0001 1.69 0.197
Residual 15 0.0004 0.0001

Total 47 0.0036 0.0001

 

Table A.19. Repeated measures analysis of variance of the dark respiration in leaves of

‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy leaves in
2004.

 

Source of Variation DF SS MS F P
Day 5 2.191 0.438 3.66 0.023
Treatment 1 0.067 0.067 0.21 0.679
Day x treatment 5 0.641 0.128 1.61 0.219
Residual 15 1 . 197 0.080

Total 47 8.380 0.178

 

Table A.20. Repeated measures analysis of variance of the assimilation rate at maximum
C02 in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to
healthy leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 1416.65 283.33 9.25 <0.001
Treatment 1 171.62 171.62 15.14 0.030
Day x treatment 5 64.65 12.93 3.80 0.020
Residual 15 51.08 3.41

Total 47 2288.40 48.689

 

145

Table A.21. Repeated measures analysis of variance of the stomatal conductance in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 53613.26 10722.65 4.93 0.007
Treatment 1 18586.67 18586.67 6.58 0.083
Day x treatment 5 5201.28 1040.26 0.85 0.539
Residual 15 18455.85 1230.39

Total 47 146071.32 3107.90

 

Table A.22. Repeated measures analysis of variance of the C02 compensation point in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 29494.60 5898.92 3. 15 0.039
Treatment 1 24245.76 24245.7 6 6.68 0.08 1
Day x treatment 5 19442.76 3888.55 2.38 0.088
Residual 15 24484.29 1632.29

Total 47 144303.37 3070.28

 

Table A.23. Repeated measures analysis of variance of the carboxylation efﬁciency in
leaves of ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 0.0022 0.00045 3.1 1 0.040
Treatment 1 0.0071 0.00714 68.82 0.004
Day x treatment 5 0.0027 0.00054 2.67 0.064
Residual 15 0.0030 0.00020

Total 47 0.0185 0.00039

 

Table A.24. Repeated measures analysis of variance of the intercellular C02
concentration in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 21901.48 4380.30 3.74 0.021
Treatment 1 20255.28 20255.28 29.90 0.012
Day x treatment 5 25036.73 5007.35 2.68 0.064
Residual 15 28078.74 1871.92

Total 47 1 19831.05 2549.60

 

146

Table A.25. Repeated measures analysis of variance of the stomatal limitation to
photosynthesis in leaves of ‘Niagara’ grapevines infected with Plasmopara viticola
compared to healthy leaves in 2004.

 

Source of Variation DF SS MS F P
Day 5 0.181 0.0362 2.56 0.067
Treatment 1 0.001 0.0003 0.04 0.852
Day x treatment 5 0.026 0.0053 0.39 0.844
Residual 15 0.161 0.0134

Total 47 0.692 0.0157

 

147

APPENDIX B

148

Figure B.1. Equation used to calculate percent uptake of l3C02 from Ba'3C02 in
‘Niagara grapevines in 2004.

Ba'3C03+2H+ ——> Ba+2+'3C02+H0H

Table B.1. Percent of pulsed l3C02 taken up by ‘Niagara’ grapevines in 2004.

 

 

 

 

 

 

 

 

 

repliiclafion Treatment % uptake
1 Healthy plant 14.67
2 Healthy plant 15.78
3 Healthy plant 14.03
4 Healthy plant 11.41
1 Infected plant 17.56
2 Infected plant 19.16
3 Infected plant 14.42
4 Infected plant 14.49

 

149

Table B.2. Analysis of variance of total dry weight of ‘Niagara’ grapevines infected with
Plasmopara viticola at the 5-mm berry stage and bunch closure compared to healthy
vines in 2003.

 

Source of Variation DF SS MS F P
Treatment 2 11192.94 5596.47 5.45 0.017
Residual 15 15399.97 1026.66

Total 17 26592.90

 

Table B.3. Analysis of variance of dry weight of individual organs of ‘Niagara’
grapevines infected with Plasmopara viticola at the S-mm berry stage and bunch closure
compared to healthy vines in 2003.

 

Source of Variation DF SS MS F P
Treatment 2 9. 113-02 4613-02 3613-02 1 .000
Tissue 2 1376.15 688.08 53.83 <0.001
Treatment x tissue 4 34.78 8.70 0.68 0.611
Residual 33 421.83 12.78

Total 41 1890.36 46.] 1

 

Table B.4. Repeated measures analysis of variance of '3C content in leaves of three-year-
old potted ‘Niagara’ grapevines infected with Plasmopara viticola compared to healthy
leaves at 1, 24, 48, and 168 h in 2004.

 

Source of Variation DF SS MS F P
Treatment 1 18.41 18.41 0.1 1 0.763
Hour 3 12099.05 4033.02 1 10.98 <0.001
Treatment x hour 3 1090.58 363.53 2.20 0.158
Residual 9 1489.36 165.49

Total 31 16094.41 519.17

 

Table B.5. Repeated measures analysis of variance of ‘3 C content in roots of three-year-
old potted ‘Niagara’ grapevines infected with Plasmopara viticola compared to roots of
healthy vines at 1, 24, 48, and 168 h in 2004.

 

Source of Variation DF SS MS F P
Treatment 1 17.93 17.93 0.09 0.787
Hour 3 8280.56 2760. 19 63.71 <0.001
Treatment x hour 3 1088.19 362.73 1.97 0.189
Residual 9 1655.99 184.00

Total 31 12708.1 1 409.94

 

150

Table B.6. Repeated measures analysis of variance of '3 C content in lesions of leaves of
three-year-old potted ‘Niagara’ grapevines infected with Plasmopara viticola compared
to healthy leaves at 1, 24, 48, and 168 h in 2004.

Source of Variation DF SS MS F P

 

Rep 3 8.38 2.79 8.69 0.005
Hour 3 4.61 1.54 4.78 0.029
Residual 9 2.89 0.32
Total 15 15.89 1.06

 

151

 

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