Title: Effects of sodium chloride and polyethylene glycol on the water relations, growth, and morphology of citrus rootstock seedlings
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Title: Effects of sodium chloride and polyethylene glycol on the water relations, growth, and morphology of citrus rootstock seedlings
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Language: English
Creator: Zekri, Mongi, 1955-
Copyright Date: 1987
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EFFECTS OF SODIUM CHLORIDE AND POLYETHYLENE GLYCOL
ON THE WATER RELATIONS, GROWTH, AND MORPHOLOGY
OF CITRUS ROOTSTOCK SEEDLINGS








By

MONGI ZEKRI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1987












In the name of God,
Most Gracious,
Most Merciful.

"It is He Who has let free the two bodies
of flowing water: one palatable and sweet
and the other salt and bitter: yet has He
made a barrier between them, a partition
that is forbidden to be passed."

Glorious Quran
Sura XXV (Furquan), or The Criterion
Verse #53


In the name of God
Most Gracious,
Most Merciful.

"It is He Who sendeth down rain from the
skies: with it We produce vegetation of all
kinds: from some We produce green (crops),
out of which we produce grain, heaped up (at
harvest); out of the date-palm and its sheaths
(or spathes) (come) clusters of dates hanging
low and near: and (then there are) gardens of
grapes, and olives, and pomegranates, each
similar (in kind) yet different (in variety):
when they begin to bear fruit, feast your eyes
with the fruit and the ripeness thereof. Behold!
in these things there are signs for people who
believe.
Yet they make the Jinns equals with God, though
God did create the Jinns; and they falsely, having
no knowledge, attribute to Him sons and daughters.
Praise and glory be to Him! (for He is) above
what they attribute to Him!
To him is due the primal origin of the heavens
and the earth: how can He have a son when He hath
no consort? He created all things, and He hath
full knowledge of all things.
That is God, your Lord! There is no god but He,
The Creator of all things: then worship ye Him:
and He hath power to dispose of all affairs."

Glorious Quran
Sura VI (An'am), or Cattle
Verses #99-102
















ACKNOWLEDGMENTS


The author expresses his deepest appreciation to his wife, Leila,

for her assistance, encouragement, and patience. He also wishes to

express his sincere gratitude to his mother and to all the family in

Tunisia for their patience and understanding through the years the

author was away from home.

The author expresses his profound gratitude to Dr. L.R. Parsons,

chairman of the supervisory committee, for his valuable advice and

helpful suggestions in the course of conducting the research and in the

preparation of the manuscript.

Sincere thanks re extended to Dr. R. C. J. Koo and to Dr. W. S.

Castle for their advice and for providing greenhouse space.

A special debt of gratitude is acknowledged to Dr. D. L. Myhre and

to Dr. A. G. Smajstrla for their helpful suggestions and comments and

for kindly serving on the supervisory committee.

The author is also grateful to Dr. J. P. Syvertsen and Mr. M. L.

Smith, Jr., for providing equipment and for the use of their laboratory

facilities.

The author's most sincere gratitude is extended to the coordinators

of the Tunisia Agricultural Technology Transfer Project for continuous

encouragement and financial support.
















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS. . . . . . . . . .. . . . iii

LIST OF TABLES . . . . . . . . ... . . . vi

LIST OF FIGURES . . . . . . . . .. . . . ix

ABSTRACT. . . . . . . . . ... . . . .... xii

INTRODUCTION. . . . . . . . . . .... 1

LITERATURE REVIEW . . . . . . . . . .. .. 3

Salts . . . . . . . . . . . . . . 3
Mechanisms of Salt Tolerance in Plants. . . . . . 3
Mechanisms of Salt Injury . . . . . . . . . 4
Osmotic Effect . . . . . . . . ... . 4
Ion Toxic Effect . . . .. . . . . . 5
Nutritional Imbalance. . . . . . . . . 6
Plant Responses to Salinity . . . . . . . . 7
Salinity and Citrus . . ... . . . . . . . 8
Citrus Salinity Research . . . . . . . . 8
Citrus Tolerance to Salinity . . . . . . . 11
Scion . . . . . . . . . . . 11
Rootstock . . . . . . . .. . 11
Salt exclusion . . . . . . . . . 12
Ion concentration ................ .12
Citrus Responses to Saline Conditions . . . ... 13
Photosynthesis . . . . . . . . . 13
Yield . . . . . . . ... . . 14
Leaf injury . . . . . . . .... . 14
Salinity and high water table . . . . .. 15
Irrigation . . . . . . . . .. .. . 15
Reducing Salt Damage . . . . . . . .... . 17
Role of Calcium. . . . . . . . .... . 17
Genetic Improvement . . . . . . . . . 18

MATERIALS AND METHODS . . . . . . . . ... . . 20

General Procedures .. . . . . . . . . . 20
Experiment 1: Effects of NaCl and PEG on the Root
Conductivity and Leaf Ion Content of Seedlings
of 7 Citrus Rootstocks ... . ... . . . . 21
Experiment 2: Water Relations of Sour Orange and Cleopatra
Mandarin Seedlings under NaC1 and PEG Stresses ... 26










Page


Experiment 3: Fibrous Root Density and Distribution of Sour
Orange Seedlings under NaCl and PEG Stresses . . .
Experiment 4: Response of Split-Root Sour Orange Seedlings
to Salinity . . . . . . . . . . .
Experiment 5: Effects of Calcium on Sour Orange Seedlings
Grown under Saline Conditions . . . . . . .

RESULTS . . . . . . . . . . . . . . .

Experiment 1: Effects of NaC1 and PEG on the Root
Conductivity and Leaf Ion Content of Seedlings
of 7 Citrus Rootstocks . . . . . . . . .
Experiment 2: Water Relations of Sour Orange and Cleopatra
Mandarin Seedlings under NaCl and PEG Stresses . . .
Experiment 3: Fibrous Root Density and Distribution of Sour
Orange Seedlings under NaCl and PEG Stresses . . .
Experiment 4: Response of Split-Root Sour Orange Seedlings
to Salinity . . . . . . . . . . .
Experiment 5: Effects of Calcium on Sour Orange Seedlings
Grown under Saline Conditions . . . . . . .
Comparison of Citrus Seedling Responses to NaC1 and PEG
Treatments . . . . . . . . . . .

DISCUSSION . . . . . . . . . . . . . .


Leaf Ion Content and Salinity Tolerance . . . .
Rootstock Tolerance . . . . . . .
Ion Exclusion and Accumulation . . . . .
Leaf Ion Content and Ion Toxicity . . . .
Importance of Calcium under Saline Conditions
Physiological Effects of NaC1 and PEG . . . .
Effect of NaC1 on Root Conductivity . . .
Effect of PEG on Root Conductivity . . . .
Effect of NaC1 on Stomatal Conductance . . .
Effect of PEG on Stomatal Conductance . . .
Effect of NaC1 and PEG on Chlorophyll . . .
Effect of NaC1 on Leaf Thickness and Succulence
Growth of Citrus Rootstock Seedlings under NaC1 and
PEG Stresses . . . . . . . . .
Relationship of Leaf Damage Symptoms to
Growth Reduction . . . . . . .
Root Growth and Distribution under NaCl
and PEG Stresses . . . . . . .
Effects of Non-Uniform Salinity and Water Stress
Comparative Effects Between NaC1 and PEG . . .


. . 82
. . 82
. . 83
. . 84
. . 85
. . 86
. . 86
. . 87
. . 88
. . 89
. . 89
. . 90

. . 90

. . 91

. . 91
. . 92
. . 93


SUMMARY AND CONCLUSIONS . . . . . . . . .. .. 95

APPENDIX . . . . . . . . ... . . . . . 100


LITERATURE CITED . . . . . . . . . .


. . 116


BIOGRAPHICAL SKETCH . . . . . . . . . . . .















LIST OF TABLES


Table Page

1. Salt treatments and chemical properties of the different
salt treatments . . . . . . . .... . 33

2. Shoot dry weight of seedlings of 7 rootstocks
grown for 5 months under different NaC1 and PEG
concentrations . . . . . . . . ... . 35

3. Root dry weight of seedlings of 7 rootstocks grown
for 5 months under different NaC1 and PEG
concentrations . . . . . . . . ... . 36

4. Specific fibrous root weight of seedlings of 7
rootstocks grown under different NaC1 concentrations 38

5. Root length, root conductivity, water flow rate, and
osmotic potential of iot exudate of seedlings of 7
rootstocks under non-stressed conditions . . ... 40

6. Visible injury in seedlings of 7 rootstocks after
5 months of NaC1 treatments . . . . . .... .42

7. Leaf sodium content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations . . . . . . . . ... . 44

8. Leaf chloride content of seedlings of 7 rootstocks
grown for 5 months under different NaCl and PEG
concentrations . . . . . . . . ... . 45

9. Ion exclusion and accumulation in leaves of citrus
rootstock seedlings . . . . . . . .... .47

10. Leaf calcium content of seedlings of 7 rootstocks
grown for 5 months under different NaC1 and PEG
concentrations . . . . . . . . ... . 48

11. Monthly new flush growth--area/leaf--of sour
orange seedlings . . . . . . . .... . 50









Table Page


12. Monthly new flush growth--leaf number--of sour
orange seedlings .. . . . . . . . . 51

13. Leaf succulence of seedlings of 2 rootstocks grown
for 6 months under different NaC1 and PEG
concentrations . . . . . . .. . . . 54

14. Total chlorophyll of seedlings of 2 rootstocks
grown for 6 months under different NaCl and PEG
concentrations . . . . . . . . . .... 55

15. Fibrous root length in the 3 compartments of the
root boxes for seedlings under different NaCl and PEG
concentrations . . . . . . . . . .... 63

16. Shoot and root dry weight of split-root sour orange
seedlings under NaCI and PEG stresses . . . .. 66

17. Midday leaf water, osmotic, and turgor potentials of
split-root sour orange seedlings under NaCl and PEG
stresses . . . . . . . . . . . 70

18. Midday stomatal conductance and transpiration of
split-root sour orange seedlings under NaC1 and PEG
stresses . . . . . . . . . . . 71

19. Root and shoot dry weight of sour orange seedlings
under different salt treatments . . . . .. 75

20. Total plant dry weight and leaf succulence of sour
orange seedlings under different salt treatments . . 76

21. Leaf mineral analysis of sour orange seedlings under
different salt treatments . . . . . . . 77

22. Summary of citrus rootstock responses to NaC1 and
PEG as compared to a no salt control . . . . .. 80

23. Shoot root ratio of seedlings of 7 rootstocks grown
for 5 months under different NaC1 and PEG
concentrations. .. . . . . . . . . 100

24. Total plant dry weight of seedlings of 7 rootstocks
grown for 5 months under different NaC1 and PEG
concentrations . . . . . . . . .. . . 101









Table Page

25. Stem cross sectional area of seedlings of 7
rootstocks grown for 5 months under different NaCl
and PEG concentrations . . . . . . .... 102

26. Leaf magnesium content of seedlings of 7 rootstocks
grown for 5 months under different NaC1 and PEG
concentrations . . . . . . . . ... . 103

27. Leaf potassium content of seedlings of 7 rootstocks
grown for 5 months under different NaCI and PEG
concentrations . . . . . . . . ... . 104

28. Leaf phosphorus content of seedlings of 7 rootstocks
grown for 5 months under different NaC1 and PEG
concentrations. ..... . . . .. . . 105

29. Leaf zinc content of seedlings of 7 rootstocks grown
for 5 months under different NaCI and PEG
concentrations . . . . . . . . .. .... 106

30. Leaf manganese content of seedlings of 7 rootstocks
grown for 5 months under different NaC1 and PEG
concentrations . . . . . . . . ... . 107

31. Seedling height of seedlings of 2 rootstocks grown
for 6 months under different NaCI and PEG
concentrations . . . . .... .. . . . . 108

32. Total leaf area of seedlings of 2 rootstocks grown
for 6 months under different NaCl and PEG
concentrations . . . . . .... . . . 109

33. Specific leaf weight of seedlings of 2 rootstocks
grown for 6 months under different NaC1 and PEG
concentrations . . .... . . . . . . . 110


viii















LIST OF FIGURES


Figure Page

1. Osmotic potential versus NaCl concentration as
determined by vapor pressure (VPD) and freezing
point depression (FPD) . . . . . . .... .22

2. Osmotic potential versus PEG concentration as
determined by vapor pressure (VPD) and freezing
point depression (FPD) . . . . . . .... .23

3. Sour orange seedlings with a split-root system .... 30

4. Effect of 3 NaCi concentrations on the total
fibrous root length, root hydraulic conductivity,
and water flow rate for seedlings of 7 citrus
rootstocks . . . . . . . . ... .. . . 37

5. Relationship between ioot hydraulic conductivity
and specific root weight of seedlings of 7 citrus
rootstocks under non-stressed conditions . . ... 41

6. Effect of NaCl at an osmotic potential of -0.35
MPa on the 7 rootstocks after 5 months of
salinity treatments . . . . . . . .... 43

7. Relationship between water flow rate and osmotic
potential of root exudate of sour orange and
Cleopatra mandarin seedlings . . . . . .... .53

8. Relationship between midday stomatal conductance
and root conductivity of sour orange and Cleopatra
mandarin seedlings . . . . . . . .... 56

9. Midday stomatal conductance of sour orange
seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG . 58

10. Relationship of time of day to stomatal
conductance of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or
with added NaCl or PEG during 2 consecutive days . 59









Figure Page

11. Growth of sour orange seedlings irrigated with
nutrient solution containing no salt (NS) or with
added NaC1 or PEG . . . . . . . . ... 60

12. Fibrous root length of sour orange seedlings
irrigated with nutrient solution containing no
salt (NS) or with added NaC1 or PEG . . . ... 61

13. Fluctuations in shoot and root growth of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG . 62

14. Root density and distribution of sour orange
seedlings growing in root boxes under
non-stressed (NS) and stressed (NaCI, PEG)
conditions . . . .. .. . . . . . 64

15. Root development of split-root sour ornage
seedlings under uniform and non-uniform NaCl and
PEG stress . . . . . . . . . . . 67

16. Leaf water, osmotic, and turgor potential of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with NaCl added to both
root halves . . . . .... . . . . . 69

17. Relationship between transpiration and stomatal
conductance of sour orange seedlings . . . ... 72

18. Cross sections of sour orange leaves . . . ... 74

19. Sour orange leaves from non-stressed (control)
and stressed (NaC1, PEG) seedlings . . . .... .81

20. Effect of 3 NaC1 concentrations on the osmotic
potential of root exudate collected from
seedlings of 7 citrus rootstocks . . . . . .. 111

21. Relationship of time of day to stomatal
conductance of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or
with added NaC1 during 3 consecutive days ...... 112

22. Relationship of time of day to stomatal
conductance of Cleopatra mandarin seedlings
irrigated with nutrient solution containing no
salt (NS) or with added NaC1 during 3 consecutive
days . . ... . . . . . . . . . . 113









Figure Page

23. Relationship of time of day to stomatal
conductance of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or
with added PEG during 3 consecutive days . . ... 114

24. Relationship of time of day to stomatal
conductance of Cleopatra mandarin seedlings
irrigated with nutrient solution containing no
salt (NS) or with added PEG during 3 consecutive
days . ..... . . . . . . . . . . 115















Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECTS OF SODIUM CHLORIDE AND POLYETHYLENE GLYCOL
ON THE WATER RELATIONS, GROWTH, AND MORPHOLOGY
OF CITRUS ROOTSTOCK SEEDLINGS

By

MONGI ZEKRI

December 1987

Chairman: Dr. Lawrence R. Parsons
Major Department: Horticultural Science (Fruit Crops)

The effects of sodium chloride (NaC1) and polyethylene glycol (PEG)

on the growth, water relations, and leaf mineral content of citrus

rootstocks were investigated. Significant growth reduction and

physiological disturbances occurred even at NaC1 and PEG concentrations

of -0.10 MPa. Growth reduction and physiological changes were found to

precede visible damage. Growth was reduced up to 30% without being

accompanied by visible leaf injury symptoms. Leaf burn symptoms

developed only after a threshold value of chloride accumulation (1%) was

reached. Leaf burn symptoms developed too slowly to accurately evaluate

salt damage. Root conductivity correlated better with salinity

tolerance among rootstocks than did total fibrous root length.

Sodium chloride usually caused less damage than PEG. Unlike PEG,

NaCl significantly increased leaf thickness and succulence along with

leaf sodium and chloride concentrations, but reduced calcium and zinc

contents in the leaves. Both NaC1 and PEG reduced leaf magnesium and

potassium contents but increased leaf phosphorus and manganese contents.









Differences in sodium and chloride exclusion capacities among

rootstocks were found. Sour orange, rough lemon, and Milam were sodium

and chloride accumulators. Poncirus trifoliata, Swingle citrumelo, and

Carrizo citrange were sodium excluders but chloride accumulators.

Cleopatra mandarin was a chloride excluder but a sodium accumulator.

Differences in NaCl sensitivity among rootstocks were also found.

Cleopatra mandarin and sour orange were the least sensitive, Milam and

Poncirus trifoliata were the most sensitive, and rough lemon, Swingle

citrumelo, and Carrizo citrange were intermediate in sensitivity.

Cleopatra mandarin tolerated high concentrations of NaCI by partial

exclusion of chloride while sour orange tolerated NaCl even though it

accumulated sodium and chloride in its leaves. Sour orange might have

the ability to compartmentalize these ions and exclude them from the

cytoplasm where they may inhibit metabolic processes.

Seedlings receiving NaC1 or PEG produced small and shallow root

systems with the majority of the roots occurring in the top layer.

Addition of calcium sulfate to saline irrigation water was found to be

beneficial in overcoming the detrimental effects of NaC1 on citrus. The

split-root experiment showed that citrus could withstand substantial

amounts of stress as long as half of the root system was growing in a

non-stressed environment.


xiii
















INTRODUCTION


It is well established that salt can impair the performance of many

agricultural plants. Salts present in the soil and irrigation water are

a serious problem for commercial agriculture particularly in arid and

semi-arid regions. However, the potential for salinity damage also

exists in humid climates. Controlling or reducing salt injury is

usually achieved either through soil management practices and irrigation

with good quality water or by combining these practices with the use of

salt-tolerant plants.

Citrus is a fruit crop of international significance. It is grown

in over 50 countries and ranks among the top 3 tree fruit crops in world

production. In certain areas where citrus is grown, salinity is already

a problem of some importance. In other areas, the future of

citriculture is threatened by salinity largely because agriculture is

being forced to use lower quality land and water for irrigation. In

agricultural areas with salinity problems, citrus is particularly

vulnerable because there is relatively little salt tolerance in the

genus.

Salinity studies have shown that among species, cultivars, and

various selections, only 2 rootstocks, Cleopatra mandarin and Rangpur

lime, have a limited capacity to tolerate certain salts. However,

rootstocks are usually selected for other attributes such as yield and

fruit quality. Rootstocks deficient in these characteristics are not

likely to be commercially used despite favorable salt tolerance.

1







2

In Florida, there are many citrus plantings located in coastal areas

where saline water is being used for irrigation. Citrus planting in

these and other southern Florida areas has been accelerated by extensive

freeze damage in more northern areas. These changes in the citrus

industry, as well as the diseases triteza and blight, have greatly

affected interest in rootstock characteristics including salt tolerance.

In the past, virtually all evaluations of citrus response to

salinity were based on visual leaf injury and correlations with leaf

chloride content. There were no root system observations recorded and

no detailed physiological studies conducted. Such observations and

measurements of physiological responses are necessary for a complete

understanding of salt injury and tolerance in plants. This information

is particularly valuable for efficient breeding and screening of new

germplasm for salt tolerance.

The objectives of this research are the following:

1. To compare the salt tolerance of citrus rootstocks commercially

important in Florida and to determine which rootstocks are salt

excluders or salt accumulators.

2. To determine the salt concentrations at which growth is

depressed, water balance is disturbed, and leaves are injured.

3. To separate specific ion effects of salts from their osmotic

effects by comparing growth, water relations, and plant chemical

analyses under NaCl and PEG stresses.

4. To measure the effects of several NaC1 and PEG concentrations on

root growth and distribution.

5. To study citrus growth and water relations under non-uniform

salinity (split-root system).

6. To examine the importance of calcium in reducing NaC1 damage.















LITERATURE REVIEW


Salts

Many hectares of land throughout the world are too saline for

profitable agriculture (Carter, 1975). Large amounts of arable lands

are being removed from crop production every year due to increasing soil

salinity (Chapman, 1975; Epstein et al., 1980). Saline irrigation water

combined with fertilizer application are the factors most responsible

for increasing soil salinity (Epstein et al., 1980; Jones et al., 1952;

Stewart et al., 1977).

The ions in soil waters which contribute significantly to salinity

problems are principal' sodium, chloride, calcium, magnesium, sulfate,

potassium, bicarbonate, carbonate, nitrate, and occasionally borate ions

(Bernstein and Hayward, 1958; Peck, 1975; Shainberg, 1975). However,

most salinity research has involved NaC1 because it is the most common

salt in saline soils and irrigation waters.

Mechanisms of Salt Tolerance in Plants

Salt-tolerant plants are generally thought to be protected from salt

stress by either ion accumulation or ion exclusion. Accumulation of

high concentrations of ions in halophyte leaves has been known to be a

salt tolerance mechanism (Flowers et al., 1977; Greenway and Munns,

1980). Salts can be tolerated because ions are compartmentalized in the

vacuole and not in the cytoplasm. Hence, metabolic processes are not

inhibited. These ions in the vacuoles balanced with neutral organic

solutes in the cytoplasm lower the leaf osmotic potential. This allows

3







4

the plant to extract water from saline solutions. However, salt

tolerance in glycophytes (nonhalophytes) is related to ion exclusion

because of the plant's inability to compartmentalize toxic ions in a

useful way and to adjust osmotically (Greenway and Munns, 1980).

Mechanisms of Salt Injury

Salt damage to plants is caused by the decrease in the water

potential of the soil solution or by the toxicity of specific ions.

Some workers attribute most of the salt damage to osmotic stress

(Bernstein, 1961, 1963; Bernstein and Hayward, 1958; Bielorai et al.,

1978, 1983; Bohn et al., 1979). Others favor the idea that toxic

effects of specific ions predominate in restricting growth and yields

(Babaeva et al., 1968; Gollek, 1973; Strogonov, 1964).

A common method of distinguishing between osmotic and ion toxic

effects of salts is to compare the effects of isosmotic solutions of the

salt with those of non-toxic organic substances. If the salt injury is

simply osmotic, all solutes should produce the same injury at the same

osmotic potential (Levitt, 1980). Polyethylene glycol (PEG), a

non-ionic compound, has been successfully used as an osmoticum for

subjecting plants and plant tissues to known levels of water stress

(Janes, 1966; Kaufmann and Eckard, 1971; Kawasaki et al., 1983a, b).

Osmotic Effect

Water is osmotically more difficult to extract from saline

solutions. Pair et al. (1975) pointed out that the addition of 0.4%

salts had the effect of reducing the total available water in the soil

by approximately 33%. Salt addition is analogous to soil drying since

both result in reduced water uptake. In extreme circumstances, salinity

can prevent water uptake even when the soil is at field capacity (Hartz,

1984). Water uptake by mature grapefruit trees, mature Valencia orange







5

trees, and Valencia orange seedlings was reduced as salinity increased

(Bielorai et al., 1983; Hayward and Blair, 1942; Plessis, 1985).

Ion Toxic Effect

Ion toxic effect of salt is attributed to excess accumulation of

certain ions in plant tissues and to nutritional imbalances caused by

such ions. Ion excess has been defined as a condition where high

internal ion concentrations reduced growth (Greenway and Munns, 1980).

In many crops, salt injury increases with increased salt uptake.

Raspberries were found to accumulate chloride ions more rapidly and

consequently were more severely injured than blackberry (Ehlig, 1964).

Tagawa and Ishizaka (1963) found that the primary cause of injury to

rice by NaC1 was chloride accumulation in the shoots. When treated with

NaC1, a less resistant barley variety accumulated higher levels of

chloride and sodium than a more resistant variety (Greenway, 1962).

Salt damage to citrus has been mainly attributed to excessive

accumulation of chloride and sodium in the leaves (Abdel-Messih et al.,

1979; Chapman et al., 1969; Cooper, 1961; Cooper et al., 1951; 1952b;

Cooper and Peynado, 1953; El-Azab et al., 1973; Furr and Ream, 1968;

Grieve and Walker, 1983). Goell (1969) suggested that salt ions such as

chloride in citrus leaves might shorten the life span of leaves by

increasing chlorosis (loss of chlorophyll and photosynthetic potential)

and by promoting senescence and abscission. Sulfate and other ions also

caused damage to citrus (Bhambota and Kanwar, 1970; Bingham et al.,

1973; Cerda et al. 1979; Hewitt and Furr, 1965a; Peynado and Young,

1964). It has been suggested that the accumulation of ions in large

amounts in the leaves is the main factor causing leaf burn and

inhibition of certain metabolic processes.







6

Sodium can also cause injury to plants through its deleterious

effect on the soil. When the proportion of exchangeable sodium is

relatively high, clay particles in the soil tend to disperse and -block

the pores through which water flows. This phenomenon decreases the

hydraulic conductivity of the soil (Bohn et al., 1979; Shainberg, 1975)

and causes poor aeration. Studies by Aldrich et al. (1945) demonstrated

that inferior performance of orange trees was caused primarily by poor

water penetration resulting from sodium accumulation on the exchange

complex.

Nutritional Imbalance

Salt can also damage plants by causing nutritional imbalances. High

sodium levels can lead to calcium and magnesium deficiencies (Bohn et

al., 1979). In spinach and lettuce, sodium salts decreased dry matter

production as well as leaf potassium, magnesium, and calcium contents

(Matar ot al., 1975). Pumpkin and sweet clover plants subjected to NaC1

showed potassium deficiency (Solov'ev, 1969). A decrease in potassium

uptake at higher concentrations of sodium was found in sugarcane

(Nimbalker and Joshi, 1975) and rice (Paricha et al., 1975). With

increased salinity, potassium and phosphorus uptake decreased in grapes,

guava, and olive plants (Taha et al., 1972), in wheat (Sharma and Lal,

1975), and in barley (Kawasaki et al., 1983b).

In citrus, nutritional imbalance has been also attributed to

depressed absorption of some nutrients. A decrease in the concentration

of calcium, magnesium, and sometimes potassium was found when salt

concentration in the irrigation water was increased (Jones et al., 1957;

Patil and Bhambota, 1980; Pearson et al., 1957).







7

Plant Responses to Salinity

Salinity has been known to adversely affect all stages of plant

development such as germination, vegetative growth, and fruiting.

Salinity has also been found to depress chlorophyll content,

photosynthesis, stomatal conductance, root conductivity, and

transpiration of many crops. For example, growth of citrus (Furr and

Ream, 1968), Vicia faba (Helal and Mengel, 1981), pepper (Hoffman et

al., 1980), alfalfa (Keck et al., 1984), bean (Meiri and

Poljakoff-Mayber, 1970), and corn (Siegal et al., 1980) was

significantly depressed under saline conditions.

Yield of grapefruit (Bielorai and Levy, 1971; Bielorai et al., 1978,

1983), orange (Bingham et al., 1973, 1974; Chapman et al., 1969), celery

(Francois and West, 1982), and muskmelon (Shannon and Francois 1978)

was severely reduced due to salinity stress. Salinity was found to

alter fruit quality by decreasing the "pack out" of oranges at a

commercial packing shed (Bingham et al., 1974) and by decreasing the

marketable yield of tomato and melon (Mizrahi and Pasternak, 1985;

Shannon and Francois, 1978). It was found that the relative amount of

the premium grade fruit decreased with use of saline water even though

there was a trend toward higher soluble solids and better taste (Bingham

et al., 1974; Mizrahi and Pasternak, 1985; Shannon and Francois, 1978).

Salinity reduced leaf chlorophyll content in grapevine, bean,

barley, citrus and mangrove (Downton and Millhouse, 1985), spinach

(Downton et al., 1985), and Acacia saligna (Shaybany and Kashirad,

1978). Leaf chlorophyll content declined only when certain amounts of

salt ions accumulated in the leaves. Salinity reduced photosynthesis in

spinach (Downton et al., 1985), rice (Flowers et al., 1985), Xanthium

strumarium (Schwarz and Gale, 1983), beans (Seemann and Critchley,







8

1985), and Acacia saligna (Shaybany and Kashirad, 1978). Under most

circumstances, photosynthetic reduction was attributed to ion

accumulation in the leaves and to reduction in stomatal conductance.

Salinity was found to reduce root conductivity in white lupin (Munns

and Passioura, 1984) and beans (O'Leary, 1969; 1974). However, salinity

did not affect root conductivity in barley (Munns and Passioura, 1984),

sunflower and tomato plants (Shalhevet et al., 1976). Reduced hydraulic

conductivity of roots has been attributed to root suberization and to

reduced root membrane permeability.

Salinity and Citrus

Citrus is generally classified as a salt sensitive crop because

physiological disturbances and growth and fruit yield reductions can

occur at relatively low salinity levels (Bernstein, 1969; Bielorai et

al., 1978, 1983; Boaz, 1978; Cherif et al., 1982; Cooper and Shull,

1953; Francois and Clark, 1980; Furr et al., 1963; Kirkpatrick and

Bitters, 1969; Marsh, 1973; Patil and Bhambota, 1980; Pehrson et al.,

1985; Walker et al., 1982).

Citrus Salinity Research

The response of citrus to salinity is a topic of concern in many

regions where citrus is grown especially the United States, Israel,

Egypt, India, Spain, and Tunisia. In the United States, salinity

studies essentially began in Texas during the 1940s. Investigations

were led by W.C. Cooper with emphasis on differences in salinity

tolerance among citrus rootstocks (Cooper, 1948; Cooper and Gorton,

1952; Cooper and Peynado, 1959; Cooper and Shull, 1953; Cooper et al.,

1951). The work was conducted mostly on young budded trees grown in the

field. Salinity treatments consisted of NaCI + CaCl2 added to Rio

Grande river water. These studies led to the observation that chloride







9

exclusion was strongly correlated with salt tolerance. Chloride

accumulation or exclusion and leaf injury symptoms were used to classify

salt tolerant and salt sensitive rootstocks.

Salinity studies on citrus were started in California in the 1950's

(Harding et al., 1958a; Janes et al., 1952; Pearson and Goss, 1953), in

Israel in the 1970s (Bielorai et al., 1973; Heller et al., 1973), and in

Australia in the 1970s (Cole and Till, 1977). Most of these studies

were conducted in the field on mature citrus trees and were focused on

yield reduction and fruit quality alteration as a function of salt

concentration in irrigation waters (Bielorai et al., 1978, 1983; Bingham

et al., 1973, 1974; Boaz, 1978; Francois and Clark, 1980; Levy et al.,

1979; Pehrson et al., 1985; Shalhevet et al., 1974).

Recent salinity work in Israel was directed to plant breeding using

cell culture techniques (Ben-Hayyim and Kochba, 1983; Ben-Hayyim et al.,

1985). Recent work in Australia was conducted mainly with young

rootstock seedlings grown in pots under glasshouse conditions

(Behboudian et al., 1986; Grieve and Walker, 1983: Walker and Douglas,

1983; Walker et al., 1982, 1983, 1984, 1986). Salinity treatments

consisted of NaCI added to a nutrient solution. These studies were

focused on sodium and chloride exclusions mechanisms, water relations,

and photosynthesis. Photosynthesis was severely reduced and

photosynthetic reduction was attributed to a loss of turgor in salt

excluder rootstocks and to chloride accumulation in salt accumulator

rootstocks.

Some salinity work on citrus conducted in Egypt (Abdel-Messih et

al., 1979; Minessy et al., 1973), India (Bhambota and Kanwar, 1970;

Patil and Bhambota, 1980), Spain (Cerda et al., 1979; Guillen et al.,

1978), and Tunisia (Cherif et al., 1981; 1982; Zid, 1975; Zid and







10

Grignon, 1985, 1986) on budded trees and rootstock seedlings involved

ion analysis and nutrient absorption. These studies showed that

salinity caused nutritional imbalance, growth reduction, and leaf burn.

Growth reduction was attributed to potassium deficiency and foliar

necrosis to sodium accumulation in the leaves.

Salinity is of increasing concern in Florida. Salt water intrusion

into groundwater in areas where citrus is grown has increased the need

for salinity studies in Florida. Many citrus rootstocks are being used

in Florida such as sour orange, Swingle citrumelo, Carrizo citrange, and

Milam without knowing their salt tolerance. As a result, there is an

incentive to study the salinity tolerance of these and other rootstocks

which are commercially important.

Physiologists often concentrate on the activities of shoots and

neglect roots because they are out of sight and more difficult to study

than shoots (Kramer, 1983). Roots play an important role in the growth

and development of the entire plant. Their health, vigor and activity

can be an index of the functioning of the above-ground parts (Crider,

1927). It is important to investigate root growth and distribution

because roots are directly in contact with salts in the soil. Detailed

information on the growth behavior and morphological development of

citrus root systems under salt conditions is not available.

The two major resistances to water movement through the

soil-plant-atmosphere continuum are the roots and the stomata (Kramer,

1969; Kriedemann and Barrs, 1981). Root conductivity and stomatal

conductance are important variables to be monitored in salinity studies

because they can provide information on the water balance disturbance

caused by salt. Root conductivity of some in citrus rootstocks under

salinity stress has not been previously studied. Furthermore,







11

information relating root conductivity to stomatal conductance and

transpiration as a function of different osmotic concentrations is

lacking.

Under field conditions, the roots of an individual plant grow in

soil which varies in water content and salt concentration both in space

and with time. In assessing the suitability of water for irrigation, it

is usually assumed that plants respond to the mean root zone salinity

(Shalhevet and Bernstein, 1968). However, some workers suggest that the

least saline part of the rooting zone controls the overall plant growth

and yield (Lunin and Gallatin, 1965). Responses of citrus to

non-uniform salinity or to zonal salinization are not known.

Citrus Tolerance to Salinity

Scion. Differences in salt tolerance among citrus varieties or

scions have been shown. Boaz (1978) concluded that Valencia orange had

a lower tolerance to salinity than grapefruit on sweet orange rootstock.

Bernstein (1969) reported that lemon was more sensitive to salinity than

orange which was more sensitive than grapefruit. Miwa et al. (1957)

also found that lemon was the most susceptible variety to foliar spray

injury from sea water. Results of Pearson and Huberty (1959) showed

that navel orange trees were more sensitive to irrigation water quality

than Valencia orange trees. Budded on rough lemon, salt tolerance

decreased in the following order: Hamlin, Valencia, Pineapple and Blood

red sweet orange (Bhambota and Kanwar, 1969). Valencia seemed to be

more sensitive to salinity than Shamouti when both were grafted on sour

orange rootstock (Shalhevet et al., 1974).

Rootstock. Some studies have indicated a wide range in salt

tolerance among citrus rootstocks (Cooper, 1948: Cooper and Edwards.

1950; Cooper et al., 1952b, 1q58). Cooper et al. (1'bl) found that







12

Cleopatra mandarin and Rangpur lime are relatively salt-tolerant

rootstocks. They classified sour orange, rough lemon, sweet lemon,

tangelo and sweet lime as sensitive rootstocks and Florida sweet.orange

and trifoliate orange as very sensitive. In another study, Cleopatra

mandarin and Rangpur lime were also found to be the most tolerant

rootstocks and Carrizo citrange was the most sensitive rootstock (Joolka

and Singh, 1979; Patil and Bhambota, 1978). Trifoliate orange and rough

lemon were found to be very salt sensitive (Bhambota and Kanwar, 1969).

Although some selections of sour orange differed in salt tolerance, Ream

and Furr (1976) found that none of them was as salt tolerant as

Cleopatra mandarin.

Salt Exclusion. Exclusion of certain ions has been demonstrated in

some citrus rootstocks. Rangpur lime and Cleopatra mandarin appear to

be chloride excluders (Cooper, 1961; Cooper and Gorton, 1952; Cooper and

Peynado, 1959; Douglas and Walker, 1983; Grieve and Walker, 1983; Hewitt

and Furr, 1965b; Walker, 1986; Walker et al., 1983; Wutscher et al.,

1973). Trifoliate orange appears to be a sodium excluder (Elgazzar

et al., 1965; Grieve and Walker, 1983; Walker, 1986) and Citrus

macrophylla a boron excluder (Cooper and Peynado, 1959; Embleton et al.

1962). This suggests the existence of a blocking mechanism in the

transport of these ions (Fernandez et al., 1977). It also indicates the

existence of apparently separate mechanisms which regulate the uptake

and transport of ions (chloride and sodium) in salt-stressed citrus

(Fernandez et al., 1977; Grieve and Walker, 1983; Walker et al., 1983).

Ion concentration. Citrus is a nonhalophyte, and its tolerance to

salinity is correlated with its ability to restrict the entry of ions

into the shoots (Greenway and Munns, 1980). Injury to citrus from NaCI

has been attributed to excess chloride accumulation (Ben-Hayyim and







13

Kochba, 1983; Cooper, 1961; Cooper and Gorton, 1952; Furr and Ream,

1969). In an effort to screen young citrus trees for salt tolerance,

Hewitt et al. (1964) found that the leaves could be analyzed for -

chloride after 3 to 4 weeks of treatment with highly saline irrigation

water. Fernandez et al. (1977) considered foliar chloride content as a

suitable index of the soil salinity status and toxicity levels.

However, Ben-Hayyim et al. (1985) showed the difficulty in determining

if any particular ion could serve as a reliable marker for salt

tolerance in citrus.

Citrus Responses to Saline Conditions

Photosynthesis. Photosynthetic rates were reduced by 50 to 75%

after 70 days of NaCl stress (Behboudian et al., 1986; Walker et al.,

1982). A decrease in photosynthesis is often caused by a drop in leaf

turgor, but studies have shown different turgor responses to salinity.

In one study with Rangpur lime, photosynthesis reduction was attributed

to low turgor pressures in rangpur lime and not to leaf chloride or

sodium concentrations since there was no significant difference in

concentrations of these ions between salt-stressed and control leaves.

In contrast to Rangpur lime, photosynthetic reduction during salt

treatment in Etrog citron was associated with a marked increase in leaf

chloride since turgor was not reduced. Their work established that a

plant's capacity for salt exclusion alone or turgor maintenance alone

was unable to protect citrus seedlings against photosynthetic reduction.

Therefore, to improve salt tolerance in citrus, studies need to be

focused not only on salt exclusing rootstocks but also on the ability of

scions to maintain turgor. It appears that the inability to osmotically

adjust and exclude toxic ions is related to citrus sensitivity to

salinity (Zid and Grignon, 1986).







14

Yield. Citrus yield has been related to salt concentration in the

soil (Bielorai et al., 1978; Harding et al., 1958b). According to Boaz

(1978) and Maas and Hoffman (1977), the threshold salinity is an

electrical conductivity of the soil saturation extract of 1.8 dS/m (1.8

mmhos/cm) for oranges and grapefruit. Above this threshold, yield is

reduced at a rate of 16% per dS/m. Pehrson et al. (1985) stated that 10

and 50% yield reductions for citrus were associated with electrical

conductivities of the soil saturation extract of 2.3 and 4.8 dS/m,

respectively.

Salinity was found to significantly reduce citrus yield without

visual symptoms (Pehrson et al., 1985). The use of moderately saline

irrigation water (2.5 dS/m) decreased orange yield by about 30% without

any visible leaf injury symptoms (Bingham et al., 1974). Within a

concentration range of 2 to 2.7 dS/m, 9 to 18% yield reduction in

grapefruit occurred without apparent t- city symptoms (Bielorai et al.,

1978, 1983). When irrigated with moderately saline water (15 to 30 mM,

CaC12 + Na2SO4 + MgSO4), Valencia orange had yield reductions of 34 to

54% with no visible leaf injury symptoms (Francois and Clark, 1980).

Leaf injury. Salinity effects develop slowly so that leaf injury

symptoms appear after a certain period of time. However, the length of

this time period is shortened by higher salt concentrations. Grown in

the field, two-year-old Ruby red grapefruit on sour orange rootstock

irrigated with salt solutions of 2500 mg/L (50:50 NaC1 and CaCl2) showed

no visible symptoms of salt injury during a one year period. Trees

irrigated with 4000 mg/L salt solution developed leaf bronzing within

1 month and marginal burning of the leaves within 2 months. Trees

irrigated with 5000 mg/L salt solution were completely defoliated within

a one year period (Cooper, 1961; Cooper et al., 1952a).







15

Salinity and high water table. Relatively few studies have been

conducted to investigate the effects of a combination of water table and

salinity on citrus even though this condition exists in many parts of

the world. Studying the effects of salinity and water table on the

growth and mineral composition of young grapefruit trees, Pearson and

Goss (1953) found that the rates of defoliation and twig dieback due to

salinity were greatly accelerated by a frequently fluctuating water

table. In a more detailed report of the same study, Pearson et al.

(1957) concluded that the salinity factor accounted for approximately

90% of the variance in growth while the water table factor accounted for

only about 4%. They found that sodium and chloride accumulated in toxic

amounts in the leaves and were responsible for the decrease in growth.

However, while investigating the effect of different water table depths

and salinity levels on sweet orange, Kanwar and Bhambota (1969) observed

that the adverse effect of water table was more pronounced than that of

salinity. Both studies agreed that the interaction of water table and

salinity affected the trees more severely than either condition alone.

The fact that Cleopatra mandarin is more sensitive to flooding

(Ford, 1964) but more salt tolerant (Cooper et al., 1951) than sour

orange raises the question about the performance of these two rootstocks

under saline conditions associated with high water table or flooding

problems.

Irrigation. Citrus is relatively sensitive to salinity, but can

withstand high salt concentrations depending on the variety, rootstock,

and irrigation management. Good irrigation management should consider

the salinity factor in the irrigation water, in the soil, and in the

root zone (Boaz, 1978). Methods of irrigation scheduling which do not

account for salinity are not sufficiently accurate for scheduling







16

irrigation in areas with a saline high water table. Irrigation water

containing about 250 mg chloride per liter reduced grapefruit yield by

28 to 32% when trees were irrigated at intervals of 40 days compared to

intervals of 18 days (Bielorai and Levy, 1971; Bielorai et al., 1973).

These studies demonstrated that the effect of salinity is more severe at

lower soil water content.

Overhead sprinkler irrigation should be avoided when using water

containing high levels of salts because salt residues can accumulate on

the foliage and seriously injure plants. Navel orange accumulated

injurious amounts of chloride and sodium from sprinkler-applied water

having 500 to 900 ppm total dissolved solids (Harding et al., 1958a).

Considerable leaf burn and defoliation of these trees were found to be

correlated with excessive amounts of sodium and chloride and lower

amounts of potassium in the leaves. Leaf injury of navel orange trees

developed at concentrations of 5 to 10 mmol/L of NaC1, CaCl2 or Na2SO4

in the sprinkler-applied waters (Ehlig and Bernstein, 1959). Salt

content of up to 1300 mg/L caused defoliation of sprinkler-irrigated

citrus trees in Texas (Lyons, 1977). In Australia, during periods of

high salinity in the irrigation water, foliar absorption of sodium and

chloride occurred when using overhead sprinklers on citrus. It was

believed that this problem caused poor tree health, low yield, and

possibly poor fruit quality in citrus (Cole and Till, 1977).

Frequency rather than duration of sprinkler irrigation is perhaps

more important in foliar absorption of salts. Salt injury was higher

under higher evaporation conditions and with short and frequent periods

of overhead sprinkling (Eaton and Harding, 1959; Ehlig and Bernstein,

1959; Harding et al., 1958a).







17

Micro-irrigation is gaining in popularity not only in arid regions

but also in humid subtropical areas. Micro-irrigation refers to both

drip and microsprinkler irrigation. Micro-irrigation enables the use of

poorer quality water that cannot be tolerated with overhead sprinklers.

Direct foliar uptake of salts, and hence leaf injury, is avoided with

drip irrigation (Calvert and Reitz, 1966). Nevertheless, saline water

cannot be used indiscriminately with micro-irrigation systems.

Comparative studies between overhead sprinklers and drip systems using

saline water showed that vegetative growth, root development, and yield

were greater with drip than with sprinkler irrigation (Goldberg and

Shmueli, 1971; Shmueli and Goldberg, 1971). In a comparison of flood

and drip systems, water high in chloride and boron was applied to young

grapefruit trees on many rootstocks (Wutscher et al., 1973). More

chloride and boron accumulation was found in trees that were flood

irrigated than in tho- that were micro-irrigated.

Drip irrigation at frequent intervals maintains a low soil water

tension and prevents salt accumulation within the wetted zone.

Consequently, water with higher salinity levels may be used without

significantly affecting the yield. Nevertheless, salt accumulation

under drip irrigation must be considered because salts may accumulate

both at the periphery of the wetted zone and on the soil surface

(Bielorai, 1977, 1985; Goldberg et al., 1976; Hoffman et al., 1985;

Yaron et al., 1973).

Reducing Salt Damage

Role of Calcium

Calcium has been known to have an ameliorating effect on the growth

of plants under saline conditions (Deo and Kanwar, 1969; Epstein, 1972;

Hyder and Greenway, 1965). This effect has been attributed to calcium







18

preventing the uptake of the sodium ion to injurious levels, and

allowing the uptake of potassium (Waisel, 1962). In the presence of

adequate concentrations of calcium, bean plants were able to exclude

sodium and to withstand the effects of relatively high NaC1

concentrations (LaHaye and Epstein, 1969, 1971). In barley, inhibition

of the absorption and translocation of potassium and phosphorus by NaCl

was found to recover dramatically in the presence of calcium (Kawasaki

et al., 1983b). Application of gypsum to the soil or in the irrigation

water markedly reduced the percentage of soluble sodium in the soil

(Harding et at., 1958b) and reduced the percentage of sodium in citrus

leaves and roots (Jones et al., 1952; Pearson and Huberty, 1959).

Calcium amendments are commonly used for replacement of exchangeable

sodium (Richards, 1954). Calcium can flocculate soil in which clay

particles and aggregates have been dispersed by sodium. Salt-affected

soils can therefore be made productive by chemical amendment, drainage,

and irrigation with high quality water, but sometimes the cost of these

operations exceeds the expected returns from the land.

Genetic Improvement

In recent years, adapting plants to saline environments through

breeding and genetic manipulation have been attempted (Epstein et al.,

1980). The genetic basis for salt tolerance, using information from

studies with whole plants, has allowed the identification of plants with

increased salt tolerance. Another approach is to increase salt

tolerance through cell culture (Croughan et al., 1981).

In some species, the variability in salt tolerance may not be

adequate for a successful breeding program because it may not be

possible to find salt-tolerant wild relatives and use them as sources of

germplasm. Suspension of cells from salt-sensitive plants in solutions











In the name of God,
Most Gracious,
Most Merciful.

"It is He Who has let free the two bodies
of flowing water: one palatable and sweet
and the other salt and bitter: yet has He
made a barrier between them, a partition
that is forbidden to be passed."

Glorious Quran
Sura XXV (Furquan), or The Criterion
Verse #53


In the name of God
Most Gracious,
Most Merciful.

"It is He Who sendeth down rain from the
skies: with it We produce vegetation of all
kinds: from some We produce green (crops),
out of which we produce grain, heaped up (at
harvest); out of the date-palm and its sheaths
(or spathes) (come) clusters of dates hanging
low and near: and (then there are) gardens of
grapes, and olives, and pomegranates, each
similar (in kind) yet different (in variety):
when they begin to bear fruit, feast your eyes
with the fruit and the ripeness thereof. Behold!
in these things there are signs for people who
believe.
Yet they make the Jinns equals with God, though
God did create the Jinns; and they falsely, having
no knowledge, attribute to Him sons and daughters.
Praise and glory be to Him! (for He is) above
what they attribute to Him!
To him is due the primal origin of the heavens
and the earth: how can He have a son when He hath
no consort? He created all things, and He hath
full knowledge of all things.
That is God, your Lord! There is no god but He,
The Creator of all things: then worship ye Him:
and He hath power to dispose of all affairs."

Glorious Quran
Sura VI (An'am), or Cattle
Verses #99-102







19

having various degrees of osmotic stress was found to be a promising

technique to select salt-tolerant cells from salt-sensitive cells. This

implies that the genetic information for growth in a saline environment

may be present in salt-sensitive cells but is not expressed. Selection

of salt-tolerant cells may provide genetic material that will help

improve our understanding of salinity resistance at the cellular level.















MATERIALS AND METHODS


General Procedures

This study consisted of 5 experiments involving citrus seedlings

grown in greenhouses in central Florida. Seeds were sown in plastic

trays composed of individual cells. The trays were filled with PROMIX

BX [60% Canadian peat, 20% perlite, and 20% vermiculite with dolomitic

limestone, superphosphate, calcium nitrate and fritted trace elements

added]. The seeds were irrigated with tap water twice a week until

emergence. Seedlings were irrigated with tap water every other day and

fertilized with 20-20-20 (N,P,K) fertilizer once a week. The

temperature and ri-ative humidity in the greenhouses were controlled by

both heating and evaporative cooling systems with conventional end-wall

air circulation fans. The minimum and maximum temperature and relative

humidity ranged from 20 to 350C and from 40 to 100%, respectively.

Three to 6 months after emergence, uniform seedlings were selected

and transplanted into pots or wooden boxes containing fine sand taken

from the top 30 cm of a citrus orchard soil. The soil was Astatula fine

sand (hyperthermic, uncoated Typic Quartzipsamments) with a pH of 6.5

and a field capacity and a wilting percentage of 7.2% and 1.2% (volume

basis), respectively. Seedlings were irrigated every 2 to 3 days with

half strength Hoagland's solution #1 (Hoagland and Arnon, 1950) for at

least one month before starting salt and polyethylene glycol (PEG)

treatments. Treatments were started by adding NaC1, PEG, or other salts

to the Hoagland solution.







21

The water holding capacity of the soil in the containers was about

18% (volume basis). The irrigation frequency was 2 to 3 days. The

amount of solution added each time was based on bringing the soil to

slightly more than the water holding capacity of the soil in the

containers to prevent salt accumulation in the growth medium and to

prevent plants from undergoing a drought stress.

Standard curves (Fig. 1, 2) of osmotic potential versus solute

concentration were developed for NaC1 and PEG 4000 by measuring vapor

pressure and freezing point depression. The values obtained were

similar to those of Steuter et al. (1981) who compared freezing point

depression and vapor pressure methods for determination of water

potential of PEG solutions. Electrical conductivities of the different

treatments were determined with a conductivity meter. Electrical

conductivity values were converted to TDS (Richards, 1954).

Sodium chloride and PEG treatments were continued for at least 4

months, after which the plants were harvested and the roots were washed

briefly with tap water to free them of sand particles. Shoots were

separated into stems and leaves, and roots were separated into taproots,

lateral roots, and fibrous roots (roots less than 2 mm in diameter).

The material was oven-dried for 3 days at 600C, weighed, ground, and

retained for ion analysis.

Analysis of variance (F-test) was used to determine significant

differences and Duncan's multiple range test was employed for mean

comparison at P < 0.05.

Experiment 1: Effects of NaCl and PEG on the Root Conductivity and Leaf
Ion Content of Seedlings of 7 Citrus Rootstocks

The objective of this experiment was to compare the growth, ion

content, and water relations of 7 rootstocks treated with different













NaCI Concentration (g L
4 6


-0.2[


-0.41


Fig. 1. Osmotic potential versus NaC1 concentration as
determined by vapor pressure (VPD) and
freezing point depression (FPD).


VPD


-0.6[







23








PEG Concentration (g L ')

50 100 150 200
--A I r


FPD


-0.6 \

\0


Fig. 2. Osmotic potential versus PEG concentration as
determined by vapor pressure (VPD) and
freezing point depression (FPD).


-0.2




-0.4







24

levels of NaCI and PEG. On October 20, 1985, 5-month-old uniform

seedlings of 7 rootstock cultivars were transplanted into 33 cm-tall

black plastic pots containing about 2.2 L of fine sand. Rootstocks

studied were the following: sour orange (Citrus aurantium), Cleopatra

mandarin (C. reshni), Swingle citrumelo (C. paradisi x Poncirus

trifoliata), Carrizo citrange (P. trifoliata x C. sinensis), rough lemon

(C. jambhiri), Milam (C. jambhiri variant) and trifoliate orange (P.

trifoliata). The plants were watered with a half strength Hoagland's

solution and were grown with this control solution for 2 months. Sodium

chloride and PEG treatments were started on December 19, 1985, and

nutrient solutions for treated plants were identical to that of the

control plants except for the addition of NaC1 and PEG. Sodium chloride

and PEG were added to the half strength Hoagland's solution to achieve

final concentrations of -0.10, -0.20, and -0.35 MPa. The basic nutrient

solution (control) had an osmotic potential (OP) of -0.05 MPa.

Treatments were as follows:

Treatment TDS OP EC NaC1


(mg L-') (MPa) (dS m- ) (mmo

1. NS control : 2 Hoagl. sol. 550 -0.05 1.1 0

2. NaC1 (0.10) : 1.0 g NaCl/L 'h Hoagl. sol. 1600 -0.10 3.1 17

3. PEG (0.10) : 55 g PEG/L b Hoagl. sol. 460 -0.10 0.9 0

4. NaC1 (0.20) : 2.2 g NaC1/L H Hoagl. sol. 3000 -0.20 5.4 38

5. PEG (0.20) : 105 g PEG/L Y Hoagl. sol. 400 -0.20 0.8 0

6. NaCl (0.35) : 4.2 g NaCl/L Y Hoagl. sol. 4900 -0.35 8.8 72

7. PEG (0.35) : 144 g PEG/L Hoagl. sol. 350 -0.35 0.7 0

Plants were adjusted to their final NaC1 and PEG concentrations

through a progression of -0.10, -0.20, and -0.35 MPa solutions at 2-day

intervals to avoid osmotic shock. Plants were then maintained at their


1)


l)







25

final osmotic levels for 5 months. The experimental unit was a single

seedling arranged in a split plot with 4 replications. The 7 salt

treatments were assigned to the main plots and the 7 rootstocks to the

subplots.

At the end of the experiment, root hydraulic properties were

evaluated while in situ on 4 seedlings of each rootstock as previously

described (Graham and Syvertsen, 1984, 1985; Levy et al., 1983;

Syvertsen and Graham, 1985). Before measuring, the soil was wetted to

field capacity to minimize possible differences in soil hydraulic

conductivity and equilibrated to 250C in the laboratory. Each pot and

intact plant were placed in a pressure chamber. The stem was then cut

10 cm above the soil and the chamber was sealed around the cut stem.

The pressure within the chamber was increased gradually to a constant

value of 0.5 MPa. After an initial equilibration time of 10 minutes,

the weight of the liquid cxuded from the cut end was measured at least 5

times at 1 minute intervals. Osmotic potential of the exudate was

measured by a Wescor vapor pressure osmometer calibrated with NaC1

solutions.

Each root system was washed free of soil, and the total length of

fibrous roots of each plant was determined by the line-intersect method

(Tennant, 1975). Water flow per root system measured in this way

included a soil conductivity component and was expressed as weight of

exudate per unit time and pressure (ug s-1 MPa- ). Root conductivity

for each rootstock was calculated by dividing the water flow by the

total fibrous root length. Thus, the root conductivity was expressed in

ug/s/MPa per meter of fibrous roots (ug m- s MPa-).

Prior to measuring root conductivity, the trunk circumference of

each seedling was measured at a point 5 cm above the soil surface and







26

converted to stem cross sectional area. Dry weights of leaves, stems,

fibrous roots, and tap roots were determined. Shoot root ratio and

specific root weight (root weight per unit length) were calculated.

Leaf chloride content was measured using a Buchler-Cotlove chloridometer

after extracting the leaf samples with a nitric-acetic acid solution.

Measurement of leaf Na, Ca, Mg, K, P, Zn, Mn, Cu, and Fe content was

performed using an inductively coupled argon plasma spectrophotometer

after a wet digestion of the samples in a nitric-perchloric acid

mixture.

Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin
Seedlings under NaC1 and PEG Stresses

The objective of this experiment was to study the effects of NaC1

and PEG on the root conductivity, plant growth, stomatal conductance,

and chlorophyll content of seedlings of 2 rootstocks differing in

chloride accumulation characteristics, sour orange and Cleopatra

mandarin (Cooper et al., 1951).

Six-month-old uniform seedlings of sour orange and Cleopatra

mandarin were transplanted on November 13, 1985, into 19-cm tall black

plastic pots containing 5.5 L of Astatula fine sand. Plants were then

watered to excess every 2 to 3 days with half strength Hoagland's

solution for one month before NaC1 and PEG treatments were started. The

treatments were the same as in Experiment 1. The treatments were

replicated 7 times in a split plot design with 2 main plots rootstockss)

and 7 subplots (solutions). All variables measured in Experiment 1 with

the exception of the chemical analysis were also measured similarly in

this experiment. Seedling height from the soil surface to the terminal

bud was measured every 2 weeks. Leaf conductance to water vapor was

measured on abaxial leaf surfaces with a Li-cor 1600 steady state







27

porometer at 2-hour intervals from 0700 to 1700 hours for 3 consecutive

days.

After 5 months of NaCl and PEG treatments, two 1-cm diameter disks

were removed from the central area of 2 mature leaves per seedling to

determine leaf chlorophyll content using N, N-dimethyl formamide as a

solvent (Moran and Porath, 1980; Syvertsen and Smith, 1984). Two

millimeters of N, N-dimethyl formamide were placed in a small bottle and

the 2 leaf disks which were removed from the same seedling were weighed

and then immersed in the solvent. The bottles were firmly closed and

stored in the dark in a freezer for 2 months. The bottles were then

removed from the freezer and left in the dark to equilibrate to the

temperature of the laboratory prior to spectrophotometer examination.

One millimeter of the chlorophyll extract from each bottle was diluted

with deionized water and examined by means of a scanning

spectrophotometer. The optical density of the extract was measured at

wavelengths of both 663 mu and 644 mu, and chlorophyll content was

calculated following the equations used by Arnon (1949). Leaf

chlorophyll content was expressed as mg of leaf chlorophyll per gram

fresh weight.

New shoot growth was determined by counting leaf number and

measuring leaf area over a 3-month period. The plants were harvested

after 6 months of NaCl and PEG treatments. Total leaf area was measured

by a Li-cor leaf area meter. Fresh and dry weights of leaves were

determined. Specific leaf weight (SLW), expressed on a fresh and dry

weight basis per unit of leaf area, was calculated. Leaf succulence was

expressed as grams of water per gram of leaf dry weight.







28

Experiment 3: Fibrous Root Density and Distribution of Sour Orange
Seedlings under NaC1 and PEG Stresses

The objective of this experiment was to determine the effect of NaCi

and PEG on the root growth and distribution of sour orange seedlings.

Five-month-old seedlings were transplanted on October 1, 1985, into root

boxes filled with Astatula fine sand. The root boxes were similar to

those described by Bevington and Castle (1982, 1985). Each container

consisted of one plexiglas sheet (6.4 mm thick) attached to the front of

a wooden box. The plexiglas was covered with a removable metal shutter

to exclude light. The internal dimensions of a root box were 87 cm

high, 27 cm wide, and 5 cm thick. The viewing surface was 23 dm and

the volume was about 11.5 L. Drainage was provided by 3 mesh-covered

outlets in the bottom of the box. The boxes were vertically oriented.

Seedlings were allowed to adjust in their containers for 2 months.

During this period, they were watered every other day with half strength

Hoagland's solution. Plants were then treated with 2 concentrations of

NaC1 and PEG (total osmotic potential equal to -0.12 and -0.24 MPa).

The experimental design was a randomized complete block with 3

replications using a single seedling per box. Treatments were as

follows:

Treatment TDS OP EC NaCl

(mg L') (MPa) (dS m-1) (mmol)

1. NS control : V2 Hoagland's sol. 550 -0.05 1.1 0

2. NaCl (0.12) : 1.1 g NaCl/L Y Hoagl. sol. 1700 -0.12 3.3 19

3. PEG (0.12) : 60 g PEG/L V' Hoagl. sol. 450 -0.12 0.9 0

4. NaC1 (0.24) : 2.8 g NaC1/L Hoagl. sol. 3300 -0.24 5.9 48

5. PEG (0.24) : 110 g PEG/L 'A Hoagl. sol. 390 -0.24 0.8 0







29

Root growth was recorded at 2-week intervals by using colored

pencils to trace the root system onto transparent acetate sheets. Plant

height was measured at 2-week intervals. Stomatal conductance was

measured about every 2 weeks and for 2 consecutive days at 2-hour

intervals from 0700 to 1700. After 6 months of NaCl and PEG treatments,

the plants were taken from their boxes by removing the plexiglas wall

and inserting a needle board to hold the root system in place. Leaves,

stems, and roots were separated and roots were divided in place into 3

equal compartments (top, middle, and bottom). Shoot and root dry

weight, shoot root ratio, leaf number, plant height, root length,

specific root weight, and stomatal conductance were determined as

described in Experiment 2.

Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity

The objective of this experiment was to determine and quantify the

growth and water relations of sour orange seedlings when only a portion

of the root system was exposed to NaCl or PEG. A split-root system was

initiated using the technique of Koch and Johnson (1984). The tap root

of each seedling at the 3-leaf stage was cut to a 1 cm length and all

other roots were removed. The remaining portion of the tap root was

dipped into a 50% ethanol solution containing 5 grams of IBA

(indolebutyric acid) per liter. Seedlings were then placed in PROMIX

BX, watered daily and fertilized weekly for 2 months. Seedlings which

had 2 uniform adventitious root systems were selected and transplanted

when 5 months old into 2.2 L square plastic containers stapled together

along one side (Fig. 3). These seedlings were left to adjust in their

double pots for 1 month before NaC1 and PEG treatments were imposed.

The treatments were replicated 4 times in a randomized complete block

design and are shown below:












































Fig. 3. Sour orange seedlings with a split-root system.
a. Root development after 2 months.
b. Container system used to grow split-root seedlings.









Treatment



NS/NS (no salt)

NS/NaC1 (0.10)

NaCI (0.10)/NaCl (0.10)

NS/NaC1 (0.20)

NaCl (0.20)/NaCl (0.20)

NS/NaC1 (0.35)

NaCl (0.35)/NaCl (0.35)

NS/PEG (0.20)

PEG (0.20)/PEG (0.20)

Water relations variable


31

TDS

(mg L l)

550/550

550/1600

1600/1600

550/3000

3000/3000

550/4900

4900/4900

550/400

400/400


es were monitored


OP

(MPa)

-0.05/-0.05

-0.05/-0.10

-0.10/-0.10

-0.05/-0.20

-0.20/-0.20

-0.05/-0.35

-0.35/-0.35

-0.05/-0.20

-0.20/-0.20

on 4 successive


EC

(dS m')

1-1/1.1

1.1/3.1

3.1/3.1

1.1/5.4

5.4/5.4

1.1/8.8

8.8/8.8

1.1/0.7

0.7/0.7

days during


the fourth month of salt treatment. Leaf water potential was measured

at sunrise and at midday on fully expanded leaves using a pressure

chamber. Leaves were then removed from the chamber, wrapped in double

plastic bags and rapidly frozen at -200C. Leaves were subsequently

thawed after 48 hours and their osmotic potential was determined with a

vapor pressure osmometer. Turgor potential was obtained by subtracting

the osmotic potential value from the water potential value. Morning and

midday stomatal conductance and leaf transpiration rates were measured

with a steady state porometer. For anatomical study, 2 mature leaves

per plant from NS/NS and NaCl (0.35)/NaCl (0.35) treatments were

selected from about half-way between the first leaf and the shoot apex.

Two small rectangles were cut at mid-lamina of each leaf, frozen

immediately, and cut by a Cryostat minot rotary microtome in sections 10

microns thick. Sections were then thawed in a phosphate buffer saline

solution. Twenty randomly selected leaf cross sections per treatment

were fixed for a light microscopy study.







32

After 4 months of NaCi and PEG treatment, the plants were harvested,

and shoot and root dry weights were determined.

Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under
Saline Conditions

The objective of this experiment was to determine if the addition of

calcium to saline irrigation water would reduce salt damage.

Three-month-old sour orange seedlings were transplanted on August 10,

1986, into the same pots used in Experiment 2. Salt treatments

(Table 1) were started after 1 month of adjustment, and seedlings were

irrigated every 2 to 3 days for 4 months. The treatments were

replicated 8 times in a randomized complete block design. The plants

were watered the night before harvest and leaves were removed the

following morning. Fresh and dry weights of leaves, stems, and roots

were recorded. The succulence of new and old leaves was computed. The

dried, mature, fully expanded leaves were ground and their mineral

content was determined as in Experiment 1.











Table 1. Salt treatments and chemical properties of the different
treatments--Experiment 5.


Sodium Osmotic
Na Ca calcium TDS EC potential
Treatment (mmol) (mmol) ratio (mg L- ) (dS m-') (MPa)


1 (NS) 0 2.5 0:2.5 550 1.1 -0.05
2 NaCi 40 2.5 16:1 3000 5.4 -0.21
3 NaC1 + 7.5 mM CaSO4 40 10.0 4:1 3500 6.3 -0.23
4 NaCi + 13.5 mM CaSO4 40 16.0 2.5:1 3800 6.8 -0.26
5 NaCI + 8.75 mM CaSO4 + 8.75 mM CaC12 40 20.0 2:1 4200 7.5 -0.26
6 NaCi + 7 mM KC1 40 2.5 16:1 3400 6.1 -0.23
7 NaCi + 7 mM KC1 + 7.5 mM CaSO4 40 10.0 4:1 3800 6.8 -0.21
8 NaCi + 17.5 mM CaC12 40 20.0 2:1 4700 8.4 -0.30



9 (NS)--no calcium 0 0 0:1 450 0.9 -0.03
10 NaC1--no calcium 40 0 40:0 2800 5.0 -0.19
11 NaCi + 1 mM CaSO4 40 1.0 40:1 2900 5.2 -0.19
12 NaCl + 5 mM CaSO4 40 5.0 8:1 3100 5.5 -0.21















RESULTS


Experiment 1: Effects of NaCl and PEG on the Root Conductivity and Leaf
Ion Content of Seedlings of 7 Citrus Rootstocks

The results of the analysis of variance showed that salt treatments

and rootstocks were significant and independent factors; i.e., the

interaction of these 2 factors was not significant.

Significant differences in growth due to NaCl and PEG treatments

were found among rootstocks. Shoot dry weight generally decreased as

NaCl and PEG concentration increased in the nutrient solution (Table 2).

Shoot dry weight at the low, medium, and high NaC1 concentrations was 18

to 36%, 30 to 55%, and 58 to 82% lower, respectively, than the control

plants. Shoot dry weight of sour orange (SO) and Cleopatra mandarin

(CM) seedlings was the least affected while Milam (ML) and Poncirus

trifoliata (PT) seedlings showed the greatest response. Sodium chloride

and PEG effects on root dry weight were similar to those on shoot dry

weight (Table 3). However, roots were less affected than shoots so that

the shoot-root ratio decreased with increasing NaCI and PEG

concentration (Table 23, Apendix). Total plant dry weight (Table 24,

Appendix) and stem cross sectional area (Table 25, Appendix) were

proportionally reduced by NaCl and PEG concentrations and reductions

were usually greater with PEG than with NaCI. Fibrous root length was

also reduced by NaCl (Fig. 4) but specific root weight (SRW, dry weight

per unit length) increased with increasing NaCl concentration (Table 4).












Table 2. Shoot dry weight (g) of seedlings of 7 rootstocks grown for 5 months under different NaCl and
PEG concentrations--Experiment 1.


Sour Cleopatra Swingle Carrizo Rough Poncirus

orange mandarin citrumelo citrange lemon Milam trifoliata
% % % % % % %
lower lower lower lower lower lower lower
Treatment than than than than than than than
(-MPa) Meanz Ns Mean NS Mean NS Mean NS Mean NS Mean NS Mean NS


NS control 21.0ay 0 22.1a 0 13.9a 0 17.8a 0 30.2a 0 24.6a 0 8.6a 0

NaCI (0.10) 17.3ab 18 17.9ab 19 9.8b 30 12.5b 30 21.Ob 30 16.7b 32 5.5b 36

NaC1 (0.20) 13.2b 37 15.4b 30 7.7bc 45 9.1c 49 14.8c 51 11.Oc 55 4.3bc 50

NaCl (0.35) 8.8c 58 9.1c 59 4.1c 70 4.5d 75 8.6d 72 4.3e 82 2.0e 76

PEG (0.10) 14.2b 32 12.5bc 44 8.4b 40 8.3c 54 15.9c 48 11.9c 52 4.3bc 50

PEG (0.20) 9.5c 55 8.8c 61 5.9c 58 6.0d 66 10.6d 65 8.0d 68 3.7cd 57

PEG (0.35) 6.7c 69 7.2c 68 4.7c 67 4.9d 72 8.0d 74 7.1d 71 2.8de 67


zMean of 4 plants.
'Mean separation within columns by


Duncan's Multiple Range Test, 0.05 level.











Table 3. Root dry weight (g) of seedlings of 7 rootstocks grown for 5 months under different NaCi and
PEG concentrations--Experiment 1.


Sour Cleopatra Swingle Carrizo Rough Poncirus
orange mandarin citrumelo citrange lemon Milam trifoliata
% % % % % % %
lower lower lower lower lower lower lower
Treatment than than than than than than than
(-MPa) Meanz NS Mean NS Mean NS Mean NS Mean NS Mean NS Mean NS


NS control 9.24ay 0 8.59a 0 6.14a 0 6.71a 0 12.01a 0 9.72a 0 3.77a 0

NaCl (0.10) 7.76ab 16 7.05ab 18 4.62ab 25 5.44ab 19 8.66ab 28 7.54ab 22 2.88ab 24

NaCl (0.20) 5.92b 36 6.32ab 26 3.60bc 41 4.14bc 38 6.25bc 48 5.09bc 48 2.28bc 40

NaCl (0.35) 4.03bc 56 3.98c 54 2.34c 62 2.44c 64 4.56c 62 2.59d 73 1.66c 56

PEG (0.10) 6.53b 29 6.02ab 30 4.70ab 24 4.57ab 32 8.73ab 27 5.50bc 43 3.00ab 20

PEG (0.20) 5.10bc 45 4.93bc 43 3.88bc 37 3.44bc 49 6.83bc 43 4.91bc 50 2.57ab 32

PEG (0.35) 3.93c 58 4.38bc 49 3.56bc 42 3.36bc 50 5.79bc 52 4.29cd 56 1.98bc 48


ZMean of 4 plants
YMean separation within columns by


Duncan's Multiple Range Test, 0.05 level.












V)
z

0
.9 80


S60
r
S40
20




Co





0 60



cD
S2


S80




S 60
60

40


- 20
8
Ir


CM
RL SC CC ML






SO

so


- CM FRL
S 1 CC n ML
| 00 H, r Pi


& & I I 6 4 4 1. .


- iU


Rootstock



Fig. 4. Effect of 3 NaC1 concentrations (a = -0.10 MPa,
b = -0.20 MPa, c = -0.35 MPa) on the total
fibrous root length, root hydraulic conductivity,
and water flow rate for seedlings of 7 citrus
rootstocks.
















Table 4. Specific fibrous root weight (mg m- ) of seedlings of 7 rootstocks
grown for 5 months under different NaCl concentrations-
Experiment 1."


Treatments Sour Cleopatra Swingle Carrizo Rough Poncirus

(-MPa) orange mandarin citrumelo citrange lemon Milam trifoliata


NS control 71 aY 70 a 68 c 59 b 68 c 71 c 54 c

NaCl (0.10) 72 a 71 a 70 bc 61 b 70 bc 74 bc 56 bc

NaCI (0.20) 73 a 71 a 73 ab 63 ab 73 b 77 ab 59 b

NaC1 (0.35) 73 a 72 a 75 a 66 a 78 a 81 a 73 a

ZMean of 4 plants.
'Mean separation within columns by Duncan's Multiple Range Test, 0.05 level.







39

The increase in SRW of rough lemon (RL), ML, and PT was greater than

that of the other rootstocks.

Root hydraulic variables were affected by rootstock and NaCl.

Significant differences in root conductivity among rootstocks were found

under non-stresed conditions (Table 5) as well as under NaCl stress

conditions (Fig. 4). Sour orange and CM had the smallest reduction in

hydraulic conductivity and ML and PT had the greatest. There was a

significant negative relationship between root hydraulic conductivity

and SRW of the 7 rootstocks studied (Fig. 5). As root weight per unit

length increased, conductivity decreased.

Water flow through the root system decreased as much as 41 to 89%

at the first NaCl level (Fig. 4). Osmotic potential of root exudate due

to NaCl stress followed the same trend as root hydraulic conductivity

(Fig. 20, Appendix). Water flow and osmotic potential of root exudate

were reduced the least in SO and CM and the most in ML and PT. However,

when NaCl was not added to the irrigation water, PT and Swingle

citrumelo (SC) had the highest osmotic potential of root exudate, and SO

and CM had the lowest potentials (Table 5).

Leaf burn symptoms appeared in the NaCl (0.35) treatment in PT and

ML after 5 weeks. In RL, SC, Carrizo citrange (CC), and SO, leaf burn

symptoms occurred after 6 weeks at the highest NaCl concentration (-0.35

MPa). Just before harvest, final evaluation of the different rootstocks

based on tree appearance and performance was made (Table 6, Fig. 6).

Leaf ion content of the seedlings of the 7 rootstocks was affected

by the NaCl and PEG concentrations. Sodium (Table 7) and chloride

(Table 8) contents in the leaves of all rootstocks increased with

increasing NaCl in the nutrient solution. Cleopatra mandarin

accumulated the least chloride while PT, SC, and CC accumulated the














Table 5. Root length, root conductivity, water flow rate, and osmotic potential of
root exudate of seedlings of the 7 rootstocks under non-stressed
conditions. Osmotic potential of the nutrient solution was
-0.05 MPa--Experiment 1.z


Rootstock Root length Root conductivity Water flow rate OP of root exudate
(m) (ug s m-i MPa ) (ug s MPa ) (MPa)

Sour orange 73.1 by 9.9 d 724 c -0.23 c

Cleopatra mandarin 78.0 b 10.4 d 811 c -0.20 c

Swingle citrumelo 45.6 c 24.2 b 1104 b -0.13 a

Carrizo citrange 53.1 c 26.6 b 1413 a -0.17 b

Rough lemon 107.4 a 14.2 c 1525 a -0.17 b

Milam 70.3 b 15.7 c 1104 b -0.17 b

Poncirus trifoliata 37.4 c 31.4 a 1174 b -0.12 a


ZMean of 4 plants.
Mean separation within columns by


Duncan's Multiple Range Test, 0.05 level.























c)
o) 30
Z)


50 60 70


Specific Root Wt


(mg m )


Fig. 5. Relationship between root hydraulic conductivity
and specific root weight of seedlings of 7 citrus
rootstocks under non-stressed conditions.











Table 6. Visible injury in seedlings
treatments--Experiment 1.


of 7 rootstocks after 5 months of NaCl


Osmotic potential of the irrigation water (MPa)

Rootstock -0.10 -0.20 -0.35


Cleopatra mandarin No leaf burn No leaf burn Leaf burn
Leaf drop


Sour orange No leaf burn Light leaf burn Leaf burn
Leaf drop


Single citrumelo No leaf burn Leaf burn Leaf burn
Carrizo citrange Leaf drop Leaf drop


Rough lemon Leaf burn Leaf burn Leaf burn
Milam Leaf drop Leaf drop
Branch die back


Poncirus trifoliata Leaf burn Leaf burn Leaf burn
Leaf drop Leaf drop Complete defoliation
Branch die back














































Fig. 6. Effect of NaC1 at an osmotic potential of -0.35 HPa on
the 7 rootstocks after 5 months of salinity treatments.














Table 7. Leaf sodium content (%) of seedlings of 7 rootstocks grown for 5 months
under different NaCi and PEG concentrations--Experiment 1.2


Treatment Sour Cleopatra Swingle Carrizo Rough Poncirus

(-MPa) orange mandarin citrumelo citrange lemon Milam trifoliata




NS control 0.02 dy 0.03 c 0.03 d 0.02 d 0.03 c 0.02 d 0.02 d

NaCI (0.10) 0.47 c 0.81 b 0.29 c 0.18 c 1.40 b 1.19 c 0.18 c

NaCl (0.20) 1.38 b 1.96 a 1.32 b 1.12 b 1.69 b 2.19 b 0.67 b

NaCl (0.35) 1.57 a 1.98 a 1.89 a 1.74 a 2.65 a 2.76 a 1.59 a

PEG (0.10) 0.03 d 0.04 c 0.03 d 0.02 d 0.03 c 0.02 d 0.02 d

PEG (0.20) 0.02 d 0.04 c 0.02 d 0.02 d 0.02 c 0.02 d 0.02 d

PEG (0.35) 0.02 d 0.03 c 0.02 d 0.02 d 0.02 c 0.02 d 0.02 d


ZMean of 4 plants.
YMean separation within


columns by Duncan's Multiple Range Test, 0.05 level.














Table 8. Leaf chloride content (%) of seedlings of 7 rootstocks
different NaCl and PEG concentrations--Experiment 1.z


grown for 5 months under


Treatment Sour Cleopatra Swingle Carrizo Rough Poncirus

(-MPa) orange mandarin citrumelo citrange lemon Milam trifoliata


NS control 0.02 dy 0.03 d 0.02 d 0.03 d 0.04 d 0.05 d 0.06 d

NaCl (0.10) 1.77 c 0.42 c 0.87 c 0.99 c 2.33 c 1.53 c 1.38 c

NaCI (0.20) 2.49 b 1.37 b 2.57 b 2.67 b 2.64 b 3.07 b 2.81 b

NaCl (0.35) 3.61 a 2.62 a 3.70 a 3.79 a 4.06 a 4.48 a 4.04 a

PEG (0.10) 0.02 d 0.02 d 0.01 d 0.03 d 0.03 d 0.03 d 0.04 d

PEG (0.20) 0.03 d 0.03 d 0.01 d 0.02 d 0.03 d 0.03 d 0.03 d

PEG (0.35) 0.04 d 0.03 d 0.02 d 0.02 d 0.03 d 0.04 d 0.03 d


ZMean of 4 plants.
YMean separation within


columns by Duncan's Multiple Range Test, 0.05 level.







46

least sodium. Even at a relatively low NaCl concentration (-0.10 MPa),

large amounts of chloride were accumulated in SO, RL, ML, and PT leaves.

Large amounts of sodium were also accumulated in RL and ML leaves. The

accumulation or exclusion characteristics of sodium and chloride for

each rootstock are summarized in Table 9.

Sodium chloride at -0.35 MPa reduced leaf calcium of all

rootstocks 10 to 40% with the exception of PT while PEG generally

increased calcium content (Table 10). Both NaCl and PEG reduced

magnesium (Table 26, Appendix). Magnesium reduction varied among

rootstocks and ranged from 28 to 50% and from 22 to 41% under NaCl and

PEG, respectively.

Potassium decreased significantly in SO, CM, RL, and ML but did not

in SC, CC, and PT with NaCl treatments (Table 27, Appendix). Potassium

seemed to be more strongly reduced in PEG treatments than in NaCl

treatments.

Both NaCl and PEG had similar effects on leaf phosphorus content

but the effect was more pronounced with PEG (Table 28, Appendix).

Sodium chloride and PEG significantly increased phosphorus in CM, SC,

CC, RL, and ML, reduced phosphorus in PT, and did not affect phosphorus

in SO.

Both zinc and manganese were significantly increased under PEG

stress. In some rootstocks, PEG more than doubled the zinc and

manganese levels. Zinc was reduced in SC, CC, RL, ML, and PT but was

not in SO and CM under NaCl stress (Table 29, Appendix). Manganese

tended to increase in the leaves of NaCl-treated plants except for RL

(Table 30, Appendix).




















Table 9. Ion exclusion and accumulation in the leaves of citrus rootstock seedlings.


Sour Cleopatra Swingle Carrizo Rough Poncirus

orange mandarin citrumelo citrange lemon Milam trifoliata




Cl accum. Cl exclu. Cl accum. Cl accum. Cl accum. Cl accum. Cl accum.



Na accum. Na accum. Na exclu. Na exclu. Na accum. Na accum. Na exclu.














Table 10. Leaf calcium content (%) of seedlings of 7 rootstocks
different NaCl and PEG concentrations--Experiment I.z


grown for 5 months under


Treatment Sour Cleopatra Swingle Carrizo Rough Poncirus

(-MPa) orange mandarin citrumelo citrange lemon Milam trifoliata


NS control 2.0 by 2.7 a 2.8 a 3.0 ab 2.3 b 3.1 b 2.3 c

NaCl (0.10) 1.7 c 2.3 c 2.2 b 2.6 c 1.8 c 2.2 c 2.3 c

NaC1 (0.20) 1.6 c 2.2 c 1.8 c 2.5 c 1.8 c 2.1 cd 2.1 c

NaCI (0.35) 1.3 d 1.4 d 1.8 c 2.5 c 1.4 d 1.9 d 2.1 c

PEG (0.10) 2.1 b 2.3 c 2.9 a 3.2 a 2.4 ab 3.2 ab 3.0 a

PEG (0.20) 2.3 a 2.7 a 2.9 a 2.9 b 2.7 a 3.5 a 2.5 b

PEG (0.35) 2.5 a 2.7 a 3.0 a 3.1 a 2.6 a 3.5 a 2.5 b


ZMean of 4 plants.
YMean separation within columns


by Duncan's Multiple Range Test, 0.05 level.







49

Experiment 2: Water Relations of Sour Orange and Cleopatra Mandarin
Seedlings under NaCI and PEG Stresses

As in Experiment 1, the results of the analysis of variance showed

significant differences among salt treatments and between rootstocks but

there were no significant interactions between these 2 factors.

The growth rate of SO and CM seedlings was significantly reduced

with increasing NaCi and PEG concentrations in the nutrient solution. A

NaCl concentration as low as -0.10 MPa (1600 mg L-1) reduced shoot and

root dry weight, root length, and stem cross sectional area by 50% after

6 months of treatment (data not presented). For both rootstocks,

seedling height was 26 to 39% and 33 to 50% lower, respectively, at the

first 2 NaCl concentrations (Table 31, Appendix). Total leaf area was

reduced by more than 40% at the -0.10 MPa NaCl level (Table 32,

Appendix). All these growth variables were more severely reduced under

PEG than under NaCi stress.

N4o significant difference in growth reduction was found between SO

and CM. Similar to Experiment 1, shoot root ratio decreased and SRW

increased with increasing NaCi and particularly PEG in the nutrient

solution (data not shown).

Sodium chloride reduced new shoot growth of SO (Tables 11, 12).

Leaf size of new shoots was smaller for salt-treated plants than for

control plants (Table 11). Sodium chloride-treated plants had 59 to 86%

fewer leaves than those grown without salt (Table 12).

Root hydraulic conductivity and water flow of the 2 rootstocks were

reduced at the first salinity level by about 50% and more than 70%,

respectively. Water flow through the root system to the shoot in the

PEG treatment was reduced by more than 95% (data not shown). Similar to

Experiment 1, no significant differences in root conductivity, water
















Table 11. Monthly new flush growth--area/leaf (cm2)--of sour
orange seedlings--Experiment 2.


February March April

Treatment % lower % lower % lower

(-MPa) Meanz than NS Mean than NS Mean than NS


NS control 16.7 ay 0 20.4 a 0 40.3 a 0

NaCl (0.10) 11.6 b 31 14.9 b 27 31.9 b 21

NaCl (0.20) 9.6 b 43 11.0 bc 46 18.0 c 55

NaCl (0.35) 8.5 b 49 9.8 c 52 16.0 c 60


ZMean of 7 plants.
YMean separation within columns


by Duncan's Multiple Range Test, 0.05 level.

















Table 12. Monthly new flush growth--leaf number--of sour orange
seedlings--Experiment 2.


February March April

Treatment % lower % Lower % lower

(-MPa) Meanz than NS Mean than NS Mean than NS


NS control 17 a' 0 27 a 0 43 a 0

NaCI (0.10) 7 b 59 10 b 63 13 b 70

NaCl (0.20) 5 b 71 7 bc 74 10 bc 77

NaCl (0.35) 4 b 77 5 c 82 6 c 86


zMean of 7 plants.
YMean separation within columns
level.


by Duncan's Multiple Range Test, 0.05







52

flow and osmotic potential of root exudate were found between SO and CM.

There was a positive correlation between water flow through the root

system and osmotic potential of root exudate (Fig. 7). With NaCl, less

water flow corresponded to higher ion concentrations in the root exudate

and consequently to a lower osmotic potential of the root exudate. Both

NaCi and PEG increased SLW when expressed on a dry weight basis (Table

33, Appendix). However, unlike NaC1, PEG decreased SLW when expressed

on a fresh weight basis. Consequently, leaf succulence was decreased by

PEG and increased by NaCl (Table 13). Among PEG treatments, succulence

was more reduced in SO than in CM seedlings.

Leaf chlorophyll content was reduced by NaCl and PEG treatments.

A significant difference in chlorophyll content due to NaCl was found

between SO and CM with a greater reduction occurring in SO (Table 14).

Polyethylene glycol generally reduced chlorophyll level in CM more than

did NaCl.

Stomatal conductance was also affected by NaC1 (Figs. 21, 22,

Appendix) and PEG (Figs. 23, 24, Appendix). No significant difference

in stomatal conductance was found between SO and CM under NaCl and PEG

stresses. Again, the effect of PEG was more pronounced on this variable

than that of NaCl. There was a significant positive linear correlation

between root hydraulic conductivity and midday stomatal conductance

(Fig. 8).

Addition of NaCl and PEG to the nutrient solution reduced seedling

water use or evapotranspiration. Water use could be approximated

because the amount of water added each time was based on bringing the

soil to slightly more than field capacity. Estimated water use for NaCl

(0.10), NaCl (0.20), NaCl (0.35), PEG (0.10), PEG (0.20), and PEG (0.35)





























1.5

c, SO
so
S0 M CM
a 1.0
4-



S0.5 -


A SO
0 CM
-0.1 -0.2 -0.3

Salt Treatment (MPa)





Fig. 7. Relationship between water flow rate and
osmotic potential of root exudate of sour
orange and Cleopatra mandarin seedlings.


0
O
3
o
-0.1 E
o
0
(D
-0.2 .

0

-0.3 :
o

m
0.
-0.4 >

(D
-0.5 "
-"
(U













Table 13.


Leaf succulence [(g water/g dry wt) x 100] of
seedlings of 2 rootstocks grown for 6 months under
different NaC1 and PEG concentrations--Experiment 2.


Sour orange Cleopatra mandarin

Treatment % difference % difference

(-MPa) Meanz than NS Mean than NS


NS control 179 by 0 125 b 0

NaCl (0.10) 190 ab +6 125 b 0

NaCI (0.20) 191 ab +7 125 b 0

NaC1 (0.35) 194 a +8 144 a +15

PEG (0.10) 63 c -65 81 c -35

PEG (0.20) 51 c -72 50 d -60

PEG (0.35) 27 d -85 52 d -58


EMean of 7 plants.
YMean separation within
0.05 level.


columns by Duncan's Multiple Range Test,













Table 14. Total chlorophyll (mg g-1 fresh wt) of seedlings
of 2 rootstocks grown for 6 months under different
NaCl and PEG concentrations--Experiment 2.


Sour orange Cleopatra mandarin

Treatment % lower % lover

(-MPa) Mean" than NS Mean than NS


NS control 1.99 ay 0 2.42 a 0

NaCl (0.10) 0.88 b 56 2.15 a 11

NaCl (0.20) 0.61 c 69 1.64 b 32

NaCl (0.35) 0.59 c 70 1.12 c 54

PEG (0.10) 0.88 b 56 1.20 c 50

PEG (0.20) 0.58 c 71 0.95 c 61

PEG (0.35) 0.56 c 72 0.83 c 66


EMean of 7 plants.
YMean separation within columns by Duncan's Multiple Range
Test, 0.05 level.


















r = 0.99


So/
-


SO
CM


A

r = 0.99










I I


2 6

Root Conductivity (ug s


m-1 MPa


Fig. 8. Relationship between midday stomatal
conductance and root conductivity of sour
orange and Cleopatra mandarin seedlings.


.50


.30






.10


I







57

treatments were, respectively, 50, 25, 17, 25, 12, and 12% of that for

the control (NS) treatment.

Experiment 3: Fibrous Root Density and Distribution of Sour Orange
Seedlings under NaCl and PEG Stresses

Plant responses in this experiment to NaCl and PEG treatments were

similar to those obtained in Experiment 2. Shoot and root dry weight,

shoot root ratio, and leaf number generally decreased with increasing

NaCI and PEG concentrations in the nutrient solution (data not shown).

Significant differences among treatments were found in stomatal

conductance during different months (Fig. 9) as well as in daily

stomatal conductance (Fig. 10). Stomatal conductance also decreased as

leaf age increased (Fig. 9). Throughout the growing period, shoot and

root growth rate increased with time, but the growth rate of stressed

seedlings was less than that of non-stressed seedlings. After 4 weeks,

measurements of seedling height (Fig. 11) and root length (Fig. 12)

showed a significant reduction in plant growth due to NaCl and PEG

treatments. Cycling between shoot and root growth was noticed under

stressed and non-stressed conditions (Fig. 13).

When the portion of the root system in each compartment (top,

middle, and bottom) of the root box was compared, root density decreased

with depth and was significantly higher in the top compartment than in

either of the lower 2 sections (Table 15, Fig. 14). Seedlings receiving

NaCl or PEG treatments developed a shallow root system as compared to

the control (Fig. 14). Stressed seedlings had a higher percentage of

the total root system in the top of the root boxes. About 65% of the

roots of the PEG-stressed seedlings, but less than 50% of the roots of

control seedlings, were located in the upper section (Table 15). In the














E .60


C .50-


C .40
o

".30-
E
0 0
) .20- E I NS
* .-----* NaCl(. 12)
a- -- NaCI(.24)
.10 -
.10-o PEG(.12)
Po PEG(.24)
Dec.4 Feb.11 Apr. 17
TIME (date)





Fig. 9. Midday stomatal conductance of sour orange seedlings
irrigated with nutrient solution containing no salt
(NS) or with added NaCl or PEG.

















E *
.20




c NS
o 10 0 NaCI(. 12)
o \
So/ o NaCI(.24)
o0 0-- ,0 0 L- o PEG(. 12)
o0
0 U\D- L e-o-o PEG(. 24)

I1 I 1 I l l I
7 11 15 7 11 15
Time (hr)






Fig. 10. Relationship of time of day to stomatal conductance of
sour orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaC1 or PEG
during 2 consecutive days. Measurements were started
on April 17, 1986. Seedlings were irrigated the day
before measurements were started and not irrigated
until after measurements were completed on Day 2.

















60


50

E
0 40

r
_ 30

I
20

u)
(I) .


Dec.4 Mar 6 Jul. 17
TIME (date)


Fig. 11. Growth of sour orange seedlings
nutrient solution containing no
with added NaC1 or PEG.


irrigated with
salt (NS) or













Sour orange
SNS


NaC(. 12)

NaCI(.24)
PEG(. 12)
PEG(.24)


Dec.4


Mar.6


Jul. 17


TIME (date)




Fig. 12. Fibrous root length of sour orange seedlings irrigated
with nutrient solution containing no salt (NS) or with
added NaC1 or PEG.































E
C

2 20


0



5


Dec Jan Feb Mar Apr May Jun
TIME (month)




Fig. 13. Fluctuations in shoot and root growth of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with added NaCl or PEG.















Table 15. Fibrous root length (m and %) in the 3 compartments of the root boxes for seedlings under
different NaC1 and PEG treatments--Experiment 3.


Top Center Bottom Total


% lower % lower % lower % lower

Treatment Meanz than NS Mean than NS Mean than NS Mean than NS


NS control 80.8 ay (46%) 0 48.1 a (28%) 0 45.1 a (26%) 0 174.0 a (100%) 0

NaCI (0.12) 68.4 ab (55%) 15 36.7 ab (29%) 24 19.4 b (16%) 57 124.5 b (100%) 29

NaCl (0.24) 54.4 bc (55%) 33 29.1 bc (29%) 39 15.5 bc (16%) 66 99.0 bc (100%) 43

PEG (0.12) 54.2 bc (65%) 33 21.6 bc (26%) 55 7.4 bc (9%) 84 83.2 cd (100%) 52

PEG (0.24) 44.8 c (65%) 45 20.5 c (30%) 57 3.1 c (5%) 93 68.4 d (100%) 61


ZMean of 3 plants.
YMean separation within columns by


Duncan's Multiple Range Test, 0.05 level.













NaCI(.24)


Fig. 14. Root density and distribution of sour orange seedlings
growing in root boxes under non-stressed (NS) and
stressed (NaCI, PEG) conditions. NaCI and PEG
treatments were at -0.24 MPa osmotic potential.


24)







65

bottom section, only 5 to 16% of the roots developed in the stressed

chambers as compared to 26% in the controls.

Fibrous root length at the plexiglas face, measured from tracings

made on acetate sheets with colored pencils, was compared to the total

fibrous root length measured at the end of the experiment. Root length

against the plexiglas represented 3 to 4%, 2 to 3%, and 4 to 5% of the

total root length in the top, middle, and bottom of the root boxes,

respectively. From the comparison of root lengths at the plexiglas and

in the box, it was concluded that growth and distribution of citrus

roots at the plexiglas-soil interface correlated satisfactorily with

growth and distribution of roots in the bulk soil.

Experiment 4: Response of Split-Root Sour Orange Seedlings to Salinity

Uniform salinity was significantly more damaging to sour orange

seedlings than non-uniform salinity (Table 16; Fig. 15). Shoot dry

weight was reduced only slightly (9 to 21%) when half of the root -ystem

was irrigated with saline solutions. When both halves of the root

system were irrigated with saline solutions, shoot dry weight was

reduced 45 to 81% (Table 16). The trend was similar with root dry

weight in that stressing one-half of the root system resulted in only a

moderate reduction (16 to 31%) in root dry weight. Stressing both

halves gave a much larger reduction in root dry weight (43 to 79%).

In the split-root test, shoot growth did not correlate well with

the average salt stress of the total root system. The average osmotic

potential of the NS/NaCl (0.20) treatment was -0.12 MPa. Even though

this was slightly greater than the average osmotic potential of the NaCl

(0.10)/NaCl (0.10) treatment, shoot dry weight was 35% (10.7 g) less in

the NaCl (0.10)/NaCl (0.10) treatment. Similarly, shoot dry weight in

the NaCl (0.20)/NaCl (0.20) treatment was 50% (14.5 g) less than that in














Table 16.


Shoot and root dry wt of split-root sour orange seedlings under NaCI and PEG
stresses--Experiment 4.


Average OP -hoot dry wt (g) Root dry wt (g)
of solution % lower than % lover than
Treatment (MPa) Meanz NS/NS Mean NS/NS


NS/NS -0.05 37.1 ay 0 9.29/9.41 a 0

NS/NaCl (0.10) -0.08 33.6 ab 9 9.64/6.12 ab 16
NaCI (0.10)/NaCl (0.10) -0.10 20.4 d 45 5.33/5.27 cde 43

NS/NaC1 (0.20) -0.13 31.1 bc 16 9.49/4.22 bc 27
NaCI (0.20)/NaCl (0.20) -0.20 14.9 f 60 3.85/3.98 ef 58

NS/NaCl (0.35) -0.20 29.4 bc 21 9.74/3.21 bcd 31
NaCI (0.35)/NaCl (0.35) -0.35 7.1 gh 81 1.97/2.01 g 79

NS/PEG (0.20) -0.13 26.9 c 27 9.79/2.15 cd 36
PEG (0.20)/PEG (0.20) -0.20 3.6 h 90 1.67/1.81 g 81


ZMean of 4 plants.
1Mean separation within


columns by Duncan's Multiple Range Test, 0.05 level.











































Split-root treatment of sour orange seedlings under
uniform and non-uniform NaCl and PEG stresses. NaCl
treatments were at -0.10, -0.20, and -0.35 MPa osmotic
potentials. PEG treatments were at -0.20 MPa osmotic
potential.


Fig. 15.







68

the NS/NaCl (0.35) treatment, even though both of these treatments had

the same average NaCl stress (-0.20 MPa).

Under uniform salinity similar to Experiments 1, 2, and 3, shoot

growth was more reduced than root growth. However, under non-uniform

salinity, root dry weight on a percentage basis appeared to be more

reduced than shoot dry weight (Table 16).

Partial leaf burn occurred after 4 weeks in the NaC1 (0.35)/NaCl

(0.35) treatment and after 5 weeks in the NaCl (0.20)/NaCl (0.20)

treatment. No leaf damage symptoms were noticed in the remaining

treatments until the end of the experiment.

Water relations variables were monitored on 4 successive days

during the fourth month of salt treatment. Data were combined because

no significant differences were found from day to day. Similar to

growth, water relations variables were also significantly more disturbed

under uniform salinity than under non-uniform salinity conditions. With

uniform salinity, leaf water and turgor potentials decreased

significantly from morning to midday, but leaf osmotic potential did not

(Fig. 16). Leaf water potential, osmotic potential, stomatal

conductance, and transpiration decreased with increasing NaC1 and PEG

concentrations in the irrigation water (Tables 17, 18). Turgor

potential significantly increased in response to NaCl treatments

particularly during the morning. A significant positive correlation was

found between stomatal conductance and transpiration (Fig. 17). Similar

to findings of the preceding experiments, PEG at -0.20 MPa was more

damaging than NaCl at the same osmotic potential.

Cross sections of leaves from control (NS/NS) and from NaCl

(0.35)/NaCl (0.35) treatments, compared by light microscopy, showed that

the number of cell layers in the epidermis, the palisade, and the spongy







69






S1.5 _
Ca
Morning e

1.0
0
0)

I- Midday 0 0
S0.5 o









Morning WP
a

S-1.0 -
SMidday WP

od

o O A ~
E A

S-2.0 -

..J


-0.1 -0.2 -0.3

Salt Treatment (MPa)



Fig. 16. Leaf water, osmotic, and turgor potential of sour
orange seedlings irrigated with nutrient solution
containing no salt (NS) or with NaC1 added to
both root halves. Solid figures are morning
values and open figures are midday values.














Table 17.


Midday leaf water, osmotic, and turgor potentials (MPa) of split-root sour
orange seedlings under NaCI and PEG stresses--Experiment 4.


Water potential Osmotic potential Turgor potential
X lower % lower % higher
Treatment Meanz than NS/NS Mean than NS/NS Mean than NS/NS


NS/NS -1.23 ay 0 -1.73 a 0 0.50 a 0

NS/NaC1 (0.10) -1.22 a -1 -1.74 a 1 0.52 a 4
NaC1 (0.10)/NaC1 (0.10) -1.32 ab 7 -1.89 bc 9 0.57 a 14

NS/NaCl (0.20) -1.26 ab 2 -1.81 ab 5 0.55 a 10
NaCl (0.20)/NaCl (0.20) -1.48 abc 20 -2.08 d 20 0.60 a 20

NS/NaC1 (0.35) -1.32 ab 7 -1.89 bc 9 0.57 a 14
NaCI (0.35)/NaCl (0.35) -1.60 c 30 -2.26 e 31 0.66 a 32

NS/PEG (0.20) -1.29 ab 5 -1.85 ab 7 0.56 a 12
PEG (0.20/PEG (0.20) -1.43 abc 16 -1.98 cd 14 0.55 a 10


ZMean of 4 plants.
YMean separation within


columns by Duncan's Multiple Range Test, 0.05 level.















Table 18.


Midday stomatal conductance and transpiration of split-root sour
orange seedlings under NaC1 and PEG stresses--Experiment 4.


Stomatal conductance (cm s-') Transpiration (ug cm s )

% lower than % lower than
Treatment Meanz NS/NS Mean NS/NS


NS/NS 0.26 ay 0 0.63 a 0

NS/NaCl (0.10) 0.22 ab 15 0.55 ab 13
NaCi (0.10)/NaCl (0.10) 0.20 ab 23 0.45 bc 29

NS/NaCl (0.20) 0.21 ab 19 0.53 ab 16
NaC1 (0.20)/NaCl (0.20) 0.15 abc 42 0.36 bc 43

NS/NaC1 (0.35) 0.21 ab 19 0.46 abc 27
NaCi (0.35)/NaCl (0.35) 0.13 bc 50 0.33 c 48

NS/PEG (0.20) 0.18 abc 31 0.37 bc 41
PEG (0.20/PEG (0.20) 0.08 c 69 0.31 c 51


ZMean of 4 plants.
7Mean separation within columns


by Duncan's Multiple Range Test, 0.05 level.


















2.50






' 2.00

E
u
o,
0)
N-I

C 1.50
o

t--
0.
C,
I-
1.00






0.50


.20 .40 .60 80


Stomatal Conductance (cm s 1)






Fig. 17. Relationship between transpiration and stomatal
conductance of sour orange seedlings.







73

mesophyll in control leaves and NaCl-treated leaves were similar.

Epidermal and palisade cells of the control and NaCl-grown leaves were

also similar in size; however, the spongy mesophyll cells of the

NaCl-treated leaves were about 3 times larger than those of the control

(Fig. 18). The overall increase in leaf thickness due to NaCl was

relatively small (23%) because the enlarged cells of the spongy

mesophyll were tightly packed with much less intercellular space. Cells

of the spongy mesophyll in NaCl-treated leaves also had fewer

chloroplasts than those in the control leaves.

Experiment 5: Effects of Calcium on Sour Orange Seedlings Grown under
Saline Conditions

Addition of NaCl to half strength Hoagland's solution significantly

reduced growth of sour orange seedlings. Shoot, root, and total plant

dry weights were reduced by about 30% (treatments 2 and 10) when 40 mM

NaCl was added to the nutrient solution (Tables 19, 20). However,

addition of 7.5 mM CaSO4 (treatment 3) to the salty solution decreased

the adverse effect of NaCl on growth. Furthermore, addition of only

5 mM CaSO4 (treatment 12) completely inhibited the adverse effect of

NaCl. Addition of either KC1 (treatments 6 and 7) or CaCl (treatments

5 and 8) to the salty solution did not improve plant growth.

In the leaves of the sour orange seedlings, addition of NaCl to the

nutrient solution significantly increased sodium and chloride, decreased

calcium, magnesium, and potassium but had little or no effect on

phosphorus, zinc, manganese, copper, and iron (Table 21). Sodium and

chloride accumulation in the leaves usually reduces growth. Addition of

CaSO (treatments 3, 4, 11, and 12) to the saline solution reduced

sodium and chloride content and, therefore, improved plant growth.

Addition of KC1 (treatment 6) did not reduce sodium and chloride; hence,














































Fig. 18. Cross sections of sour orange leaves.
a. Leaf cross section of non-stressed seedling.
b. Leaf cross section of NaCl-stressed seedling.
i.s. = intercellular space.











Table 19. Root and shoot dry wt of sour orange seedlings under different salt treatments--Experiment 5.


Sodium Root dry wt (g) Shoot dry wt (g)
calcium % lower % lower
Treatment ratio Meanz than NS Mean than NS


1 (NS) 0:2.5 9.44 ay 0 36.62 a 0
2 NaCl 16:1 6.80 a 28 25.91 cde 29
3 NaC1 + 7.5 mM CaSO4 4:1 9.15 a 3 33.41 ab 9
4 NaCl + 13.5 mM CaSO4 2.5:1 7.54 a 20 29.22 bcd 20
5 NaCl + 8.75 mM CaSO4 + 8.75 mM CaCl2 2:1 6.94 a 26 25.06 de 32
6 NaCl + 7 mM KC1 16:1 6.86 a 27 27.04 cd 26
7 NaCl + 7 mM KC1 + 7.5 mM CaSO4 4:1 7.65 a 19 28.34 cd 23
8 NaCl + 17.5 mM CaC12 2:1 6.84 a 28 23.17 e 37


9 (NS)--no calcium 0:0 9.45 a 0 35.27 ab 0
10 NaCl--no calcium 40:0 7.36 a 22 26.12 cde 26
11 NaCl + 1 mM CaSO4 40:0 8.18 a 13 32.15 ab 9
12 NaCl + 5 mM CaSO4 8:1 9.32 a 1 36.99 a -5


ZMean of 8 plants.
YMean separation within columns by Duncan's Multiple


Range Test, 0.05 level.











Total plant dry wt and leaf succulence (water content as
under different salt treatments--Experiment 5.


% dry wt) of sour orange seedlings


Succulence =
Total plant dry wt (g) (g water/g dry wt) x 100

OP % lower
Treatment (MPa) Meanz than NS New leaves Old leaves


1 (NS) -0.05 46.06 ay 0 341 210
2 NaC1 -0.21 32.71 cde 29 307 222
3 NaCl + 7.5 mM CaSO4 -0.23 42.56 ab 8 322 215
4 NaCl + 13.5 mM CaSO4 -0.26 36.76 bcd 20 319 217
5 NaCl + 8.75 mM CaSO4 + 8.75 mM CaCl2 -0.26 32.00 de 31 320 244
6 NaCI + 7 mM KC1 -0.23 33.90 cde 26 315 258
7 NaCI + 7 mM KC1 + 7.5 mM CaSO4 -0.21 35.99 cd 22 339 241
8 NaCI + 17.5 mM CaCl2 -0.30 30.01 e 35 304 252


9 (NS)--no calcium -0.03 44.72 ab 0 357 222
10 NaCl--no calcium -0.19 33.48 cde 25 319 238
11 NaCl + 1 mM CaSO4 -0.19 40.33 ab 10 321 236
12 NaC1 + 5 mM CaSO4 -0.21 46.31 a -4 321 215


ZMean of 8 plants.
YMean separation within columns by Duncan's


Multiple Range Test, 0.05 level.


Table 20.













Leaf mineral analysis of sour orange
treatments--Experiment 5.z


seedlings under different salt


Treatment Ca (%) Mg (%) Na (%) Cl (%) K (%)



1 (NS) 2.1 cy 0.30 b 0.02 d 0.02 g 2.8 b
2 NaC1 1.7 d 0.21 c 0.47 ab 0.97 cd 2.0 c
3 NaC1 + 7.5 mM CaSO4 2.7 b 0.20 cd 0.24 c 0.43 f 1.9 c
4 NaCl + 13.5 mM CaSO4 2.7 b 0.20 cd 0.24 c 0.39 f 1.9 c
5 NaC1 + 8.75 mM CaSO4 + 8.75 mM CaC12 3.4 a 0.20 ed 0.26 c 0.73 de 2.0 c
6 NaCl + 7 mM KCl 1.3 ef 0.15 e 0.43 b 1.21 b 3.6 a
7 NaCl + 7 mM KC1 + 7.5 mM CaSO4 2.1 c 0.17 de 0.20 c 0.56 ef 2.9 b
8 NaC1 + 17.5 mM CaC12 3.5 a 0.21 c 0.20 c 1.52 a 1.9 c


9 (NS)--no calcium 1.6 de 0.36 a 0.02 d 0.04 g 2.9 b
10 NaC1--no calcium 1.1 f 0.23 c 0.61 a 1.13 bc 2.2 c
11 NaCl + 1 mM CaSO4 1.7 d 0.22 c 0.43 b 0.48 f 2.1 c
12 NaCI + 5 mM CaSO4 2.4 be 0.21 c 0.27 c 0.41 f 1.9 c


(Cont'd)


Table 21.











Table 21. (Cont'd)


Treatment P (%) Fe (ppm) Mn (ppm) Zn (ppm) Cu (ppm)



1 (NS) 0.13 ab 113 a 9 b 32 a 7 a
2 NaC1 0.13 ab 87 ab 11 ab 30 a 6 a
3 NaC1 + 7.5 mM CaSO4 0.13 ab 97 ab 10 ab 27 a 6 a
4 NaC1 + 13.5 mM CaSO4 0.12 abc 73 ab 9 b 28 a 5 a
5 NaC1 + 8.75 mM CaSO4 + 8.75 mM CaCl, 0.11 c 90 ab 10 ab 24 a 6 a
6 NaCl + 7 mM KC1 0.13 ab 113 a 9 b 25 a 5 a
7 NaC1 + 7 mM KC1 + 7.5 mM CaSO4 0. 3 ab 90 ab 10 ab 31 a 6 a
8 NaCI + 17.5 mM CaC12 0.11 c 103 ab 12 a 24 a 6 a


9 (NS)--no calcium 0.12 abc 83 ab 10 ab 35 a 6 a
10 NaC1--no calcium 0.11 bc 63 b 11 ab 27 a 6 a
11 NaC1 + 1 mM CaSO4 0.13 ab 97 ab 12 a 29 a 7 a
12 NaCl + 5 mM CaSO4 0.14 a 80 ab 12 a 30 a 5 a


SMean
YMean


of 8 plants. (


- I.- <


separation within columns by Duncan's Multiple Range Test, 0.05 level.







79

growth was not improved. Addition of CaC1, (treatments 5 and 8) reduced

sodium but did not reduce chloride sufficiently to improve growth.

Significant growth reduction occurred without any visible symptoms

of salt damage. Although total plant dry weight was reduced by more

than 28% in some treatments after 4 months of salinity stress, none of

these treatments caused any apparent leaf damage symptoms.

Comparison of Citrus Seedling Responses to NaCl and PEG Treatments

The effects of NaC1 and PEG on citrus seedlings differed in the

degree and the type of damage. When considering NaC1 and PEG at similar

osmotic potentials, the damaging effects on all measured variables

generally appeared to be larger in the PEG treatment than in the NaCI

treatment. Citrus seedling responses to NaC1 and PEG compared to the no

salt control are summarized in Table 22. The higher salinity damage

occurring in Experiment 2 in comparison to Experiment 1 was thought to

be mainly due to the more rapid onset of salt treatment and to the

longer duration of salt treatment.

Differences in damage and leaf burn symptoms were also found

between NaC1 and PEG. Leaves from NaCl-treated seedlings appeared

abnormally thickened. Leaf symptoms in the NaCl treatment were

initially similar to nitrogen dificiency (uniform loss of the green

color over the entire leaf). Later, leaf burn occurred as large spots

merged together. Leaf scorch and areas of dead tissue extended inward

from the margins of the leaf. Sodium chloride-damaged leaves readily

abscised and dropped as soon as visual burn symptoms appeared.

Sometimes leaves fell off before they reached this stage. Leaf symptoms

in PEG treatment first appeared similar to iron-manganese deficiency

(intervenal chlorosis). Then, leaf burn appeared at the edges and

particularly at the tip of the leaf. Later, the dead area extended

inward from the tip (Fig. 19).














Table 22.


Summary of citrus rootstock responses to NaCl and PEG as-
compared to the no salt control.


Variable NaCI PEG


Growth


Total fibrous root length
Leaf number
New shoot growth
Total root dry weight
Total shoot dry weight
Seedling height
Stem cross sectional area
Total leaf area

Water Relations

Root conductivity
Water flow rate
OP of root exudate
Stomatal conductance
Transpiration
Water use
Leaf water potential
Leaf osmotic potential
Leaf turgor potential
Leaf succulence

Leaf Mineral Analyses


Chloride
Sodium
Calcium
Magnesium
Potassium
Phosphorus
Zinc
Manganese
Copper
Iron


Other Variables

Shoot root ratio
Specific root weight
Specific leaf wt (Dry wt basis)
Specific leaf wt (Fresh wt basis)
Leaf chlorophyll


decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease


decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
increase
increase


increase
increase
decrease
decrease
decrease
increase
decrease
increase
no change
no change



decrease
increase
increase
increase
decrease


decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease



decrease
decrease
decrease
decrease
decrease
decrease
decrease
decrease
increase
decrease


no change
no change
increase
decrease
decrease
increase
increase
increase
no change
no change



decrease
increase
increase
decrease
decrease










Sour orange


Control NaCI-damaged leaves


NaCI


PEG
PEG


Control

Fig. 19. Sour orange leaves from non-stressed (control)
and stressed (NaCl, PEG) seedlings.















DISCUSSION


Leaf Ion Content and Salinity Tolerance

Rootstock Tolerance

Important differences in salt tolerance among citrus rootstocks

were demonstrated in this study. Based on various measurements of plant

growth (shoot, root, and total plant dry weight), on plant water

relations factors (root hydraulic conductivity, water flow rate, and

osmotic potential of root exudate), and on seedling appearance and

performance (leaf burn, leaf drop, and dieback), SO and CM seedlings

were the least affected while the most damage occurred in ML and PT

seedlings. Rough lemon, SC, and CC had an intermediate response. From

these results, SO as well as CM were classified as relatively tolerant

rootstocks, RL, SC, and CC were sensitive, and ML and PT were very

sensitive rootstocks to NaCl. Cooper et al. (1951) and Ream and Furr

(1976), who based their conclusions on visual leaf burn symptoms and

leaf chloride content, found that CM appeared to be more salt tolerant

than SO. The current study, which was mainly based on growth and water

relations measurements, showed that SO was as tolerant as CM. This

overall classification agreed with Cooper et al. (1951) who reported

that PT was a very salt sensitive rootstock and with others who found

that CC was a salt sensitive rootstock when compared to CM (Joolka and

Singh, 1979; Patil and Bhambota, 1978).









Ion Exclusion and Accumulation

Sour orange seemed to behave differently from the other rootstocks.

Even though SO accumulated higher amounts of sodium and chloride-than PT

and its hybrids (SC and CC) at the first salinity level (Tables 7, 8),

plant growth and physiological activities of SO were relatively

unaffected as compared with those of RL, ML, SC, CC, and PT. Rough

lemon and ML were sodium and chloride accumulators similar to SO.

However, salt damage was more severe and tree growth and appearance were

poorer in RL and ML than in SO. Since excess accumulation of both

chloride and sodium in SO leaves caused relatively minor damage to this

rootstock, SO might have the ability to partially exclude these ions

from the cytoplasm where they could inhibit metabolic functions.

Salinity studies at the cellular level could further clarify ion

exclusion and compartmentalization ability in citrus.

The citrus rootstocks tested in this study are considered to be

salt sensitive because no rootstock has the ability to exclude both

chloride and sodium. Rough lemon, ML, and SO are chloride and sodium

accumulators. Poncirus trifoliata, SC, and CC are chloride accumulators

but sodium excluders. Cleopatra mandarin is a chloride excluder but a

sodium accumulator. Furthermore, sodium exclusion capacity in PT, SC,

and CC and chloride exclusion capacity in CM are limited. This study

(Tables 7, 8) showed the inability of PT, SC, and CC to exclude sodium

and the inability of CM to exclude chloride at moderate salinity levels

(-0.2 MPa). It is suggested that in any program where plants are being

screened for salt tolerance on the basis of salt exclusion, chloride

exclusion as well as sodium exclusion should be considered because the

chloride and sodium accumulating properties of a particular species are

quite different (Grieve and Walker, 1983).









Leaf Ion Content and Ion Toxicity

Sodium chloride was found to reduce potassium in SO, CM, RL, and ML

but not in SC, CC, and PT leaves (Table 27, Appendix). Sour orange, CM,

RL, and ML are sodium accumulators (Table 9). Sodium accumulation in

these rootstocks might be the main factor which depressed leaf

potassium. Poncirus trifoliata, SC, and CC are sodium excluders

(Table 9). The unaffected leaf potassium in these rootstocks might be

attributed to sodium exclusion.

There was an inverse relationship between chloride ion accumulation

in the leaves and salt tolerance. Usually chloride accumulation was

associated with more damage. Since PT and its hybrids (SC and CC)

accumulated large amounts of chloride, their ability to exclude sodium

(particularly at -0.10 MPa) and to maintain adequate potassium did not

help prevent these rootstocks from showing severe growth reduction and

water relation disturbances. Furthermore, although CM accumulated

excess sodium in its leaves, growth and water relations of CM were not

as severely affected at -0.10 MPa since it was a chloride excluder.

The levels of chloride and sodium accumulation at which leaf burn

symptoms developed were found to be higher than the upper limits set by

earlier investigators. Such differences are mainly attributed to

different experimental conditions. Comparison between leaf chloride

content (Table 8) and visual symptoms (Table 6) shows that a leaf

chloride content of about 1% in SC and CC and even 1.7% in SO did not

cause any leaf burn symptoms. Similarly, when comparing leaf sodium

content (Table 7) to visual symptoms (Table 6), leaf sodium content up

to 1.9% in CM did not cause visible leaf burn. However, growth and

water relations were severely altered.







85

Leaf chloride and sodium analysis is thought to provide useful

information on toxicity limits as well as on rootstock tolerance.

Harding and Chapman (1951) recommended that a leaf chloride content

exceeding 0.25% be considered indicative of chloride toxicity.

Bernstein (1969) stated that although 0.25% might not lead to obvious

chloride toxicity symptoms, it might affect the longevity of leaves and

reduce the yield. Chapman et al. (1969) suggested that 0.30% chloride

in the dry matter was regarded as the threshold value of injury and leaf

levels over 0.75% chloride would be indicative of serious growth

retardation and yield reduction. According to Abdel-Messih et al.

(1979), sodium leaf content higher than 0.36% would be critical for

developing burn symptoms in citrus leaves. These threshold values were

lower than those found in this study because of the higher degree of

stress under field and dry climate conditions.

Importance of Calcium under Saline Conditions

Under salinity conditions, addition of calcium to irrigation waters

resulted in different responses in citrus. The present study on SO

seedlings showed that the beneficial effect of calcium depended on the

anion associated with the calcium salt. Calcium sulfate was found to be

significantly more effective than calcium chloride in reducing the

deleterious effect of NaCl on growth (Tables 19, 20). Walker and

Douglas (1983) did not observe any improvement in citrus growth by

increasing calcium chloride in the growth medium. However, the earlier

work on citrus by others showed the effectiveness of calcium sulfate,

calcium nitrate, and calcium carbonate on reducing sodium concentrations

in plant tissues, in preventing the deflocculation effect of sodium and

in improving tree appearance and growth (Cooper, 1961; Harding et al.,

1958b; Jones et al., 1952). LaHaye and Epstein (1969, 1971)







86

demonstrated that an increase in calcium levels by adding either calcium

sulfate or calcium chloride protected bean plants from salt injury by

restricting sodium absorption and translocation to the leaves. Failure

in effectiveness of calcium chloride in our work might have been due to

the chloride accompanying the calcium and to the sensitivity of citrus

to chloride.

Physiological Effects of NaCl and PEG

Effect of NaCl on Root Conductivity

The present study showed that root hydraulic conductivity in citrus

seedlings was severely reduced due to NaC1 stress and that root

conductivity varied significantly among rootstocks under stressed and

non-stressed conditions. Under non-stressed conditions, these results

were consistent with data obtained by others (Graham and Syvertsen,

1985; Syvertsen and Graham, 1985; Syvertsen et al., 1981). Under

salinity stress, root conductivity of different citrus rootstocks has

not been thoroughly studied.

Reduced hydraulic conductivity of roots has been attributed to

several factors. Bielorai et al. (1983) suggested that reduced water

uptake by mature citrus trees irrigated with saline water was a result

of soil solute potential reduction and to root suberization. Hayward

and Blair (1942) observed a condition resembling dormancy caused by

suberization of epidermal and root cap cells of Valencia orange

seedlings irrigated with NaCl solutions. They also noted reductions in

water uptake and development of lateral roots and root hairs as salinity

increased. Walker et al. (1984) studied the anatomy and the

ultrastructure of roots from two citrus genotypes (Rangpur lime and

Etrog citron) with different abilities for chloride exclusion. They

found that NaC1 increased suberization of the hypodermis and endodermis




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