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This is to certify that the
dissertation entitled

EVALUATION OF NITROGEN-FERTILIZER UPTAKE, NITROGEN-
USE AND WATER-USE EFFICIENCY IN SWEET CHERRY
(Prunus avium L.) ON DWARFING AND STANDARD
ROOTSTOCKS

presented by
Costanza Zavalloni

has been accepted towards fulfillment
of the requirements for the

Ph.D degree in Horticulture

 

 

 

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EVALUATION OF NITROGEN—FERTILIZER UPTAKE, NITROGEN-USE AND
WATER—USE EFFICIENCY IN SWEET CHERRY (Prunus avium L.)
ON DWARF ING AND STANDARD ROOTSTOCKS
By

Costanza Zavalloni

A DISSERTATION
Submitted to
Michigan State University

in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Horticulture

2004

 

ABSTRACT
EVALUATION OF N ITROGEN-F ERTILIZER UPTAKE, NITROGEN-USE AND
WATER—USE EFFICIENCY IN SWEET CHERRY (Prunus aw'um L.)
ON DWARF INC AND STANDARD ROOTSTOCKS
By
Costanza Zavalloni

Optimum management ofnitrogen (N) and water is of critical importance in order
to maintain growth and high production of fruit trees in modem orchards. The objectives
of this dissertation were to evaluate in sweet cherry on dwarﬁng and standard rootstocks:
1) the N-fertilizer uptake efﬁciency, and nitrogen-use efﬁciency (NUE), at different
phenological stages; 2) the water—use efﬁciency (WUE) under non-limiting water
availability and water deﬁcit condition, and 3) the effect of water deﬁcit on growth and
physiological parameters. N-fertilizer uptake efﬁciency, NUE, and WUE were evaluated
ﬁve times during the growing season on one-year-old potted sweet cherry cv. ‘Rainier’,
grafted on the dwarﬁng rootstock ‘Gisela 5’, the semi-dwarﬁng rootstock ‘Gisela 6’, and
the standard rootstock ‘Mazzard’. Also the same rootstocks without scion were
compared. N-fertilizer uptake was inﬂuenced by the accumulation of dry matter and was
higher from rapid shoot growth until the beginning of leaf senescence. Overall, there
were no differences in N-fertilizer uptake between dwarﬁng and standard rootstocks.
NUE was signiﬁcantly higher in ‘Mazzard’ compared to either the dwarﬁng or the semi-
dwarﬁng rootstocks without scion. Values of NUE were similar for ‘Mazzard’, and cv.
‘Rainier’ grafted on dwarﬁng, semi-dwarﬁng, and standard rootstocks, in all the periods

considered. WUE was higher in the standard rootstock without scion, compared to both

dwarﬁng rootstocks without scion. N-fertilizer uptake and NUE were also evaluated in

ﬁeld-grown, ﬁve—year-old sweet cherry cv. ‘Sam’ grafted on ‘Mazzard’ and ‘Gisela 5’.
KISNOg was applied at full bloom, rapid shoot growth, and at the beginning of leaf
senescence. N-fertilizer was absorbed in greater amounts when applied at bloom or at
rapid shoot growth than at the beginning of leaf senescence. When N-fertilizer was
applied at bloom, the percent of N—fertilizer was higher in leaves of sweet cherry on
dwarﬁng than standard rootstocks indicating that N-fertilizer contribute more to the total
N of dwarﬁng trees than standard trees. NUE, as well as N retranslocation from
senescent leaves, did not differ between the rootstocks. Plant growth and gas exchange
parameters, water—use efﬁciency and leaf carbon isotope composition were evaluated on
one-year—old potted sweet chen'y cv. ‘Rainier’ grafted on ‘Mazzard’ and ‘Gisela 5’ under
two different water treatments: a) well-watered (control), which received 100% of the
amount of water lost by ET, and b) water deﬁcit treatment, which received 50% of the
water applied to the control. Gas exchange parameters were affected earlier than growth
parameters. Growth parameters measured in sweet cherry on standard and dwarﬁng
rootstocks were affected similarly. Cumulative leaf area was the ﬁrst growth parameter
to be affected by water deﬁcit. WUE was not signiﬁcantly different between rootstocks,
and did not appear to increase under water deﬁcit condition, indicating that irrigation
should be considered as an important practice in sweet cherry orchards, especially when

dwarﬁng rootstocks are selected.

Copyright by
COSTANZA ZAVALLONI
2004

DEDICATION

This thesis is dedicated to my husband Daniele and my mother Piera

for their continuous support and encouragement.

ACKNOWLEDGMENTS

I would like to thank my major professor and mentor Dr. Jim Flore for his
guidance, support, and for believing in me over these years. I am especially thankful for
his friendship, his enthusiasms and creativity that have been an incredible source of
encouragement during my graduate experience at MSU.

I would like to express my gratitude to the members of my graduate committee
Dr. Phil Robertson, Dr. Ken Poff, Dr. Ron Perry, and Dr. Eric Hanson. In particular, I
would like to thank Dr. Phil Robertson for the valuable discussions, for his advises, and
for giving me conﬁdence throughout the project; Dr. Ken Poff for his friendship and his
constant reminders to ‘think outside the box’; Dr. Ron Perry for his friendship, insight
and support throughout the years, and Dr. Eric Hanson for his help during the
experiments and for keeping his door always open for advises. I’m also very grateful to
Dr. Martin J. Bukovac for the numerous valuable discussions and Prof. Bruno Marangoni
for encouraging me to pursue a Ph.D. at MSU.

Special thanks must be extended to Adriana Nikoloudi for her friendship and her
tireless help throughout the years — thank you.
I would like to thank Dario Stefanelli, Roberto Zoppolo, Marlene Ayala, Randall Vos,
Judith Nyiraneza, John Sorochan, and Royal Fader for their friendship and their help
during my graduate studies at MSU.

My sincere gratitude goes also to my family and my husband Daniele for their

understanding and support.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... xii
KEY TO SYMBOLS OR ABBREVIATIONS ................................................. xix
LITERATURE REVIEW ............................................................................ 1
Introduction ................................................................................... 2
Imponance of cherry production in United States and the World .................... 3

Cherry rootstocks and physiology of dwarﬁng process ................................. 4

Cherry rootstocks ................................................................... 4
Characteristics of dwarﬁng rootstocks .......................................... 5

Hypotheses on dwarﬁng mechanism ............................................. 6

Effect of cultivar and rootstock on leaf nutrient concentration ............... 8

Nitrogen in the plant ....................................................................... 10

Forms of nitrogen absorbed by plants .......................................... 10

Assimilation and translocation of nitrate and ammonium ................... 12

Movement and accumulation of nitrogenous compounds ................... 13

Internal cycling of N in trees ..................................................... 14

Nitrogen fertilization in fruit trees .............................................. 15

Carbohydrates and N metabolism ............................................... 16
Environmental problems related to the utilization of mineral N... . . . . l 7

Nitrogen-use efﬁciency: deﬁnition and importance ................................... 18
Physiology of water deﬁcit ............................................................... 20

Plant water relations .............................................................. 21

Effect of water deﬁcit on plant growth parameters ........................... 22

Effect of water deﬁcit on physiological and morphological parameters...23

Water-use efﬁciency: deﬁnition and importance ....................................... 24
Leaf 13C composition: effect of plant water deﬁcit .................................... 26
Rationale and objectives of the research ................................................ 28
Literature cited .............................................................................. 30

vii

C_HALTEL1_
N-FERTILIZER UPTAKE, NITROGEN-USE AND WATER-USE EFFICIENCY OF
ONE-YEAR-OLD SWEET CHERRY (Prunus avium L.) CV. ‘RAINIER’ ON

STANDARD AND DWARFING ROOTSTOCKS ............................................ 44
Abstract ...................................................................................... 44
Introduction .................................................................................. 45
Materials and methods ..................................................................... 47

‘5 Nitrogen applications .......................................................... 48

Plant growth measurements, harvests, and samples collection ............. 48

Soil sampling ...................................................................... 49

'5 Nitrogen analysis ................................................................ 49
Nitrogen-use efﬁciency ........................................................... 50
Water-use efﬁciency .............................................................. 50
Experimental design ............................................................... 51
Results ....................................................................................... 53
Plant growth ........................................................................ 53

Plant total nitrogen content ...................................................... 54
Seasonal efﬁciency of N-fertilizer plant uptake .............................. 54
Partitioning of absorbed N-fertilizer ............................................ 55
Nitrogen-use efﬁciency .......................................................... 56
Water-use efﬁciency .............................................................. 57
Discussion ................................................................................... 72
Seasonal efﬁciency of N-fertilizer plant uptake ............................. 72
Partitioning of absorbed N-fertilizer ............................................ 73
Nitrogen-use efﬁciency .......................................................... 74
Water-use efﬁciency .............................................................. 75
Conclusions ................................................................................. 76
Literature cited .............................................................................. 77

viii

CHAPTER2
NITROGEN UPTAKE AND NITROGEN-USE EFFICIENCY OF FIELD-GROWN
SWEET CHERRY (Prunus avium L. ‘SAM’) ON DWARFING AND STANDARD

Abstract ...................................................................................... 82
Introduction ................................................................................. 83
Materials and methods ..................................................................... 86
15 Nitrogen applications ........................................................... 86
Plant measurements and samples collection ................................... 87
15 Nitrogen analysis ............................................................... 89
Nitrogen-use efﬁciency ........................................................... 89
Experimental design ............................................................... 90
Results ....................................................................................... 90
Trunk cross sectional area ....................................................... 90
N-fertilizer uptake efﬁciency .................................................... 90
N-fertilizer partitioning ............................................................ 91
Nitrogen-use efﬁciency ........................................................... 92
Nitrogen in abscised leaves ...................................................... 93
Discussion ................................................................................. 103
N-fertilizer uptake efﬁciency ................................................... 103
N-fertilizer partitioning .......................................................... 106
Nitrogen-use efﬁciency ......................................................... 107
Retranslocation of N from senescent leaves ................................. 108
Conclusions ................................................................................ 109
Literature cited ............................................................................ l 1 l

CHAPTER 3
WATER-USE EFFICIENCY OF ONE-YEAR-OLD SWEET CHERRY (Prunus avizrm
L. ‘RAINIER’) ON DWARFING AND STANDARD ROOTSTOCKS, UNDER WELL

WATERED AND WATER-DEFICIT CONDITIONS ...................................... 117
Abstract .................................................................................... 1 17
Introduction ................................................................................ 1 18
Materials and methods ................................................................... 121

Water treatments ................................................................. l 21

Soil water content ............................................................... 122

Plant growth measurements .................................................... 122

Gas exchange measurements ................................................... 123
Photosynthetic water-use efﬁciency ........................................... 124

Plant harvest ...................................................................... 124

Leaf carbon isotope determination ............................................. 124
Experimental design ............................................................. 125
Results ...................................................................................... 126
Soil water content ................................................................ 126

Plant growth parameters ........................................................ 126

Shoot elongation ........................................................ 126
Leafemergence ratel27

Cumulative leaf area ................................................... 127

Trunk cross sectional area ............................................. 128

Plant dry weight ......................................................... 128

Gas exchange parameters ....................................................... 128

Net carbon dioxide assimilation rate ................................. 128

Transpiration rate ....................................................... 129

Stomatal conductance .................................................. 129

Internal carbon dioxide ................................................ 130

Water-use efﬁciency ............................................................ 130

Daily trend of gas exchange parameters ...................................... 131

Leaf carbon isotope composition .............................................. 132
Discussion ................................................................................. 149
Conclusions ................................................................................ 152
Literature cited ............................................................................ 154

SUMMARY AND CONCLUSIONS ............................................................ 158

APPENDIX A ...................................................................................... 163
APPENDIX B ...................................................................................... 166
APPENDIX C ...................................................................................... 169

xi

LIST OF TABLES
CHAPTER 1

Table 1.1. Plant phenological stages during ’SN application, dates of 15N application, and
dates of plant harvests .................................................................... 52

Table 1.2. Amount of Nitrogen Derived From Fertilizer (NDFF) in plant and soil, and
recovery of NDFF in plant and soil (percent of the applied N, average per
harvest). Plants were fertilized with 1’N at different Days After Bud Break
(DABB). Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6) and ‘Mazzard’
(M) .......................................................................................... 64

Table 1.3. Amount of Nitrogen Derived From Fertilizer (NDFF) in plant and soil, and
recovery of NDFF in plant and soil (percent of the applied N, average per harvest)
Plants were fertilized with 15N at different Days After Bud Break (DABB).
Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainier/Mazzard’ (R/M) ................................................................. 65

Table 1.4. Increase in dry weight (DW at 94 - DW at 36 DABB, g), evapo-transpiration
(ET, L) and water use efﬁciency [ADW(g)/AET(L)] of plants, calculated between
36 and 94 Days After Bud Break (DABB). Comparison between ‘Rainier/Gisela
5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6) and ‘Rainier/Mazzard’ (R/M) and between
‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6) and ‘Mazzard’ (M) ................................ 71

W

Table 2.1. Trunk cross sectional area (TCSA, cm2) of sweet cherry ‘Sam/Mazzard’
(S/M) and ‘Sam/Gisela 5’ (S/Gi5) measured 15 May, 2002, and 1 1 May, 2003 and
percent increase of area .................................................................. 94

Table 2.2. N concentration (mg N g'1 dry weight) in sweet cherry ‘Sam/Mazzard’ (S/M)
and ‘Sam/Gisela 5’ (S/Gi5) ﬁne roots, coarse roots, 2002 wood (current season
wood), and leaves, harvested 40 days after each 15N application. 15N-fertilizer
was applied at bloom (11 May), rapid shoot growth (20 June), and beginning of
leaf senescence (11 September) ......................................................... 97

Table 2.3. N concentration (mg N g'1 dry weight) in sweet cherry ‘Sam/Mazzard’ (S/M)
and ‘Sam/Gisela 5’ (S/Gi5) 2001 wood harvested 40 days after each 15N
application. 15N-fertilizer was applied at bloom (11 May), rapid shoot growth (20
June), and beginning of leaf senescence (11 September) ............................ 98

Table 2.4. Percent of N-fertilizer (0/0 NFF, percent of total N) in sweet cherry
‘Sam/Mazzard’ (S/M) and ‘Sam/Gisela 5’ (S/Gi5) ﬁne roots, coarse roots, 2001
and 2002 wood, and leaves, harvested 40 days after each 15N application. 15N-

xii

 

 

fertilizer was applied at bloom (11 May), rapid shoot growth (20 June) and
beginning of leaf senescence (11 September) ......................................... 99

Table 2.5. Nitrogen concentration (mg N g"l dry weight) in sweet cherry ‘Sam/Mazzard’
(S/M) and ‘Sam/Gisela 5’ (S/Gi5) buds from current season shoots and spurs,
harvested 40 days after 15N application. 15N-fertilizer was applied at on the 20
June (rapid shoot growth) and on the 11 September (beginning of leaf
senescence) .............................................................................. l 00

Table 2.6. Percent of N-fertilizer (0/0 NFF, percent of total N) in sweet cherry
‘Sam/Mazzard’ (SM) and ‘Sam/Gisela 5’ (S/Gi5) buds from current season shoot
and spur, harvested 40 days after 15N application. 15N—fertilizer was applied on
the 20 June (rapid shoot growth) and on the 11 September (beginning of leaf
senescence) ............................................................................... 100

Table 2.7. Total dry weight (DW), total nitrogen (N), C :N ratio, Nitrogen Derived From
Fertilizer (NDF F), and percent of NDF F content compared to the total N content,
measured in abscised leaves of sweet cherry ‘Sam/Mazzard’ (S/M) and
‘Sam/Gisela 5’ (S/Gi5). 15N-fertilizer was applied at bloom (11 May), rapid shoot
growth (20 June) and at the beginning of leaf senescence (11 September). . .....102

CHAPTER 3

 

Table 3.1. Leaf area regression equations, number of leaf measured (n), and R2 for leaf
area prediction. Leaf area is calculated using leaf width (cm) and length (cm).
Different equations were developed for ‘Rainier/Mazzard’ (R/M) and
‘Rainier/Gisela 5’ (R/Gi5) ............................................................. 125

Table 3.2. Percent of increase in trunk cross sectional area from bud break (15 May)
until tree harvest (31 July). Comparison between R/M and R/Gi5 under well-
watered (W and R/Gi5 Wrooyo) and W and R/Gi5 under water deﬁcit
conditions (R/M and R/Gi5 Wsoo/O) .................................................... 137

Table 3.3. Carbon isotope composition (613C, %o) and atom % of leaves of one-year-old
sweet cherry ‘Rainier/Mazzard’ under well-watered (R/M W100%) and water
deﬁcit conditions (R/M W50%) and ‘Rainier/Gisela 5’ under well-watered (R/Gi5
W100%) and water deﬁcit conditions (WGiS W50%). Leaves were collected when
trees were harvested ..................................................................... 148

APPENDIX C

 

Table C.1. Summary of statistical signiﬁcant differences between control and water
deﬁcit plants of ‘Rainier/Mazzard’ and ‘Rainier/Gisela 5’ of plant growth and gas
exchange parameters measured during the experiment ............................ 171

xiii

 

 

Table C.2. Summary of statistical signiﬁcant differences of growth and gas exchange
parameters measured during the experiment. Comparison between control plants
of ‘Rainier/Mazzard’ and ‘Rainier/Gisela 5’ (control) and between water deﬁcit
plants of ‘Rainier/Mazzard’ and ‘Rainier/Gisela 5’(water deﬁcit), calculated as
percent of their controls during the experiment. ................................... 172

xiv

LIST OF FIGURES
CHAPTER 1

Figure 1.1. Total shoot length per plant at different Days After Bud Break (DABB). A.
Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M).
B. Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’
(R/Gi6) and ‘Rainier/Mazzard’ (R/M) ............................................ 58

Figure 1.2. Total leaf dry weight (DW) per plant at different Days After Bud Break
(DABB). A. Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and
‘Mazzard’ (M). B. Comparison between ‘Rainier/Gisela 5’ (R/Gi5),
‘Rainier/Gisela 6’ (R/Gi6), and ‘Rainier/Mazzard’ (R/M) ..................... 59

Figure 1.3. Total dry weight (DW) per plant at different Days After Bud Break (DABB).
A. Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’
(M). B. Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’
(R/Gi6), and ‘Rainier/Mazzard’ (R/M) ........................................... 60

Figure 1.4. Root to shoot ratio at different Days After Bud Break (DABB). A.
Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M).
B. Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’
(R/Gi6), and ‘Rainier/Mazzard’ (R/M) ........................................... 61

Figure 1.5. Total N content per plant at different Days After Bud Break (DABB). A.
Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M).
B. Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’
(R/Gi6), and ‘Rainier/Mazzard’ (R/M) ........................................... 62

Figure 1.6 Nitrogen Derived From Fertilizer (NDFF, % of the applied) at different
harvests during the growing season (Days After Bud Break, DABB). A.
Comparison between ‘Gisela 5’ (G15), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M).
B. Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’
(R/Gi6), and ‘Rainier/Mazzard’ (R/M) .......................................... 63

Figure 1.7. Partitioning of absorbed N-fertilizer (in percent) in different plant organs, at
different harvests during the growing season (Days After Bud Break,
DABB). Comparison between A. ‘Gisela 5’ (Gi5), B. ‘Gisela 6’ (Gi6), and
C. ‘Mazzard’ (M) ................................................................... 67

Figure 1.8. Partitioning of absorbed N-fertilizer (in percent) in different plant organs, at
different harvests during the growing season (Days After Bud Break,
DABB). Comparison between A. ‘Rainier/Gisela 5’ (R/Gi5), B.
‘Rainier/Gisela 6’ (R/Gi6), and C. ‘Rainier/Mazzard’ (R/M) ................. 69

XV

Figure 1.9.

Figure 2.1.

Nitrogen use efﬁciency (NUE) expressed as whole plant C:N ratio at
different Days After Bud Break (DABB). A. Comparison between ‘Gisela
5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M). B. Comparison between
‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainier/Mazzard’ (R/M) ........................................................... 70

W

Percent of nitrogen from fertilizer (0/0 NFF) in leaves of sweet cherry
‘Sam/Gisela 5’ (S/Gi5) and ‘Sam/Mazzard’ (S/M), collected at different
Days After Full Bloom (DAFB). Arrows indicate time of [SN applications.
A. Application of 15N at full bloom. B. Application of 15N at rapid shoot
growth. C. Application of 15N at the beginning of leaf senescence .......... 95

Figure 2.2. Nitrogen use efﬁciency (NUE) expressed as the C :N ratio of leaves in sweet

Figure 3.1.

cherry cv. ‘Sam’, measured at different Days After Full Bloom (DAFB).
Comparison between ‘Sam/Gisela5’ (S/Gi5) and ‘Sam/Mazzard’ (S/M)..101

cw

Soil Water Content (SWC) measured with Time Domain Reflectometry
(TDR). A Comparison between ‘Rainier/Gisela 5’ (R/Gi5 W100%) under
well-watered and ‘Rainier/Gisela 5’ (IUGiS W50%) under water deﬁcit
conditions, and between ‘Rainier/Mazzard’ (R/M WIOOO/o) under well-
watered and ‘Rainier/Mazzard’ (R/M W50%) under water deﬁcit conditions.
B SWC of R/Gi5 W50% and WM W50% expressed as % of their controls..133

Figure 3.2. Relative Shoot Growth Rate (RSGR, cm day") of sweet cherry cv. ‘Rainier’

Figure 3.3.

Figure 3.4.

plants. Comparison between A ‘Rainier/Gisela 5’ under well—watered
(R/Gi5 W100%) and water deﬁcit (R/Gi5 W5o%) conditions, and B
‘Rainier/Mazzard’ under well-watered (R/M W100%) and water deﬁcit (R/M
W50%) conditions .................................................................. 134

Relative Leaf Emergence Rate (RLER, leaves day") of sweet cherry
cv.‘Rainier’. Comparison between A ‘Rainier/Gisela 5’ under well-watered
(R/Gi5 W100%) and water deﬁcit (R/Gi5 W50%) conditions, and B
‘Rainier/Mazzard’ under well-watered (R/M WWW/0) and water deﬁcit (R/M
W50%) conditions ................................................................... 135

Cumulative leaf area (cmz) of newly formed leaves of sweet cherry
cv.‘Rainier’. Comparison between A ‘Rainier/Gisela 5’ under well-
watered (R/Gi5 W100%) and water deﬁcit (R/Gi5 W50%) conditions, and B
‘Rainier/Mazzard’ under well-watered (R/M WWW) and water deﬁcit (R/M
W50%) conditions ................................................................... 136

xvi

Figure 3.5. Plants net assimilation rate (A, umol COZ m'2 8"). Comparison between A

Figure 3.6.

‘Rainier/Gisela 5’ under well-watered (R/Gi5 Wrooo/o) and water deﬁcit
(R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M WSW) conditions ...................... 138

Plants transpiration rate (E, mmol HZO m'2 5"). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/Gi5 Wr00%) and water deﬁcit
(R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M Wjoo/O) conditions ...................... 139

Figure 3.7. Plants stomatal conductance (g5, mmol HZO m'2 s"). Comparison between A

Figure 3.8.

Figure 3.9.

Figure 3.10.

Figure 3.11.

Figure 3.12.

Figure 3.13.

‘Rainier/Gisela 5’ under well-watered (R/Gi5 WWW/0) and water deﬁcit
(R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M WSOU/o) conditions ...................... 140

Leaf intercellular C02 concentration (C,, uL L"). Comparison between A
‘Rainier/Gisela 5’ under well—watered (R/Gi5 Wroocyo) and water deﬁcit
(R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M W509.) conditions ...................... 141

Water-use efﬁciency (WUE, pCOz mmol"1 H20). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/Gi5 W100%) and water deﬁcit
(R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M W50%) conditions ...................... 142

Water—use efﬁciency (WUE, umol COZ mmol'| H20). A Comparison
between ‘Rainier/Gisela 5’ (R/Gi5 W100%) and ‘Rainier/Mazzard’ (R/M
W100%) under well-watered conditions. B WUE of ‘Rainier/Gisela 5’
(R/Gi5 W50%) and ‘Rainier/Mazzard’ (R/M W50%) under water deﬁcit
conditions expressed as % of their controls .................................... 143

Daily trend of 1. net carbon dioxide assimilation rate (A), 2. transpiration
rate (E), and 3. water-use efﬁciency (WUE) of sweet cherry cv.‘Rainier’.
Comparison between ‘Rainier/Gisela 5’ under well-watered (R/Gi5 W100%)
and water deﬁcit (R/Gi5 W50%) conditions and ‘Rainier/Mazzard’ under
well-watered (R/M W100%) and water deﬁcit (R/M W50%) conditions. . ....145

Daily trend of stomatal conductance (gs) of sweet cherry cv. ‘Rainier’.
Comparison between ‘Rainier/Gisela 5’ under well-watered (R/Gi5 W100%)
and water deﬁcit (R/Gi5 W50%) conditions and ‘Rainier/Mazzard’ under
well-watered (R/M W100%) and water deﬁcit (R/M W50%) conditions. . ....146

Daily trend of internal CO; (Ci) of sweet cherry cv. ‘Rainier’. Comparison
between ‘Rainier/Gisela 5’ under well-watered (R/Gi5 W100%) and water
deﬁcit (R/Gi5 W50%) conditions and ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M W509» conditions ...................... 147

xvii

APPENDIX A

 

Figure A. 1. Maximum and minimum temperature (°C), and precipitation (mm) recorded
daily during the experiment at the MSU Horticulture Teaching and Research
Center during 2001. Data were collected by an automated weather station of
the Michigan Automated Weather Network. Arrows indicate 15N-fertilizer
application times .................................................................. 164

Figure A 2. Nitrogen—use efﬁciency (NUE) expressed as C:N ratio of leaves during the
growing season (Days After Bud Break, DABB). A. Comparison between
‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M). B. Comparison
between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainier/Mazzard’ (R/M) ........................................................ 165

APPENDIX B

 

Figure 8.1. Maximum and minimum temperature (°C) and precipitation (mm) daily
recorded at the USDA National Resources Conservation Service of Bear
Lake, during 2002. Data were collected by an automated weather station of
the Michigan Automated Weather Network. Arrows indicate time of N-
fertilizer applications ............................................................. 167

Figure 8.2 Figure B.2. Leaf nitrogen concentration (mg of N g"1 dry weight) in leaves of
sweet cherry cv. ‘Sam’, measured at different Days After Full Bloom
(DAFB). Comparison between ‘SarrUGiselaS’ (S/Gi5) and ‘Sam/Mazzard’
(S/M) ............................................................................... 168
APPENDIX C

 

Figure C. 1. Maximum and minimum temperature (°C) and precipitation (mm) recorded
daily during the experiment at the MSU Horticulture Teaching and Research
Center during 2002. Data were collected by an automated weather station of

the Michigan Automated Weather Network. Arrow indicates beginning of

water treatments ................................................................... 170

Figure C.2. Total shoot length (cm) per plant of sweet cherry cv. ‘Rainier’ measured at

different Days After Bud Break (DABB). Comparison between
‘Rainier/Gisela 5’ (R/Gi5) and ‘Rainier/Mazzard’ (R/M) ................... 173

xviii

KEY TO SYMBOLS OR ABBREVIATIONS

Symbol Parameter Unit

A ............... net carbon dioxide assimilation rate .................... umol COz m’zs'1
ABA. . . abscissic acid

ATP. . . . . .. adenosine tri-phosphate

C3. . . . .. three-atom carbon cycle

C4. . . . . . . . .. four-atom carbon cycle

Ca ............... ambient air COz concentration .......................... pL L'l

CAM. . . . . crassulacean acid metabolism

C,- ............... leaf internal COz concentration ......................... uL L'1

5 13C. . . . . . . . plant carbon isotope composition ....................... %o

A 13C. . . plant carbon isotope discrimination .................... %o

DABB ......... days after bud break

DAFB ......... days after full bloom

DW ............ dry weight

E ................ transpiration rate .......................................... mol H20 in2 s"
ET .............. evapotranspiration ........................................ L plant'1 day'l
Gi5............. Gisela5

Gi6.............. Gisela6

GOGAT. . glutamate synthase

GS. . . . . . . . glutamine synthetase

gS ............... stomatal conductance .................................... mol HzO rn'2 s'1

xix

LSMEANS. .. Least-Squares means

M ............... Mazzard

NDFF .......... nitrogen derived from fertilizer .......................... g
NFF ............ nitrogen from fertilizer ................................... O/o
NUE. . . . . . .. nitrogen-use efﬁciency ...................................

pa ............ leaf external partial pressure ............................ MPa
pi. . . . . . leafintemal partial pressure ............................ MPa

PDB. . . . . . . . PeeDee belemnite limestone

PEP. . . . . . . . phosphoenolpyruvate

Ra ............... l3C/ 12C atmospheric ratio

Rp ............... l3C/IZC plant ratio

R/Gi5 .......... Rainier/Gisela 5

R/Gi6 .......... Rainier/Gisela 6

RLER .......... relative leaf emergence rate ............................. leaves day'1
R/M ............ Rainier/Mazzard

RS ............... ‘3 C/ 12C PeeDee belemnite ratio

RSGR ......... relative shoot growth rate ................................ cm day‘l
RuBP ......... ribulose—l,5-bisphosphate

Rubisco. . . . . ribulose-1,5-bisphosphate Carboxilase/Oxigenase

i/JL ................... leaf water potential ....................................... MPa
SE .............. standard error
S/Gi5 ........... Sam/Gisela 5
S/M ............ Sam/Mazzard

XX

STD............

SWC ...........

TCSA ..........

WUE ...........

standard deviation
soil water content .........................................
trunk cross sectional area ................................

water-use efﬁciency

xxi

o/o (V/V)

2
C m

 

LITERATURE REVIEW

LITERATURE REVIEW

Introduction

Sweet cherries (Prunus avium L.) are one of the commercial fruit trees with the
highest economical returns per acre (Lang 2000; USDA, 2002, www.usda.gov).
Together with traditional standard rootstocks, new selections of sweet cherry dwarﬁng
rootstocks are commercially available. Selection of the appropriate rootstock is
important for maximizing the proﬁtability of the orchard, since the vigor of the rootstock
has a major impact on orchard costs such as harvest, pruning and spraying. Little is
known on how rootstocks cause a dwarﬁng growth habit and on the effects of dwarﬁng
rootstocks on nitrogen (N) uptake and use efﬁciency, and on water use efﬁciency.
Nitrogen is a major component of amino acids and proteins and represents 2 to 5% of the
plants dry weight (Marschner, 1995). Understanding the seasonal pattern of N uptake in
fruit trees can be of great value for optimizing the timing ofN fertilization. Physiological
base process that influence plant N utilization such us the nitrogen use efﬁciency (NUE)
could also be affected by the rootstock since NUE is based on the relation between plant
N content and growth rate (Small, 1972). Optimization of N-fertilizer uptake efﬁciency,
as well as the utilization of plants with high NUE, could reduce fertilizer costs and
amount of nutrient loss, lowering the negative effects of N application on soil, water and
air quality. Understanding how rootstocks respond to water deﬁcit is essential for
selecting the proper rootstock, or the proper management practices when drought stress is
likely to occur. Plant parameters such as growth, gas exchange and water relations are
affected by water deﬁcit conditions and among them, leaf formation and expansion were

one of the most sensitive plant parameters to water stress, in peach trees (Olien and Flore,

1990). Leaf carbon isotope composition is also influenced by water stress and in
particular is correlated with water use efﬁciency as shown in several crops (Farquhar et
al., 1982; Knight et al., 1994). The main objective of this research is to compare standard
and dwarﬁng sweet cherry rootstocks in terms of N-fertilizer uptake efﬁciency, NUE and

WUE.

Importance of cherry production in United States and the World

Among fruit trees and nut, sweet cherry fruit production is ranked as the eighth
most valuable production in US (USDA, 2002). The largest producer of sweet cherries is
Iran (13% of the world total production), followed by United States (12%), Turkey (12
%), Italy (8%), and Germany (7%) (USDA, 2002). During 1997 to 2001, US sweet
cherry production averaged 210,000 tons. Sweet cherries are primarily grown in
Washington State, Oregon and California, followed by Michigan, which produces about
10% of the yearly total US production (NASS, USDA 2002,

www.nass.usdagmr/wa/swtcherv.pdt). The average value of sweet cherry production in

 

Michigan was $17 million between 1996 and 1999 (NASS, USDA 2002). Many of the
fruit crops in US are becoming less proﬁtable resulting from increasing labor costs and
foreign competition. Using vigorous trees with long establishment period before
signiﬁcant fruiting occurs can reduce the proﬁtability of orchards (Lang, 2000). Together
with traditional standard rootstocks, several selections of dwarﬁng or semi-dwarﬁng
sweet cherry rootstocks are now available and can be used to increase the proﬁtability of

sweet cherry orchards.

Cherry rootstocks and physiology of dwarfing process
Cherry rootstocks

The primary commercial sweet cherry rootstocks are seedling or clonal selections
of P. avr'um L., known as ‘Mazzard’, or P. Malia/ab L., known as ‘Mahaleb’ (Perry,
1987). ‘Mazzard’ and ‘Mahaleb’ are vigorous trees and not precocious (Webster and
Schmidt, 1996). The main advantages of these rootstocks are their graft compatibility
with both sour and sweet cherry, and the large availability of plant material (Webster and
Schmidt, 1996).

Reduction of tree size on sweet cherry has been recently achieved with the use of
intra- and interspeciﬁc hybrid clones. A successful program for breeding dwarﬁng
rootstock was initiated by W. Gruppe in 1965, at Giessen University, Germany (Franken-
Bembenek, 1996). Approximately 6000 hybrids were obtained from the cross of more
than 10 different Prunus species (Franken-Bembenek, 1996). After several rootstock
trials at different locations, 25 GI-clones were selected and among them were the clones
GI 148/2, later introduced in the market as ‘Gisela 5’ (Gi5), and GI 148/l, introduced in
the market as ‘Gisela 6’ (Gi6). Both Gi5 and Gi6 are hybrids between P. cerasus (cv.
‘Schattenmorelle’) x P. canescens (Franken-Bembenek, 1996). Franken-Bembenek
(1996) reported that trunk cross sectional area of four to ﬁve-year-old Gi5 was 40 to 65
0/o of ‘Mazzard’, while the one of Gi6 was 65 to 150% of ‘Mazzard’. The high variability
in vigor of rootstocks tested was determined by the different climatic conditions of the
locations of the trial (Franken-Bembenek, 1996). In another ﬁeld trial Gi5, grafted with
15 sweet cherry cultivars, was compared in terms of vigor to ‘Mazzard F12/ 1’, grafted

with ﬁve cultivars (Franken-Bembenek, 1998). After nine years, trunk cross sectional

area of Gi5 was always lower than ‘Mazzard F12/ 1’, but the intensity of the reduction
depended on the cultivar considered (Franken-Bembenek, 1998). Franken-Bembenek
(1998) concluded that, beside trunk cross sectional area, cultivar had a high influence on
cumulative yield and yield efﬁciency. An extensive rootstock trial in East Malling, UK,
included Gi6 as one of the genotypes tested with sweet cherry cultivars ‘Van’ and
‘Merton Glory’ (Webster and Lucas, 1997). Trees on Gi6 were precocious with high
yield efﬁciency and, only with cv. ‘Van’, smaller than the standard ‘Colt’ (Webster and
Lucas, 1997). Webster and Lucas (1997) concluded that Gi6, although very promising in
terms of precocity and total yield, may exhibit different dwarﬁng potential depending on
the scion cultivar used. Gi6 and Gi5 have been reported to have low suckering intensity,
medium frost hardiness, and hiin waterlogging tolerance for Gi6, while only medium
waterlogging tolerance for Gi5 (Franken-Bembenek, 1996). Besides Gi5 and Gi6, other
promising dwarﬁng or semi-dwarﬁng rootstocks include Edabriz, and the series Maxma

and Weiroot (Webster and Schmidt, 1996; Lang, 2000)

Characteristics of dwarﬁng rootstock

Tree size can be controlled by dwarﬁng rootstocks or compact scion cultivars
(Bargioni, 1996). Dwarﬁng rootstocks offer flexibility in growth control with various
scion cultivars (Bargioni, 1996), and have several advantages: precocity, the possibility
of intensive plantings (1000 to 1700 tree ha"), and reduced costs associated with pruning,
spraying, and harvest. Dwarﬁng trees have a limiting number of growing points and
short duration of shoot extension, but can maintain high fruit yields as the tree mature.

Reducing the tree size may improve the distribution of light throughout the canopy and

therefore minimize the internal shading in the tree; better interception of light is usually
correlated with higher dry matter productions (Jackson, 1980). The capability of roots to
acquire nutrients and water affect scion perfomiance in temis of total production and fruit
quality. The root system of dwarﬁng rootstocks explores a more restricted soil volume
compared to standard rootstocks, and therefore dwarf trees have higher probability to be
limited in water, nutrients and oxygen than standard ones. Dwarﬁng rootstocks may
determine early senescence, reduced shoot elongation and declining number of flowering

spurs on older wood with tree aging (Webster and Schimidt, 1996).

Hypotheses on dwarﬁng mechanism

Several hypotheses have been proposed to explain how rootstocks cause a
dwarﬁng growth habit, but none has been fully able to explain the dwarﬁng mechanism.
The majority of studies have been carried out on dwarﬁng apple rootstocks. There is a
strong interdependence between rootstock and scion. Rootstock and scion before
combination as one plant may have different growth rates. After grafting, the two
genotypes develop a uniform growth rate, and the low growth rate of one of the two
genotypes can result in a dwarf tree (Lockard and Schneider, 1981). The
interdependence between rootstock and scion growth has been shown in peach seedlings
by physically restricting the root volume (Richards and Rowe, 1977). Root dry weight
was reduced of 39% when roots were physically restricted and at the same time top dry
weight was reduced of 34% (Richards and Rowe, 1977).

Phenols have also been proposed as an important factor in the dwarﬁng

mechanism in apple, although results have been contradictory. The amount of phenols

required to reduce growth varies with the phenol type and the plant tissue. Scholz (1957)
found that the inhibiting effect of phenols extracts from interstock bark of apple trees
with different vigor was correlated with the vigor of the plant sampled and with the
interstock dwarﬁng capability. On the other side, Martin and Williams (1967) found that
certain phenols like phloridzin, were higher in the bark of the vigorous M16 than the
dwarﬁng M9 indicating that phenols in general did not have a direct effects on the
reduced growth rate of M9.

The proposed mechanisms for dwarﬁng dwarﬁng growth habit induced by
rootstock in apple tree by Lockard and Schneider (1981) is based on the communication
between shoot and root through plant hormones. Auxins produced in the shoots move to
the roots, and promote root growth (Goodwin et al., 1978). Part of auxins flowing
basipetally through the phloem are degraded in the bark by indoleacetic acid oxidase,
peroxidase, and phenols present in the phloem and cambial cells. An inadequate supply
of auxin to the roots decreases root growth (Beever and Woolhouse, 1975), reducing the
amount of cytokinins synthesized by the root system. Cytokinins produced in the roots,
are translocated to the shoots, where they inﬂuence shoot growth (Goodwin et al., 1978)
and auxins production, in proportion to their amount. Since bark of different plants
contain different amounts of indoleacetic acid oxidase, peroxidase, and phenols, different
quantities of auxins will reach the root system, causing different tree size. Cytokinins
exert some controls over gibberellins metabolism in the shoots, which also inﬂuence
shoot growth (Lockard and Schneider, 1981). In general, no consistent relationship has
been found between gibberellin content (Graebe and Ropers, 1978) and abscisic acid

(ABA) content in different plant tissues and the dwarﬁng mechanism in plants, therefore

Lockard and Schneider (1981) considered the involvement of gibberellins and ABA to be
less important than the one played by auxins and cytokinins in the dwarﬁng mechanism.

In general, besides the use of dwarﬁng rootstocks to control tree growth, dwarl
plants can be obtained by using dwarﬁng rootstocks as interstocks between scions and
rootstocks, or by increasing the height of budding and grafting (Parry, 1986). Dwarﬁng
interstocks are used when soil conditions are not optimal for the dwarﬁng rootstock itself
or to allow a better tree anchorage.

The use of dwarﬁng cherry rootstock clones as interstock was less successful and
effective than for apple (Webster, 1998, Webster and Schmidt, 1996). Inconsistency on
the effect of height of budding on obtaining dwarf plants also suggests that dwarﬁng
effect on scion in sweet cherry could have a different mechanism of growth control
compared. to the one operating in apple trees (Webster, 1998). Webster (1998) suggested
that rootstock control on scion vigor in sweet cherry is perhaps more related to the

physiology of the root system than to the stem of the rootstock.

Effect of cultivar and rootstock on leaf nutrient concentration

Foliar mineral concentrations can be affected by scion and rootstock (Ferree,
1998; Kruczyr’iska et al., 1990; Ponchia et al., 1997; Rom et al., 1995; Tagliavini et al.,
1992). Comparison of plant nutritional status, yield efﬁciency (kg of fruit cm'2 of trunk
cross sectional. area), and graft compatibility of ‘Bartlett’ pears grafted on rootstocks with
different vigor evidenced little differences on leaf nutrient content, including N (Chaplin

and Westwood, 1980).

Year-to-year variations of leaf mineral concentration were more important than
different rootstock and interstem combinations in determining ﬁnal leaf mineral
concentration of ‘Golden Delicious Smoothee’ (Ebel et al., 2000). Little differences in
N, P, Fe, Zn, and B were found in leaves of dwarﬁng and semi-dwarﬁng apple rootstocks
in the different years, while K, Ca, and Mn varied during the ﬁve-years experiment, but
not consistently (Ebel et al., 2000). Lord et al. (1985) evaluated leaf nutrient level of
different apple rootstocks and interstock/rootstock combinations, with different vigor.
Although nutrient levels differed in the combinations tested, it was impossible to draw
any conclusion regarding a speciﬁc combination effect on leaf mineral concentration due
to the inconsistency of results between years (Lord et al., 1985).

Leaf nutrient content of ‘Montmorency’ sour cherry grafted on standard
rootstocks ‘Mazzard’ and ‘Mahaleb’ was compared in a four-year study, in two locations,
in Michigan (Hanson and Perry, 1989). Leaf N, K, Ca, Mg, B, and Mn concentrations
were affected by rootstocks and locations (Hanson and Perry, 1989). Leaf nutrient levels
did not appear to be related to crop load or tree vigor (Hanson and Perry, 1989). Hanson
and Perry (1989) speculated that although root distribution pattern played a role in
nutrient uptake, it could not fully explain the differences. Differently, crop load seemed
to be the cause of different leaf mineral concentrations obtained in sweet cherry cv.‘Bing’
on rootstocks with different vigor, in a four-year study, in two locations (Nielsen and
Kappel, 1996). Yield efﬁciency was higher in dwarﬁng trees GM 9, GM 61/1, Gi6, Gi
195/1 and Gi196/4 than standard trees (Neilsen and Kappel, 1996). Rootstocks with
higher yield efﬁciency than ‘Mazzard’ had lower K and Mg than. ‘Mazzard’, while the

effect of higher yield efﬁciency on leaf N concentration was less evident than for K and

Mg (Nielsen and Kappel, 1996). Among the genotypes with lower N concentration than
‘Mazzard’, only GM 61/1 and GM 9 had also a lower trunk cross sectional area than
‘Mazzard’ (Nielsen and Kappel, 1996). Nielsen and Kappel (1996) speculated that the

inadequate nutrition could have been the reason, in part, for lower tree size.

Nitrogen in the plant

Forms of N absorbed byplants

 

The three major fomis of inorganic N present in soils are nitrate (NOg'),
ammonium (NH4+), and organic nitrogen. Fruit trees satisfy their N requirement mostly
by taking up NO3' and NH4+. The reliance of plants on one form or another varies with
the relative availability of the two forms and transformation of N in the soil, with
environmental conditions, and plant species characteristics (Titus and Kang, 1982).
Nitrogen absorption by trees is affected by light (Frith and Nichols, 1975), soil pH,
temperature and mineral composition, as well as carbohydrate supply to the roots
(Marschner, 1995). Attempts to compare the importance of NO3' and NH4+ nutrition
under ﬁeld and controlled environment conditions have not been conclusive, and results
are often contradictory. Uptake and assimilation of NO3‘ and NH4+ require metabolic
energy in the form of ATP. However, there is a different demand of ATP and carbon
skeletons for the uptake of the two N forms: two ATP are required per NH4+ absorbed,
while assimilation of NO3' requires 12 ATP (Bloom, 1997). Uptake of NO3' by roots
depends on the concentration of N03" in soil solution, on the volume of soil exploited by

roots, and on the efﬁciency of roots to absorb NO3' (Engels and Marschner, 1995). At

low NO3' concentrations, high afﬁnity NO3‘ transporters are required, together with more
transporters per unit root surface, and with greater root density (Lawlor, 2002).

The form of N absorbed by the plants has a great influence on the cation/anion
uptake ratio and thus on the rhizosphere pH. Nitrate uptake is correlated with a higher
rate of HCO3' or OH" net release (or H+ consumption) that causes an increase in the pH of
the rhizosphere while NH4+ uptake is correlated with a higher rates of H+ release, which
causes a decrease in the pH of the rhizosphere (Marschner, 1995). As a result, in neutral
or alkaline soils, rhizoshere acidiﬁcation in plants fed with NH4+ may enhance
mobilization of sparingly soluble calcium phosphate, and favor the uptake of P, Fe, Mn,
and Zn. On the other side, in acidic soils the increase of pH induced by NO3' uptake may
enhance P uptake. Rhizosphere pH may differ from the bulk soil pH up to two units,
depending on root-induced changes and soil factors like pH buffering capacity
(Marschner, 1995).

There are several organic N forms taken up by plants, such as urea and several
amino acids. Shim et al. (1973a) demonstrated that young apple trees can absorb 14C-
urea after only two hours from its application. Urease is the enzyme responsible for the
hydrolysis of urea in ammonia and carbon dioxide. Shim et al. (1973b) found urease in
apple leaves, roots, and bark, and roots showed the highest urease activity. In general,
the uptake of amino acids is considered an adaptation to ecosystems with limited
availability of N. Spencer and Titus (1971) showed that glutamate and aspartate were
readily taken and metabolized by one-year-old apple trees. Recent studies on non-
mycorrhizal Pinus sylvestris have conﬁrmed that the amino acid uptake rate was

comparable to the uptake rates of N03" and NH4+ (Persson and Nasholm, 2001). Little is

11

known about the concentration of amino acids in the soil solution therefore it is difﬁcult

to estimate its contribution to the total N in plants.

Assimilation and translocation of nitrate and ammonium

In order to be assimilated, NO3' needs to be reduced to NOz' via nitrate reductase,
which is localized in the cytoplasm (Marschner, 1995). Furthermore, NOZ' needs to be
reduced to NH4+ by the enzyme nitrite reductase, localized in chloroplasts in leaves, or in
proplastids in roots (Marschner, 1995). Nitrate can be assimilated in the root or
translocated via xylem into the shoot. The amount of NO3' translocated from the root to
the shoot depends on the site of its reduction. Nitrate reduction in apple trees occurs
preferentially in roots (Frith, 1972), and more speciﬁcally in ﬁne roots (Eckerson, 1931).
Nitrate has been detected in apple leaves under high availability of NO3' in the soil or
medium, or under high concentrations of NH4+ in the medium. Under high availability of
NOg’ nitrate reductase in the roots is saturated and NO3' is translocated to the aerial part
of the plant (Titus and Kang, 1982). Under high availability of NH4+ there is a feedback
inhibition of the activity of root enzymes but not of the leaf nitrate reductase activity
(Frith, 1972). Nitrate reductase has also been detected in leaves of apricot, sour and
sweet cherry, plum, pear, grapevine, and walnut (Leece et al., 1972; Perez and Kliewer,
1978). When pear, sweet cherry, plum, walnut and grapevine were compared in terms of
leaves nitrate reductase activity, walnut resulted to have the greatest activity while sweet
cherry the least (Perez and Kliewer, 1978).

Unlike NO3’, NH4+ is assimilated only in the roots since in plant cells even low

concentrations are toxic. Ammonium absorption is a function of its assimilation into

12

complex compounds (Barker and Mills, 1980; Marschner, 1995). Ammonium
assimilation in roots has a large requirement for carbon skeletons; therefore NH4+
absorption is often carbon limited. These carbon skeletons are provided by the
tricarboxylic acid cycle and the removed intermediates are replenished by increased
activity of PEP carboxylase (Lawlor, 2002). The NH4+ is converted into amino acids by
the GS/GOGAT enzyme reaction (Marschner, 1995). The ﬁrst amino acid product of
NH4+ assimilation is glutamine, synthesized by the enzyme glutamine synthetase, GS
(Titus and Kang, 1982). Synthesis of glutamate proceeds from glutamine in the presence

of glutamate synthase, GOGAT (Titus and Kang, 1982).

Movement and accumulation of nitrogenous compounds

Upward movement of nitrogenous compounds in fruit trees occurs through the
xylem (Tromp and Ovaa, 1976). Movement of N compounds in plants occur also from
the xylem to the phloem tissue, in the radial direction, through ray cells and cambium
(Shim et al., 1973b). The major amino acids mobilized from the roots to the shoots are
aspartate and glutamate, their amide, and arginine (Titus and Kang, 1982).

Nitrate can be mobilized from the roots to the shoots via xylem and can
accumulate in the leaves. Nitrate does not appear to be phloem mobile (Pate, 1980),
therefore tissues with low transpiration rate will not contain high quantity of NO3'
(Millard, 1988). Most ofthe NO3' in leaves will be accumulated in the vacuole (Smirnoff

and Stewart, 1985).

13

Internal cycling of N in trees

Important factors that regulate year-to-year tree N status are the tree storage and
remobilization capabilities. Millard (1988) considers N to be stored if it can be
remobilized from one tissue and used for growth of another tissue. In deciduous trees, N
is stored over the winter in roots and bark as amino acids and proteins (Millard and Proe,
1991; Tromp, 1983; Titus and Kang, 1982) while over the summer N is stored in leaves
as Rubisco (Titus and Kang, 1982; Millard, 1996). During senescence, N is withdrawn
from leaves and stored in woody tissue over the winter and subsequently it is remobilized
and used for spring growth (Millard, 1996). RuBP carboxylase and other enzymes
related to photosynthesis are the major proteins broken down during leaf senescence
(Titus and Kang, 1982). This decline in enzyme leads to the general decrease in plant
leaf protein that occurs during senescence (Titus and Kang, 1982).

Early season growth and bloom of deciduous trees, like grape (Conradie, 1991),
apple (Nielsen et al., 1997 and 2001 a and b; Titus and Kang, 1982) and peach
(Tagliavini et al., 1999; Munoz et al., 1993) are supported by N remobilized from plant
storage organs (Weinbaum et al., 1984; Millard and Nielsen, 1989). Millard (1996)
reported that the ability of trees to remobilize N in spring is related to the previous year N
supply, and is independent of the current season N supply. Root N uptake becomes
increasingly important as the season progresses. Weinbaurn et a1. (1978) found that in
non-bearing prune (P. domestica L.) trees N—fertilizer was absorbed in greater amount
when supplied after the beginning of rapid shoot growth. In grape, N absorption is most
rapid between bloom and veraison (Williams, 1987 a and b) while between veraison and

harvest, N uptake tends to decline (Conradie, 1991).

14

Nitrogen fertilization in fruit trees

Application of nitrogen (ground or foliar) inﬂuences plant growth and
development during the current and subsequent season (Weinbaum, 1987). Several
factors affect fertilizer use efﬁciency such as plant nutrient demand (Weinbaum et al.,
1992), form of nutrient applied (Barker and Mills, 1980), application method (Sanchez et
a1, 1995), and the rate and timing of application (Taylor et al., 1975; Conradie, 1991).

Fertilizer use efﬁciency is deﬁned as the fraction of applied fertilizer that is
absorbed and used by a speciﬁc plant (Weinbaum et al., 1978). Fertilizer not absorbed by
the target plant can be taken up by weeds, lost in the atmosphere as gases, incorporated
into stable organic fractions in soil, or leached below the rooting zone (Robertson, 1997).

The synchrony between plant N demand and N soil availability is of critical
importance for increasing fertilizer use efﬁciency. The seasonal pattern of uptake and N
accumulation in trees reﬂects N demand and can be used for timing N application in
order to maximize N-fertilizer uptake (Weinbaum et al., 1992). N uptake during the
season, and partitioning of N to different plant organs, has been investigated in several
fruit tree species (Weinbaum et al., 1978, 1984; Munoz et al., 1993; Policarpo et al.,
2002; Sanchez et al., 1992). During spring, bud break takes place when conditions for
root uptake are not always optimal and there is a lack of new carbon skeletons necessary
for the synthesis of amino acids. At this time, N remobilization is of critical importance
in supplying N to the developing tissues. In a study on 9-year old walnut (Juglans regia
L.) trees, Weinbaum and van Kessel (1998) determined that approximately 60% of the
annual N demand derived from N redistribution from internal pools, and the remaining

part was met by N from fertilizer-soil pool. Nitrogen applied after bloom or during rapid

15

shoot growth satisﬁed the N demand of new growth in grapes (Conradie , 1991), prune
(Weinbaum et al., 1978), peach (Policarpo et al., 2002), pears (Sanchez et al., 1990) and
apple (Neilsen et al., 2001 a and b). Kinetic of N uptake was assessed in late and early
maturity peach (P. persica) cultivars under a Mediterranean climate (Policarpo et al.,
2002). Uptake was relatively low for the ﬁrst month after bud burst (approximately 30%
of the N applied), and then it increased to 80-90% of the N applied during the rest of the
season, until leaf senescence when it slightly decreased (Policarpo et al., 2002). Leaves
were the major N sink during the majority of the season but after summer N was
partitioned mainly to tree permanent organs, and speciﬁcally to the coarse roots
(Policarpo et al., 2002). Even if the two cultivars were characterized by different
phenology, they absorbed a similar amount of N over the season and they had similar N
partitioning (Policarpo et al., 2002). It has been reported in many fruit crops that after
shoot growth termination, leaves switch from being a strong sink, to be a source of N for
reproductive organs (Millard, 1988). Crop load of the tree determine the potential size of
the fruit sink. Late season applications increase the N storage pool that will be
remobilized in the following year (Millard and Thomson, 1989; Sanchez et al., 1991).
Nitrogen fertilization during summer or post-harvest was more efﬁcient than N

application at bud break for ‘Thompson Seedless’ grape (Peacock et al., 1989).

Carbohydrate and N metabolism
Carbohydrate and N metabolism are closely related in all phases of plant growth
and development. Neilsen et al. (2001a) in young apple trees on M9 dwarﬁng rootstock

indicate that crop load effects on N partitioning to fruit and shoot leaves were related to

16

dry matter partitioning and therefore N uptake was considered closely coupled with
carbon supply.

The major leaf protein is Rubisco, which catalyses the reaction between CO; and
RuBP giving rise to triose phosphate, and also oxygenate RuBP through photorespiration
in C3 plants (Lawlor, 2002). Rubisco has low catalytic rate therefore C3 plants required it
in large amount (Lawlor, 2002). Lawlor et a1. (1989) evaluated that the amount of
Rubisco in wheat ﬂag leaves was approximately 7 g m"2 of leaf, which constituted 30% of
the total N and up to 50% of the soluble leaf protein.

The pentose phosphate pathway, glycolysis, and the tricarboxylic acid cycle are
closely coupled to amino acids biosynthesis with regards to the supply of carbon
skeletons and energy. Because carbon and N are acquired separately by leaves and roots
respectively, the study of how their metabolism is coupled provide information about

physiological integration at the whole plant level (Gamier and Roy, 1994).

Environmental problems related to the utilization of mineral N

Of all the essential elements, N is the most commonly applied in orchards, and at
the greatest rate. Excess of N stimulates vigorous growth, and therefore shading within
the tree, which negatively affects ﬂower bud development, fruit set, fruit quality, and
sometimes delays fruit maturity (Weinbaum et al., 1992). Excess N causes late season
growth, resulting in higher winter injury for the tree. Overfertilization is associated with
high levels of residual nitrate in the soil, which potentially contribute to groundwater and

atmospheric pollution, as a result of leaching and denitriﬁcation (Weinbaum et al., 1992;

17

 

Mervvin et al., 1996). Nitrogen is widely regarded as responsible for the hypoxia (low
oxygen) zone in the Gulf of Mexico (Keeney and Hatﬁeld, 2001).

Efﬁcient use of N fertilizer in crop production can reduce costs and minimize the
detrimental effects of N movement into surface or ground water (Throop and Hanson,
1997). High leaching losses of nitrate occur when the N fertilizer rate is not adjusted to
the crop N demand (Weinbaum et al., 1992) and does not consider the level of available

nitrogen in soil.

Nitrogen-use efﬁciency: definition and importance

Nitrogen-use efﬁciency (NUE) is deﬁned as the amount of dry matter produced
per unit of N taken up (Robertson, 1997). Several indicators have been used to evaluate
NUE in plants and among them are the plant C:N ratio (Maranville and Madhavan,
2002), the inverse of N concentration in plant biomass (Chapin, 1980; Nakarnura et al.,
2002; Tateno and Kawaguchi, 2002), or the amount of utilizable plant material (seed,
grain, fruits, forage) per amount of absorbed N (Baligar et al., 2001; Maranville et al.,
1980). High plant C:N ratio indicates high plant NUE. In non-cultivated ecosystems
NUE appears to be higher where soil N availability is low (Robertson, 1997). Nitrogen-
use efﬁciency has been evaluated in different plants such as barley (Gonzales Ponce et
al., 1993), rice (Cassman et al., 1993), and wheat (van den Boogaard et al., 1995).
Nitrogen fertilization in general leads to lower plant C:N ratios as shown by the failure to
increase the crop yield with addition of N above a plant-speciﬁc saturation level

(Robertson, 1997).

18

In deciduous trees NUE could also be deﬁned as the amount of dry matter loss per
unit of N lost in the litterfall. The withdrawal of N from senescent leaves allows the use
of the same unit of N used during the current year, to be reused for new plant organs
successively (Clark, 1977; Turner, 1977). Nitrogen-use efﬁciency calculated in litterfall
of forested ecosystem appears to be correlated with the availability of N in the ecosystem
(Vitousek, 1982). Sites where symbiotic nitrogen ﬁxers are dominant, have a relatively
low NUE of litterfall since N-ﬁxers have potentially unlimited availability of N
(Vitousek, 1982). Sites relatively poor in N have relatively low N concentration in the
litterfall and therefore high NUE; in such sites, NUE normally decrease when N is
supplied by fertilization (Turner, 1977; Vitousek, 1982).

Several hypotheses have been formulated to explain the higher NUE in litterfall of
trees on low-nutrient sites. Trees may be able to ﬁx more carbon per unit of N and this
could be achieved by using the same unit of N to ﬁx carbon over time like in case of
evergreen (Shaver, 1981). However, trees with higher NUE may have a higher
retranslocation of N from leaves prior to abscission, which would determine a higher
litterfall massznitrogen ratio (Vitousek, 1982). Another important aspect of the litterfall
C:N ratio regards its effect on availability of N in the ecosystem: with high C:N ratio
(higher than 20:1), decomposers are N limited and retain N in their biomass while with
low C:N ratio (between 12-20:1), decomposers tend to release N into the soil solution
(Vitousek, 1982). This mechanism is considered the cause of the reduced N availability
in low-nitrogen site, where litterfall has the tendency to have a high C:N ratio (Vitousek,

1982).

19

Photosynthetic NUE is calculated as the ratio between net photosynthesis (C02
assimilated) and leaf nitrogen content (Field et al., 1983). In general C4 plants have a
higher photosynthetic NUE than C3 plants (Morison, 1989). There are substantial
differences between C 3 and C4 plants in the photosynthetic components content of leaves.
In C4, Rubisco functions only as a carboxylase and therefore these have a higher rate of
C02 ﬁxation and require less Rubisco compared to C3 plants. In C3 plants, Rubisco
functions as both carboxylase and oxygenase and part of the carbon ﬁxed is lost through
photorespiration. In C3 plants 30 to 60% of the soluble proteins in leaves is Rubisco,
while in C4 plants only 5-10% of soluble protein is Rubisco (Marschner, 1995).
Consequently, the N-content per unit of leaf is smaller in C4 than C3 plants, and the N—

requirement is less to achieve the same production.

Physiology of water deﬁcit

Water deﬁcit, or water stress, refers to conditions in which plant water potential
and turgor are reduced and affect normal functioning of the plant (Kramer, 1983); it
develops in situations where water loss by transpiration exceeds absorption by the root
system. Plant water deﬁcit can be described on a daily basis, as a midday water deﬁcit
(Kramer, 1983; Werber and Gates, 1990). The increase in transpiration rate during the
day causes a decrease in leaf water potential (ll/r-) and turgor; during the afternoon,
stomata begin to close, transpiration rate decreases and absorption of water continue until
leaf parenchyma cells are reﬁlled and their water potential rises again (Kramer, 1983).
Long-term water deﬁcit begins with a daily cycle, which is altered by the inability of the

plant to recover the water lost during the day. Plant pemianent wilting occurs when soil

20

water potential values decreases to —1.5 MPa (Kramer, 1983). Plants can experience
drought when soil water content is limiting, when atmospheric water content declines, or
when both condition are present. Water deﬁcit have implications for plant growth,

physiological process, and water relations (Hsiao, 1973; Jones at al., 1985).

Plant water relations

Plant water balance is determined by water lost during transpiration and water
absorbed from the soil (Lawlor and Comic, 2002). Two driving forces that play a role
when water moves from the soil into the roots are: 1) osmotic movement, in slowly
transpiring plants, and 2) mass ﬂow, in rapidly transpiring plants caused by tension or
negative pressure in the xylem sap (Kramer and Boyer, 1995). As the rate of
transpiration increases, the increasing in mass ﬂow of water through the roots dilutes the
root xylem sap until the osmotic mechanism becomes ineffective and absorption is
controlled by the pressure potential in the xylem sap. In rapidly transpiring plants the
xylem water potential can fall as low as —l .5 to —2.0 MPa (Kramer and Boyer, 1995).

Water potential, osmotic potential and turgor potential are the major contributors
to cell growth (Boyer, 1988). When transpiration rate exceeds absorption, cell turgor
falls, concentration of cellular content increases causing an increase in osmotic potential
and water potential falls (Lawlor and Comic, 2002). Within a cell, the pressure potential
or turgor measures differences between internal and external pressure (Jones et al., 1985).
Turgor potential is important for stomatal functioning. Low turgor potential negatively
effects plant growth and stomatal conductance (Lawlor and Comic, 2002). Maintenance

of turgor under water deﬁcit is a condition that has been associated with increasing

21

 

 

osmotic potential (Jones et a1. 1985; Lakso, 1983). Plants can adjust their osmotic
potential in a passive way e.g. by changing the partitioning of water between symplast
and aposplast or in an active way by increasing the number of solute molecules (Jones et
al., 1985). Osmotic adjustment is found to play a bigger role in maintaining the turgor of
cells when plants undergoes slowly to water stress, like in situations of ﬁeld-grown plants
(Jones et al., 1985 ; Ranney et al., 1991 a and b). Osmotic adjustments have been
observed in leaves and roots of cherry (Ranney et al., 19913), and leaves of peach (Young

et al., 1982).

Effect of water deﬁcit on plant growth parameters

Processes affected by water deﬁcit in fruit trees include cell division and
elongation, ﬂower bud differentiation, and partitioning of carbohydrates among organs
(Faust, 1989). In general, under water deﬁcit, leaf area is reduced but speciﬁc leaf
weight (weight per unit area), thickness of cutin, and amount of wax on the leaf surface
sometimes increase (Kramer, 1983). Shoot growth is reduced more than root growth,
therefore root to shoot ratio is usually increased (Kramer, 1983). Shoot and leaf
expansion are among the most sensitive plant parameters to be affected by decreases in
04,. Andersen and Brodbeck (1988) found that as water deﬁcits developed in peach trees,
shoot length and leaf expansion rate are inhibited before reduction in assimilation rate
(A) and stomatal conductance (gs) takes place. In another study on peach trees, growth

parameters, like leaf expansion and leaf emergence rates were more sensitive than. gS and

r//L to water stress in peach trees (Olien and Flore, 1990). In plants with commercial

22

importance, the effects of water deﬁcit have negative impact on yield and fruit quality

(Webster and Looney, 1996).

Effect of water deﬁcit on physiological and morphological parameters

Closure of stomata during water stress can be responsible for the observed
reduction in photosynthetic rate (Flore and Layne, 1996). Stomatal movements are
regulated by the turgor of the guard cells which are affected by environmental factors
such as light intensity, COZ atmospheric concentration, humidity, wind, and temperature,
and by endogenous factors such as plant hormones, leaf water status, and internal COz
(Jones et al., 1985; Kramer and Boyer, 1995; Hetherington and Woodward, 2003). Root-
produced ABA transported to leaves is believed to be the signal from drying roots that
causes stomatal closure under water deﬁcit (Davies et al., 1994; Davis and Zhang, 1991).
Wartinger et a1. (1990) showed that in almond trees under a drying cycle, as xylem ABA
increased, leaf conductance decreased. At the same time, daily courses of ABA
concentration in xylem sap in almond trees did not appear related to stomata
conductance, perhaps because of the narrow range of ABA concentration able to affect
stomata conductance (Wartinger et al., 1990). Not all authors agree on the role of ABA
in stomata closure. It has been observed that stomata stay open in leaves with high ABA
or remain closed even after ABA concentration has decreased (Beardsell and Cohen,
1975). Severe drought stress in kiwifruit determined reduction in stomatal aperture and
decrease in overall growth (Buwalda and Smith, 1990). At the biochemical level, water

deficit results in a decrease in starch and increase in sugar content of the cells. Nitrogen

23

metabolism is disrupted; protein hydrolysis occurs and amino acids, especially proline,
accumulate (Kramer and Boyer, 1995).

A wide range of morphological and physiological characteristics in response to
water deﬁcit is affected by rootstock, scion, and their interaction (Lockard and Schneider,
1981). Rootstocks inﬂuenced transpiration rate and WUE in peach (Bongi et al., 1994),
and leaf water potential in peach (Young and Houser, 1980) and apple (Olien and Lakso,
1984). Root ability to uptake water is a function of morphological and physiological
characteristics of the root system. Increasing the volume of soil explored by roots leads
to higher resistance of plant against stresses such as nutrient deﬁciency, drought and
anoxia (Buwalda and Smith, 1990). Factors that inﬂuence plant hydraulic resistance are
the total length and density ofthe root system (Jones et al., 1985). Rootstocks can affect
hydraulic resistance as observed in apple and citrus (Jones et al., 1985). Cherry trees are
reported to respond. to low water potential by dropping leaves and by substantial osmotic
adjustments (Longstrogh and Perry, 1996). Seedling rootstocks like ‘Mazzard’ and
‘Mahaleb’ are deep-rooted and can tolerate drought conditions. In areas where drought
prevails, ‘Mahaleb’ should be preferred to ‘Mazzard’ for its higher tolerance to water

deﬁcit (Longstrogh and Perry, 1996).

Water-use efficiency: definition and importance
The availability of water for irrigation will be reduced in the future because of
increasing competition for water for urban use, increasing depth of water table due to

over pumping aquifers, and increasing problems of salinization of soils due to irrigation.

24

Water-use efﬁciency of plants will be of critical importance in agricultural systems with
limited water availability.

Tanner and Sinclair (1983) reviewed different relationships that can be used to
characterize plant WUE. Most researchers describe WUE as the yield of crop produced,
either the total biomass or only the marketable biomass, per unit of water evapotranspired
(Kramer, 1983; Harﬁeld et al., 2001).

Physiologists prefer to express W 11E as milligram of COZ per gram of water transpired:

WUE = net CO; uptake in mg or g (A)
H20 transpired in g or kg (E)

 

Sometimes the term ‘intrinsic water-use efﬁciency’ is used when WUE is calculated by
the ratio between the instantaneous rates of C02 assimilation (A) and transpiration (E)
(Condon at al., 2002; Field et al., 1983).

There is extensive evidence that WUE varies among species in the same
environment and among climate for the same species (Tanner and Sinclair, 1983;
Loornis, 1983). A genotype may have a greater WUE than another if it has: 1) higher A
and similar E; 2) similar A and lower E; 3) higher A and E, but proportionally higher A
than E; 4) lower A and E, but proportionally lower E than A (Patterson et al., 1997).

Water-use efﬁciency is also affected by leaf age as shown in a study on young
grapefruit trees (Syvertsen, 1985). When young grapefruit leaf expanded and matured,
net photosynthesis and rnesophyll conductance declined while little changes occurred in

leaf stomatal conductance, determining a decline in WUE (Syvertsen, 1985).

25

Leaf 13’C composition: effect of plant water deﬁcit

Approximately 1.11% of the carbon in the biosphere is in the form of the stable
isotope l3C (Boutton, 1991). Carbon isotope composition of plant tissue (5 13C) is a
measure of the 13C/IZC ratio in a plant, relative to the value of the same ratio in the

international standard, the limestone PeeDee Belemnite (PDB). Thus:

6'3CPDB (%o) =(R,/R,-1) x 1000

where Rp is the 13C/ 12C ratio measured in plant material and R8 is the ratio of the standard
(Farquhar et al., 1982). Plants have negative values of 513C due to the fact that l3C/IZC
ratio in the atmosphere is less than that of PDB, and there is a net discrimination against
l3C by plants during COz ﬁxation (Farquhar et al., 1989). The discrimination of 13C can
occur during diffusion of C02 through the stomata and C02 ﬁxation by the primary COz-
ﬁxing enzyme (O‘Leary, 1988). Also some downstream fractionations associated with
plant metabolic pathways and respiration are reported (O‘Leary, 1988). The rate of
diffusion of l3C02 across the stomatal pore is lower than that of l2COz by a factor of 4.4
per mill (%o) (Farquhar et al., 1982). Higher plants with the conventional C3 pathway of
carbon assimilation reduce C02 to phosphoglycerate via the enzyme RuBP carboxylase.
Rubisco discriminates against l3C due to its intrinsically lower reactivity with 13C,
determining a 513C of about -28%o on average in C3 plants (Boutton, 1991; O‘Leary,
1988). C4 plants reduce COz to aspartic or rnalic acid through the enzyme PEP

carboxylase, which does not discriminate against 13C02 as much as RuBP carboxylase.

26

Therefore C4 plants have relatively high value of 6'3C with a mean around -11%o
(Boutton, 1991). Plants that exhibited a CAM methabolisrn have intermediate values of
513C (Farquhar et al., 1982). Carbon isotope discrimination is modiﬁed by several
environmental and physiological variables such as light intensity, humidity, water
availability, and photosynthesis (Farquhar et al., 1989).

Carbon isotope discrimination (ADC) is a measure of the l3C/I‘ZC ratio in plant

material relative to the value of the same ratio in the air on which plants feed (Farquhar

and Richards, 1984):

NC = Ra/Rp — 1

where R2, is the 13C/‘ZC ratio measured in the atmosphere and Rp is the l3C/I‘ZC ratio
measured in plant material. Carbon isotope discrimination has positive values, which
reﬂect the fact that C3 plants actively discriminate against 13C during photosynthesis.
A13 C and WUE are related to the ratio between concentration of C02 in the leaf
intercellular space and the ambient air (Ci/Ca) (Farquhar et al., 1982). In C3 plants, ABC
decreases as WUE increases when soil water is limited, resulting in more positive BBC
values of plant tissues under drought stress (Knight et al., 1994; Stewart et al., 1995).
Problems associated with the use of ABC to measure WUE is that it provides no
information on the magnitudes of either photosynthetic rate (A) or transpiration (E) or
whether variation in A13 C is driven by stomatal conductance or photosynthetic capacity

(Condon et al., 2002).

27

Rational and objectives of the research

Nitrogen fertilizer application inﬂuences tree growth and development during the
current year, and in the following growing season (Weinbaum, 1987). The seasonal
pattern of N uptake in trees reﬂects their N demand and can be used for timing N
application in order to maximize fertilizer uptake (Weinbaum, 1992). N uptake during
the season has been investigated in grape (Conradie, 1991), prune (Weinbaum et al.,
1978), peach (Policarpo et al., 2002), pear (Sanchez et al., 1990), and apple (Neilsen et
al., 2001b) but little is known on the efﬁciency ofN uptake of sweet cherry at different
times during the season, especially when grown on dwarﬁng rootstocks.

Genetic and physiological traits of plants determine their ability to utilize N under
different environmental and ecological conditions (Baligar et al., 2001). Nitrogen use
efﬁciency has been evaluated in several crops (Gonzales Ponce et al., 1993; Cassrnan et
al., 1993; van den Boogaard et al., 1995) but there is a lack of information on the
inﬂuence of dwarﬁng and standard rootstocks on NUE. The utilization of plants with
high NUE, together with best management practices, would achieve a more sustainable
agricultural system and reduced the detrimental effect of N application on soil, water and
air quality.

Understanding how rootstocks adapt and respond to drought stress is essential in
selecting the proper rootstock for situation where drought stress is likely to occur.

In sweet cherry, although deep-rooted rootstocks, like ‘Mazzard’, are considered to be
more tolerant to drought than clonally propagated rootstocks, such as ‘Gisela 5’ (Webster

and Schmidt, 1996), little is known on the effect of the vigor of the rootstock on growth

28

and physiological parameter when water deﬁcit conditions occur. Also there is a lack of

knowledge on the effect of dwarﬁng and standard rootstocks on plant WUE.

The overall objectives of this research are to:

1. Evaluate N-fertilizer uptake efﬁciency at different phenological stages in

dwarﬁng and standard sweet cherry rootstocks, and determine if dwarﬁng

rootstocks inﬂuence N-fertilizer uptake;

l\)

Compare NUE of dwarﬁng and standard rootstocks at different phenological

stages, under non-limiting availability of nitrogen;

3. Compare WUE of dwarﬁng and standard rootstocks under non-limiting water
availability and under water deﬁcit conditions; and

4. Determine if growth and physiological parameters of dwarﬁng and standard trees

are affected differently under water deﬁcit conditions.

29

 

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42

CHAPTER 1

43

CHAPTER 1

N-fertilizer uptake, nitrogen-use and water-use efﬁciency of one-year-old swee

cherry (Prunus avium L.) cv. ‘Rainier’ on standard and dwarﬁng rootstocks.

Abstract

Plant demand for water and nitrogen (N) vary during the season, and is stror
dependent upon weather conditions, soil moisture, stage of plant development, and c
load. This research was initiated to determine if dwarﬁng rootstocks have an inﬂuc
on N fertilizer uptake efﬁciency, N use efﬁciency (NUE), and water use efﬁcic
(WUE) when compared to standard rootstocks used in the industry. We investigated
N—fertilizer uptake efﬁciency, NUE and WUE on one-year-old potted sweet cherry
‘Rainier’ grafted on the dwarﬁng rootstocks ‘Gisela 5’, on the semi-dwarﬁng ‘Giselz
and on the standard rootstock ‘Mazzard’. ‘Gisela 5’, ‘Gisela 6’, and ‘Mazzard’ witl
scion were also compared. N-fertilizer uptake efﬁciency and NUE were evaluated
times during the growing season. ”N was applied to the soil at a rate of 0.5 g ofN
container as KISNO3. Plants were harvested 10-12 days after 15N applications. Tota
percent of 15N, and dry weight of the current season growth, trunk, and roots V
evaluated. N-fertilizer uptake was inﬂuenced by the accumulation of dry matter and
higher from rapid shoot growth until the beginning of leaf senescence. Overall, t2
were no differences in N-fertilizer uptake efﬁciency between dwarﬁng and stan<
rootstocks. NUE was signiﬁcantly higher in ‘Mazzard’ without scion compared to l

dwarﬁn g and semi-dwarﬁng rootstock without scion, and had comparable values with

44

all the rootstocks with cv. ‘Rainier’. W 1 1E was not affected by the vigor of the rootstocks
when grafted with cv. ‘Rainier’, but it was higher in ‘Mazzard’ without scion, comparec

to both dwarﬁng and semi-dwarﬁng rootstock without scion.

Introduction

Optimum management of water and N is of critical importance in order tc
maintain growth and high production of fruit trees in modern orchards. Several factors
affect N-fertilizer uptake efﬁciency, including plant N demand (Weinbaum et al., 1992)
form of N applied (Baker and Mills, 1980), application methods (Sanchez et al., 1995:
and the timing of application (Taylor et al., 1975). The seasonal pattern of uptake and l\
accumulation in trees reﬂects their N demand and can be used for timing N-fertilizer
application (Weinbaum et al., 1992). Application of N-fertilizer in excess often causes
high levels of nitrate in soils, which represents a risk for groundwater contamination, anc
can contribute to increase atmospheric pollution as a result of denitriﬁcation process
(Weinbaum et al., 1992). Nitrogen uptake during the season has been evaluated in grape
(Conradie, 1991), prune (Weinbaum et al., 1978), peach (Policarpo et al., 2002), pear
(Sanchez et al., 1990) and apple (Neilsen et al., 2001) but little is known about the
efﬁciency of N uptake during the season in sweet cherry, especially when grown or
dwarﬁng rootstocks.

Sweet cherries are grown mainly on rootstocks that results in full sized or
‘standard sized’ trees. The most common sweet cherry rootstocks are ‘Mazzard’
seedlings (P. avium L.), the clonal ‘Mazzard F.12/1’, or ‘Mahaleb’ (P. mahaleb L.)

(Webster and Schmidt, 1996). Reduction of tree size on sweet cherry has been recently

45

achieved with the use of intra- or interspeciﬁc hybrid clones, such as ‘Gisela 5’ ant
‘Gisela 6’ (Webster and Schmidt, 1996). Dwarﬁng rootstocks allow high tree densities
reduction of harvest and pruning costs, and promote precocity and yield capacity of tree:
(Webster and Schmidt, 1996).

Differences in plants physiological traits may determine their ability to utilize 1‘
under different environmental and ecological conditions (Baligar et al., 2001). Nitroger
use efﬁciency (NUE) is deﬁned as the amount of dry matter produced per unit of 1‘
absorbed (Robertson, 1997). Several indexes have been used to evaluate NUE in plant;
such as the plant C:N ratio (Maranville and Madhavan, 2002), the leaf C :N ratio (Tatenr
and Kawaguchi, 2002) or the C:N ratio of other tissues of plants (Nakamura et al., 2002)
Also, NUE be expressed as the amount of utilizable plant material (e. g. seed, grain, fruits
forage) per amount of absorbed N (Maranville et al., 1980; Baligar et al., 2001). Higl
C:N ratios indicates high plant NUE. Nitrogen use efﬁciency has been evaluated ir
barley (Gonzales Ponce et al., 1993), rice (Cassman et al., 1993), and wheat (van der
Boogaard et al., 1995) but little is known on the inﬂuence of dwarﬁng and standard swee
cherry rootstocks on NUE.

Tanner and Sinclair (1983) summarized different relationships that can be used tr
characterize plant water use efﬁciency (WUE). Most researchers describe WUE as thr
yield produced per unit of water evapotranspired (Hatﬁeld et al., 2001). The higher thi
biomass produced per unit of water evapotranpired, the higher the WUE. Rootstocks car
inﬂuence transpiration rate, WUE, and leaf stomatal conductance (Webster and Schmidt
1996). Besides plant physiological characteristics, morphology of the root system suel

as depth and the number of ﬁne roots are important factors that inﬂuence wate

46

absorption. Little is known on the effect of the vigor of rootstocks on WUE in
cherry.

A better knowledge of sweet cherry tree N requirement during the sea
needed in order to synchronize application of N with tree N demand, and overall in
the N-fertilizer uptake efﬁciency. Also, a better understanding of the tree use efﬁr
of water and N, as well as N-fertilizer uptake efﬁciency, could be of great val
decision on the best management practice to adopt when dwarﬁng rootstocks are us

The objectives of this study were to evaluate, on standard, semi-dwarﬁr
dwarﬁng sweet cherry rootstocks, either grafted or ungrafted: 1) the N-fertilizer r
efﬁciency at different phenological stages, and 2) NUE and WUE at di:

phenological stages.

Materials and methods

One-year-old ‘Rainier’ sweet cherry grafted on the standard rootstock ‘Ma;
the dwarﬁng rootstock ‘Gisela 5’ (P. cerasus (cv. Schattenmorelle) x P. canescen:
the semi-dwarﬁng rootstock ‘Gisela 6’ (P. cerasus (cv. Schattenmorelle) x P. cane:
and one-year-old ‘Mazzard’, ‘Gisela 5’, and ‘Gisela 6’ rootstocks without scion
maintained outdoors, in 11 L containers, at the Horticulture Teaching and Re
Center, Michigan State University, East Lansing, MI. Dormant trees were potted 0
16m, 2001 in a mixture of 10% silt and clay, and 90% coarse sand, were pruned to
ﬁve buds and subsequently trained to three branches. Trees were watered as neede
a drip irrigation system with two emitters per pot. Nutrient solution with all es

macro— and micro-nutrients (13-2-13, N-P-K, 6% Ca, 2% Mg and micronutrient:

47

applied weekly to supply 0.5 g N per container per week. Foliar iron was periodically
applied (Iron ehelate DP). Daily maximum and minimum air temperature and
precipitation were recorded by an automated weather station located 300 in SW of the
experimental plot (Michigan Automated Weather Network,
http://www.agweather.geo.msu.edu, Figure A], Appendix A).
’5 Nitrogen applications

Labeled 15N fertilizer was applied ﬁve times during the season, at different
phenological stages (Table 1.1). Four plants per genotype were used at each application
time. 15N was applied to the soil at a rate of 0.5 g ofN per container (K’SNO3, 10 atom %
excess, ICON Services, Mt. Marion, N.Y.). Each of the ﬁve 15N applications was applied
to four plants per genotype. The fertilizer was applied in 200 mL water, followed by 100
mL water in order to ensure uniform distribution into the root zone. To reduce loss of
15NO3', leachate was collected in a saucer, and re-applied to the plant. Five additional
trees per rootstock with scion were maintained with standard fertilizer without 15N
enrichment to evaluate the 15N natural abundance (MN/”N control ratio) at every harvest.
Plant growth measurements, harvests, and samples collection

Shoot length was measured from the base of the shoot to the terminal leaf, and
was recorded at 18, 25, 28, 35, 42, 52, 73, 82 and 94 Days After Bud Break (DABB).
Sets of four trees per genotype were destructively harvested at six different times during
the season (Table 1.1). After washing the roots to remove the potting medium, trees were
divided into the following components: ﬁne roots (32mm diameter), coarse roots (>2mm
diameter), trunk, current season shoots, and leaves. Fine roots were carefully separated

from the rest of the root system and only the white, actively growing roots were included

48

in the ﬁne roots component. Plant tissues were dried to constant weight at 60°C
(approximately for 72 hours), and DW was recorded. Root to shoot ratio was expressed
as the ratio between the belowground and the aboveground biomass. For l5N analysis, a
sub-sample of the different plant tissues collected at each harvest, was freeze-dried, and
DW was recorded. The sub-samples were ground to pass a 40-mesh screen in a Wiley
Mill and analyzed for total N and 15N enrichment with mass spectrometry analysis.

Soil sampling

 

Soil samples were taken at every plant harvest in order to evaluate the 15N present
in the soil. A total of two soil cores per pot were taken with a 2.5-cm soil probe to a
depth of 25 cm. Soil samples were air-dried, ground with a mortar and pestle, sieved
with 500 um screen, and analyzed for total N and 15N enrichment. Soil 15N natural
abundance was determined from untreated soil (0.371 atom %).

15Nitrogen analysis

 

Between 5-12 mg of dry plant tissue, depending on the plant organ, and 50 mg of
dry soil were weighed into tin capsules for mass spectrometry analysis (Automated
Carbon and Nitrogen Analyzer, RobOprep, Europa Scientiﬁc, Cheshire, England). Atom
enrichment values were converted to percentage of N From Fertilizer (% NFF) according

to the following equation (Cabrera and Kissel, 1989):

% NFF = (gtom % 15N in the tissue) — (atom % 15N natural abundance) x 100
(atom % 15N in the fertilizer) — (atom % 15N natural abundance)

49

Natural abundance of 15N of untreated trees was measured, and was equivalent to
0.367%. The amount of N Derived From Fertilizer (NDFF) in different tree organs was

calculated with the following equation (Millard and Nielsen, 1989):

NDFF = %NFF x DW of organ x N concentration of organ

NDFF in the soil per each pot was calculated in a similar way, used for tree organs, and
expressed on total soil dry weight bases.
Nitrogen-use efﬁciency

Nitrogen-use efﬁciency was calculated as C:N ratio of the plant, and as C :N on
the leaf bases. Total plant or leaf carbon was estimated by multiplying the DW of the
plant or leaf by 0.45 (Gifford, 2000). Total N in the whole plant was calculated by
adding the amount of N content of the different plant organs. N concentration of
different plant tissue was measured by mass spectrometry analysis. Higher values of C:N
ratio indicate higher plant NUE.
Water-use efﬁciency

Water-use efﬁciency was calculated in the period between 36 and 94 DABB.
Evapo-transpiration of plants was estimated gravimetrically, by weighing the potted
plants (METTLER, TOLEDO, SB32001 DeltaRange) and changes in weight were
calculated (Tan and Buttery, 1982). Water was manually applied to the trees, on a daily
base, and the amount was recorded. Precipitation was taken into account in the

calculation of the water applied to the trees.

50

WUE was calculated as:

WUE = A dry matter produced (g)
H20 evapotranspired (L)

(Hatﬁeld et al., 2001)

where ‘A dry matter produced’ stands for increment in plant DW calculated as the
difference in DW of plants harvested at 36 and at 94 DABB, and ‘HZO evapo-transpired’
stands for the total water applied to trees between 36 and 94 DABB.
Experimental design

The experiment was a randomized complete block design with six treatments
(genotypes) and four single-tree replications, per genotype, at each harvest time.
Analysis of variance was performed to compare the rootstocks with scion separately from
the rootstocks without scion. The N application times were analyzed separately, and
differences within each application time are presented. Analysis of variance was
perfomred using the MIXED procedure (SAS, Version 8, SAS Institute, Cary, NC, USA)
to detect treatment effects. Separation of signiﬁcant treatment effects was performed

using Least-Squares means test (LSMEANS test) with pS0.05.

51

Table 1.1. Plant phenological stages during 15N application, dates of 15N application, and

dates of plant harvests.

 

Phenological stage between day of Day of 1TN application Day of tree harvest

 

15N application and plant harvest (Days After Bud Break)

5 to 10 fully expanded leaves _ June 7 (20 DABB)

Rapid shoot growth June 11 June 22 (35 DABB)

Rapid shoot growth June 23 July 9 (52 DABB)

End of shoot growth July 16 July 30 (73 DABB)
Terminal bud set August 6 August 20 (94 DABB)
Beginning of leaf senescence September 12 September 21 (126 DABB)

 

52

Results

Plant growth

 

Total shoot length per plant increased from approximately 5 to 55 cm in ‘Gisela
5’ (Gi5), ‘Gisela 6’ (Gi6) and ‘Mazzard’ (M) between 20 and 73 DABB and there were
no signiﬁcant differences among the genotypes (Figure 1.1. A). At 82 and 94 DABB,
total shoot length of Gi6 continued to increase, and was signiﬁcantly higher than in Gi5
or M (Figure 1.1. A). Total shoot length increased from approximately 5 cm to 80, 100
and 120 cm for ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainier/Mazzard’ (R/M), respectively, between 20 and 94 DABB (Figure 1.1. B). Total
shoot length was not different between R/M, R/Gi5 and R/Gi6 in any of the dates
measured, except at 35 DABB, where R/Gi6 and R/M shoot length was higher than
R/Gi5 (Figure 1.1. B).

Total leaf dry weight (DW) per plant of Gi5, Gi6 and M increased. during the
season from approximately 0.2 to 4 g for Gi5 and M, and to 7 g for Gi6 (Figure 1.2. A).
Gi6 and Gi5 had lower leaf DW than M at 73 DABB, Gi6 and M had higher leaf DW
than Gi5 at 94 DABB, while at 126 DABB Gi6 leaf DW was higher than both G15 and M
(Figure 1.2. A). Total leaf DW of R/Gi5, R/Gi6, and R/M increased during the season
from approximately 2 to 32 g, and differed only at 52 DABB, where R/GiS and IUGi6
had a higher leaf DW than R/M (Figure 1.2. B).

Total plant DW of ungrafted rootstock increased from approximately 3 to 25g
between 20 and 126 DABB (Figure 1.3. A). M had a higher DW than Gi5 and Gi6 at 20
DABB, while Gi5 and M had a higher DW than Gi6 at 35 DABB (Figure 1.3. A). At 73

DABB, M had a higher DW compared to Gi5 and Gi6 (Figure 1.3. A). Instead, at 94

53

DABB, M and Gi6 had a higher DW than Gi5, while at 126 DABB Gi6 had a higher DW
compared to both M and Gi5 (Figure 1.3. A). Total plant DW was not signiﬁcantly
different at any of the harvest times for R/Gi5, R/Gi6, and RM and it increased during
the season from approximately 55 to 200 g (Figure 1.3. B).

Root to shoot ratio was not signiﬁcantly different between Gi5, Gi6 and M in any
of the harvest times (Figure 1.4. A). The ratios were higher than one at 20 and 35 DABB,
decreased to values around or lower than one at 52, 73 and 94 DABB, and were again
higher than one, at 126 DABB (Figure 1.4. A). Root to shoot ratio was signiﬁcantly
higher for R/M than R/Gi5, R/Gi6 at 20 and 126 DABB, and was higher or around one at
20, 35 and 126 DABB (Figure 1.4. B). R/Gi5 had ratios lower than one and similar to
R/Gi6, except at 35 DABB, where R/Gi6 has a ratio higher than 1 (Figure 1.4. B).

Plant total nitrogen content

Total N content per plant progressively increased from 0.05 to 0.2 g of N for Gi5
and M, and from 0.05 to 0.4 g ofN for Gi6, from 35 DABB to 126 DABB (Figure 1.5.
A). At 35 DABB Gi5 had a higher plant N content than Gi6 and M, while at 126 DABB
Gi6 had higher plant N content than Gi5 and M (Figure 1.5. A). Total N content per
plant increased approximately from 1 to 2 g of N for R/Gi6, R/Gi5 and R/M from 35
DABB to 126 DABB (Figure 1.5. B). R/Gi5 had a higher plant N content than R/Gi6 and
W at 94 DABB, reaching values of 2.5 g ofN per plant (Figure 1.5. B).

@sonal efﬁciency of N-fertilizer plant uptake

At 35 DABB, Gi5 absorbed more N-fertilizer than both Gi6 and M. The percent

Of N-fertilizer recovery for Gi5, Gi6 and M increased from 1 to 3% during early shoot

growth, to 10 to 15 % at rapid shoot growth and end of shoot growth (Figure 1.6. A).

54

When N-fertilizer was applied at terminal bud set and at the beginning of leaf senescence,
percent of N-fertilizer recovery in Gi6 increased to 24 and 27% of the applied,
respectively, and it was signiﬁcantly higher than Gi5 and M (Figure 1.6. A).

N-fertilizer percent recovery in R/Gi5, R/Gi6 and R/M was between 10 and 15%
of the applied at 35 DABB, and it increased to approximately 60 to 80% at 52, 73, and 94
DABB (Figure 1.6. B). At 94 DABB, R/Gi5 and R/Gi6 absorbed signiﬁcantly greater
amount of N-fertilizer compared to RM. At the beginning of leaf senescence, the
recovery of N-fertilizer decreased to approximately 40% of the applied in R/Gi5, R/Gi6
and R/M (Figure 1.6. B).
Percent of recovery of applied N-fertilizer in plant and soil

Total recovery of N-fertilizer for Gi5, Gi6 and M, accounting both plants and soil,
was approximately 35% at 35 DABB, and it increased to 51%, 61 %, and 80% at 52, 73,
and 94 DABB, respectively, while at 126 DABB was 42% of the total N-applied (Table
1.2). Total recovery of N-fertilizer for R/Gi5, R/Gi6, and R/M in both plants and soil,
was approximately 66% at 35 DABB, and reached values as high as 90 % of the total N
applied, in the subsequent harvests (Table 1.3).
Partitioning of absorbed N-fertilizer

Absorbed N-fertilizer was allocated in higher percent to current season growth
(leaves and shoots) during the shoot growth period, in all the genotypes tested (Figure 1.7
and 1.8). At terminal bud set (94 DABB), approximately 50% of the total N-fertilizer
absorbed was partitioned to roots and trunk in Gi5, Gi6 and M, while 70% of the
absorbed N-fertilizer was partitioned to roots and trunk in R/Gi5, R/Gi6, and R/M (Figure

1.7 and 1.8). At the beginning of leaf senescence, roots and trunk contained

55

 

approximately 70% of the N-fertilizer absorbed, in all genotypes tested (Figure 1.7 and
1.8).

Gi5 and Gi6 harvested at 35 DABB partitioned a signiﬁcantly higher percent of
N-fertilizer to current season growth, while M partitioned more N-fertilizer to roots
(coarse and ﬁne) than both Gi5 and Gi6 (Figure 1.7). At 73 DABB, Gi6 and M
partitioned a higher percent of the absorbed N-fertilizer to current season growth than
Gi5 (Figure 1.7). Higher percent of absorbed N-fertilizer was allocated in the trunk of M
than Gi6 at 94 DABB, while at the beginning of leaf senescence M had more absorbed N-
fertilizer partitioned to the trunk than Gi5 and Gi6 (Figure 1.7).

Higher percent of the absorbed N-fertilizer was allocated to the trunk of R/Gi5
and R/Gi6 than R/M at 35 DABB (Figure 1.8). N-fertilizer was partitioned in higher
percent to the trunk of R/Gi5 than R/Gi6 and R/M at 52 DABB (Figure 1.8). R/M
presented higher amounts of N—fertilizer in roots than R/Gi5 or R/Gi6 at 52 and 73
DABB (Figure 1.8). At 94 DABB, the absorbed N-fertilizer was allocated in greater
amounts to the trunk of R/Gi5 than IUGi6 or R/M (Figure 1.8).

Nitrogen—use efﬁciency

 

Nitrogen-use efficiency, expressed as plant C:N ratio, was signiﬁcantly higher in
M than Gi5 and Gi6, in all the times measured (Figure 1.9. A). At 126 DABB, Gi6 had a
lower C :N ratio than Gi5 (Figure 1.9. A). There were no signiﬁcant differences between
R/Gi5, R/Gi6, and R/M in plant C:N ratio, except at 94 DABB, where R/M had a
signiﬁcantly higher C:N ratio than R/Gi5 (Figure 1.9. B). C:N ratio of leaf showed a
trend similar to plant C:N ratio, at all times measured and for all the genotypes tested

(Figure A.2, Appendix A).

56

Water-use efﬁciency

 

Between 36 and 94 DABB Gi6 and M had a greater increase in DW than Gi5
(Table 1.4). Evapo-transpiration of Gi5, Gi6 and M was approximately 12 L and was not
different between genotypes (Table 1.4). WUE was higher in M than Gi5 (Table 1.4).
Although R/Gi5 had higher evapo—transpiration than R/Gi6 and R/M, there were no

signiﬁcant differences in WUE between rootstocks with scion (Table 1.4).

57

160 - A

 

 

 

 

 

140 _ --O--G15
— U- Gi6
120 - P M
E ***
3
5 100 - “0,, a
CD
5
: 80 J
O
O
'5.
E 60 -
’5
t—
40 -
20 -
O I I I I I I I
0 20 40 60 80 100 120 140
160 - B
140 -
120 ~
E
Z 100 -
’51)
E
j: 80 -
O
52
'5 6O "'
‘5
r-
40 ~
20 ~
0 I I I
0 100 120 140

 

DABB

Figure 1.1. Total shoot length per plant at different Days After Bud Break (DABB). A.
Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M). B.
Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6) and
‘Rainier/Mazzard’ (R/M). Each point represents the mean (iSE) of four replications.
Asterisks indicate the signiﬁcance level of the plant effect; * and *** stand for
signiﬁcance at pS0.05, or 0.001, respectively. Letters indicate differences between
genotypes at pS0.05 (LSMEAN S test).

58

9 . --<D-'-Ch5 a
-{3-'Ch6

Total leaf DW (g)
Ur

 

 

 

140

401

35 4

 

30 a

25 -

20 -

15 -

Total leaf DW (g)

 

 

 

0 20 40 60 80 100 120 140
DABB

Figure 1.2. Total leaf dry weight (DW) per plant at different Days After Bud Break
(DABB). A. Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M).
13. Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainier/Mazzard’ (R/M). Analysis of variance was canted out separately for each
harvest. Each point represents the mean (iSE) of four replications. Asterisks indicate the
Signiﬁcance level of the plant effect; * and ** stand for signiﬁcance at p50.05, or 0.01,
respectively. Letters indicate differences between genotypes at pS0.05 (LSMEANS test).

59

40-

--O--Gi5 a

35~

30-

25-

20-

15-

Total plant DW (g)

10*

 

 

250 q

200 '4

150 -

100 -

Total plant DW (g)

 

501

 

 

0 20 40 60 80 100 120 140
DABB

Figure 1.3. Total dry weight (DW) per plant at different Days After Bud Break (DABB).
A. Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M). B.
Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (PJGi6), and
‘Rainier/Mazzard’ (R/M). Analysis of variance was carried out separately for each
harvest. Each point represents the mean (i SE) of four replications. Asterisks indicate
the signiﬁcance level of the plant effect. * and ** stand for signiﬁcance at pS0.05, or
0.01, respectively. Letters indicate differences between genotypes at [930.05 (LSMEANS
test).

60

2.0- ”OUGIS

1.8- —'Cl—'Gi6

1.6- M ‘
1.4- ._
1.21 ,' “ ."
1‘0“ %\\ ‘~ .” "'u. " //

O.8* \ ‘, ,r' /
I

 

Root:Shoot ratio
/
I
D
\

0.4 a
0.2 .

 

 

0.0 I I I I I I I
0 20 40 60 80 100 120 140

2"" nonmcrs
1.8 - -l—-R/Gi6
+R/M

1.6 ~ *
1.4 ~
1.2 -
1.0 ~
0.8 -
0.6 -
0.4 -
0.2 ,

RootzShoot ratio

 

 

 

0.0 I I I I 6 I ﬁ

0 20 40 60 80 100 120 140
DABB

Figure 1.4. Root to shoot ratio at different Days After Bud Break (DABB). A.
Comparison between ‘Gisela 5’ (Gi5), ‘Gisela 6’ (Gi6), and ‘Mazzard’ (M). B.
Comparison between ‘Rainier/Gisela 5’ (R/Gi5), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainjer/Mazzard’ (R/M). Dotted lines indicate reference for root to shoot ratio equal to
one. Analysis of variance was carried out separately for each harvest. Each point
represents the mean (i SE) of four replications. Asterisks indicate the signiﬁcance level
of the plant effect; * and ** stand for signiﬁcance at pS0.05, or 0.01, respectively.
Letters indicate differences between genotypes at 1230.05 (LSMEAN S test).

61

06- ,
nouoo w A

-{3 - CH6 a
0.5 ~

011-

0L34

Total N (g)

OLZ‘

OJ -

 

 

 

0.0 I I I I I I I
0 20 40 60 80 100 120 140

SA)
0
J

neume ; B
—i—me "

Total N (g)
3. 2 if.

p—d
O
l

 

9’
Ur
r

 

 

9’
C)

I I I I I

0 20 40 60 80 100 120 140
DABB

Figure 1.5. Total N content per plant at different Days After Bud Break (DABB). A.
Comparison between ‘Gisela 5’ (G15), ‘Gisela 6’ (G16), and ‘Mazzard’ (M). B.
Comparison between ‘Rainier/Gisela 5’ (R/GiS), ‘Rainier/Gisela 6’ (R/Gi6), and
‘Rainier/Mazzard’ (R/M). Analysis of variance was carried out separately for each
harvest. Each point represents the mean (i SE) of four replications. Asterisks indicate
the signiﬁcance level of the plant effect; * and ** stand for signiﬁcance at p50.05, or
0.01, respectively. Letters indicate differences between genotypes at p_<_0.05 (LSMEANS
test).

62

 

 

---O---G15 u

 

 

 

 

 

 

35—
—-r:1——616 * a A
A 30- —A—M a
"C
.2 _,...
g25- ’,-—--—
(U
‘8
°\° 20—
1.;
g 15—
E.
E 10-
LL.
LL
D
Z 5-
0 r 1
0 20 140
100 — -----0- W615 ** B
90, —-I-—R/Gi6 i
g +R/M ." "
.2 80d ’/’é_~“;—i “.
E.
g- 70— a
“—4
2 604
e.
g 50"
S:
E 40-
Q.
.E 30-
E
Q 20-
Z 104
O I I l I l T ‘1
0 20 40 60 80 100 120 140

DABB

Figure 1.6. Nitrogen Derived From Fertilizer (NDFF, % of the applied) at different
harvests during the growing season (Days After Bud Break, DABB). A. Comparison
between ‘Gisela 5’ (G15), ‘Gisela 6’ (G16), and ‘Mazzard’ (M). B. Comparison between
‘Rainier/Gisela 5’ (R/G15), ‘Rainier/Gisela 6’ (R/Gi6), and ‘Rainier/Mazzard’ (R/M).
Analysis of variance was carried out separately for each harvest. Each point represents
the mean (i SE) of four replications. Asterisks indicate the signiﬁcance level of the plant
effect; * and ** stand for signiﬁcance at p30.05, or 0.01, respectively. Letters indicate
differences between genotypes at pS0.05 (LSMEANS test).

63

Table 1.2. Amount of Nitrogen Derived From Fertilizer (NDFF) in plant and soil, and
recovery of NDFF in plant and soil (percent of the applied N, average per harvest). Plants
were fertilized with 15N at different Days After Bud Break (DABB). Comparison
between ‘Gisela 5’ (G15), ‘Gisela 6’ (Gi6) and ‘Mazzard’ (M).

 

 

 

 

 

 

Rootstock N-ferttirllizatron H3212“ Plant Soil EZZIZInydosfoiiIE’ZFofn
(DABB) (DABB) (mg) (mg) the applied N)
Gi5 24 35 16.7 ay 167 35
G16 24 35 8.8 b 124
M 24 35 4.6 b 193
SigniﬁcanceZ * NS
Gi5 36 52 56.8 226 a 51
G16 36 52 48.9 265 a
M 36 52 35.8 136 b
Signiﬁcance NS *
Gi5 59 73 53.9 240 61
G16 59 73 80.4 268
M 59 73 58.1 209
Significance NS NS
Gi5 80 94 54.8 b 398 82
G16 80 94 120.8 a 334
M 80 94 25.8 b 297
Significance * NS
Gi5 117 126 57.3 b 160 a 42
Gi6 117 126 137.5 a 81 c
M 117 126 39.5 b 147 b
Signiﬁcance ** **

 

2 Analysis of variance was carried out separately for each harvest. NS, *, and “‘2 Non Signiﬁcant or
signiﬁcantly different at p30.05 or 0.01, respectively.

" Means within columns followed by different letters are signiﬁcantly different by LSMEANS test at
pS0.05

64

 

 

Table 1.3. Amount of Nitrogen Derived From Fertilizer (NDFF) in plant and soil, and
recovery of NDFF in plant and soil (percent of the applied N, average per harvest) Plants
were fertilized with 15N at different Days After Bud Break (DABB). Comparison
between ‘Rainier/Gisela 5’ (R/G15), ‘Rainier/Gisela 6’ (R/Gi6), and ‘Rainier/Mazzard’
(R/M).

 

 

 

 

 

 

H222“ 2“)
(DABB) (DABB) (mg) (mg) applied N)
R/Gi5 24 35 47 283 66
R/G16 24 35 83 253
R/M 24 35 44 276
SigniﬁcanceZ NS NS
R/Gi5 36 52 345 77 80
R/Gi6 36 52 362 55
W 36 52 291 72
Signiﬁcance NS NS
R/Gi5 59 73 319 32 76
R/Gi6 59 73 404 42
W 59 73 293 47
Signiﬁcance NS NS
R/Gi5 80 94 440 ay 60. 90
R/Gi6 80 94 392 a 82
R/M 80 94 272 b 113
Signiﬁcance ** NS
R/Gi5 1 1 7 I26 204 192 78
R/Gi6 117 126 184 175
R/M 117 126 190 222
Signiﬁcance NS NS

 

Z Analysis of variance was carried out separately for each harvest. NS, *, and **: Non Signiﬁcant or
signiﬁcantly different at pS0.05 or 0.01, respectively.

y Me ans within columns followed by different letters are signiﬁcantly different by LSMEANS test at
pSOOS

65

 

 

Figure 1.7. Partitioning of absorbed N-fertilizer (in percent) in different plant organs, at
different harvests during the growing season (Days After Bud Break, DABB).
Comparison between A. ‘Gisela 5’ (G15), B. ‘Gisela 6’ (Gi6), and C. ‘Mazzard’ (M).
Each bar represents the average of four replications. Analysis of variance was carried out
separately for each harvest. Letters represent differences between organs of different
genotypes, in each harvest date, at ps0.05 (LSMEAN S test).

66

 

 

 

 

 

 

 

 

 

-
0 0 0 0
00 6 4 2

pen—cogs coNzgﬂ- Z we e\o

 

126

 

 

 

 

 

 

 

 

0 0 0 O O
00 6 4 2

UQDLOWDN u®N_:t®,wrZ,wO o\o

126

94

73

52

35

 

 

 

 

 

 

 

 

O
O
1

O 0 0 0 0

00 6 4 2

895% esseeze so

 

126

94

 

m
D

k
n
m

T

&

I New growth

 

[:1 Root

67

 

 

Figure 1.8. Partitioning of absorbed N-fertilizer (in percent) in different plant organs, at
different harvests during the growing season (Days After Bud Break, DABB).
Comparison between A. ‘Rainier/Gisela 5’ (R/Gi5), B. ‘Rainier/Gisela 6’ (R/Gi6), and C.
‘Rainier/Mazzard’ (R/M). Each bar represents the average of four replications. Analysis
of variance was carried out separately for each harvest. Letters represent differences
between organs of different genotypes, in each harvest date at pS0.05 (LSMEANS test).

68

A. R/Gi5

 

 

100]

_ _ _ _
0 0 0 O
00 6 4 2

888% Ezeei do .x.

B. R/Gi6

 

 

100 -

_ a _ _
O O 0 0
00 6 4 2

895%? SN:E£-ZE e\e

 

C. R/M

 

 

_ _ a _
0 0 O 0
00 6 4 2

100 -

eopcomna SNEEEZCO e\o

126

94

52

35

I New growth

I Trunk

'r

[3 Root

69

80 -

 

 

 

 

 

 

-<D --(h5 [X
70 . C] —~Ch6
+M
60‘ ***
i: 50 -
U
3,340- b .—
v b
E“ 30' Q2“ b C b D
L. K. '
4 b \3 Cl c
20- G b ‘ ‘ 8 "
b b
10 J
O I I I I I I I
0 20 40 60 80 100 120 140
80’ --O--R/GiS B
+R/M
60 -
7 50-
u
'52
E401
E?
if 30 ~
20 4
10 .
O I I If I I I I
0 20 40 60 80 100 120 140

DABB

Figure 1.9. Nitrogen use efﬁciency (NUE) expressed as whole plant C:N ratio at
different Days After Bud Break (DABB). A. Comparison between ‘Gisela 5’ (G15),
‘Gisela 6’ (G16), and ‘Mazzard’ (M). B. Comparison between ‘Rainier/Gisela 5’ (R/G15),
‘Rainier/Gisela 6’ (IUG16), and ‘Rainier/Mazzard’ (R/M). Analysis of variance was
carried out separately for each harvest. Each point represents the mean (i SE) of four
replications. Asterisks indicate the signiﬁcance level of the plant effect; *, ** and ***
stand for signiﬁcance at p50.05, 0.01, or 0.001, respectively. Letters indicate differences

between genotypes at [230.05 (LSMEANS test).

70

Table 1.4. Increase in dry weight (DW at 94 — DW at 36 DABB, g), evapo-transpiration
(ET, L) and water use efﬁciency [ADW(g)/AET(L)] of plants, calculated between 36 and
94 Days After Bud Break (DABB). Comparison between ‘Rainier/Gisela 5’ (R/GiS),
‘Rainier/Gisela 6’ (R/Gi6) and ‘Rainier/Mazzard’ (R/M) and between ‘Gisela 5’ (G15),
‘Gisela 6’ (G16) and ‘Mazzard’ (M).

 

 

 

Rootstock ADW Total ET WUE
(DW at 94 — DW at 36 DABB, (L) [ADW(g)/ET(L)]
g)

R/G15 100 18.4 a2 5.45
IVG16 75 17.2 b 2.68
W 83 16.7 b 4.95
Signiﬁcancey NS * NS
G15 7.8 b 12.5 0.60 b
Gi6 12.7 a 12.2 1.04 ab
M 13.9a 12.7 1.10a
Signiﬁcance * NS *

 

2 Means within columns followed by different letters are signiﬁcantly different by LSMEANS

test at pS0.05

yNS, and *: Non Signiﬁcant or signiﬁcant at pS0.05.

71

Discussion
Seasonal efﬁciency of N-fertilizer plant uptake

Understanding the seasonal efﬁciency of N-fertilizer uptake in sweet cherry
grafted on dwarﬁng and standard rootstocks is an important step for optimizing the
fertilization practice in modern orchards. N-fertilizer uptake efﬁciency was low during
early shoot growth, and increased considerably when plants were fertilized during the
second period of rapid shoot growth. During early shoot growth, plants had still a limited
canopy, as shown by the root to shoot ratio higher than one and by the total leaf DW
(Figure 1.4 and 1.1), and they were probably still in the process of developing an active
root system, since they were potted approximately 24 days prior to the ﬁrst 15N
application. Similar results have been observed in non-bearing, two-year-old prune trees,
where N-fertilizer uptake signiﬁcantly increased during rapid shoot growth compared to
bud break or earlier N applications (Weinbaum et al., 1978). Based on the low uptake
rate of N-fertilizer during the early stages of plant growth obtained in all the genotypes
tested, we can speculate that early growth was sustained by the N reserves accumulated
in the previous year in storage organs. Early spring growth of deciduous fruit trees is
supported by remobilization of N from storage organs, such as woody tissue (Tagliavini
et al., 1998; Titus and Kang, 1982; Weinbaum et a1. 1978 and 1984), and is independent
from the current season N availability (Millard, 1996). In apple, Neilsen et a1. (1997)
reported that spring remobilization of N provided the majority of N required for spur leaf
growth, and half of the N used for shoot leaf growth. Fertilizer uptake followed the
pattern of plant DW accumulation and more speciﬁcally of leaf DW accumulation. Plant

leaf DW increased until terminal bud set (Figure 1.2, 94 DABB) and it coincided with

72

 

higher N uptake (50 to 80% of the N-fertilizer applied) while during the beginning of leaf
senescence N-fertilizer uptake decreased considerably in all the genotypes tested.
Absorption of N paralleled the pattern of DW accumulation in ‘Sevin blanc’grapevines
(Hanson and Howell, 1995), and in ‘Maycrest’ peach trees (Muﬁoz et al., 1993). The
different rootstock vigor did not inﬂuence the N-fertilizer uptake efﬁciency. This is
supported by the lack of signiﬁcant differences between the rootstocks with scion, with
the exception of the period of terminal bud set, where R/G15 and R/Gi6 were more
efﬁcient in N-fertilizer uptake than R/M (Figure 1.8).

In general, rootstocks without scion recovered less fertilizer than the same
rootstocks with cv. ‘Rainier’. Total plant DW was 7-fold higher in R/G15, R/Gi6 and
R/M compared to the same rootstocks without scion, and they presented a more
developed root system. In G15 and the standard rootstock ‘Mazzard’ the N uptake was
between 5 and 10 % of the N-fertilizer applied from rapid shoot growth until the
beginning of leaf senescence while in Gi6 N uptake increased from 10 % at rapid shoot
growth to 25 % of the N-fertilizer applied at the beginning of leaf senescence. The
prolonged shoot growth period of Gi6 compared to G15 and M caused a higher increase
in plant DW for Gi6 than both M and G15, which drove the higher N uptake of Gi6
during the last periods of the growing season.

Partitioning of absorbed N—fertilizer

N-fertilizer partitioning in different plant organs is an important aspect to consider
when N-application time is evaluated in regards to the use of N-fertilizer in the plants,
eg. for promoting growth, or increasing N reserves. At rapid shoot growth, between 40

and 60% of absorbed N was partitioned to the current season growth, indicating that

73

 

during rapid shoot growth leaves act as a major sink for N-fertilizer absorbed during the
current season. Current season growth became a less important N sink at terminal bud
set, where N was partitioned in higher amounts to roots than to current season growth,
especially in the rootstocks with scion. At the beginning of leaf senescence the amount
of N-fertilizer partitioned to the roots was approximately 60 to 80% of the absorbed N,
similar to percent recovered in roots of nectarine trees (P. persica var. nectarina)
(Tagliavini et al., 1999). A similar trend of N-partitioning at different phenological
stages has been reported in a study with two-year-old prune trees (Weinbaum at al.,
1978). During rapid shoot growth, prune trees partitioned about 70% of N-fertilizer
absorbed into new shoots and leaves (Weinbaum at al., 1978), while in mid-September
the major sink for N were roots, with 67% of the N-fertilizer present in the plant
(Weinbaum et al., 1978). Also in young bearing peach trees, during plant growth, N was
mainly partitioned to leaves, shoots and new fruits; instead, at the beginning of leaf fall,
N was partitioned mainly to bark of branches, trunk and roots (Muﬁoz et al., 1993). N-
fertilizer partitioning depends on the relative sink strength of plant organs. As previously
demonstrated in other fruit trees, sink strength during the season in non-bearing sweet
cherry, on dwarﬁng and standard rootstocks and the same rootstocks without scion
switched from leaves during shoot growth, to roots and trunk, during leaf senescence.
Nitrogen-use efﬁciency

High NUE is a characteristic of plants adapted to low N availability, and represent
plant N requirement to produce a gram of dry weight. Nitrogen use efﬁciency was higher
in M than in both G15 and Gi6 while there were no differences in NUE between the

rootstocks with scion. Overall, absolute values of NUE were similar for M, R/M, R/G15,

74

and R/Gi6 in all the periods considered during the season. The similar values of NUE
obtained for the rootstocks with scion and the ungrafted ‘Mazzard’ appeared determined
by the species of the aerial part of the plant rather than caused by the different rootstocks.
This is supported by the fact that both the sweet cherry cv. ’Rainier’ and the rootstock
‘Mazzard’ belong to the P. avium species. Leaf N composition was also affected by two
apple cultivars when grown on rootstocks with different vigor (Tagliavini et al., 1992).
Graft union has been indicated as one of the possible reason for alteration of leaf mineral
composition (Granger and Looney, 1983), but under our experimental conditions, this
was not responsible for different N leaf composition.
Water-use efﬁciency

Water use efﬁciency between 36 and 94 DABB was not different among the
rootstocks with scion, while it was higher for M than G15 and Gi6 (Table 1.4).
Considering that WUE is the result of the ratio between assimilation rate and
transpiration rate, a genotype may have a greater WUE than another if it has either a
higher assimilation rate and similar transpiration, or a similar assimilation rate and lower
transpiration rate (Patterson et al., 1997). In the period considered, ‘Mazzard’ had a
higher increase in DW than G15 and Gi6, which determined the higher WUE of M
compared to G15 and Gi6. There were no differences in evapo-transpiration between the
M, G15 and Gi6 therefore transpiration rate did not play a signiﬁcant role in determining

the higher WUE of ‘Mazzard’.

75

 

Conclusions

1) Nitrogen uptake followed the accumulation of dry matter in the tree. N-
fertilizer uptake was higher from rapid shoot growth, and was fairly constant until the
beginning of leaf senescence, when it decreased considerably. Although the absolute
values of fertilizer-N uptake obtained with potted tree are difﬁcult to extrapolate to ﬁeld-
grown mature trees due to differences in magnitude of tree N demand, and availability of
N from internal cycling at different tree age, the relative comparisons of genotypes N-
fertilizer uptake obtained in this study, are applicable to non bearing ﬁeld grown trees
and can be used to optimize time of application of N fertilizer.

2) Overall, there was no difference in N uptake efﬁciency between dwarﬁng and
standard trees with the exception of terminal bud set, where dwarﬁng and semi-dwarﬁng
trees were more efﬁcient in N-fertilizer uptake than standard trees. Rootstocks without
scion differed in N uptake at terminal bud set and beginning of leaf senescence, and Gi6
was more efﬁcient than both G15 and M due to its higher increase in plant DW.

3) NUE was higher in M than G15 and Gi6 while rootstocks with cv. ‘Rainier’
grafted on had similar NUE. Considering that ‘Mazzard’ had similar values of NUE of
R/GiS, R/Gi6 and R/M, the aerial part of the plant appears to have a higher incidence in
determining plant NUE rather than the root system of the plant.

4) There was no difference in WUE between R/G15, R/Gi6 and R/M although
there was a higher evapo-transpiration of R/G15 compared to R/Gi6 and R/M. M had a
higher WUE compared to both Gi5 and Gi6 due to the higher increase in biomass in the

period considered.

76

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80

CHAPTER 2

81

CHAPTER 2

Nitrogen uptake and nitrogen-use efﬁciency of ﬁeld-grown sweet cherry (Prunus

avium L. ‘Sam’) on dwarﬁng and standard rootstocks.

Abstract

This research was initiated to determine if the dwarﬁng rootstock ‘Gisela 5’
inﬂuences nitrogen (N)-fertilizer uptake and N use efﬁciency (NUE), when compared to
the standard rootstock ‘Mazzard’. The objectives were to evaluate in dwarﬁng and
standard rootstocks: l) efﬁciency of N-fertilizer uptake, at different times during the
growing season; 2) the NUE, expressed as C:N ratio of leaves; and 3) retranslocation of
N from senescent leaves. The experiment was conducted in 2002, on ﬁve-year-old sweet
cherry trees of cv. ‘Sam’ grafted on the standard rootstock ‘Mazzard’ and the dwarﬁng
rootstock ‘Gisela 5’. The orchard was located in Cepemish, Michigan on a loamy-sand
soil. During the growing season, a total of21g ofN as K15NO3 was applied three times:
at full bloom (11 May), at rapid shoot growth (40 days after full bloom, DAFB), and at
the beginning of leaf senescence (126 DAFB). Shortly after bloom a severe frost
eliminated all the ﬂowers in the trees resulting in total crop loss. Leaves were sampled at
one, ﬁve, 10 days following the 15N applications, and then every week thereafter, until
leaf senescence occurred. N content and % of 15N were determined in the leaves sampled
during the season. Before leaf senescence, trees were enclosed in netting bags to collect
abscised leaves in order to evaluate total amount of N and N derived from fertilizer lost

during leaf fall. N-fertilizer was absorbed in the greatest amount when applied at bloom

82

or at rapid shoot growth. When N-fertilizer was applied at bloom and rapid shoot
growth, the percent of N-fertilizer was higher in leaves of sweet cherry on dwarﬁng than
standard rootstocks. Therefore it appears that dwarﬁng trees rely more on N-fertilizer
than standard trees. NUE was lower at bloom, and increased progressively during the
season until 60 DAF B. Overall, NUE as well as N-retranslocation from senescent leaves

did not differ between dwarﬁng and standard trees.

Introduction

Application of N inﬂuences tree growth and development during the current year
and in the following growing season (Weinbaum, 1987). Several factors affect
N-fertilizer use efﬁciency including, plant demand for N (Weinbaum et al., 1992), the
form of N applied (Barker and Mills, 1980), the application method (Sanchez et al, 1995),
and the rate and timing of application (Taylor et al., 1975; Conradie, 1991; Sanchez et al.,
1 992).

The seasonal pattern of uptake and N accumulation in trees reﬂects their N
demand and can be used for timing N application in order to maximize fertilizer uptake
(Weinbaum et al., 1992). The synchrony between plant N demand and N soil availability
is an important factor to consider, in order to increase the efﬁciency of N-fertilizer
applications. N uptake during the season, and partitioning of N to different plant organs,
has been investigated in several fruit tree species (Weinbaum et al., 1978, 1984; Muf’roz
et al., 1993; Neilsen et al., 2001 a and b; Policarpo et al., 2002). Little is known about the
pattern of N uptake in sweet cherry trees, particularly when grown on the new dwarﬁng

rootstocks. Sweet cherries in the United State are grown mainly on standard size

83

rootstocks like ‘Mazzard’ seedling (Prunus avium L.), the clonal ‘Mazzard F.12/ 1’ or
‘Mahaleb’ (P. mahaleb L.) (Perry, 1987). Recently, sweet cherry on dwarﬁng rootstocks
(i.e. ‘Gisela 5’, P. cerasus (cv. Schattenmorelle) x P. canescens), are being extensively
planted. The main advantages of a dwarﬁng rootstock are the precocity of production,
the possibility of intensive plantings, and lowering production costs of pruning, spraying,
and harvest. When dwarﬁng rootstocks are used, an appropriate management of N is
needed in order to maintain a proper balance between reproductive and vegetative growth
in trees, and to sustain high quality fruit production (Lang, 2000).

A better knowledge of sweet cherry tree N requirements during the season is
important in order to avoid problems related to overfertilization. Excess N-fertilizer in
soils creates high risk for groundwater contamination as a result of nitrate leaching
(Merwin et al., 1996). A large number of sweet cherry orchards in Michigan are located
on sandy and sandy-loam textured soils characterized by rapid drainage and low nutrient
retention. Overfertilization has been also associated with the risk of atmospheric
pollution from NZO production in soil (Weinbaum et al., 1992). Excess N in fruit trees is

detrimental for fruit quality (Faust, 1989), detemrines vigorous growth (Weinbaum,
1992), and decreases tree cold hardiness.

Nitrogen use efﬁciency is deﬁned as the amount of dry matter produced per unit
0f N absorbed (Robertson, 1997). Several indicators have been used to evaluate Plants
NUE and among them are the plant C:N ratio (Maranville and Madhavan, 2002), the
inverse of leaf N concentration (Tateno and Kawaguchi, 2002) or other plant tissue N
Concentration (Nakamura et al., 2002), and the amount of utilizable plant material (seed,

grain, fruits, forage) per amount of absorbed N (Maranville et al., 1980; Baligar et al.,

84

 

2001). High plant C:N ratio indicates high plant NUE. In non-cultivated ecosystems
NUE appears to be higher where soil N availability is low (Robertson, 1997). Nitrogen
use efﬁciency has been evaluated in several crops (Gonzales Ponce et al., 1993; Cassman
et al., 1993; van den Boogaard et al., 1995) but little is known on the effects of dwarﬁng
and standard sweet cherry rootstocks on NUE.

Nitrogen-use efﬁciency can also be deﬁned as the amount of litterfall dry matter
lost per unit of N lost. The withdrawal of N from senescent leaves allows the reuse of the
same unit of N to build new plant organs, the following season (Clark, 1977; Turner,
1 977). Nitrogen use efﬁciency calculated in forest litterfall appears to be correlated with
the availability of N in the ecosystem (Vitousek, 1982). Differences in N retranslocation
from senescent leaves as affected by dwarﬁng and standard sweet cherry rootstocks has
not been previously evaluated and will be important for an overall understanding of plant
NUE. Plants with high NUE or high fertilizer uptake efﬁciency could reduce fertilizer

costs, and nutrient loss. A better understanding of the trees NUE, as well as of N-
fertilizer uptake efﬁciency, will be of great value for deciding the best management
practices to adopt in terms of timing of fertilization in sweet cherries, on dwarﬁng or
standard rootstocks.

The objectives of this study were to compare: 1) N-fertilizer uptake efﬁciency and
partitioning to different plant organs, in standard and dwarﬁng sweet cherry rootstocks, at
three different phenological stages: bloom, rapid shoot growth, and beginning of leaf

senescence, and 2) to compare standard and dwarﬁng sweet cherry trees NUE and N

retranslocation from senescent leaves.

85

Materials and methods

The experiment was carried out in 2002 in a ﬁve-year-old sweet cherry orchard
(P. avium L. ‘Sam’) grafted on either the dwarﬁng rootstock ‘Gisela 5’ (P. cerasus (cv
Schattenmorelle) x P. canescens) or the standard rootstock ‘Mazzard’ (P. avium L.). The
orchard, located in Copemish, MI, (Lat. 44°29' N, Long. -86°08' W) was on a loamy-sand
soil (84.4 % sand, 7.9% silt and 7.7% clay) with pH = 6.8, CEC = 3.1 meq/100 g, and
1.47 % of organic matter. The trees were trained as a vase, with a spacing of4 X 4.8 m,
between trees and rows for ‘Sam/Mazzard’ (S/M), and 3.5 X 4.8 m, between trees and
rows for ‘Sarn/Gisela 5’ (S/G15). Natural grass sod was maintained between the rows,
and an herbicide strip of a total width of approximately 2 m was maintained under the
trees. Standard production practices were used to control insects and diseases (MSU
Fruit management guide, E154). In previous years the orchard was annually fertilized
with urea at the rate of 85 kg ha'1 of N, distributed in two equal applications, at bloom
and at pit hardening. Daily maximum and minimum air temperature, and precipitations
during the growing season were recorded with an automated weather station of the
Michigan Automated Weather Network, located at the USDA National Resources
Conservation Service of Bear Lake, approximately 25 km west of the orchard
(http://www.agweather.geo.msu.edu, Figure B.1, Appendix B).
15Nitrogen Applications

Twelve trees for each rootstock with similar trunk cross sectional area and
blossom density were selected at full bloom (10 May) in six adjacent rows, three rows
with S/M and three rows with S/GiS. Approximately seven days after full bloom

(DAF B), a severe frost caused a complete loss of the cherry ﬂowers in the orchard.

86

Nitrogen was applied three times during the season: at full bloom (11 May), at
rapid shoot growth (20 June, 40 DAFB) and at the beginning of leaf senescence
(11 September, 120 DAFB). At each application time, four trees of each rootstock
received 21 g ofN as KISNO3 (7.5 % atom, ICON Services, Mt. Marion, N.Y.) while the
other eight trees for each rootstock were fertilized with an equal amount of non-enriched
KN O3. Each tree received ‘SN only one time during the season.

To increase the efﬁciency of N-fertilizer applications, the fertilizer was applied in
a targeted method. A strip 2 x 4 m wide below the treated trees (corresponding to the
location of higher root density) was kept vegetation-free with the use of herbicide. The
location of higher roots density was identiﬁed with the help of soil cores, taken at 0-30
and 30-60 cm depths, from non-treated trees. The fertilizer was dissolved in 18 L of
water and applied uniformly with watering cans to the vegetation-free area under each
tree. Another 18 L of water were applied after the N application, to ensure uniform
distribution of the fertilizer into the root zone. Considering only the treated area
(8 m2 tree") the three N-applieations provided to every tree a total of 63 g of N,
equivalent to approximately 80 kg N ha”. Only at bloom, to prevent leaching of the N-
fertilizer due to heavy rain, the treated area was covered with black polyethylene after the
distribution of 15N for approximately 10 days
Plant measurements and samples collection

Tree trunk circumference was measured 011 the 15 May, 2002 and on the 11 May,
2003 at 45 cm from the ground with a metal measuring tape to evaluate the yearly

increase in trunk cross sectional area.

87

Leaf samples were collected at one, ﬁve and 10 days after each 15N applicatior
and then every week thereafter until leaf senescence started. Each sample consisted c
eight well-exposed fully expanded leaves, collected from the middle portion of shoots c
the current season growth, from every 15N treated tree. For the initial 15N applicatio
until the end of May each leaf sample consisted of "four to ﬁve shoot leaf clusters sine
leaves from shoots of the current season’s growth were not present yet. All leaf sample
were freeze-dried, and dry weight (DW) was recorded. All samples were ground in
Wiley Mill to pass a 40-mesh screen.

Partitioning of newly absorbed N in the tree, approximately 40 days after each 151
application was assessed by collecting ﬁne roots (S 2mm diameter), coarse roots (> 2mr
diameter), 2001 wood (one-year-old wood), 2002 wood (current season shoot), buds fror
shoot, buds from spur, and leaves. Buds from shoots were separated from 5 to 6 currer
season shoots, while buds from spurs were separated from spurs on one-year-old wooc
In samples collected 40 days after the initial 15N application new buds were not full
formed and could not be separated. from the wood itself. Roots were sampled by takin
two to three soil cores with a 5-cm soil probe to a depth of 30 cm. Samples were freeze
dried, ground in a stainless steel mill to pass a 40-meslr screen, and ﬁnally analyzed fc
total N and 15N enrichment with mass spectrometry analysis.

At 120 DAFB tree canopies were enclosed with netting bags to collect th

senescent leaves as they abscised from the trees. Abscised leaves were collected in tw
times, 160 DAFB and 180 DAFB, dried at 60°C for approximately 72 hours until ther

was no change in weight, and DW was recorded. A leaf sub-sample was ground in

88

Wiley Mill to pass a 40-mesh screen and analyzed for total N and 15N enrichment with

mass spectrometry analysis.

’5 Nitrogen analysis

Between 5-12 mg of dry sample were weighted into tin capsules for mass
spectrometry analysis (Automated Carbon and Nitrogen Analyzer, Roboprep, Europa
Scientiﬁc, Cheshire, England). Atom ’5N enrichment values were converted to

percentage of N From Fertilizer (% NFF) according to the following equation (Cabrera

and Kissel, 1989):

0/o NFF = (atom % 15N in the tissue) ~ (atom % 15N natural abundance) x 100
(atom % 15N in the fertilizer) - (atom % ‘5 N natural abundance)

15N natural abundance of plant tissues was obtained from untreated trees and was equal to
0.366%. The value of 15 N natural abundance subtracted from the N-fertilizer was also
considered equal to 0.366%. The amount of N Derived From Fertilizer (NDFF) present

in the abscised leaves was calculated with the following equation (Millard and Nielsen,

1 989):
NDFF = DW ofleaves x N concentration ofleaves x %NFF

Nitrogen-use efﬁciency

Nitrogen-use efﬁciency was expressed as leaf C:N ratio. Dry weight was
converted to total carbon by multiplying by 0.45 (Gifford, 2000), while total N in leaves

was measured by mass spectrometry analysis.

89

Experimental design

The experiment was a two-way factorial randomized design, rootstock genotypr
(two genotypes) x timing of 15N application (three times). Four replicate trees of eacl
genotype were treated with 15N at each N application time. Analysis of variance wa
performed using MIXED procedure with SAS (Version 8, SAS Institute, Cary, NC, USA
to detect treatment effects. Repeated-measure analysis was used to detect treatrnen
effects for NUE and percent of 15 N in leaves. When treatments effects were signiﬁcant

means were separated using Least-Squares means test (LSMEANS test) with pS0.05.

Results
Trunk cross sectional area

Trunk cross sectional area was signiﬁcantly higher in ‘Sam/Mazzard’ (S/M) thar
‘ Sam/Gisela 5’ (S/G15) in both 2002 and 2003 (Table 2.1). The yearly increase in trunl
cross sectional area was approximately two-fold greater in S/M than in S/G15 (Table 2.1)
N- fertilizer uptake efﬁciency

N-Fertilizer was detected in leaves ten days after the 15N application at bloom
five days after the 15N application at rapid shoot growth, and 12 days after the 151‘
application at the beginning of leaf senescence (Figure 2.1.). When 15N-fertilizer wa
applied at bloom and at rapid shoot growth it accounted for a maximum of 12% and 6°/
in leaves of S/G15 and S/M, respectively (Figure 2.1. A and B), whereas when it wa
applied at the beginning of leaf senescence accounted for only a maximum of 4 % in botl
S/GiS and S/M (Figure 2.1. C). Leaf percent of NFF from trees treated with 15N at bloon

was signiﬁcantly higher in S/G15 than S/M from 10 until the 38 DAFB, and at 52 DAFI

90

(Figure 2.1. A). When 15N was applied at rapid shoot growth similar concentrations c
N-fertilizer were found in leaves of both genotypes except at 80 DAFB, when leaves 0
S/G15 had a signiﬁcantly higher percent of N-fertilizer than S/M (Figure 2.1. B). Afte
the 15N application at the beginning of leaf senescence the percent of fertilizer-N wa
higher in S/Gi5 than S/M at 136 and 143 DAFB (Figure 2.1. C).
N-fertilizer partitioning
Nitrogen concentration in ﬁne roots of S/G15 and S/M was higher 40 days after 1
application at the beginning of leaf senescence than 40 days after N application at fu‘
bloom and rapid shoot growth while coarse roots had higher N concentration at th
beginning of leaf senescence than rapid shoot growth (Table 2.2). Nitrogen concentratio
of 2002 wood and leaves was higher at 40 days after 15N application at full bloom than 2
40 days after 15N application at rapid shoot growth and beginning of leaf senescence 1
both SM and S/G15 (Table 2.2). S/M had higher N concentration in 2001 wood whe
sampled at 40 days after full bloom than rapid shoot growth and beginning of lee
senescence, while 2001 wood N concentration in S/G15 was higher 40 days after fu
bloom and beginning of leaf senescence than rapid shoot growth (Table 2.3). While % c
NFF was similar in ﬁne roots, in either rootstock, in S/M the percent of N-fertilizer i
coarse roots was higher at 40 days after the 15N application at the beginning of lee
senescence than rapid shoot growth and full bloom (Table 2.4). In S/GiS, the percent c
NFF in coarse roots was higher 40 days after rapid shoot growth and beginning of lee
senescence 15N applications than after at 40 days after full bloom (Table 2.4). N-fertilize

was higher in S/GiS than S/M in 2001 wood, 2002 wood, and leaves at 40 days after fu'

91

bloom and rapid shoot growth while it was similar at the beginning of leaf senescenc
(Table 2.4).

Buds from shoots and spurs had a higher N concentration in trees 15N fertilized a
the beginning of leaf senescence than rapid shoot growth in both trees considered (Tabl
2.5). N-fertilizer contributed in higher percent to the total N in buds from shoots in tree
treated at rapid shoot growth than at the beginning of leaf senescence in S/G15 than S/l\
(Table 2.6). Differently, S/M buds from shoots had higher percent of N-fertilizer thar
S/G15 in trees fertilized at the beginning of leaf senescence (Table 2.6). N-fertilizer i:
buds from spurs accumulated in greater amounts in S/M at 40 days after ‘5 N applicatio:
at the beginning of leaf senescence than at 40 days after rapid shoot growth (Table 2.6]
N-fertilizer in buds from spurs accumulated in similar amount in S/G15 in both period
considered, and in signiﬁcantly higher amount than in S/M spurs buds at 40 days afte
15N application at rapid shoot growth (Table 2.6).

Nitrogen—use efﬁciency

 

Leaf N concentration during the season decreased from approximately 50 mg N g
lDW at full bloom to approximately 20 mg N g'lDW at 40 DAFB, and then remainer
fairly constant until the end of the season (Figure 2B. Appendix B).

Leaf C:N ratio (Figure 2.2) was low at the beginning of the season (approximatel;
8), and it increased progressively until 60 DAFB (approximately 20) and remained fairl;
constant until the end of the season. S/G15 had a signiﬁcantly higher C :N ratio than Sﬂ\
at 5, 38, 42, 52, and 74 DAFB (Figure 2.2). C:N ratio was higher for S/M than S/Gi

toward the end of the season 136 and 150 DAF B (Figure 2.2).

92

Nitrom in abscised leaves

Approximately 35% of the total DW of leaf abscised at senescence was collected
at 160 DAFB and the other 65% at 180 DAFB. Total DW, amount of total N, and N-
fertilizer present in leaves collected at 160 and 180 DAFB did not differ between
rootstocks (data not shown). Dry weight, total N, total NDFF, and percent of NDFF
compared to total N present in abscised leaves were signiﬁcantly higher in S/M compared
to S/G15 in all the fertilization timing (Table 2.7). Total N, NDFF, and percent of NDFF
compared to total N were signiﬁcantly higher in leaves abscised from S/M trees fertilized
with 15N at rapid shoot growth than at full bloom and at the beginning of leaf senescence
(Table 2.7). Total N, NDFF, and percent of applied N-fertilizer in leaves abscised from
S/G15 trees were not signiﬁcantly different among trees fertilized in different times

(Table 2.7).

93

 

Table 2.1. Trunk cross sectional area (TCSA, cm2) of sweet cherry ‘Sam/Mazzard’
(SM) and ‘Sam/Gisela 5’ (S/GiS) measured 15 May, 2002, and 11 May, 2003 and

percent increase of area.

 

 

Rootstock TCSA 2002 TCSA 2003 Increase in area
(cm’) (cm’) 1%)

S/M 82.9 i12Z 112.2 i17 29.3 i9

S/G15 67.6 i 8 80.2 i 9 12.4 i3

Signiﬁcancey ** *** **

 

Z 3: standard deviation of the means
3’ .. and m stand for signiﬁcant within column at p300] and 0.001, respectively

94

 

Figure 2.1. Percent of nitrogen from fertilizer (% NFF) in leaves of sweet cherry
‘Sam/Gisela 5’ (S/Gi5) and ‘Sam/Mazzard’ (S/M), collected at different Days After Full
Bloom (DAF B). Arrows indicate time of [SN applications. A. Application of 15N at full
bloom. B. Application of 15N at rapid shoot growth. C. Application of 15N at the
beginning of leaf senescence. Each point represents the mean of four replications (i
STD). *, **, and *** stand for signiﬁcant at p_<_0.05, 0.01, and 0.001, respectively.

95

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97

Table 2.3. N concentration (mg N g'1 dry weight) in sweet cherry ‘Sam/Mazzard’ (S/M)
and ‘Sam/Gisela 5’ (S/GiS) 2001 wood harvested 40 days after each 15N application.
15N-fertilizer was applied at bloom (11 May), rapid shoot growth (20 June), and
beginning of leaf senescence (11 September).

 

N concentration
(mg N g" dTLWCighl)

 

 

 

 

Plant Time of N application 2001 wood

S/M Full bloom 14 a2
Rapid shoot grth 8 b
Beginning of leaves senescence 9 b

S/G15 Full bloom 12 a
Rapid shoot growth 8 b
Beginning of leaves senescence 12 a

Plant (Pf NS

Fertilization time (F) ***

P x F :1:

 

2 Means within columns followed by different letters are signiﬁcantly different by LSMEANS test at
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98

 

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99

Table 2.5. Nitrogen concentration (mg N g'1 dry weight) in sweet cherry ‘Sam/Mazzard’
(SM) and ‘Sam/Gisela 5’ (S/G15) buds from current season shoots and spurs, harvested
40 days after 15N application. 15N-fertilizer was applied at on the 20 June (rapid shoot
growth) and on the 11 September (beginning of leaf senescence).

 

N concentration (mg N g" dry weight)

 

 

 

 

Time of N application Bud (Shoot) Bud (Spur)
S/M S/G15 S/M S/G15

Rapid shoot growth 10 b2 10 b 9 b 9 b

Beginning of leaves senescence 13 a 13 a 12 a 12 a

Plant (P)y NS NS

Ferilization time (F) *** ***

P x F NS NS

 

2 Means within columns followed by different letters are signiﬁcantly different by LSMEANS test at
p50.05
y NS, and m stand for Non Signiﬁcant or signiﬁcant at pS 0.001, respectively

Table 2.6. Percent of N-fertilizer (% NFF, percent of total N) in sweet cherry
‘Sam/Mazzard’ (SM) and ‘Sam/Gisela 5’ (S/GiS) buds from current season shoot and
spur, harvested 40 days after 15N application. 15N-fertilizer was applied on the 20 June
(rapid shoot growth) and on the 11 September (beginning of leaf senescence).

 

 

 

 

 

 

% NFF (% of total N)
(40 days after N-application)

Plant Time of N application Bud (Shoot) Bud (Spur)
S/M Rapid shoot growth 5.0 b2 4.4 b

Beginning of leaves senescence 10.3 a 10.1 a
S/G15 Rapid shoot growth 10.7 a 8.8 a

Beginning of leaves senescence 7.6 b 9.5 a
Plant (P)y NSy *
Ferilization time (F) NS *
P X F ** >l<

Z Means within columns followed by different letters are signiﬁcantly different by LSMEANS test at

p50.05
y NS, * and H stand for Non Siginﬁcant or signiﬁcant at pS0.05, and 0.01, respectively

100

 

 

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Figure 2.2. Nitrogen use efﬁciency (NUE) expressed as the C:N ratio of leaves in sweet
cherry cv. ‘Sam’, measured at different Days After Full Bloom (DAF B). Comparison
between ‘Sam/GiselaS’ (S/Gi5) and ‘Sam/Mazzard’ (S/M). Each point represents the
mean of four replications (i STD). *, **, and *** stand for signiﬁcant at pS0.05, 0.01,
and 0.001, respectively.

101

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Discussion
N-fertilizer uptake efﬁciency

Based on leaf 15N concentration, N-fertilizer was absorbed more rapidly and i1
higher quantity when applied at bloom and at rapid shoot growth than at the beginning 0
leaf senescence, for scions on either dwarﬁng or standard rootstocks (Figure 2.1]
Similar results were obtained in two-year-old potted prune trees (Weinbaum et al., 1978]
as well as in one-year-old potted sweet cherry trees (Chapter 1, Figure 1.6) where N
fertilizer was absorbed in high amounts at rapid shoot growth, and decrease consistentlj
in all the genotypes during leaf senescence. Nitrogen applied after bloom or during rapi¢
shoot growth satisﬁed the N demand of new growth in grape (Conradie , 1991'), peacl
(Policarpo et al., 2002), pear (Sanchez et al., 1990) and apple (Neilsen et al., 2001b)
Early N applications were associated with higher bloom density and fruiting compare<
with summer N applications, in young ‘Golden Delicious’ apples on the dwarﬁn;
rootstock M9 (Neilsen et al., 2001a). Neilsen et al. (2001a) hypothesized that tree
treated early in the season were able to develop a larger root system, which in genera
caused greater N uptake during the season (Neilsen et al., 2001a).

N-fertilizer applied at the beginning of leaf senescence was also absorbed b;
Sweet cherry trees, since leaf percent of N-fertilizer reached values of 4% (Figure 2.1. C]
Late season N applications increase tree N storage pool, which is remobilized and used i]
the following year (Millard and Thomson, 1989; Sanchez et al., 1991). Deciduous plant
Such as grape (Conradie, 1991), apple (Titus and Kang, 1982) and peach (Tagliavini 6
al., 1999; Muﬁoz et al., 1993) are highly dependent on storage N for early spring growtl

and bloom. Even if the efﬁciency of N-fertilizer uptake at the beginning of lea

103

 

senescence was low, N-fertilizer applied in fall could significantly contribute t
reserves in sweet cherry. More studies are needed to evaluate the effects of late seasc
applications on cold hardiness, under cold climate like the one of the Great Lakes Reg
N-fertilizer contributed more to the total N content of leaves of sweet cherrie
dwarﬁng than standard rootstocks, particularly following the N-fertilization at blc
Similar results were found in spur-type and standard ‘Golden delicious’ apple t:
under low N and high N supply (Sanchez et al., 1995). Spur-type apple trees had 3
and 72% higher percent of N-fertilizer in leaves than standard trees at low N and hig
applications, respectively (Sanchez et al., 1995). Based on their higher N-ferti
percent in leaves and fruit, spur-type apple trees were considered more dependent on
supplies than standard trees (Sanchez et al., 1995). Several hypotheses can be formul
to explain the higher percent on N—fertilizer in leaves of dwarf compared to stan
trees. A possible reason for the higher percent of N-fertilizer in leaves of dwarf
standard trees, could be due to the different extents of the root systems. Dwar
rootstocks may explore smaller soil volumes than deep-rooted standard rootstocks, w
explore larger area of soil and could uptake more naturally available N than dwa1
rootstocks. The root distribution pattern was also considered to play an important ro
determining differences in leaf mineral contents in a four-year study on sour cherr)
‘Montmorency’ grafted on ‘Mazzard’ and ‘Mahaleb’ (Hanson and Perry, 1989). Am
hypothesis for the higher percent of N-fertilizer in leaves of dwarf than standard tre
the effect of the bloom density of trees on N-fertilizer uptake. Even if bloom density
not evaluated by counting prior to frost, dwarf trees had a very high bloom density.

the other side, standard trees presented very few ﬂower clusters indicating that the ‘-

104

were just transitioning from the vegetative to the reproductive stage. Even if the se'
frost determined the complete loss of ﬂowers shortly after the N-fertilization at bloom
fertilizer uptake may have been higher in dwarﬁng than standard trees in order to sue
ﬂowers, and subsequently the potential production of trees. Another possible specula
on the high percent of N-fertilizer in leaves of dwarﬁng than standard trees is that di
trees could contain fewer N reserves in storage organs than standard trees, and de}:
more on N-fertilization during the early stage of shoot growth than standard trees.
Due to the different tree size, it is difﬁcult to speculate on the total amount 0
fertilizer absorbed by the two rootstocks, since the lower percent of N-fertilizer in 1e:
of standard than dwarf trees may reﬂect, in part, a dilution of 15N fertilizer due to
larger biomass of the standard trees. In general, the recommended rate for mature s
fruits in Michigan is approximately of 80 kg N ha'1 per year (Hanson, 1996). Lee
concentration is considered to be adequate for growth when it is approximately betwe
and 3.4 % (Nielsen and Kappel, 1996; Hanson, 1996). Under our experimental condit
application of 80 kg ha" of N supported growth in both standard and dwarﬂng trees 8
leaf N concentration was always equal or higher than 1.9% (Figure 2.B, Appendix
The study was conducted on trees without fruits and it will be important to consider
effect of fruits as N sink. N—fertilizer may have a smaller contribution to leaf grc
when fruit competition is present, as also shown in fertilization studies on almond 1
(Weinbaum et al., 1984, 1987). It will be important to evaluate both rate and timing .
applications in standard and dwarﬁng trees at full production. In conclusion, applicz
of N at bloom and rapid shoot growth in sweet cherry trees on dwarﬁng and stan

I‘Ootstocks was more effective than N application at the beginning of leaf senesce

105

 

When dwarf and standard trees were compared in terms of utilization of N-fertilizer, it
appeared that N-fertilizer contributed more to the total N content of leaves of sweet
cherries on dwarﬁng than standard rootstocks, especially when N was applied at bloom.
N-fertilizer partitioning

N-fertilizer absorbed at full bloom and rapid shoot growth was distributed mainly

to new growth (leaves and current season shoot), in both standard and dwarf trees as a
consequence of their active growth. Similar results were found in another study on two-
year-old fruiting peach trees (Munoz et al., 1993). When N-fertilizer was applied at rapid
shoot growth, peach trees allocated 78% of the N-fertilizer into current season growth
(Munoz et al., 1993).

N—fertilizer applied at the beginning of leaf senescence was partitioned in greater
amount in coarse roots than when applied at bloom, in both rootstocks (Table 2.4).
Coarse roots represent the major storage organ for N as reported in peach (Baﬁados et al,
1997; Muﬁoz et a1, 1993; Policarpo et a1, 2002), sweet cherry (Chapter 1, Figure 1.7 and
1 .8), prune (Weinbaum et al., 1978), and pear (Sanchez et al., 1992).

In standard trees, N-fertilizer in spur and shoot buds was higher when N was
applied at the beginning of leaf senescence than at rapid shoot growth (Table 2.6).
Differently, dwarf trees had similar percent of N-fertilizer in spur buds at rapid shoot
growth and at the beginning of leaf senescence, and higher percent of N-fertilizer in shoot
buds at rapid shoot growth than beginning of leaf senescence (Table 2.6). In both
rootstocks, accumulation of N-fertilizer in buds followed the same pattern of N-fertilizer
accumulation in current season shoot (2002 wood) and in one—year-old wood (2001

Wood) (Table 2.4). Nitrogen status of developing buds is important for fruit set

106

(Williams, 1965). Fall N applications appear beneﬁcial for increasing the amount of N-
fertilizer in buds and therefore they may be an important practice for improving bud
quality and maintaining high fruit set in trees.

Nitrogen—use efﬁciency

Dwarf and standard trees had similar NUE during the growing season (Figure
2.2). In the present study signiﬁcant differences in NUE appeared only in a few sampling
periods during the season. In a potted-tree experiment with one-year—old sweet cherry
trees, similar values of NUE were obtained in ‘Mazzard’ rootstock, ‘Rainier/Mazzard’,
‘Rainier/Gisela 6’, and ‘Rainier/Gisela 5’ (Chapter 1, Figure 1.9). The values of NUE
obtained with one-year-old potted trees seemed related to the presence of the same
genotype, P. avium L., in the aerial part of the tree. Higher NUE of dwarf compared to
standard trees measured during the rapid increase in leaf C:N ratio may have been caused
by different grth rates of the trees. Ingestad (1979) showed a close linear relationship
between relative growth rate and N concentration, in birch seedlings,. More studies are
needed to evaluate the effect of growth rate of dwarﬁng and standard rootstocks on NUE
of sweet cherry scions. Also, it will be important to compare NUE in fruiting dwarf and
standard trees considering the yield produced per absorbed N.

The effect of different rootstocks or scions on leaf N concentration has been
previously compared in fruit trees (Tagliavini et al., 1992; Rom et al., 1995; Ferree,
1998). Nielsen and Kappel (1996) compared leaf N concentration in several fruiting
standard and dwarf sweet cherry trees. Leaf N concentration of sweet cherries on
dwarﬁng rootstocks was occasionally lower than the ones on standard rootstock

‘Mazzard’. The major difference between trees was the higher cumulative yield

107

efﬁciency of dwarf compared to standard trees. Decrease in leaf N, P, and K
concentrations with increasing crop loads were also observed in apple trees (Hansen,
1980)
Retranslocation of N from senescent leaves

Nitrogen remobilization rate of sweet cherry on dwarﬁng and standard rootstocks
did not differ as shown by the similar C:N ratio of abscised leaves. Standard trees had
two times higher amounts of NDFF, calculated on the total N, in abscised leaves than
dwarf trees. Since the remobilization rate of the two trees was similar, based on N-
fertilizer quantity in the abscised leaves we can infer that the total amount of N-fertilizer
absorbed by standard trees was higher than in dwarf trees. Based on the trunk diameter,
standard trees trunk cross sectional area increased 29% while dwarﬁng trees trunk cross
sectional area increased 12% (Table 2.1). The higher growth rate of standard trees can
explain the higher N-fertilizer uptake of standard than dwarﬁng trees. In several studies
the amount of N-fertilizer in senescent leaves was considered as part of the evaluation of
plant N-fertilizer use efﬁciency (Weinbaum et al., 1998; Nielsen et al., 2001b). In mature
walnut trees treated at the beginning of the growing season, the amount of N-fertilizer
found in senescent leaves was approximately 0.2% of the N applied (Weinbaum et al.,
1998). In another study on three-years-old fertigated ‘Elstar’ apple trees N-fertilizer in
senescent leaves was 6.8% of the N applied (Nielsen et al., 2001 b). The amount of N-
recovered in the abscised leaves in standard trees was comparable with the amount
recovered in ‘Elstar’ apple trees, while in dwarf trees only 3% of the total N applied was

recovered in abscised leaves.

108

The percent of N-fertilizer recovered in abscised leaves may be different than the
one found in this study if fruiting trees are evaluated. Fruits represented a strong sink for
N-fertilizer in six-year-study on walnut trees (Weinbaum et al., 1998). During the ﬁrst
ﬁve years, total amount of N-fertilizer recovered in leaves litter was approximately 1% of
the fertilizer applied while fruits exported approximately 18% of the N-fertilizer applied

(Weinbaum et al., 1998).

Conclusions

1) N-fertilizer applied during bloom and rapid shoot growth in sweet cherry on
dwarﬁng and standard rootstocks was absorbed in greater amounts than N-fertilizer
applied at the beginning of leaf senescence. Fertilization practices for sweet cherry
production should be optimized considering application of fertilizer at bloom and rapid
shoot grth (40 days after bloom).

2) N-fertilizer contributed in higher amount to the total N content of leaves of
sweet cherries on dwarﬁng than standard rootstocks, especially when N was applied at
bloom. N-fertilization may play an important role to sustain growth when sweet cherry
are grown on dwarﬁng rootstocks, especially during the early stage of growth, and in soil
with low organic matter. Nevertheless, N-fertilizer requirements of sweet cherry grown
on dwarﬁng rootstocks need to be carefully evaluated to avoid risks associated to
overfertilization since previous studies on apple, on the dwarﬁng rootstock M9, indicated
low N requirements (between 8 and 44 kg N ha!) (Neilsen and Neilsen, 2002).

3) Dwarﬁng and standard rootstocks did not differ in terms of NUE.

109

 

 

4) Retranslocation of N from senescent leaves was also similar between dwarf and
standard trees. Since retranslocation of N from senescent leaves is another aspect of trees
NUE, it can be concluded that also based on this process, standard and dwarﬁng

rootstocks did not affect NUE.

110

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115

CHAPTER 3

116

CHAPTER 3

Water-use efficiency of one-year-old sweet cherry (Prunus avium L. ‘Rainier’) on

dwarﬁng and standard rootstocks, under well-watered and water deﬁcit conditions

Abstract

Physiological and mmphological parameters of scions may be affected by the
degree of adaptation to water deﬁcit condition of a particular rootstock. This study was
conducted to determine whether standard and dwarﬁng sweet cherry rootstocks under
water deﬁcit conditions responded differently, relative to plant growth and gas exchange
parameters, water-use efﬁciency (WUE), and leaf carbon isotope composition. One-year-
old potted sweet cherry cv. ‘Rainier’ grafted on the standard rootstock ‘Mazzard’ and on
the dwarﬁng rootstock ‘Gisela 5’ were compared under two different water treatments: 1)
well-watered, which received daily 100% of the amount of water lost by ET, and 2) a
water deﬁcit treatment, which received 50% of the water applied to the control. Relative
shoot growth rate, leaf emergence rate and cumulative leaf area were recorded every
three to seven days during the experiment. Leaf net carbon dioxide assimilation rate,
stomatal conductance, transpiration rate, internal CO; concentration, and WUE were
measured daily for the duration of the experiment. At the end of the experiment, leaf
samples were collected to determine leaf carbon isotope composition. The growth
parameters measured were affected similarly in the two rootstocks, indicating a similar
degree of sensitivity to water deﬁcit in the genotypes tested. Cumulative leaf area was

affected earlier by water deﬁcit than relative shoot growth and leaf emergence rate. Gas

117

exchange parameters were affected earlier than growth parameters. Overall, WUE was
not signiﬁcantly different between dwarﬁng and standard rootstocks, and did not appear
to increase under water deﬁcit condition, indicating that irrigation should be considered
as an important practice in sweet cherry orchards, especially when dwarﬁng rootstocks

are selected.

Introduction

Understanding how rootstocks adapt and respond to water stress is essential for
selecting the proper genotype and irrigation method for situations where drought stress is
likely to occur. Unlike other stone fruits, e.g. almonds, which can often withstand
drought and still produce fruit adequately, cherries require frequent supplies of water
during the growing season to sustain tree growth, especially during fruit set and fruit
development (Webster and Looney, 1996). In addition, availability of irrigation water
will be reduced in the future because of increasing competition for urban use, therefore
irrigation strategies able to increase water use, or cultivation of plants with high water-
use efﬁciency (WUE) are becoming more important.

Water-use efﬁciency is deﬁned as the amount of dry matter produced per unit of
water used. It can be calculated by the instantaneous ratio of assimilation rates of C02
(A) and transpiration (E) (Bongi et al., 1994; Field et al., 1983) or it can be calculated on
a seasonal basis (Hatﬁeld et al., 2001). Water-use efﬁciency is affected by plant factors
(scion and rootstock, Bongi et al., 1994) and by the environment (atmospheric C02
concentration, humidity, and soil water availability). Water use efﬁciency is an

important component of drought tolerance and usually high WUE indicates the

118

 

adaptation of plants to limited water availability. Although WUE is found to increase
under water deﬁcit conditions (Flore et al., 1985), Glenn et al. (2001) reported a decline
in WUE in water stressed peach.

Processes affected by water deﬁcit in fruit trees include: cell division and
expansion, ﬂower bud differentiation, accumulation of carbohydrates (Faust, 1989), and
photosynthesis (Flore and Lakso, 1989; Flore et al, 1985). Closure of stomata during
water stress can be responsible for the observed reduction in photosynthetic rate (Flore
and Layne, 1996). Stomatal movements are regulated by the turgor of the guard cells,
which are affected by external factors such as light intensity, C02, humidity, wind, and
temperature, as well as by endogenous factors such as plant hormones, leaf water status,
and internal C02 (Kramer and Boyer, 1995; Hetherington and Woodward, 2003). Root-
produced ABA transported to leaves is believed to regulate stomatal behavior (Davies et
al., 1990). Wartinger et a1. (1990) showed that in almond under a drying cycle, as xylem
ABA increased, leaf conductance decreased.

Carbon isotope composition (5 ’3 C) is a measure of the 13C/IZC ratio in plant
tissues, relative to the ratio of an accepted international standard, the limestone Pee Dee
belemnite (PDB). Plants have negative values of 513C because the l3C/IZC ratio in the
atmosphere is less than that of PDB and there is a net discrimination against 13 C by plants
during C02 ﬁxation. During water stress, the ability of plants to discriminate against the
heavier isotope l3C decreases, therefore the isotopic composition. (5 1" C) of water stressed
plants becomes more positive than the one of well-watered plants (Farquhar et al., 1982;
Farquhar and Richards, 1984). Farquhar and Richards (1984) showed that carbon isotope

composition is correlated with WUE in a study with different wheat genotypes. Also

119

 

carbon isotope composition was correlated with WUE in several interspeciﬁc hybrid of
peach rootstock (Bongi et al., 1994).

Plants can adapt to water deﬁcit conditions by improving water uptake or
reducing water loss (Wang et al., 1998). Rootstocks have been shown to affect
photosynthetic rate (F erree and Barden, 1971), growth rate (Tubbs, 1973), and
transpiration rate and WUE (Bongi et al., 1994). Besides plant physiological
characteristics, morphology of the root system such as depth, and the number of ﬁne
roots are important factors for water absorption. Several reports on apple rootstocks have
shown contradicting results on their ability to tolerate water stress as inﬂuenced by the
vigor of the rootstock. Landsberg and Jones (1981) reported that dwarﬁng rootstocks,
and especially M9 were more tolerant to water stress than vigorous ones. Ferree and
Carlson (1987) showed that M9 was less tolerant to water stress than the vigorous
MM111. Fernandez et al. (1997 a and b) showed that the dwarﬁng rootstock ‘Mark’ was
the most drought sensitive, followed by the standard rootstock MM 1 1 1, while the most
drought resistant was the dwarﬁng rootstock M9 (Fernandez et al., 1997 a and b). In
sweet cherry ‘Mahaleb’ is considered to tolerate drought better than ‘Mazzard’ (Lang,
2000). Little is known on the effect of dwarﬁng and standard sweet cherry rootstocks on
growth, physiological parameters, and WUE when grown under water deﬁcit conditions.

Periods of water shortage can occur even in regions with high annual rainfall. If
water shortage coincides with a critical stage of tree and fruit growth and development,
reduction in yield could take place. In order to obtain high yields, the selection of the
most suitable rootstock in non-irrigated orchards is of critical importance. The objective

of this study was to compare WUE of standard and dwarﬁng rootstocks under

120

 

well-watered and water deﬁcit conditions. Also, we evaluated the performances of
standard and dwarﬁng rootstocks affected by water deﬁcit in terms of plant growth, gas

exchange parameters, and leaf carbon isotope composition.

Materials and methods

One-year-old ‘Rainier’ sweet cherry (P. avium L.) grafted on Mazzard (P. avium
L.), and Gisela 5 (P. cerasus (cv. Schattenmorelle) x P. canescens) rootstocks, were
maintained outdoors, in 11 L containers at the Horticulture Teaching and Research
Center, Michigan State University, East Lansing, MI. Trees were potted at dormant bud
on 13 May, 2002 in a mixture of 10% silt and clay, and 90% coarse sand, they were
pruned at three to ﬁve buds and subsequently trained to two to three branches. Nutrient
solutions with all the essential macro- and micro-nutrients (13-2-13, N-P-K, 6% Ca, 2%
Mg and micronutrients) was applied weekly in order to supply 0.5 g of N per plant, per
week. Foliar iron was periodically applied (Iron chelate DP). The pots were wrapped
with aluminum foil, to limit heat stress on the root system. Daily maximum and
minimum air temperature and precipitation during the experiment were recorded by an
automated weather station, located 300 m SW of the experimental plot, at the
Horticulture Teaching and Research Center (Michigan Automated Weather Network,
http://www.agweather.geo.msu.edu, Figure C.1 Appendix C).

Water treatments

 

Water applied during the experiment was based on the evapotranspiration (ET) of
trees, measured every two to three days. ET was estimated gravimetrically by weighting

the potted trees on a scale (METTLER, TOLEDO, SB32001 DeltaRange) and recording

121

 

their weight changes (Tan and Buttery, 1982). Two different water treatments were
applied: 1) well-watered (control, Wioov/o), which received 100% of the amount of water
lost by ET during the previous day, and 2) a water deﬁcit treatment (W50%), which
received 50% of the water applied to the control. All plants were initially grown for a
period of six weeks under well-watered condition where water was applied as needed
through a drip irrigation system, with two emitters per pot. Before starting the
application of the different water regimes, all pots were irrigated to ﬁeld capacity. Water
treatments were imposed from 48 to 69 days after bud break (DABB). Water was applied
each day, in the late afternoon. Every pot was covered with an aluminum lid to exclude
rain water.

Soil water content

 

Soil water content (SWC, % v/v) was determined with a time-domain
reﬂectometer (Mini Trace TDR, Soilmoisture Equipment Corp. Santa Barbara, CA),
using two 15-cm steel rods per pot (pots were approximately 22 cm in height).
Measurements were made 2, 5, 8, l3 and 20 days from the beginning of the treatments
(Topp and Davis, 1985; Topp et al., 1982).

Plant growth measurements

Shoot length and number of leaves per plant were recorded at 1, 7, 10, 14, and 21
days after the beginning of the treatments. Shoot length was measured from the base of
the shoots and relative shoot growth rate (RSGR, cm day”) was calculated from shoot
length of sequential measurements, according to the following equation:

RSGR : L2 — 101/At

122

where L1 and L2 stand for plant shoot length at time 1 and 2, respectively and At stands
for time between the two measurements, in days. The leaves were counted from the base
of each shoot, and the count included every unfolded leaf.

Three days after the beginning of the experiment, the three most recently unfolded
leaves per shoot were tagged, and width and length were measured. Increment in tree
leaf area during the experiment was estimated by measuring width and length of the three
original leaves plus every new unfolded leaf until no increment in either width or length
per leaf was detected. For each genotype, a regression equation was used to estimate the
cumulative leaf area when only width and length were known (Table 3.1). Measurements
of leaf width (point of maximum width) and length (from the petiole base to the leaf tip)
were taken at 3, 7, 11, 14, and 20 days after the beginning of the treatment.

Trunk diameter (cm) was measured at 10 cm above the graft union with a digital
caliper recording NS and EW dimensions twice during the experiment, at bud break (15
May) and at tree harvest (31 July).

Gas exchange measurements

 

Net carbon dioxide assimilation rate (A), stomatal conductance (gs), transpiration
rate (E), and internal C02 concentration (C,-) were measured at 1, 3, 4, 6, 7, 9, 10, ll, 12,
13, 14, 15, 16, 19, and 21 days after the beginning of the experiment, on three leaves per
plant with CIRAS-2 portable infrared gas analyzer (PP systems, Haverhill, MA). Fully
expanded leaves were selected and tagged at the beginning of the experiment and
photosynthetic measurements were made on the same leaves throughout the experiment.
Gas exchange measurements were taken in the morning between 9:00 am to 12:00 pm.

During gas exchange measurements, VPD ranged from 12 to 28 mbar and leaf

123

 

temperature ranged from 22 to 34 °C on different days, PPFD > 900 umol m'zs‘l, ﬂow
rate into the cuvette was 200 mL min], while C02 concentration into the cuvette was
held at 370 ppm.

On day 24 from the beginning of the treatment, gas exchange was evaluated four
times during the day, in order to detect any differences in the diurnal pattern of
photosynthetic rate between treatments and/or genotypes. Photosynthesis was measured
between 9:00 to 11:00 am, 11:00am to 1:00 pm, 1:00 to 3:00 pm, and 3:00 to 5:00 pm.

Photosynthetic water—use efﬁciency

 

Photosynthetic water use efﬁciency (WUE) was calculated as the ratio between
C02 assimilated (A, umol CO; m'2 s") and water loss through transpiration (E, mmol

H20 111'2 s“) (Wang et al., 1998; Zhang and Marshall, 1994).

Plant harvest

 

Trees were destructively harvested at the end of the experiment (day 30 from the
beginning of the experiment) to evaluate dry weight (DW) differences between control
and water deﬁcit. After washing the roots to remove the potting medium, trees were
divided into the following components: ﬁne roots (S 2mm diameter), coarse roots (>2mm
diameter), trunk, current growth shoots, and leaves. Plant components were dried to
constant weight for approximately 72 hours at 60°C, and DW was measured.

Leaf carbon isotope determination

 

Leaf samples collected at plant harvest were freeze-dried, and ground in a Wiley
Mill to pass a 40-mesh screen. Approximately 1 mg of DW of leaves was weighted into
tin capsules for mass spectrometry analysis (Automated Carbon and Nitrogen Analyzer,

Roboprep, Europa Scientiﬁc, Cheshire, England) to determine total C and 5'3 C.

124

 

 

 

Carbon isotope composition (8 '3 C) is calculated as follow:

5‘3ch8 (%o) = [(RS/RPDB) —1] x 1000

where RS is the 13C/IZC ratio measured in plant material and RPDB is the 13C/ 12C ratio of
the PDB standard, equal to 0.0112372 (Farquhar et al., 1982).
Merimental design

The experiment was a two—way factorial complete randomized block design,
analyzed as a repeated-measurement design. One factor was represented by the rootstock
(two rootstocks), and the other factor was the water treatment (two water treatments).
Five replications per rootstock per each water treatment were used (total of 20 trees).
Analysis of variance was performed using the MIXED procedure (SAS, Version 8, SAS
Institute, Cary, NC, USA) to detect treatment effects. When treatments effects were
signiﬁcant, means were separated using the Least-Squares means test (LSMEANS test)
with [930.05.
Table 3.1. Leaf area regression equations, number of leaf measured (11), and R2 for leaf

area prediction. Leaf area is calculated using leaf width (cm) and length (cm). Different
equations were developed for ‘Rainier/Mazzard’ (W) and ‘Rainier/Gisela 5’ (R/Gi5).

 

2

 

Plant Number of leaves used Regression equation for area R
_ (11)
R/M 148 -20.5067+ 5.20138 >< width + 3.16405 x length 0.95
R/Gi5 168 -25.7269+ 9.17772 x width + 1.93198 x length 0.94

‘

125

3e_w_lt§
Soil water content

Maximum soil water content (SWC) calculated at ﬁeld capacity was 16% (v/v).
SWC of the control trees was maintained between 10-12% (v/v) during the experiment
(Figure 3.1. A). SWC of the W50% trees was signiﬁcantly lower than the controls starting
8 days after the beginning of the experiment, when it was approximately 80% of the
SWC of the controls and continued to decrease until the end of the experiment (Figure
3.1. A and B). There were no signiﬁcant differences between SWC in R/Gi5 and R/M
W100% and between R/Gi5 and R/M W500, at any of the measurement times.

Plant growth parameters

 

Shoot elongation

Total shoot length increased from 10 to 90 cm per plant, between 25 and 60 Days
After Bud Break (DABB) (Figure C.2 Appendix C). No differences were detected
between the two rootstocks, on any of the measurement times (Figure C.2 Appendix C).

Table Cl and C2 , Appendix C, give a summary of occurrence over time of
signiﬁcant differences between WWW, and W500, of each rootstock, and differences
between controls or between water deﬁcit plants (expressed as percent of their controls)
of all growth parameters measured during the experiment.

Relative shoot growth rate (cm day") increased steadily until 48 DABB with a
maximum of 3 cm day" between 37 and 41 DABB and gradually decreased until 69
DABB (data not shown). Relative shoot growth rate started to decreased in W50% plants
from 7 days after the beginning of the treatments, but was not signiﬁcantly lower than

controls until the 14 through 21 days period. During this period of time, both R/GiS and

126

 

R/M W50% showed a signiﬁcant lower relative shoot growth rate compared to the
corresponding controls (Figure 3.2). The decrease in relative shoot growth rate was
similar in R/M and R/Gi5 W50%, when values where express as a percent of their controls
(data not shown).
Leaf emergence rate

Relative leaf emergence rate (leaves day'l) of R/Gi5 W100% plants was four times
higher than R/Gi5 W50% plants, between 14 and 21 days after the beginning of the
treatments (Figure 3.3. A). W W500, had a rate equal to zero between 14 to 21 days
from the beginning of the experiments and was signiﬁcantly lower than the W100% plants
(Figure 3.3. B). Relative leaf emergence rate of R/Gi5 Wsoo/o plants, expressed as a
percent of their controls, was higher than that of R/M W50% plants between 14 and 21
days from the beginning of the treatments (data not shown).
Cumulative lea area

Estimated cumulative increment in leaf area of R/Gi5 W50% was signiﬁcantly
lower than Wmoo/o plants from 11 days after the beginning of the treatments (Figure 3.4.
A). Estimated cumulative increment in leaf area of RM W50%was signiﬁcantly lower
than W100% plants ﬁ‘om 14 days after the beginning of the experiment (Figure 3.4. B).
Increment in leaf area was statistically higher for R/Gi5 W100% plants than R/M W100%
plants in all the measured dates (data not shown). When expressed as a percent of their
corresponding controls, no difference was detected between R/Gi5 and R/M W5o% plants

(data not shown).

127

 

Trunk cross sectional area

Although trunk cross sectional area of W100% and W500, plants was not statistically
different at the end of the experiment (data not shown), the percent of increase in trunk
cross sectional area from budbreak until tree harvest was signiﬁcantly higher in W100%
than W50% plants, in both rootstocks (Table 3.2).
Plant dry weight

Dry weight of ﬁne roots, coarse roots, trunk, shoot and leaves were not
signiﬁcantly affected by the two different water treatments, although there was a
tendency for the W50% plants of both genotypes to have a lower amount of total DW than
the W100% plants (data not shown). Root to shoot ratio was not signiﬁcantly affected by
the water treatments and there was no difference between the two rootstocks. The root to
shoot ratio was approximately 0.5 and 0.7 for R/Gi5 and R/M, respectively.

Gas exchange parameters

 

Table C1 and C2 , Appendix C, gives a summary of occurrence over time of
signiﬁcant differences between W100% and W500, of each rootstock, and differences
between controls or between water deﬁcit plants (expressed as percent of their controls)
of all gas exchange parameters measured during the experiment.

Net carbon dioxide assimilation rate

Values of net carbon dioxide assimilation rate (A) for W100% plants during the
experiment varied between 16.9 and 10.3 and between 14.3 and 9.05 umol COz-m'Z-s'1 for
R/GiS and R/M, respectively (Figure 3.5). Statistically signiﬁcant differences between
W100% and W50% plants were detected on day 7 for R/M and on 9 day for R/Gi5 (Figure

3.5). On day 8, A of W50% plants was approximately 80% of the control values, in both

128

 

genotypes, and it progressively decreased until it reached 20 % of the controls, on day 21.
Control plants of R/Gi5 had a signiﬁcantly higher A than R/M on days 3, 4, 9, 10, 13, 14,
15 and 21. Nevertheless, the two rootstocks under water deﬁcit conditions did not show
signiﬁcant differences when A was expressed as a percent of their controls, except on day
9 of the experiment, where R/M had a higher A then R/Gi5.
T ranspiration rate

Transpiration rate (E) for W100% plants throughout the experiment varied between
3.7 and 2.2, and between 2.2 and 1.7 mmol HZO‘m'Z-s'1 for R/Gi5 and IUM, respectively
(Figure 3.6). Control plants of R/M and of R/Gi5 had a signiﬁcantly higher E than R/M
W50% and R/Gi5 W50% plants starting from day 7 and day 6, respectively until the end of
the experiment (Figure 3.6). On day 8, E of W50% plants was approximately 80% of the
W100% plant values, in both genotypes, and it progressively decreased until it reached 20
% of W100% plant values, on day 21. Control plants of R/Gi5 had a higher E than R/M on
days 3, 4, 9, 15 and 21. Transpiration of the two genotypes under water deﬁcit conditions
did not differ, when expressed as a percent of their controls, except on days 3, 10 and 11
from the beginning of the treatments, where R/M had a higher transpiration rate than
R/Gi5.
Stomatal conductance

Values of stomatal conductance (gs) for W100% plants during the experiment
varied between 116 and 246 and between 118 and 219 mmol HzO-m'Z-s'l for R/Gi5 and
R/M, respectively (Figure 3.7). Statistically signiﬁcant differences between W100% and
W50% plants were detected at day 7 for R/M and at day 6 for R/Gi5 (Figure 3.7). Control

plants of R/Gi5 had higher gS than R/M on days 3, 4, l3, and 16. The two genotypes

129

under water deﬁcit conditions did not show any signiﬁcant differences when gs was
expressed as a percent of their controls except on day 3 and 11, where R/M had a higher
gS of R/Gi5.
Internal carbon dioxide

Values of internal COz (C) for W100% plants during the experiment varied
between 181 and 218, and between 192 and 233 uL L’1 for R/Gi5 and R/M, respectively
(Figure 3.8). Internal CO2 was statistically higher in IUGiS WlOOO/o than R/Gi5 W500),
plants on days 3, 9, 10, 11, l2, l4 and 15 (Figure 3.8. A). For R/M, it was statistically
higher in WSW, plants than Wlooo, plants on days 3 and 4, while it was higher for W100%
than W50% plants on days 9, 12, 14, '15, and 16 (Figure 3.8. B). The percent of C,- of W50%
plants during the experiment was between 80 and 110% of the W100% trees, for both
genotypes. Comparing the controls of the two genotypes, C was statistically higher for
R/M than R/Gi5 on days 3, 6, 9, l2, 14, 15, and 16. When expressed as percent ofthe
control, R/M W50% plants had a higher C,- than R/Gi5 W50% on days 3, 4, 10, and 12,
while on days 13 and 16 R/Gi5 W500, had higher values of C, than R/M W50% plants.

Water-use efﬁciency

 

Values of water-use efﬁciency (WUE) for Wmoo/o plants during the experiment
vary approximately between 3.4 and 6.5 umol C02 mmol"1 HzO, for both R/Gi5 and R/M
(Figure 3.9). On days 13 and 21, WUE was statistically higher in R/Gi5 W100% than
W50% plants while on day 14 R/Gi5 W50% plants had a higher WUE than W100% plants
(Figure 3.9. A). On days 3 and 21 of the experiment, WUE was statistically higher in
W100% than W50% plants for R/M while on days 14 and 16 R/M W50% plants had a higher

WUE than Wmoo/O (Figure 3.9. B). On days 9, 11, 12, 13, and 14 R/GiS WWW, plants had

130

 

a higher WUE than R/M Wlooo/0 plants (Figure 3.10. A). The percent of WUE of the
W50% plants was approximately between 110 and 90% of the controls with a few times
around 70% of the controls (Figure 3.10. B). On days 3, 10, ll R/Gi5 WSW, plants had a
higher WUE than R/M W50% plants, when expressed as a percent of their controls (Figure
3.10. B).

Daily trend of gas exchangggarameters

 

Assimilation rate was higher in W100% than W500, plants, during all the intervals
measured (Figure 3.11. 1). R/Gi5 WIOOU/o plants had higher A than R/M Wlooiyo between
9:00 and 11:00 am and 3:00 and 5:00 pm, while R/Gi5 Wsoo/0 plants had higher A than
R/M W50% between 9:00 and 11:00 am and 1 and 3 pm (Figure 3.11. 1). Transpiration
rate was higher for IUGiS and R/M controls than the same genotypes under water deﬁcit,
in all the intervals measured (Figure 3.1 l. 2). WUE decreased from approximately 8 to 4
umol COz mmol'1 HZO from 9:00 to 11:00 am to 3:00 to 5:00 pm, respectively, in both
treatments and genotypes (Figure 3.1 l. 3). WUE was different between W100% and W500,
plants only between 9:00 to 11:00 am (Figure 3.11. 3). Stomatal conductance decreased
from 9:00 to 11:00 am to 3:00 to 5:00 pm and was higher in W100% than W500, plants, in
both genotypes (Figure 3.12). Internal C02 was higher in R/M W100% plants compared to
the rest, between 9:00 to 11:00 am and l l :00 am to 1:00 pm (Figure 3.13). Between 1:00
and 3:00 pm, C, was signiﬁcantly higher in R/M and R/Gi5 controls than IUGiS W500,
plants (Figure 3.13). Also, between 1:00 and 3:00 pm, Ci was signiﬁcantly higher in R/M

W500, than R/Gi5 W50% plants (Figure 3.13).

131

 

Leaf carbon isotope composition

 

The 513C of leaves was affected by the different water treatments (Table 3.3).
Leaves of W50% plants of both genotypes had a more positive 513 C and a higher atom "/0
than their corresponding controls (Table 3.3). Comparing the two genotypes, R/M had

more negative values of 5'3 C than R/Gi5 (Table 3.3).

132

  
  
   

 

 

 

 

 

2 1
0 +R/GiS w100°o
18 ‘ - - o - - R/GiS ws0%
164 +R/MW100%
14- "'A"R/MW50%
9
g 12-
%, 10- _
o
a 8~ o-- --- .. -o...
W ‘---.5..‘ .-
4. iii
2..
O I I I I I I I I I
0 2 4 6 8 10 12 14 16 18 20
100- .“3 ﬁns 2’; *j’“ *:* B
90. é‘zizpné
E 80-
8
.9
“'5 70-
O\0 s i
3 60' +controls
m - - A - - R/GiS ws0% " ”*2...
50— --O--R/MW50% “v.5
40 I I I I I I I I I I
0 2 4 6 8 10 12 14 16 18 20

Days from the beginning of the experiment

Figure 3.1. Soil Water Content (SWC) measured with Time Domain Reflectometry
(TDR). A Comparison between ‘Rainier/Gisela 5’ (R/GiS Wlooo/o) under well-watered
and ‘Rainier/Gisela 5’ (IUGiS W50%) under water deﬁcit conditions, and between
‘Rainier/Mazzard’ (R/M Wmoo/o) under well-watered and ‘Rainier/Mazzard’ (R/M W50%)
under water deﬁcit conditions. B SWC of R/Gi5 WSW, and W W50% expressed as % of
their controls. Vertical bars indicate standard errors (SE, n=5) of means. ** and ***

stand for signiﬁcant at p S 0.01 and 0.001, respectively.

133

 

I R/Gi5 W100% A. R/Gi5
i lR/GiS W50%

,_‘
LA
I

RSGR (cm day'l)
S

0.5 ~

 

 

 

0.0 -

   

I R/M W100% B. R/M
1 lR/M W50%

  

RSGR (cm day")

NS. z ~‘X\\ “WWW «W\\\\\\“ $WN§§>X~W\X§R\WW§\W€ \1“ \\“\“§‘ “ 4 :

 

  

l-7 7-10 10-14 l4-2l

Days from the beginning of the experiment

Figure 3.2. Relative Shoot Growth Rate (RSGR, cm day'l) of sweet cherry cv. ‘Rainier’
plants. Comparison between A ‘Rainier/Gisela 5’ under well-watered (IUGiS W100%) and
water deﬁcit (R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-watered
(R/M W100%) and water deﬁcit (R/M W50%) conditions. Vertical bars indicate standard
errors (SE, n=5) of means. * stands for signiﬁcant at p S 0.05.

134

2.0 - . A. R/Gi5
I R/G15 w100%

1 l R/GiS W50%

‘N

RLER (leaves day'l)
.0
00

.0
4S

 

 

 

 

0.0
7—10 10-14 14—21
2-0 ‘ B. R/M
lRfM w100%
HR/M ws0%

RLER (leaves day“)

 

 

l-7 7-10 10—14 l4-21

Days from the beginning of the experiment

Figure 3.3. Relative Leaf Emergence Rate (RLER, leaves day'l) of sweet cherry
cv.‘Rainier’. Comparison between A ‘Rainier/Gisela 5’ under well-watered (R/G15
W100%) and water deﬁcit (R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well—
watered (R/M W100%) and water deﬁcit (R/M W50%) conditions. Vertical bars indicate
standard errors (SE, n=5) of means. *** stands for signiﬁcant at p S 0.001.

135

1200 7
I R/Gi5 W100%

' 0
1000 _ llR/GIS W504

800 -

600 ~

400 -

Cumulative leaf area (cmz)

200 -

 

 

 

   

1200 1
IR/M W100% B'R/M

1000 , {HUM w50%

800 -

600 ~

  
   

400 -

Cumulative leaf area (cmz)

200 -

awﬂﬁiﬁwmxwam .1;

 

  

.mgsgsmwm” A-z-ii‘mh'ﬁ

 

 

Days from the beginning of the experiment

Figure 3.4. Cumulative leaf area (cm2) of newly formed leaves of sweet cherry
cv.‘Rainier’. Comparison between A ‘Rainier/Gisela 5’ under well-watered (R/Gi5
W100%) and water deﬁcit (R/Gi5 W50%) conditions, and B ‘Rainier/Mazzard’ under well-
watered (R/M W100%) and water deﬁcit (R/M W50%) conditions. Vertical bars indicate
standard errors (SE, n=5) of means. *, **, and *** stand for signiﬁcant atp S 0.05, 0.01
and 0.001 , respectively.

136

Table 3.2. Percent of increase in trunk cross sectional area from bud break (15 May)
until tree harvest (31 July). Comparison between R/M and R/Gi5 under well-watered

(R/M and R/Gi5 W100%) and R/M and R/Gi5 under water deﬁcit conditions (WM and
R/Gi5 W50%).

 

 

 

 

 

Rootstock Increase in area
(%)

R/M W100% 17.5 i lZ

R/M W50% 7.1 i 2

R/G15 W100% 11.3 i 3

R/Gis W50% 4.7 i 3

Signiﬁcance

Planty NS

Treatment *

Plant x Treatment NS

 

2 Standard Error (n=4) of means

"’NS and " Non Signiﬁcant and signiﬁcant atpS0.05, respectively

137

 

A. R/Gis
+ R/GiS w100%
18 . - - <> - -R/Gi5 ws0% **

Hut:

A (umol C02 m'2 s")
S

 

 

 

B. R/M
+ R/M WlOO‘Vo

‘8 ‘ ”AH-R/MWSOO/o

 

A (itmol C02 in2 s")

 

 

 

0 I l I I f l I
0 3 6 9 12 15 18 21

Days from the beginning of the experiment

Figure 3.5. Plants net assimilation rate (A, umol COz m'2 3"). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/GiS W100%) and water deﬁcit (R/GiS W50%)
conditions, and B ‘Rainier/Mazzard’ under well-watered (R/M Wmoo/O) and water deﬁcit
(R/M W50%) conditions. Vertical bars indicate standard errors of means of ﬁve plants
(three leaves per plant). *, **, and *** stand for signiﬁcant at p S 0.05, 0.01. and 0.001,
respectively.

138

-2 -1
E (mmol H30 in s )

.— .—‘ I" E" 'r" 3” P P

O U1 0 UI O U} O U}

l l l l L 1 l J

9
LI!
1

 

+R/G15 W100%
- - O - - R/Gi5 W50%

 

A. R/GiS

***

 

 

.0
o

s")

l”
O
1

’3

E (mmol HZO m"
N .N
O U]
L l

in
J

+R/MW1000/o
- - ﬂ - - R/M W50%

***

**
**

***

‘5 an: . 5
§ I \
‘ , s
‘

***

B. R/M

 

 

I I I

6 9 12

15

Days from the beginning of the experiment

Figure 3.6. Plants transpiration rate (E, mmol HZO m'2 8"). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/Gi5 W100%) and water deﬁcit (R/Gi5 W50%)
conditions, and B ‘Rainier/Mazzard’ under well-watered (R/M Wmm) and water deﬁcit
(R/M W50%) conditions. Vertical bars indicate standard errors of means of ﬁve plants
(three leaves per plant). *, **, and *** stand for signiﬁcant at p S 0.05, 0.01 and 0.001,

respectively.

139

 

 

300 _ +R/GiS W100%

 

 

 

 

 

A. R/GiS
- - O ' ' R/GiS W50%
Hunt:
250 -
a” 200 -
E
Q.
I 150 .
'5
E
E 100 ~
613
50 e
0 I I I I I I
O 3 6 9 12 15 18 21
300 - B. R/M
+R/M W100%
250'4 ""A"R/MW50% ***
A *** ***
-—'- ***
ﬁlm 200 ‘ *** ***
E ** ** II: ***
C2. . *
I 150 - ,. " ‘.
_. A ~
52 . .
E 100 ' 5mg“;
t i.
‘. ,5- '5.
50 - Z' 23 """"" A.
‘n
0 r I I I7 I I I
0 3 6 9 12 15 18 21

Days from the beginning of the experiment

Figure 3.7. Plants stomatal conductance (gs, mmol HZO m'2 5"). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/Gi5 Wmoo/o) and water deﬁcit (R/GiS W50%)
conditions, and B ‘Rainier/Mazzard’ under well—watered (R/M W100%) and water deﬁcit
(R/M W50%) conditions. Vertical bars indicate standard errors of means of ﬁve plants

(three leaves per plant). *, **, and *** stand for signiﬁcant atp S 0.05, 0.01 and 0.001,

respectively.

140

 

 

A. R/GiS

 

 

 

260 -
+ R/Gi5 WlOOO/o
- . . . "' " 0
240 ‘ 0 W013 WDO /0
. * ***
220 - ‘.
_'__1 \X‘ Mai-
:11 200 _ §~ *** "
I: ‘ .~ . *** Hut ' I’
U 7 7 . ' ‘.. ~ ' I §. I
180.. ‘.‘§ '1 I éa‘ ‘~~‘ I
\‘\ é: 6“ ’1' ‘ . ~.1
160 « § § . 9
I40 I I I II I I 1
0 3 6 9 12 15 18 21
B. W
260 ' +R/M W100%
** -- a - - R/M ws0%
240 - 1% **,, 4n
\ ***
1’ \ * *** ‘

220 4 . . ,
I ‘ ‘ I
A ~.__ , r ‘ ,
u I ~ -- l
A\ A \ ,
200 . .% é ~\ .

1804 ii% ..-%

160‘

C. (11L L“)

 

140 I I 1 1 f i a
0 3 6 9 12 15 18 21

 

Days from the beginning of the experiment

Figure 3.8. Leaf intercellular CO; concentration (C i, 11L L'l). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/G15 W100%) and water deﬁcit (R/Gi5 W50%)
conditions, and B ‘Rainier/Mazzard’ under well-watered (R/M W100%) and water deﬁcit
(R/M W50%) conditions. Vertical bars indicate standard errors of means of ﬁve plants
(three leaves per plant). *, **, and *** stand for signiﬁcant atp S 0.05, 0.01 and 0.001,
respectively.

141

 

 

A. R/GiS
+ R/G15 W100°/o

." -- o - - R/GiS ws0%

"
‘

WUE (timol CO 2 mmol '1 HzO)

 

 

+R/MW100%
7. .. ---A--R/MW50%

WUE (pmol COleIanl-I 1130)
D

 

 

2 I I I I I I
0 3 6 9 12 15 18

Days from the beginning of the experiment

Figure 3.9. Water-use efﬁciency (WUE, uCOz mmol'] H20). Comparison between A
‘Rainier/Gisela 5’ under well-watered (R/Gi5 Wmoo/o) and water deﬁcit (R/GiS W50%)
conditions, and B ‘Rainier/Mazzard’ under well-watered (R/M W100%) and water deﬁcit
(R/M W50%) conditions. Vertical bars indicate standard errors of means of ﬁve plants
(three leaves per plant). * and ** stand for signiﬁcant at p S 0.05 and 0.01, respectively.

142

 

 

 

8-
+R/G15WIOOO/o
-1I-R/N1VV10096
6 7- an:
I
'25 6-
E
E * u- :1:
Q” 5~
U I
E v ‘
55.. 4~ *
1:; / l,
:3
3 3-
2 ﬁ I I ﬁ f I I
0 3 6 9 12 15 18 21
140* B
"(D"I{K3ﬁSVV5096
130- --ﬁ"R/MW50%
3|:
1204

110‘ % $ ‘% ,’ .~§‘ I. ‘g ~.“~g
100 § +’ "i" 6" SE “ -------- f

90 - i l
80 - §

70-

WUE (% of controls)

 

 

 

60 I I I I I I I
O 3 6 9 12 15 18 21

Days from the beginning of the experiment

Figure 3.10. Water-use efﬁciency (WUE, umol COz mmol'l H20). A Comparison
between ‘Rainier/Gisela 5’ (R/Gi5 Wloocyo) and ‘Rainier/Mazzard’ (R/M Wloocyo) under
well-watered conditions. B WUE of ‘Rainier/Gisela 5’ (R/G15 W50%) and
‘Rainier/Mazzard’ (R/M W50%) under water deﬁcit conditions expressed as °/o of their
controls. Vertical bars indicate standard errors of means of ﬁve plants (three leaves per
plant). * and ** stand for signiﬁcant at p S 0.05 and 0.01, respectively.

143

 

 

 

 

Figure 3.11. Daily trend of 1. net carbon dioxide assimilation rate (A), 2. transpiration
rate (E), and 3. water-use efﬁciency (WUE) of sweet cherry cv.‘Rainier’. Comparison
between ‘Rainier/Gisela 5’ under well-watered (R/GiS W100%) and water deﬁcit (R/GiS
W50%) conditions and ‘Rainier/Mazzard’ under well-watered (R/M W100%) and water
deﬁcit (R/M W50%) conditions. Vertical bars indicate standard errors of the means.
Treatment means with different letters are signiﬁcantly different at p S 0.05 (Least—square
means test). * and ***: signiﬁcant atp S 0.05 and 0.001, respectively.

144

 

A (pmol CO: in.2 s'I )

E (mmol 1120 m-2 s-I )

-0)

-1
mmol 11».

WU E (timol CO~

—
J]
I

.—
IQ
L

‘0
1

C‘
1

 

+R/Gi5 W100%
- - O --R/GiS W50%
+R/MW1000/o
- - 1A - - R/M W50%

altt

a**#

 
  
   
 

 

 

9:00 AM

1 1:00 AM 1:00 PM 3:00 PM 5:00 PM

at“
a*** a

 

 

 

 

00
9:00AM

10-1

 

 

11:00 AM 1:00 PM 3'00 PM 5:00 PM

 

 

 

9:00 AM

I I I I

11:00 AM l:00 PM 3:00PM 5:00PM

Dayti me

145

 

 

 

 

- _ + W615 W100%
130 a***
”"O' R/GlS W50%
+ R/M W'lOOo/o
----A--- W wso‘ro
12() ‘
7U}
("IE 90 —
9.
E 60 -
.5
&)
30 ‘
O x . .
9:00 AM 11:00 AM 1300 PM 3100 PM 5300 PM

Daytime

Figure 3.12. Daily trend of stomatal conductance (g) of sweet cherry cv. ‘Rainier’.
Comparison between ‘Rainier/Gisela 5’ under well-watered (R/GiS W100%) and water
deﬁcit (R/Gi5 W50%) conditions and ‘Rainier/Mazzard’ under well-watered (R/M W100%)
and water deﬁcit (R/M W50%) conditions. Vertical bars indicate standard errors (SE, n=5)
of the means. Treatment means with different letters are signiﬁcantly different at p S
0.05 (Least-square means test). * and ***: signiﬁcant atp S 0.05 and 0.001, respectively.

146

250 _ + R/GiS W1000/o

 

 

 

 

mom R/GiS ws0%
a*** +R/M WlOO°/o
225 _ a... a ----A--- W ws0%
A 200 -
'_.J
._1
3
U 175 -
150 .
125 . . . .—
9:00AM 11:00AM 1:00 PM 3:00PM 5:00PM

Daytime

Figure 3.13. Daily trend of internal COZ (Ci) of sweet cherry cv. ‘Rainier’. Comparison
between ‘Rainier/Gisela 5’ under well-watered (R/GiS W100%) and water deﬁcit (R/Gi5
W50%) conditions and ‘Rainier/Mazzard’ under well-watered (R/M Wlooo/o) and water
deﬁcit (R/M W50%) conditions. Vertical bars indicate standard errors (SE, n=5) of the
means. Treatment means with different letters are signiﬁcantly different at p S 0.05
(Least-square means test). * and ***: signiﬁcant atp S 0.05 and 0.001, respectively.

147

 

Table 3.3. Carbon isotope composition (513C, %o) and atom % of leaves of one-year-old
sweet cherry ‘Rainier/Mazzard’ under well-watered (R/M W 100%) and water deﬁcit
conditions (R/M W50%) and ‘Rainier/Gisela 5’ under well-watered (R/Gi5 W100%) and
water deﬁcit conditions (IUGiS W50%). Leaves were collected when trees were harvested.

 

 

 

 

Rootstock 513C (%o) Atom %
R/M W100% -28.6 a2 1.79 d
R/M W50% -28.0 b 1.80 c
R/G15 W100% -27.5 C 1.81 b
R/Gi5 W50% -26.7 d 1.82 a

Signiﬁcance

Plant” * *

Treatment ** **

Plant x Treatment NS NS

 

7' Means within columns followed by different letters are signiﬁcantly different by LSMEANS test at
pS0.05.

yNS'i‘ and": Non Signiﬁcant and signiﬁcant at pS0.05, 0.01, respectively

 

148

 

 

Discussion

The ﬁrst growth parameter affected by water deﬁcit in both R/G15 and R/M was
the cumulative leaf area. Shoot growth rate and leaf emergence rate were also affected
by water deﬁcit, but only during the last seven days of the experiment. Shoot growth rate
and leaf emergence rate were not affected to the same extend of cumulative leaf area,
perhaps because the experiment was done during the last period of shoot growth (Figure
C.2, Appendix C). The growth parameter more affected was the length of the less
vigorous shoots, in a comparison between apple rootstocks with different vigor, exposed
to water stress (Fernandez et al., 1997a). In addition, trunk cross sectional area and leaf
emergence rate were affected differently depending on the genotype of the rootstock, but
the observed differences did not follow a certain trend based on tree vigor, as it may have
been expected (Fernandez et al., 1997a). In our experiment, the growth parameters
measured on the R/Gi5 and R/M under water deﬁcit conditions were affected in a similar
way and to the same extend in the two genotypes when compared to their controls. This
ﬁnding indicates that the two genotypes have a similar degree of sensitivity to water
deﬁcit in terms of growth.

Leaf expansion rate and leaf emergence were more sensitive than stomatal
conductance and leaf water potential to water stress in peach trees (Olien. and Flore,
1990). In our experiment, gas exchange parameters were affected earlier than all growth
parameters measured. Stomata closure affected A and E in a similar way in standard
plants, since reduction in A and E was detected at the same time, while in dwarf plants
assimilation rate was affected three days later than E, indicating that A in dwarf plants

was less influenced by water deﬁcit. This is further supported by a larger decrease in C.

149

 

 

in dwarf than in standard plants. Higher internal COz of standard than dwarﬁng controls
indicated a limitation in the use of C02 in standards, which can be explained by their
lower assimilation rate. We can speculate that stomatal conductance was not the limiting
factor for assimilation rate in standard plants. In both genotypes under progressively less
soil water availability, C,- generally decreased but when soil water availability decreased
further, internal C02 showed a sharp increase. This is in accordance with observations of
C. in conifers (Bodribb, 1996), in wheat and sunflower (Lawlor, 1995), under decreasing
relative water content in leaves. The sharp increase in C.- perhaps indicates also an effect
on the biochemical processes of photosynthesis.

Water-use efﬁciency was not signiﬁcantly different between the two genotypes in
most of the days measured. From day 9 to day 14 from the beginning of the treatments,
control plants of dwarﬁng rootstocks showed a higher WUE than control plants of
standard rootstocks (Figure 3.10 A). The differences in WUE were mainly due to the
higher photosynthetic rate of the dwarf compared to standard plants, even though
transpiration was also higher in dwarf than in standards plants. WUE has been found to
be higher on vigorous than dwarﬁng interspeciﬁc hybrid peach rootstocks but it was due
to lower transpiration in vigorous than dwarf plants (Bongi et al., 1994). On the contrary,
Fernandez et a1. (1997 b) found similar WUE in apple rootstocks with different vigor. In
our experiment, plants of both rootstocks did not show any increase in WUE compared to
their controls when challenged with water deﬁcit (Figure 3.9). Similarly, in peach trees
grown at elevated COZ, WUE was not improved in water stressed plants (Centritto et al.,
2002). Since both rootstocks did not improve their WUE under water deﬁcit conditions,

other plants characteristics such as root system mmphology may play an important role

150

 

 

under water deﬁcit conditions. Alternatively, the values obtained in this experiment,
were already high enough so any further decrease in stomatal conductance to reduce
transpiration would have negatively affected the photosynthetic rate as well. In general,
WUE during the experiment ranged from 3.4 to 6.4 umol COz mmol'l 1120. These values
are higher than the ones obtained for kiwifruit plants that are considered quite inefﬁcient
in the use of water, where WUE ranged from 2.8 to 4.7umol COz mmol'I H20 from 90 to
200 days after bud break (Buwalda and Smith, 1990).

Daily measurements of gas exchange parameters indicated a general decrease in
A and gS during the day, independent from the water regimes of the plants. Werber and
Gates (1990) suggested that diurnal reduction in A resulted from increased water loss and
a more negative water potential, which leads to stomatal closure. Neither Gucci et al.
(1991) in a study on defruited plums, nor Layne and Flore (1993) studying continuously
illuminated cherry, were able to show that diurnal decline in A was caused by a change in
water potential. Flore and Layne (1996) proposed that the diurnal decrease in gs is a
result of reduction in A due to feedback inhibition and not to water stress. Although leaf
water status was not evaluated during this experiment, based on the observed decline of
A independently from the water availability of plants, we can also speculate that
reduction of gS was due to a reduction in assimilation rate. The diurnal trend of gas
exchange measurements evidenced that differences between controls and plants under
water deﬁcit were independent from the time of the day where gas exchange parameters
were evaluated. Water use efﬁciency decreased from mid morning until late afternoon

due to both a decrease in A and a slight increase in E (Figure 3.1 1).

151

 

 

In general, when soil moisture level decreases, the ratio of intercellular to
atmospheric partial pressure of C02 (pi/pa) will decrease, if stomata close and
photosynthesis continue to operate (Farquhar et al., 1989). This could be measured by an
increased in ’3 C isotopic composition (813 C) (Farquhar et al., 1989). The higher 513C
values observed in W50% plants of both genotypes were caused by the increased use of
intercellular COz. Internal COZ of W50% plants was signiﬁcantly lower than in controls
from day 9 to day 16 (Figure 3.8); this was caused by a higher uptake of internal C02 and
less discrimination against the heavier isotope ’3 C. Duranceau et al. (1999) found that
under decreasing relative water content (RWC) in leaves, 13C enrichment was correlated
with decreased pi/pa in Phaseolus vulgaris. When leaf RWC decreased, sucrose and
starch became enriched in 13C, since A was supported by the CO; present in the

intercellular spaces (Duranceau et al., 1999).

Conclusions

1) Growth parameters measured on dwarﬁng and standard trees under water
deﬁcit conditions were affected in a similar way, and to the same extent, when compared
to their controls. These ﬁndings indicate that the two genotypes have a similar degree of
sensitivity to water deﬁcit in terms of growth.

2) Gas exchange parameters like assimilation and transpiration rate, stomatal
conductance, and internal C02 were affected earlier than all growth parameters measured,
perhaps because the water deﬁcit treatments were applied during the last period of shoot

growth.

152

 

 

3) Leaves 513 C values of dwarﬁng and standard trees under water deﬁcit
conditions increased compared to their controls. 513C values were higher in plants under
water deﬁcit due to their increasing use ofintercellular C02.

4) Water use efﬁciency ranged from 3.4 to 6.4 umol COZ mmol'l H20 and was
not signiﬁcantly different between dwarﬁng and standard trees, in most of the days
measured.

5) Dwarﬁng and standard trees under water deﬁcit conditions did not show any

increase in W 1 1E compared to their controls.

These ﬁndings indicate that irrigation is an important agronomic practice to be
considered in sweet cherry orchard, especially when trees are grown on dwarﬁng
rootstocks characterized by shallow root systems, or when orchards are established on

sandy, and sandy-loam textured soils, with limited water holding capacity.

153

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185.

Bongi, G., A. Palliotti, P. Rocchi, and G. Roselli. 1994. Evaluation of WUE in peach
grafted on different interspeciﬁc hybrid rootstocks. Plant Physiol. Biochem.
32:149—157.

Buwalda, J.G. and GS. Smith. 1990. Acquisition and utilization of carbon, mineral
nutrients and water by the kiwifruit vine. Hort. Rev. 12:307-347.

Centritto, M., M.E. Lucas, and PG. Jarvis. 2002. Gas exchange, biomass, whole-plant
water use efﬁciency and water uptake of peach (Prunus persica) seedlings in
response to elevated carbon dioxide concentration and water availability. Tree
Physiol. 22:699-706.

Davies, W.J., T.A. Mansﬁeld, and AM. Hetherington. 1990. Sensing of soil water status
and the regulation of plant growth and development. Plant Cell Environ. 13:709-
719.

Duranceau, M., J. Ghashghaie, F. Badeck, E. Deleens, and G. Comic. 1999. 51" C of C02

respired in the dark in relation to 513C of leaf carbohydrates in Phaseolus vulgaris
L. under progressive drought. Plant Cell Environ. 22:515-523.

Farquhar, G.D., J .R. Ehleringer, and KT. Hubick. 1989. Carbon isotope discrimination
and photosynthesis. Ann. Rev. Plant Physiol. Plant Mol. Biol. 40:503-537.

Farquhar, G.D., M.H. O’Leary, and J.A. Berry. 1982. On the relationship between
carbon isotope discrimination and the intercellular carbon dioxide concentration
in leaves. Austral. J. Plant Physiol. 9:121-137.

F arquhar, OD. and RA. Richards. 1984. Isotopic composition of plant carbon correlates
with water-use efﬁciency of wheat genotypes. Austral. J. Plant Physiol. 11:539—
552.

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Field, C., J. Merino, and HA. Mooney. 1983. Compromises between water—use

efﬁciency and nitrogen-use efﬁciency in ﬁve species of California evergreen.
Oecologia 60:384-389.

Fernandez, R.T., R.L. Perry, J .A. Flore. 1997a. Drought response of young apple trees
on three rootstocks: growth and development. J. Amer. Soc. Hort. Sci. 122214-19.

Fernandez, R.T., R.L. Perry, J .A. Flore. 1997b. Drought response of young apple trees
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Ferree, DC. and RF. Carlson. 1987. Apple rootstocks. In: ‘Rootstocks for fruit crops.’
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Ferree, ME. and J.A. Barden. 1971. The influence of strains and rootstocks on

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157

 

SUMMARY AND CONCLUSIONS

158

SUMMARY AND CONCLUSIONS

This research was initiated to detemiine if dwarﬁng and standard rootstocks
differed in N-fertilizer uptake efﬁciency, N use efﬁciency (NUE), and water-use
efﬁciency (WUE). Application ofN influences tree growth and development during the
current year and in the following growing season (Weinbaum, 1987). The seasonal
pattern ofN uptake in trees reﬂect their N demand and can be use for timing N-fertilizer
application in order to maximize fertilizer uptake (Weinbaum et al., 1992).

N uptake in one-year-old sweet cherry trees followed the accumulation of dry
matter in trees. N-fertilizer uptake was high at rapid shoot growth, and was fairly
constant until the beginning of leaf senescence, when it decreased considerably. Overall,
there was no difference in N-fertilizer uptake efﬁciency between dwarﬁng and standard
trees with the exception of terminal bud set, where dwarf and semi-dwarf trees
‘Rainier/Gisela 5’ and ‘Rainier/Gisela 6’ were more efﬁcient in N-fertilizer uptake than
standard trees ‘Rainier/Mazzard’. Rootstocks without scion differed in N-fertilizer
uptake at terminal bud set and at the beginning of leaf senescence, and ‘Gisela 6’ was
more efﬁcient than both ‘Gisela 5’ and ‘Mazzard’ due to its higher increase in plant DW.
Comparisons of genotypes N-fertilizer uptake efﬁciency obtained in this study are
applicable to non-bearing ﬁeld grown trees and can be used to optimize time of
application ofN fertilizer.

N-fertilizer applied at bloom and rapid shoot growth in ﬁve-year-old ﬁeld-grown
sweet cherry trees on dwarﬁng and standard rootstocks was absorbed in greater amount
than N—fertilizer applied at the beginning of leaf senescence. Based on these results,

fertilization practices for sweet cherry should be Optimized considering application of N-

159

fertilizer at bloom and rapid shoot growth (40 days after bloom). N-fertilizer contributed
in higher amount to the total N content of leaves of sweet cherries on dwarﬁng than
standard rootstocks, especially when N was applied at bloom. When dwarﬁng rootstocks
are utilized in sweet cherry orchard, N-fertilization may play an important role to sustain
the growth of the tree, particularly during the early stages of growth. Even if the
contribution of N-fertilizer was higher in dwarf than standard trees, the overall amount of
N-fertilizer absorbed by dwarf trees may be limited. Previous studies on apple grown on
the dwarﬁng rootstock M9 indicated low N requirements, between 8 and 44 kg N ha'1
(Neilsen and Neilsen, 2002). Therefore N-fertilizer requirements of sweet cherry on
dwarﬁng rootstocks need to be carefully evaluated to avoid risks associated to
overfertilization.

Nitrogen-use efﬁciency evaluated on one-year-old potted sweet cherry was higher
in the standard rootstock ‘Mazzard’ than in the dwarﬁng rootstocks ‘Gisela 5’ and the
semi-dwarﬁng rootstock ‘Gisela 6’, while when cv. ‘Rainier’ was grafted as scion, the
genotypes had similar NUE. Considering that ‘Mazzard’ had a similar value of NUE of
‘Rainier/Gisela 5’, ‘Rainier/Gisela 6’ and ‘Rainier/Mazzard’, the aerial part of the plant
appears to have higher incidence in determining the differences in plant NUE rather than
the root system. Dwarﬁng and standard rootstocks did not differ in terms of NUE when
evaluated on the base of leaf N concentration, under ﬁeld conditions. Also
retranslocation of N from senescent leaves was similar between dwarﬁng and standard
rootstocks. Since retranslocation of N from senescent leaves is another aspect of trees
NUE, it can be concluded that even based on this process, standard and dwarﬁng trees

did not differed in terms of NUE.

160

Understanding how rootstocks adapt and respond to water stress is essential in
order to select the proper genotype and irrigation method for situations where drought
stress is likely to occur. When WUE was evaluated under well-watered conditions in
one-year-old potted trees over a period of 60 days, there was no difference in temis of
WUE between ‘Rainier/Gisela 5’, ‘Rainier/Gisela 6’ and ‘Rainier/Mazzard’, although
‘Rainier/Gisela 5’ had a higher evapotranspiration compared to ‘Rainier/Gisela 6’ and
‘Rainier/Mazzard’. ‘Mazzard’ rootstocks had a higher WUE compared to both ‘Gisela 5’
and ‘Gisela 6’ due to the higher increase in biomass during the period considered.

Growth parameters measured on dwarf trees ‘Rainier/Gisela 5’ and on standard
tree ‘Rainier/Mazzard’ under water deﬁcit conditions were affected in a similar way, and
to the same extend in the two genotypes, when compared to their controls. These
ﬁndings indicate that dwarf and standard trees have a similar degree of sensitivity to
water deﬁcit in terms of growth. Gas exchange parameters like assimilation and
transpiration rate, stomatal conductance, and internal COZ were affected earlier than all
growth parameters measured, perhaps because the water deﬁcit treatments were applied
during the last period of shoot growth. Leaves 813C values of dwarf and standard trees
under water deﬁcit conditions increased compared to their controls. 5’3 C values were
higher in plants under water deﬁcit due to their increasing use of intercellular C02.
Water use efﬁciency ranged from 3.4 to 6.4 umol COZ mmol'I H20 and was not
signiﬁcantly different between the two genotypes, in most of the days measured. The
few differences in WUE observed during the experiment indicated a higher WUE of
dwarf than standard trees, and were mainly due to the higher photosynthetic rate of the

dwarf compared to standard trees. Water-use efﬁciency of standard and dwarf trees

161

under water deﬁcit conditions was not higher compared to their controls. Overall, these
ﬁndings indicate that irrigation is an important agronomic practice to be considered in
sweet cherry orchard, especially when sweet cherry are grown on dwarﬁng rootstocks,
characterized by shallow root systems or when orchards are established on sandy, and

sandy-loam textured soils, with limited water holding capacity.

Literature cited

Neilsen, D. and G.H. Nielsen. 2002. Efﬁcient use ofnitrogen and water in high-density
apple orchard. HortTechnology 12:19-25.

Weinbaum, S.A., R.S. Johnson, and TM. DeJong. 1992. Causes and consequences of
over fertilization in orchards. HortTechnology 2:1 12-121.

Weinbaum, S.A., I. Klein, and Muraoka T.T. 1987. Use of nitrogen isotopes and a light-
textured soil to assess annual contributions of nitrogen from soil and storage pools
in mature almond trees. J. Amer. Soc. Hort. Sci. 112:526-529.

162

 

 

APPENDIX A

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165

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168

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Total shoot length (cm)

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DABB

Figure C.2. Total shoot length (cm) per plant of sweet cherry cv. ‘Rainier’ measured at
different Days After Bud Break (DABB). Comparison between ‘Rainier/Gisela 5’
(R/GiS) and ‘Rainier/Mazzard’ (R/M). Vertical bars indicate standard errors of means
(SE, n=3 0). Arrow indicates the beginning of different water treatments.

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