Aluminum effect on growth of citrus roots in solution and soil systems

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Aluminum effect on growth of citrus roots in solution and soil systems
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Lin, Zhongyan, 1946-
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Thesis (Ph. D.)--University of Florida, 1989.
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Includes bibliographical references (leaves 129-136).
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by Zhongyan Lin.
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University of Florida
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ALUMINUM EFFECT ON GROWTH OF CITRUS ROOTS
IN SOLUTION AND SOIL SYSTEMS



by



ZHONGYAN LIN


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


1989















ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation and deep

gratitude to Drs. Donald L. Myhre, chairman, and Tzu L. Yuan, co-

chairman of his supervisory committee, for their guidance, super-

vision, assistance, encouragement and personal counseling throughout

the graduate program and during the preparation of this dissertation.

Appreciation and gratitude are also extended to Drs. John G.A.

Fiskell, R. Dean Rhue, and Kenneth M. Portier, for serving on the

supervisory committee, and for their constructive suggestions and

efforts to improve the content of his dissertation.

The author is also indebted to Dr. Brian L. McNeal for his

friendship, concern, and assistance during the author's application

and study in the department. Deep appreciation is also given to

Drs. Edward A. Hanlon, Jr. and Donald A. Graetz for their assistance

and for providing laboratory equipment.

The author is grateful to the following people: Mr. L.E. Hudson

for providing access to the Immokalee soil, Mr. H.W. Martin for his

statistical assistance, Mr. Joseph H. Nguyen for his assistance in

the laboratory, and Ms. An T. Nguyen for typing the dissertation.

Special thanks are given to Dr. and Mrs. D.L. Myhre and their

family, and Dr. and Mrs. T.L. Yuan for their love, concern, and help

extended to the author and his family.

Finally, the author sincerely acknowledges his wife, and his two

sons whom the author has not seen for four and a half years, and his










father-in-law and mother-in-law who have taken care of the author's

two sons, for their deep love, patience, understanding, encourage-

ment, support, and sacrifice.
















TABLE OF CONTENTS
Page

ACKNOWLEDGEMENTS............................................. ii

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

LIST OF FIGURES.............................................. viii

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

CHAPTERS

I GENERAL INTRODUCTION... ............................ 1

II LITERATURE REVIEW................................... 4

Beneficial Effects of Aluminum on Plant
Growth................................... 4
Phytotoxicity of Aluminum................... 5
Differential Aluminum Tolerance of Plants... 6
Study Methods for Aluminum Phytotoxicity.... 8
Studies of Aluminum Effects on Citrus....... 10

III SUPERNATANT SOLUTIONS CONTAINING VARIOUS LEVELS OF
ALUMINUM AND SIMILAR CONCENTRATIONS OF PHOSPHORUS
AS CULTURE SOLUTIONS FOR THE ALUMINUM STUDY...... 13

Introduction............................... 13
Materials and Methods....................... 15
Results and Discussion...................... 19
Summary and Conclusions.................... 33

IV DIFFERENTIAL RESPONSE OF CITRUS ROOTSTOCKS TO
ALUMINUM LEVELS IN SOLUTION CULTURE.............. 35

Introduction............................... 35
Materials and Methods....................... 36
Results and Discussion....................... 41
Summary and Conclusions..................... 76

V GROWTH OF CITRUS ROOTS AS AFFECTED BY ALUMINUM LEVEL
IN SOILS UNDER FIELD CONDITIONS.................. 78

Introduction............................... 78
Materials and Methods....................... 79
Results and Discussion...................... 84
Summary and Conclusions..................... 94











VI EFFECTS OF LIME AND PHOSPHOGYPSUM ON FIBROUS CITRUS-
ROOT GROWTH AND PROPERTIES OF THE Bh HORIZON OF A
SPODOSOL........................................ 96

Introduction............................... 96
Materials and Methods....................... 98
Results and Discussion...................... 103
Summary and Conclusions..................... 112

VII OVERALL SUMMARY AND CONCLUSIONS.................... 114

APPENDIX...................................................... 118

LITERATURE CITED............................................. 129

BIOGRAPHICAL SKETCH.......................................... 137















LIST OF TABLES


Table Page

3-1 Effects of pH, and additions of Al and P, on the
concentrations of Al and P, and on EC in filtrates
(aged for 7 days at 250C)................................ 21

3-2 Equations describing effects of pH(x ), Al addition (x ),
and P addition (x3) on concentration of Al (Y ), concen-
tration of P (Y ) and EC (Y ) in filtrates obtained during
Experiment 1 (aged for 7 days at 250C)................... 22

3-3 Elemental additions to solution and concentrations in
filtrates at two pH values after aging for 7 days at 25C
(mixed solution with magnetic stirrers)................... 26

3-4 Elemental additions to solution and concentrations
in supernatants at pH 4.0 after aging for 7 days at room
temperature (mixed solution manually).................... 27

3-5 Shoot height and new-growth shoot height of Rough
lemon in the 3rd 20-day growth period in the supernatant
solution................................................. 30

3-6 Elemental concentrations and EC of supernatant solution
after growing five 7-month-old citrus seedlings in ten
liters of supernatant solution for 20 days................ 32

4-1 Linear regression equations for prediction of new-growth
root length ( c, cm plant ), new-growth shoot height
(H, cm plant ), and new-growth fresh weight (W, g p ant )
of citrus seedlings from Al concentration (Al, mg L ) in
nutrient solution. (A = Carrizo citrange; C = Cleopatra
mandarin; 0 = Sour orange; R = Rough lemon; and S =
Swingle citrumelo)...................................... 51

4-2 Linear regression equations for prediction of relative
new-growth root length (RL, %), relative new-growth shoot
height (RH, %), and relative new-growth shoot weight (RW, %)
of citrus seedlings from Al concentration (Al, mg L ) in
nutrient solution. (A = Carrizo citrange; C = Cleopatra
mandarin; 0 = Sour orange; R = Rough lemon; and S = Swingle
citrumelo).............................................. 55

5-1 Relevant characteristics of the E horizon of the Immokalee
fine sand used for implants.............................. 81











Table Page

5-2 Relevant characteristics of saturation extracts for
soils from five treatments............................... 85

5-3 Fibrous citrus-root growth in implant bags of soil
(3.17 dm ) after 46 d as related to treatments.......... 87

5-4 Concentrations of elements in fibrous citrus-root tissues. 93

6-1 Selected chemical characteristics for the Bh horizon
of the Smyrna fine sand used for implants................ 100

6-2 Contrasts for root-length density as affected by lime
and phosphogypsum (PG) amendments to the Bh horizon of
a Smyrna fine sand...................................... 105

6-3 Contrasts of selected chemical properties of the soils
(four dates) ............................................ 107

6-4 Contrasts of some ions in the saturation extract of soils
(four dates) ........................................... 109

6-5 Coefficients of determination (r2) between acidity and
exchangeable Al (four dates)............................. 110















LIST OF FIGURES
Figure Page

3-1 Phosphorus concentration in filtered nutrient solution
as affected by P addition at two pH values and two
levels of Al addition (aged for 7 days at 25C).
Vertical bars indicate standard deviations............ 24

3-2 Eight-month-old Rough lemon seedlings grown for
60 days in supernatant solution with various
concentrations of Al. From left to right: 0.1, 2.7,
4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L .............. 31

4-1 The root systems of five citrus seedlings which were
submerged in nutrient solutions in pails which were in
turn placed in a water pool........................... 39

4-2 Water circulation in the pool by a pump, which was
maintained at 2510C by passing through a cooling
system................................................ 39

4-3 Citrus seedlings growing in nutrient solutions
in pails which were randomly assigned positions
in the water-filled pool.............................. 40

4-4 Thickened root tips of Sour orange seedlings grown
in solution with 24.4 mg Al L ....................... 43

4-5 Stubby new-growth roots of Rough lemon seedlings
grown in nutrient solution with 24.4 mg Al L ........ 43

4-6 Root tip covered by a root cap with black gelatinous
material for Cleopatra mandarin seedlings grown in
solution of 24.4 mg Al L ............................ 44

4-7 Young leaves of Swingle citrumelo seedlings grown in
nutrient solutions with various concentrations of Al.
From left to right: 0.1, 2.7, 24.4, and 28.4 mg Al
L ................................................... 45

4-8 Shoot with yellow, mottled, and withered young leaves
and aborted terminal of Swingle citrumelo seedling
grown in nutrient solution with 44.6 mg Al L for
60 days............................................... 45

4-9 Effects of increasing Al concentrations in the nutrient
solution on root and shoot growth of 8-month-old
Carrizo citrange seedlings. From left to right: 0.1,
2.7, 4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L ......... 47


viii










Figure Page

4-10 Effects of increasing Al concentrations in the nutrient
solution on root and shoot growth of 8-month-old
Cleopatra mandarin seedlings. From left to right:
0.1, 2.7, 4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L ..... 47

4-11 Effects of increasing Al concentrations in the nutrient
solution on root and shoot growth of 8-month-old
Sour orange seedlings. From left to right: 0.1, 2.7,
4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L ............... 48

4-12 Effects of increasing Al concentrations in the nutrient
solution on root and shoot growth of 8-month-old
Rough lemon seedlings. From left to right: 0.1, 2.7,
4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L ............... 48

4-13 Effects of increasing Al concentrations in the nutrient
solution on root and shoot growth of 8-month-old
Swingle citrumelo seedlings. From left to right: 0.1,
2.7, 4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L .......... 49

4-14 Effects of Al concentrations (Al, mg L- ) in nutrient
solution on new-growth root length (L, cm plant )
of 8-month-old citrus seedlings grown for 60 days..... 50

4-15 Effects of Al concentrations (Al, mg L-1) in nutrient
solution on relative new-growth root length (RL, %)
of 8-month-old citrus seedlings grown for 60 days..... 53

4-16 Effects of Al concentrations (Al, mg L- ) in nutrient
solution on new-growth shoot height (H, cm plant ) of
8-month-old citrus seedlings grown for 60 days........ 56

4-17 Effects of Al concentrations (Al, mg L- ) in nutrient
solution on relative new-growth shoot height (RH, %)
of 8-month-old citrus seedlings grown for 60 days..... 57
-1
4-18 Effects of Al concentrations (Al, mg L ) in nutrient
solution on new-growth fresh weight (W, g plant ) of
8-month-old citrus seedlings grown for 60 days........ 59

4-19 Effects of Al concentrations (Al, mg L- ) in nutrient
solution on relative new-growth fresh weight (RW, %)
of 8-month-old citrus seedlings grown for 60 days..... 60

4-20 Aluminum concentration of 8-month-old citrus seedlings
grown for 60 days in nutrient solution with various
concentrations of Al. (A = Carrizo citrange; C =
Cleopatra mandarin; 0 = Sour orange; R = Rough lemon;
and S = Swingle citrumelo)............................ 62











Figure


4-21 Calcium concentration of 8-month-old citrus seedlings
grown for 60 days in nutrient solution with various
concentrations of Al. (A = Carrizo citrange; C =
Cleopatra mandarin; 0 = Sour orange; R = Rough lemon;
and S = Swingle citrumelo)............................ 64

4-22 Magnesium concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)........... 66

4-23 Potassium concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)........... 67

4-24 Phosphorus concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)........... 68

4-25 Zinc concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo).......... 71

4-26 Manganese concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo).......... 72

4-27 Copper concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo).......... 73

4-28 Iron concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo).......... 75
-1
5-1 Effects of Al concentration (Al, mg L ) in soil
saturation3extract on fibrous root-length density
(D, cm dm ). Critical 1 concentration was
23 mg Al L [i.e., (Al) = 4.8]..................... 90











Figure Page

6-1 Mean root-length densities for three treatments
at four sampling periods............................. 104















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


ALUMINUM EFFECT ON GROWTH OF CITRUS ROOTS
IN SOLUTION AND SOIL SYSTEMS


by

Zhongyan Lin

December 1989


Chairman: Dr. Donald L. Myhre
Cochairman: Dr. Tzu L. Yuan
Major Department: Soil Science

Aluminum phytotoxicity may be a growth-limiting factor for

citrus roots growing in acid soils. Four experiments were conducted

to investigate the effects of Al on citrus root growth in solution

and soil systems.

In a laboratory study, two sets of supernatant nutrient solutions

were prepared and evaluated for Al phytotoxicity studies. For the

pH 4.0 and pH 4.5 sets, actual Al concentrations ranged from 0.1 to

171 mg Al L and from 0.1 to 10 mg Al L and P concentrations were

about 1 mg P L and 0.2 mg P L respectively.

In a greenhouse study, five 6-month-old citrus rootstock

seedlings were grown in supernatant nutrient solutions containing

seven levels of Al at pH 4.0 for 60 days. According to the response

of new-growth fresh weight of whole plants to Al concentrations in

solution, relative Al-tolerances were Cleopatra mandarin (C. reshni

Hort. ex Tan.) > Rough lemon (C. jambhiri Lush.) = Sour orange










(C. aurantium L.) > Swingle citrumelo (C. paradisi x P. trifoliata) >

Carrizo citrange [C. sinensis (L.) Osbeck x Poncirus trifoliata (L.)

Raf.]. The critical Al concentrations in solution for toxic effects

were 12.2, 5.1, 5.1, 4.5, and 1.8 mg Al L-1, respectively, for the

above rootstocks. Concentrations below or above the critical Al

levels caused either beneficial or toxic effects, respectively. When

Al concentrations in nutrient solution increased from 0.1 to 4.8 mg
-1
Al L Al, K, Mg, and P concentrations in roots and Al, K, and P

levels in shoots increased; whereas Ca, Zn, Cu, Mn, and Fe in roots

and Ca, Mg, Cu, and Fe in shoots decreased. Aluminum-tolerant

rootstocks accumulated more Al in their roots than did Al-sensitive

rootstocks. The more Al-tolerant rootstocks contained higher Fe

concentrations in their roots than did the less tolerant ones when

Al concentrations in solution were lower than 8.3 mg Al L-1

In a field experiment, E-horizon soil was treated with either

lime or four levels of Al, placed in porous bags, and then implanted

in the surface horizon of a citrus grove for 46 days. Results

indicated that the critical Al concentration for toxicity in the

saturation extract of soils was 23 mg Al L-1 for root growth of

mature trees of Sour orange rootstock.

In another similar field experiment, Bh-horizon soil was amended

with either lime or phosphogypsum, implanted, and collected after 55,

84, 113, and 139 days. Application of lime significantly increased

fibrous citrus-root growth while phosphogypsum did not. The soil

amended with phosphogypsum had a lower pH, higher salinity and

exchangeable Al; higher Ca2+ and Mg2+ and lower P (H2PO and HPO-)
and C contents in the saturation extract than the non-amended soi.
and Cl contents in the saturation extract than the non-amended soil.


xiii















CHAPTER I

GENERAL INTRODUCTION

Aluminum toxicity is probably the most important growth-limiting

factor for plants in most strongly acid soils. A number of crops

have been studied with respect to their response to Al phytotoxicity.

Citrus grows widely in tropical and subtropical areas in which soils

are highly weathered and generally acidic. Aluminum toxicity may be

an important factor limiting citrus growth in these acid soils.

Few studies have been conducted on the effects of Al on citrus

growth (Haas, 1936; Liebig et al., 1942; Yokomizo and Ishihara, 1973;

Worku et al., 1982). These researchers found that low Al concentra-

tions in solution stimulated, but high concentrations depressed, root

growth of some citrus species. However, no experimental results have

been reported in the literature for screening and evaluation of

citrus rootstocks for Al tolerance. The Al phytotoxicity levels are

still not known for many citrus rootstocks, and few data exist on the

effects of Al on the mineral nutrition of citrus.

Solution culture has been frequently used for Al phytotoxicity

studies. Aluminum effects on roots are confounded by many factors,

such as pH, temperature, concentrations of P, Ca, and Mg (Rhue and

Grogan, 1977). The common problems in the previous studies with

nutrient solution were the confusion of added levels of Al with

actual concentrations of Al in the nutrient solution, and the con-

founding effects of P (Bollard, 1983; Marschner, 1986).










Research on the effects of Al on citrus growth has been mainly

limited to nutrient-solution studies. Field studies are highly

desirable to evaluate Al effects on citrus root growth under field

conditions.

In Florida, Spodosols have been increasingly used for citrus

production. The spodic horizons of these soils are generally very

acid and have high Al contents (Myhre et al., 1987), so it is

probable that Al toxicity problems occurs in these subsoils. Perhaps

it is worthwhile to determine whether phosphogypsum could be used as

an ameliorant for the subsoil acidity syndrome.

The overall objectives of this dissertation research were to

develop a better understanding of Al effects on citrus growth and

nutrient uptake, both in nutrient solution and under field

conditions, and to evaluate citrus rootstocks used in Florida for

Al-tolerance. In addition, lime and phosphogypsum were tested as an

ameliorant for the acidity syndrome of spodic horizon soils.

This dissertation is divided into six parts. Chapter II is a

review of the literature for the entire set of studies. Chapter III

describes a supernatant solution containing various levels of Al and

similar concentrations of P that was prepared as a culture solution

for Al studies. In Chapter IV, five rootstocks were studied in

nutrient solution for their Al-tolerance, and their elemental

composition of roots and shoots as affected by Al concentrations.

In Chapter V, a field experiment was conducted using the implanted

soil-mass technique to evaluate the critical Al concentration in

saturation extracts of soils for toxicity and elemental compositions

in roots as affected by Al concentration under field conditions.





3




In Chapter VI, the effects of lime and phosphogypsum on fibrous

citrus-root growth and properties of Bh horizon soil were studied in

the field using the implanted soil-mass technique. Finally, in

Chapter VII, the studies are summarized and recommendations are

provided for further work.















CHAPTER II

LITERATURE REVIEW

Beneficial Effects of Aluminum on Plant Growth

Aluminum is not regarded as an essential nutrient, but low

concentrations can sometime increase plant growth or produce other

desirable effects. An early report of the stimulation of plant

growth was made by Maz4 (1915), and similar reports have continued

to appear from a number of laboratories. Plants that have shown

positive growth response to Al include rice, maize inbreds,

eucalyptus, tea, peach, sugar beet, tropical legumes, wheat, and pea.

For a more-thorough review on this subject, see Bollard (1983) and

Foy (1984). The growth stimulus is greater for Al-tolerant cultivars

than for Al-sensitive cultivars (Howeler and Cadavid, 1976; Clark,

1977). The stimulating Al concentrations are usually about 1 mg L-

or less. In the tea plant, however, growth stimulation is observed

at Al concentrations as high as 27 mg L-.

The mechanisms of Al beneficial effects are debatable and may be

different for different plant genotypes and growth media. Possible

explanations (Foy, 1984) include (1) increasing P, Fe, and Ca

uptake; (2) preventing toxicities of Cu, Mn, and P; (3) altering

the distribution of growth regulators; and (4) serving as a

fungicide.

The beneficial effects are the exception, however, and toxic

effects of Al on plant growth in soils of low pH are the rule.










Phytotoxicity of Aluminum

Aluminum toxicity is probably the most important growth-limiting

factor for plants in most strongly acid soils and mine spoils (Foy,

1974; McLean, 1976).

The symptoms of Al toxicity are not easily identified. Aluminum

toxicity effects are first detected in the root system, in which

there is reduced growth of the main axis, resulting in short thick

roots, and in the inhibition of lateral root formation (Alam and

Adams, 1979; Bollard, 1983). In plant tops restricted growth is

often the main symptom of toxicity, but sometimes mottling and

necrotic symptoms can appear on leaves as well (Cate and Sukhai,

1964). In some plants the foliar symptoms resemble those of P

deficiency. In others, Al toxicity appears as an induced Ca or Fe

deficiency (Foy, 1984). Young seedlings are generally more

susceptible to Al toxicity than are old plants (Thaworuwong and Van

Diest, 1974).

Several distinct positive modes of Al-toxic mechanisms have been

investigated. Excess Al has been reported to interfere with cell

division in root tips and lateral roots; increase cell-wall rigidity

by cross-linking pectins; reduce DNA replication by increasing the

rigidity of the DNA double helix; fix P in less-available forms in

soils and on root surfaces; decrease root respiration; interfere with

enzymes governing sugar phosphorylation and the deposition of cell

wall polysaccharides; and interfere with the uptake, transport, and

use of several essential nutrient elements, including Ca, Mg, K, P,

and Fe (Foy, 1984).










Excess Al may reduce the uptake of certain essential elements

and increase that of others (Ali, 1973; Alam, 1981; Duncan et al.,

1980). Aluminum toxicity is often associated with Al-induced P

toxicity (McCormick and Borden, 1972) or Al-induced P deficiency

(James et al., 1978). Aluminum-induced Fe deficiency is frequently

mentioned in the literature (Alam, 1981; Clark et al., 1981), and

aluminum x Ca interactions are important in acid soils. Lance and

Pearson (1969) showed that reduced Ca uptake was the first externally

observed symptoms of Al damage on cotton seedling roots. Lund (1970)

found that Ca reduced the detrimental effects of Al in nutrient

solution. However, the data for effects on nutrient uptake are

difficult to interpret in terms of Al toxicity mechanisms. No one

pattern of elemental accumulation applies to all cases of Al injury

(Foy, 1984), with the entire array of elements in the tops of

Al-injured plants probably representing the accumulated systematic

effects of initial root injury by Al. Such effects are generally too

far removed from the initial root injury to reveal Al-toxicity

mechanisms (Foy, 1984). The reduction in levels of some elements is

also a result of reduced root surface area rather than a specific

effect of Al (Clarkson, 1966).

Differential Aluminum Tolerance of Plants

Different plant species and varieties differ widely in their

tolerance to excess Al in the growth medium. There is now consider-

able activity devoted to breeding crop cultivars better adapted to

acid-soil conditions, with work in this field having resulted in the

detection of certain differences between susceptible and tolerant

cultivars. However, the exact physiological mechanisms of Al










tolerance are still being debated; tolerance may be controlled by

different genes, acting through different biochemical pathways in

different plants (Foy, 1984).

Three major mechanisms are involved in Al tolerance: (1)

exclusion from uptake excluderr plants); (2) inactivation in the

roots excluderr, include plants); and (3) accumulation in the

shoots includer plants). Mechanism (3) exists mainly in highly Al

tolerant species of natural vegetation, with only a few cultivated

species being Al includes. In crop species, mechanisms (1) and (2)

predominate, and it is often difficult to differentiate between the

two (Marschner, 1986) The following factors may be of primary

importance in the exclusion mechanism:

1. Rhizosphere pH. When Al is present, some tolerant cultivars

tend to raise the external pH faster than sensitive cultivars, both

in the nutrient solution (Foy et al., 1967) and in the rhizosphere of

soil culture (Mugwira and Petel, 1977). A slight pH increase at the

root surface or in the free space is probably sufficient to lower the

charge of Al, which leads to the formation of Al polymer species.

These polymer species may facilitate P uptake.

2. Aluminum uptake and distribution. Some Al-tolerant plants

have a lower Al concentration in roots than do Al-sensitive plants.

In this case, Al tolerance apparently involves an exclusion

mechanism. Other Al-tolerant plants have either more or less Al in

their tops than do Al-sensitive plants. Such plants have higher

internal tolerance to Al. Tea, certain Hawaiian grasses, pine trees,

and mangrove are examples of Al accumulators (Foy et al., 1978), but

little is known about the forms in which Al may exist in the foliage










of accumulator species (Bollard, 1983). Some effort has been made

to establish critical levels of Al for toxicity in plant tops

(Wallace and Romney, 1977; Duncan, 1982).

+
3. Nutrient uptake. Some Al-tolerant plants are also NH -
+
tolerant. This NH4-tolerance is important in strongly acid soils,

where high concentrations of NH4 may be present (Foy and Fleming,

1982). Aluminum tolerance in certain cultivars of wheat, barley,

soybean, and snapbean has been associated with the ability to resist

Al-induced Ca deficiency (Foy et al., 1978). In many plants, Al

tolerance also appears to be closely related to P-use efficiency

(Foy et al., 1978).

4. Organic Al complexes. Naturally occurring organic acids

in Al-tolerant species chelate Al and thereby reduce the Al-P pre-

cipitation expected at normal pH levels in plant sap (Jones, 1961).

Klimashevskii and Chernysheva (1980) found that the roots of Al-

tolerant varieties of pea, maize and barley contained substantially

higher concentrations of citric acid than did those of Al-sensitive

varieties of the same species. Complexation of Al by organic acids

not only provides protection against the harmful effects of free

Al on root growth, but is also important for the uptake of P

(Marschner, 1986).

Study Methods for Al Phytotoxicity

Study methods for Al phytotoxicity normally include solution

culture, soil culture in the greenhouse, and field experiments.

Solution culture has been used most frequently.

Solution culture (nutrient solution or soil solution) has

been used for studying the relationship between Al speciation and










phytotoxicity. Attempts have been made to relate the concentrations

of Al species in solution (Al3+, hydroxy-Al monomers, hydroxy-Al

polymers, AlSO et al.) as obtained by calculation with GEOCHEM or

other programs to plant-growth parameters in order to find out what

species is most toxic to plant growth. However, there is no clear

consensus as to the species) predominantly responsible for phyto-

toxicity (Blamey et al., 1983; Alva et al., 1986; Parker et al.,

1988, 1989).

Rapid screening methods have been developed, mainly in solution

culture systems. Most workers have found that the primary damage

caused by Al occurs in the roots (Bouma et al., 1981; Bollard, 1983).

In most crop species, the relative root length of plants exposed

versus those not exposed to Al is the most appropriate parameter

(Marschner, 1986). Because Al toxicity to roots is affected by many

factors, such as pH and concentrations of P, Ca, and Mg, the biggest

problem in developing rapid screening techniques is finding an

appropriate combination of these factors to use (Rhue and Grogan,

1977). In some instances the classification of genotypes based on

their Al tolerance via the rapid screening methods correlates well

with the growth response of these genotypes in acid soils (Howeler

and Cadavid, 1976). However, the correlations often are quite poor

(Nelson, 1983). These discrepancies are not surprising and indicate

that a) factors such as rhizosphere pH have been insufficiently

considered, and b) factors other than excessive Al levels may have

been involved and may have had an even more harmful effect on growth.

There has been much work with plants grown in nutrient solution,

on the effects of Al concentration and possible modifying factors on










the growth and elemental composition of plants. There are consid-

erable technical difficulties with such experiments. There is

often confusion about what level of Al is actually present in

solution. A general problem in most studies on the beneficial

effects of Al on plants is the contamination of the nutrient solution

with Al. Reasonably high levels are frequently reported in plants

growing in conventional culture solutions without any added Al

(Wilkinson and Gross, 1967). Moreover, the reduced solubility of

aluminum phosphate with increasing pH greatly restricts the combina-

tions of Al and phosphate concentrations and pH of culture solution

which can be compared. The other problem in some studies is the

confounding effects of P and Al. When zero or small amounts of Al

are added, excessive P levels are quite common (Marschner, 1986).

When large amounts of Al are added, P is often deficient in the

nutrient solution due to the precipitation of aluminum phosphate.

Field experiments are very important in Al-phytotoxicity and

screening studies for Al tolerance (Foy et al., 1974; Mugwira et al.,

1981). However, field experiments are labor-intensive, require

several months or more for completion, and are often influenced by

secondary factors such as the variation of soil properties. Applica-

tion of Al to a large field area is not practical. It also is very

difficult to study root systems of crops in field experiments without

disturbance of the soils and the root system, particularly for large

plants such as trees.

Studies of Aluminum Effects on Citrus

Citrus species are grown widely in tropical and subtropical

areas of high annual rainfall in which the soils are almost always





11




acid. Citrus trees were domesticated from wild ancestors in Eastern

and Southern Area (Hill, 1937), where most soils are highly acid.

Aluminum phytotoxicity may be an important factor limiting citrus

growth in the more acid of these soils. However, only a few studies

have been reported in this field.

An early report of Al effects on citrus growth was made by Haas

(1936). He used leafy-twig cuttings of some citrus in a nutrient

solution and found that low Al concentrations stimulated root growth

while high Al concentrations were toxic. He also found that addition

of Al increased P uptake. Liebig et al. (1942) made similar

findings. They also found that addition of Al reduced Cu toxicity.

Yokomizo and Ishihara (1973) conducted a solution culture with a wide

range of Al additions. They found that, at low Al additions, citrus

root growth increased. At 100 mg Al L-1 addition, however, citrus

root growth was extremely depressed. Worku et al. (1982) conducted a

study with highly weathered Oxisols in Hawaii. They found that high

levels of Al and Mn were toxic to some citrus species. The effects

of Al and Mn, however, were confounded. Other researchers (Sekiya

and Aoba, 1975; Huang, 1983) have linked high Al concentration to

poor citrus growth.

Additional information about Al effects on citrus is needed

and the following aspects are in particular need of further study:

1. Comparing the effects of Al on several citrus species,

to find out the critical Al concentrations for toxicity in solution

culture.

2. Screening Al tolerance of citrus species systematically.











3. Study of the relationship between Al effects and macro- and

micro-nutrients.

4. Study of the mechanisms of beneficial and toxic effects of

Al on citrus.

5. Investigation of the effects of Al on citrus growth in

soils, particularly in the field, and assessment of the critical Al

concentrations in soil solution or in citrus leaves which reflect

toxicity.

6. Testing amendments which may be used practically in citrus

groves to ameliorate Al toxicity.















CHAPTER III

SUPERNATANT SOLUTIONS CONTAINING VARIOUS LEVELS OF ALUMINUM
AND SIMILAR CONCENTRATIONS OF PHOSPHORUS AS CULTURE
SOLUTIONS FOR THE ALUMINUM STUDY

Introduction

Solution culture has been widely used to study the effects of Al

concentration on the growth and elemental composition of plants and

to screen crop species for Al tolerance. Normally, authors report

the amounts of Al added to the solution but not the actual Al concen-

tration in the growth solution. Many nutrient culture studies have

employed high Al additions (up to 7.4 mM or more) (Tanaka and

Navasero, 1966; Yokomizo and Ishihara, 1973), high P additions (up to

0.48 mM) (Moore et al., 1976; Nelson, 1983; Williams, 1982) and high

solution pH (4.8 or higher) (Malavolta et al., 1981; Tanaka and

Navasero, 1966; Williams, 1982). These conditions have probably

resulted in the precipitation of Al(OH)3 and aluminum phosphate

(Blamey et al., 1983; Yokomizo and Ishihara, 1973). Such losses

of Al from the test solutions would be expected to cause an over-

estimation of the threshold concentration for Al toxicity (Asher,

1981). Another general problem in most studies on the effect of

low levels of Al on plant growth has been the contamination of the

nutrient solution with Al even where the Al solution level was

assumed to be zero (Marshner, 1986; Tanaka and Navasero, 1966;

Wilkinson and Gross, 1967). Therefore, there are good reasons to

report the actual Al concentrations of the growth solution.










The precipitation of aluminum phosphate also causes the decrease

of actual P concentration in the solution (Munns, 1965; Tanaka and

Navasero, 1986). For a given pH and P addition, if the amount of

added Al is zero or small, there may be a P-toxicity problem

(Marschner, 1986). If the amount of added Al is large, there may be

a P deficiency problem. Either of these problems may confound the

effects of Al on plant growth. In many previous studies, beneficial

or toxic effects of Al were reported to be related to P (Foy, 1984).

Some researchers also found that P toxicity or deficiency affected

the Al toxicity symptoms and critical concentrations in solution

culture (Tanaka and Navasero, 1966). Therefore, it is necessary to

report the actual concentration of P in the growth medium. It is

also important to get similar concentrations of P in nutrient

solutions with different Al concentrations, although there are

considerable technical difficulties because of uncertainty which

extends even to the prediction of precipitation.

In nutrient solutions with amorphous precipitates of Al(OH)3,

aluminum phosphate, and other compounds, it is difficult to estimate

the actual concentrations of Al, P and other elements which may react

with Al and P to form precipitates during the growth periods. Such

precipitates may become a sink or source for the elements in the

solution. The actual concentrations of the elements in the solution

may dynamically change as well. Furthermore, with continuous

aeration amorphous precipitates may deposit on root surfaces and this

coverage may affect the physiological function of the roots. It is

preferable to use supernatant solution (filtered or siphoned) instead

of turbid solution to grow plants in Al studies.










In earlier work, some techniques were used to avoid precipi-

tation problems. Munns (1965) suggested comparing the effects of

Al concentration in culture nutrient solution only at phosphate

concentration of 19 uM (0.59 mg P L-1) or less, Al concentrations on

the order of 100 uM (2.7 mg Al L-1), and pH values of 4.0-4.2 to

avoid precipitation problems. Nonetheless, many experiments have

been carried out with treatments exceeding such narrow limits. In

order to avoid precipitation of aluminum phosphate, phosphorus has

been omitted (Moore et al., 1976), or plant roots have been

alternately exposed to culture solutions containing either Al or P,

or split-root techniques have been used. These modifications,

however, impose their own constraints on the interpretation of

experimental results (Pierre et al., 1932; Wright, 1937).

The objectives of this study were two-fold: (1) to investigate

the actual concentrations of Al and P in nutrient solution under

different pH and different Al and P additions; and (2) to develop

and test a supernatant-solution method for Al studies, in which the

supernatant solutions contain various levels of Al and similar

concentrations of P.



Materials and Methods

General

All reagents were of analytical grade and double-deionized

water was used. The basal nutrient solution used for this study

contained about one-fourth of the macronutrient concentrations,

except for P, of no. 1 Hoagland and Arnon solution (1950). This

basal nutrient solution has been used previously for some other Al










studies (MacLeod and Jackson, 1967; Yokomizo and Ishihara, 1973).

The basal nutrient solution contained the following elements in mg
-1
L-1: 50 N from NH4NO3, 50 K from K2SO4, 50 Ca from CaC12, 15 Mg from

MgSO4.7H20, 2.0 Fe from FeSO4*7H20, 0.2 Mn from MnSO4 H20, 0.1 Zn

from ZnSO 4*7H20, 0.02 Cu from CuSO 45H20, 0.2 B from H3BO3, and 0.02

Mo from (NH4)6MO7024'4H20. Phosphorus and Al were not included in

the basal nutrient solution. In preparation of mixed solution

aluminum was added from Al 2(SO4 ) 318H2 0 and P from NaH2PO4*H20.

The filter paper used was Whatman 42, which was ashless and had a

minimum particle-retention diameter of 2.5 um. The pH of solution

was measured using a combination glass electrode, and the electrical

conductivity (EC) was measured using a conductivity bridge.

Elemental composition of solution was determined by ICAP (Inductively

coupled argon plasma) emission spectroscopy. In Experiments 1 and 2,

all treatments were replicated three times.

Experiment 1. Effects of pH and Additions of Al and P on EC and
Concentrations of Al and P in Filtrated Nutrient Solution

Six levels of Al addition (0, 5, 25, 50, 100, and 500 mg L-)

were used in factorial combination with two levels of P addition

(3 and 15 mg L-1) and four pH levels (3.5, 4.0, 4.5 and 4.8).

Aluminum or P salt was added separately to each 200 mL of the basal

nutrient solution, and dissolved. The solution containing P then was

added to the Al solution slowly with vigorous stirring by magnetic

stirrer. Then the pH of the mixed solution (400 mL) was adjusted

with 0.5 or 1 M first and then with 0.1 M HC1 or NaOH. The HCl or

NaOH was added to the solution drop by drop, with vigorous stirring

by magnetic stirrer. After aging for seven days at 250C, the solution










was filtered and the filtrate was analyzed for EC and concentrations

of Al and P.

In addition, four trials were conducted to investigate a) the

composition differences between filtered nutrient solution and

supernatant solutions obtained by siphon, b) the effect of equili-

bration time and temperature, and c) the effect of storage

temperature on composition of filtrate.

Experiment 2. Supernatant Solutions Containing Several Levels of Al
and Similar Concentrations of P

Two trials were conducted. The first was to develop one set of

supernatant solutions which contained various levels of Al (0 to 150

mg L- ), with a similar P concentration (1 mg L-1) and at the same pH

(4.0). There were a number of Al-addition levels (0 to 320 mg L )

in factorial combination with a number of P-addition levels (0 to 320

mg L- ). The pH of all mixed solutions was adjusted to 4.0. The

second trial was to develop a second set of supernatant solutions

which contained various levels of Al (0 to 10 mg L-1) with similar P

concentration (0.2 mg L- ) and at the same pH (4.5). There were a

number of Al-addition levels (0 to 160 mg L-1) in factorial combina-

tion with a number of P-addition levels (0 to 160 mg L-1). The pH of

all mixed solutions was adjusted to 4.5. The procedure of mixing,

aging, and filtering were the same as described in Experiment 1. The

filtrates were analyzed for EC, and for concentrations of Al, P, and

other elements.

Experiment 3. Elemental Composition of Large-volume Supernatant
Solutions Prepared Manually in the Greenhouse

In order to make 100 liters of the supernatant solution

developed in Experiment 2 as the first set, 55 liters of basal











nutrient solution was prepared in a 120-liter plastic container.

Aluminum and P solutions were prepared with water, each in separate

20-liter plastic containers. The aluminum solution was mixed with

the basal nutrient solution in a 120-liter container. Then the P

solution was added. The solution was mixed by hand-stirring with a

plastic bar. The pH was adjusted with additions of 3.5 M HC1 or NaOH

from a wash bottle, and the solution was mixed. The mixed solution

was then made up to 100 liters with basal nutrient solution. After

the solution was aged for 7 days at room temperature in the green-

house, the supernatant liquid was siphoned. Electrical conductivity,

and Al and P concentrations in the supernatant solution, were deter-

mined. The supernatant solution was used for Experiment 4 also.

Experiment 4. Test of Supernatant Solutions as Culture Solutions
Using Citrus Seedlings

The objective of this experiment was to use citrus seedlings to

test the first set of supernatant solutions developed in Experiment 2

and prepared in Experiment 3.

Five citrus rootstocks were used: Carrizo citrange [C. sinensis

(L.) Osbeck x Poncirus trifoliata (L.) Raf.], Cleopatra mandarin (C.

reshni Hort. ex Tan.), Rough lemon (C. jambhiri Lush.), Sour orange

(C. aurantium L.), and Swingle citrumelo (C. paradisi x P. trifoliata).

Six-month seedlings (liners) were obtained from nurseries. Uniform

seedlings were selected and their roots were thoroughly washed with

tap water, and then given a final rinsing with deionized water. The

seedlings were transferred to the supernatant solutions prepared in

Experiment 3. Ten-liter pails (25 cm diam. x 21 cm height) were used

to hold the nutrient solution. Five holes were made in the plywood










lid of the pails, one for each of the five rootstock seedlings.

The entire root system of the five seedlings was submerged in the

solution, and the solution was continuously aerated. This also

served to keep the solution uniformly mixed. Air-conditioning was

used in the greenhouse to maintain the air temperature in a range

from 25 to 35C. The pails were put inside pools filled with water

which was circulated by a pump and passed through a cooling system.

The water temperature in the pools, as well as the solution

temperature in the pails, was maintained at 25 IC.

Seven Al concentrations were used. Eight pails contained the

same Al concentration and five rootstock seedlings. A total of 56

pails containing treatment solutions were randomly assigned positions

in the pools. The pH levels were checked every 2 to 3 days and

adjusted to 4.0 by additions of diluted HC1 or NaOH as necessary.

The solution level was maintained by addition of deionized water in

a quantity sufficient to offset loss due to evapotranspiration every

two days. The treatment solutions were renewed every 20 days. The

seedlings grew in the solution for 60 days. At the end of the last

20-day growth period, samples of the solutions were taken to determine

concentrations of Al, P, and other elements. At the beginning and

end of the last 20-day growth period, shoot heights of the seedlings

were measured. Photographs were taken at the end of the experiment.



Results and Discussion

Experiment 1. Effects of pH and Additions of Al and P on EC and on
Concentrations of Al and P in Filtrated Nutrient Solution

Aluminum concentrations increased with increased levels of Al

addition but decreased with increased pH or increased levels of P










addition (Tables 3-1 and 3-2). As pH increased or P addition

increased, more precipitate was found in the mixed solution. When pH

increased from 3.5 to 4.5, Al concentration decreased drastically,

and, when pH increased to 4.8, Al concentration became extremely low,

averaging only a few mg Al L1. Even when 500 mg Al L-1 and only 3

mg P L- were added, the actual Al concentration was only 3.6 mg L-
-1
With small amounts of Al addition, such as 5 mg Al L- together with

15 mg P L-1, Al concentration was essentially zero in the filtered

nutrient solution. The large difference between the amounts of Al

added and actual Al concentrations in the filtered nutrient solution

suggests that it is necessary to report the actual Al concentrations

in the growth nutrient solution for Al studies.

Phosphorus concentrations increased with increasing levels of P

addition, but decreased with increasing pH and addition levels of Al.
-1
It was noteworthy that at pH 4.5 or higher, when 25 mg Al L or more

were added, the P concentration was zero, whether 3 or 15 mg P L-

had been added. These treatments could result in extreme P

deficiency, and would confound the Al-toxicity effects. In contrast,
-1
when Al addition was zero or small, addition of 15 mg P L or more

would be toxic to some plants, and this toxicity would reduce any

beneficial Al effects. Phosphorus supply has been associated

historically with root growth (Tisdale and Nelson, 1975). In Al

phytotoxicity studies, many researchers have used root elongation as

a main parameter. Therefore, levels of P supply may have been a very

important factor influencing the conclusions of Al phytotoxicity

studies. Ideally, it should be best to have the same P concentration

for all levels of Al.









Table 3-1.


Effects of pH, and additions of Al and P, on the concentrations
of Al and P, and on EC, in filtrates (aged for 7 days at 250C).


-i
Al addition, mg L1
0 5 25 50 100 500
-I
pH P addition, mg L
3 15 3 15 3 15 3 15 3 15 3 15


Al concentration, mg L-

3.5 0.1 0.1 4.7 5.0 25.1 22.5 50.2 46.6 93.5 85.3 493.2 487.5
4.0 0.1 0.1 2.9 1.3 17.5 8.6 46.7 26.4 78.0 66.5 385.0 270.9
4.5 0.1 0.1 0.9 0.2 5.1 1.6 7.1 4.0 8.6 8.0 21.0 19.2
4.8 0.1 0.0 0.2 0.0 0.4 0.2 0.7 0.3 1.7 0.9 3.6 2.1

P concentration, mg L-

3.5 3.1 15.1 3.0 14.5 2.9 9.6 2.7 8.5 2.5 8.6 2.3 8.8
4.0 3.1 15.1 2.1 13.0 0.1 1.1 0.3 0.5 0.4 0.5 0.3 0.5
4.5 3.1 15.2 0.6 8.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
4.8 3.0 14.9 0.1 7.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
-1
EC, dS m-1

3.5 0.80 0.83 0.89 0.91 0.99 1.02 1.06 1.09 1.26 1.30 2.56 2.64
4.0 0.78 0.81 0.81 0.81 0.97 1.02 1.05 1.10 1.30 1.33 3.19 3.28
4.5 0.74 0.82 0.78 0.80 0.96 1.05 1.10 1.14 1.58 1.60 4.10 4.14
4.8 0.78 0.79 0.87 0.94 0.98 1.14 1.16 1.18 1.60 1.66 4.38 4.47









Table 3-2. Equations describing effects of pH(x ), Al addition (x ),
and P addition (xq) on concentration of Al (Y ),
concentration of P (Y ) and EC (Y3) in filtrates obtained
during Experiment 1 (aged for 7 days at 250C).


Regression equations


R2


Y1 = 0.36 + 4.048x2 0.853x1x2 0.023 x 2x3 + 0.004 x x 3 0.97t
2
Y = 2.06 + 1.9062x 0.0720x + 0.0001x2 0.3367x x
2 3 2 2 13 0.63
0.0002x1X2X3 0.63
2 2
Y = 2.80 0.956091x 0.005863x + 0.111680x + 0.000228x2
3 2.80 .959 2 1 3
0.000002x2 + 0.002962x1x2 0.99



t All values of R2 are significant at P < 0.001.










The EC increased with increasing levels of Al and P addition.

Increased pH caused a rapid increase of EC when Al or P additions

were large. Also, Blamey et al. (1983) reported that the EC of basal

solutions affected the concentrations of Al and P in solution.

After aging for 7 days, the Al and P concentrations and EC in

the filtered nutrient solutions and the supernatant solutions were

the same. Therefore, in the greenhouse studies with large volumes of

culture solution, supernatant liquid in the mixture could be siphoned

instead of filtered for convenience. There were no significant

differences in Al and P concentrations and EC between 7 and 14 days

of aging in the range of pH, Al and P additions of this experiment.

The 7-day aging temperatures of 5, 25, and 450C had no significant

effect, either. The Al and P concentrations and EC in the filtered

nutrient solution did not change after storage in tightly closed

bottles for 20 days.

Experiment 2. Supernatant Solution Containing Several Levels of Al
and Similar Concentrations of P

At certain pH levels, and when Al addition was small, the P

concentrations in the filtered nutrient solution increased

continuously with increased levels of P addition. When large amounts

of Al were added, the P concentrations went up and down several times

with increased P addition (Fig. 3-1). At a certain P concentration,

there might be more than one P addition and more than one corres-

ponding Al concentration which decreased continuously. At different

pH values, however, the upper limits of Al addition for continuous

increases of P concentrations were different (i.e., the higher the

pH, the smaller the upper limit of Al addition). For example, at pH

4.0 and with a 20 mg Al L-1 addition, the P concentration still


























pH 450
Tad 00mg Al L'
T


PH 4.00
' oad 160mg Al L'


II

i


I /
1 '


80 100


160 180


P ADDITION (mg L- )


Figure 3-1.


Phosphorus concentration in filtered nutrient
solution as affected by P addition at two pH
values and two levels of Al addition (aged
for 7 days at 250C). Vertical bars indicate
standard deviations.










continuously increased with increased P additions. However, at pH

4.5 when Al addition was only 15 mg Al L-1, the P concentration was

not increased continuously with increased P additions. An attempt to

interpret these results in terms of the prevailing concept of Al3+

hydrolysis, and of complexation by Al and P, has not been successful.

According to results from mixing the solutions having a number

of levels of Al addition and a number of levels of P addition at two

pH values, two sets of filtered nutrient solutions for the Al study

were found (Table 3-3). In Set A, the filtered solution contained

various levels of Al (0.1 to 171.3 mg Al L-1), but all P concentra-
-1
tions were about 1 mg L The concentrations of other nutrients,

and EC of the filtered solution, were adequate for plant growth.

This set of solutions may be suitable for large seedlings (e.g., tree

seedlings) which are more tolerant to Al and which need more P. In

Set B, the pH was 4.5. Aluminum levels ranged from 0.1 to 10.2 mg Al

L and P concentrations were kept at 0.2 mg P L- in all treatments.

The concentrations of other nutrients and the value of EC were also

adequate. This set of supernatant solutions may be suitable for

small seedlings which are more sensitive to Al, need less P, and

require higher pH. Seedlings of some cereals and vegetables may

adapt to this set of solutions.

Experiment 3. Elemental Composition of Large-volume Supernatant
Solutions Prepared Manually in the Greenhouse

The nutrients added to solutions and their concentrations in

supernatant solutions are shown in Table 3-4. The pH and additions

of Al and P were the same as in Set A of Experiment 2. However, Al,

P, and Fe concentrations, and EC values, were different from those of



























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Set A. Electrical conductivities were slightly lower and Fe concen-

trations slightly higher than those in Set A. When levels of Al

addition were large, P concentrations slightly decreased. The big

change from Set A in Experiment 2 was in Al concentrations, which

ranged only from 0.1 to 44.8 mg Al L-1. The main causes for the big

difference in Al concentrations between the supernatants prepared in

Experiments 2 and 3 were the methods used in adjusting the pH of the

solution and mixing the solution. In the preparation of the

equilibrium solution for Experiment 3, more concentrated NaOH

solution was used. Furthermore, a larger amount of NaOH was added

each time in Experiment 3 than in Experiment 2, and the added NaOH

could not be mixed immediately and thoroughly. The local higher

concentration of NaOH reacted with Al to form greater amounts of

amorphous hydroxy-aluminum and, thus, soluble monomeric Al decreased.

The large amount of amorphous hydroxy-aluminum in solutions

representing high Al treatments reacted almost immediately with

phosphate to form precipitates. In contrast, more Al in the Set A

solutions of Experiment 2 was reacted with phosphate to form soluble

complexes (Hsu, 1968). Therefore, the formation of large amounts of

amorphous hydroxy-aluminum caused decreased Al and P concentrations

in the supernatant solution. However, the P-concentration decrease
-1
was small and all of the P concentrations were about 1 mg L This

supernatant solution containing various Al levels and yet similar P

concentrations should be suitable for Al phytotoxicity studies.

Experiment 4. Test of Supernatant Solutions as Culture Solutions
Using Citrus Seedlings

Citrus seedlings were grown in the supernatant solution prepared

in Experiment 3 for 60 days. The seedlings grew rapidly during the










last 20-day period. The data for Rough lemon were chosen to show the

shoot height at the beginning and the new-growth shoot height at the

end of the third 20-day period (Table 3-5). The 2.7 and 4.8 mg Al
-1
L- treatments appeared to have had a beneficial effect, but 8.3 mg
-1
Al L- or more had a toxic effect on shoot growth. Figure 3-2 shows

that the beneficial and toxic effects of different Al concentrations

on Rough lemon were obvious. Growth for the other four rootstocks

was similar to that for Rough lemon. All seedlings responded signifi-

cantly to Al concentrations and no symptoms of nutrient deficiencies

or excesses were found, except that some seedlings (Swingle citrumelo)

showed yellow color, and mottled and withered young leaves and

aborted terminals in the high-Al treatments near the end of 60 days.

These symptoms may have been caused by Al toxicity.

The pH of the supernatant solution during the growth period

normally changed 0.2 units every 3 days. When the roots grew

vigorously at low Al levels near the end of 60 days, the pH decrease

of the solution was greater. This was probably due to the acid

exudate of the citrus roots. During this period, the pH was adjusted

daily.

The nutrient composition of the supernatant solution was

analyzed at the end of the last 20-day period, with results as shown

in Table 3-6. Aluminum concentration changed very little. The

amounts of nutrients remaining in the supernatant solution indicated

that the supernatant solution had the capacity to support large

seedlings growing for 20 days. For 7-month-old citrus seedlings,
+ -1
it appears that more than 25 mg NH4-N L should be applied.










Table 3-5.


Shoot height and new-growth shoot height of Rough lemon in
the 3rd 20-day growth period in the supernatant solution.


Al concentration
Treatment in supernatant Shoot height New-growth shoot
solution at the beginning height at the end

mg L ------- cm plant -------

Al-0 0.1 26.62.7+ 10.71.1

Al-I 2.7 29.63.0 13.71.6

Al-2 4.8 28.42.6 12.51.4

Al-3 8.3 21.71.3 5.80.5

Al-4 24.4 16.80.8 0.90.1

Al-5 28.4 16.70.7 0.80.1

Al-6 44.6 16.70.7 0.80.1



t Aluminum concentration taken as the average of Al concentrations at
the beginning and at the end of the 3rd 20-day growth period.


+ Standard deviation.
























































Figure 3-2.


Eight-month-old Rough lemon seedlings grown for
60 days in supernatant solution with various
concentrations of Al. From left to right: 0O1,
2.7, 4.8, 8.3, 24.4, 28.4, and 44.6 mg Al L .










Table 3-6.


Elemental concentrations and EC of supernatant solution
after growing five 7-month-old citrus seedlings in ten
liters of supernatant solution for 20 days.


Treat-
ment Al P NH NO Ca Mg K Fe Zn Cu Mn B EC
ment 4 3
---------------------mg L- ------------------------- dS m

Al-0 0.1 0.7 9 18 48 14 42 0.6 0.1 0.01 0.1 0.1 0.64

Al-I 2.5 1.0 6 14 45 14 38 0.3 0.2 0.01 0.2 0.1 0.82

Al-2 4.6 0.9 7 16 46 14 39 0.4 0.2 0.01 0.2 0.1 1.47

Al-3 8.2 0.7 11 18 47 14 46 0.5 0.2 0.01 0.2 0.1 2.25

Al-4 24.0 0.7 14 19 48 14 48 0.5 0.2 0.02 0.2 0.2 2.47

Al-5 28.2 0.7 16 21 48 14 49 0.5 0.2 0.02 0.2 0.2 2.60

Al-6 44.4 0.7 18 22 48 14 50 0.6 0.2 0.02 0.2 0.2 2.64











Summary and Conclusions

Precipitation of Al(OH)3 and aluminum phosphate may occur in

nutrient solution if a large amount of Al and P have been added at a

relatively high pH. The objective of this study was to investigate

the actual concentrations of Al and P in nutrient solution under

different pH conditions and varied levels of Al and P addition, and

to develop and test a supernatant-solution method for Al studies in

which the supernatant solutions contained various levels of Al and

similar concentrations of P. The aluminum concentration in super-

natant solutions was greatly reduced when pH was adjusted to 4.5 or

higher. Phosphorus concentration became negligible when pH was 4.5

or higher and Al addition was 25 mg L-1 or more, even when 15 mg P
-1
L was added to the solution. The large changes in P concentration

of the supernatant solution may confound the apparent effects of Al

on plant growth. Two sets of supernatant solutions which contained

various levels of Al and similar concentrations of P at two pH levels

were developed. One set of the supernatant solutions with pH 4.0 was

used in the greenhouse study to test suitability of the supernatant

solutions as culture solutions for Al phytotoxicity studies. Results

showed that the supernatant-solution technique was successful.

Two sets of supernatant solutions are recommended for Al phyto-

toxicity studies. In the pH 4.0 set, Al additions as A12(SO4)3'18H20

are 0, 20, 80, 160, 220, 270, and 320 mg Al L-1 and corresponding P

additions as NaH2PO4 H20 are 0.9, 16, 60, 90, 85, 75, and 27 mg P

L-. The maximum Al concentration will be 171 mg Al L and the P

concentration will be about 1 mg P L1 in all treatments. This set











is suitable for larger seedlings. In the pH 4.5 set, Al additions
-1
are 0, 1, 30, 60, 100, 130, and 160 mg Al L-1 and corresponding P
-1
additions are 0.25, 0.50, 12, 32, 28, 26, and 25 mg P L-1. The
-1
maximum Al concentration will be 10 mg Al L- and the P concentration
-1
will be about 0.2 mg P L- in all treatments. The concentrations of

Al and P are affected by the preparation procedure, such as the

concentrations of alkali and acid used to adjust pH and the speed of

mixing for this solution. This supernatant-solution method makes it

possible to avoid the confounding effects of P on Al, and to report

the actual concentration of Al in solution. Also, this method and

the use of regression procedures make it possible to obtain critical

values of Al concentration of toxic effects to plant growth.















CHAPTER IV

DIFFERENTIAL RESPONSE OF CITRUS ROOTSTOCKS TO ALUMINUM
LEVELS IN NUTRIENT SOLUTIONS


Introduction

Few researchers have studied the effects of Al on citrus root-

stocks in nutrient solutions. Haas (1936) used leafy-twig cuttings

of lemon, Lisbon, and Valencia orange in a nutrient solution and

found that, when Al was present the citrus roots were healthy, more

extensive, and root caps were numerous, but the shoots usually were

retarded. He concluded that "a concentration of 15 to 20 mg L-I of

Al was rather high for the production of the greatest growth" (tops

and roots). His data showed that the addition of Al to the solution

increased the percentage of P in root dry matter. Liebig et al.

(1942) found that the addition of 2.5 to 5 mg Al L- to nutrient

solutions greatly stimulated root development but depressed shoot

growth of Valencia orange and lemon cuttings. Lower concentrations

of Al, i.e., 0.1 and 0.5 mg L-1, did not produce this effect. They

found an antagonistic effect of Al on Cu. Yokomizo and Ishihara

(1973) concluded that root growth of Natsudaidai (C. Natsudaida

Hayata) seedlings improved at low concentration of Al but began to

decrease at an Al addition of 20 mg L and was extremely depressed

at 100 mg L-1 in nutrient solution. Root growth of Satsuma mandarin

trees was apparently increased by the supply of 10 and 20 mg Al L .

However, no results have been reported in the literature for










systematic screening of citrus rootstocks for Al tolerance. The Al

phytotoxicity levels are still not known for many citrus rootstocks,

and few data exist on effects of Al on mineral nutrition of citrus.

A general problem with previous work was that the authors only

reported the amounts of Al added to the solution, not the actual

concentrations in the nutrient solution. The actual concentrations

of Al in the nutrient solution were always lower than the original

concentration due to precipitation of Al(OH)3 and aluminum phosphate

(Yokomizo and Ishihara, 1973; Blamey et al., 1983; Bollard, 1983).

Another problem is the confounding effects of P on Al effects on

plant growth. The use of a supernatant-nutrient-solution method

developed in Chapter III might help to resolve these problems.

The objectives of this study were (1) to evaluate root and

shoot growth responses of citrus rootstocks to different Al concen-

trations in nutrient solution; (2) to estimate the critical Al

phytotoxicity concentrations for citrus rootstocks grown in Florida;

and (3) to evaluate the relationships between elemental concen-

trations in plant tissue and Al effects on plant growth.



Materials and Methods

Plant Material

Five citrus rootstocks were evaluated which account for more

than 90% of the citrus rootstocks used in Florida (Fisher, 1988).

They included: (1) Carrizo citrange [C. sinensis (L.) Osbeck x

Poncirus trifoliata (L.) Raf.], (2) Cleopatra mandarin (C. areshni

Hort. ex Tanaka), (3) Rough lemon (C. jambhiri Lush.), (4) Sour

orange (C. aurantium L.), and (5) Swingle citrumelo (C. paradisi x











P. trifoliata). Six-month-old seedlings were obtained from nurseries.

Uniform seedlings were selected, washed thoroughly with tap water,

and rinsed with deionized water. The root system of each seedling

was spread on a 1-cm grid background and photographed. The photo-

graphs were enlarged and used to measure the original root length

(Tennant, 1975). The fresh weight of whole plant and shoot height

of seedlings were recorded. The seedlings were then transferred to

nutrient solutions.

Nutrient Solution

The first set of supernatant nutrient solutions recommended in

Chapter III was used. The procedure of nutrient-solution preparation

was the same as described in Experiment 3, Chapter III. The

elemental additions and concentrations in the supernatant solutions

were as shown in Table 3-4. The seven Al concentrations in the

supernatant solutions ranged from 0.1 to 44.8 mg Al L-1, with P
-i
concentrations of about 1 mg P L in all treatments. The nutrient

solution was replaced each 20 days (three total replacements).

Before the plants were put into the solutions, and after each 20-day

growth period, aliquots of the solutions were taken for Al analysis.

The initial and final Al concentrations were averaged and were

assumed to represent the Al concentration during this 20-day growth

period. Three 20-day Al concentrations were averaged and the mean

was taken as the Al level during the 60-day growth period.

Equipment

Ten-liter pails were used to hold the nutrient solution. Five

holes were made in the plywood lids of the pails, one for each

seedling. A seedling was held in the hole by a rubber stopper cut










into two parts, with a small hole in the middle. The entire root

system of a seedling was submerged in the solution. One small hole

was also made in the lid for inserting an aeration tube. Continuous

aeration was supplied by an air pump hooked up to plastic tubes which

were attached to airstones in the solution. This also served to keep

the solution uniformly mixed (Fig. 4-1).

The toxicity of a given concentration of Al is highly

temperature-dependent (Konzak et al., 1976; Aniol, 1983). In the

present study, air-conditioning was used in the greenhouse to main-

tain the air temperature between 25 and 380C. Plastic-lined pools

were also set up on the benches in the greenhouse, with the pails

then put inside the pools filled with water. The water in the pools

was circulated by a pump and passed through a cooling system. The

temperature of the water in the pools and in the nutrient solution

in the pails was maintained at 2510C (Fig. 4-2).

Procedure

The study was conducted using a split-plot design with 7 Al

levels as the whole plot, completely randomized in 8 replications

with 5 rootstocks as the subplot. Eight pails contained the same

concentration of Al and 5 rootstock seedlings. A total of 56 pails

containing treatment nutrient solutions were randomly assigned to

positions in the pools (Fig. 4-3).

The pH levels were checked every 2 or 3 days and adjusted to 4.0

by HC1 or NaOH additions when necessary. The solution level was

maintained by addition of deionized water in quantity sufficient to

offset loss due to transpiration every two days. The seedlings grew

in the solution from June 2 to August 2, 1989.



























Figure 4-1.


The root systems of five citrus seedlings which
were submerged in nutrient solutions in pails
which were in turn placed in a water pool.


fr
Id
** T^:


Figure 4-2.


Water circulation in the pool by a pump, which
was maintained at 25t1C by passing through a
cooling system.

























'" 1



r d4-:'


Figure 4-3.


Citrus seedlings growing in nutrient solutions
in pails which were randomly assigned to
positions in the water-filled pool.











At the end of the experiment, plants were washed thoroughly with

tap water and given a final rinsing with deionized water. The root

and shoot morphology was assessed visually and from photographs.

Total root length, shoot height, and fresh weight of whole seedling

were measured. The differences between initial and final

measurements were considered as new-growth root length, new-growth

shoot height, and new-growth fresh weight. Roots and shoots were

dried and analyzed for elemental concentrations. Because of the

small quantities of some seedlings, the roots or shoots of the eight

replications of a given treatment were randomly combined into four

samples, respectively. The roots or shoots were ground to pass a

0.85-mm sieve. Tissue samples of 0.500 g were dry-ashed at 500C in

a muffle furnace for 4 h; the ash was then dissolved in 10 mL of 6 M

HC1, evaporated to dryness, and the temperature increased slightly to

dehydrate SiO2. The residue was dissolved in 6.7 mL of 2 M HC1,

heated to near-boiling, and then filtered. Elemental concentrations

in the solution were determined by inductively coupled argon plasma

(ICAP) emission spectroscopy.

Regression/correlation techniques were employed to relate growth

to Al concentrations in nutrient solution. The growth data were

transformed to natural logs and the Al concentrations to square-root

values.

Results and Discussion

Morphology of Roots and Shoots as Affected by Al Concentration

At 2.7 and 4.8 mg Al L-1 the roots of all rootstocks except

Carrizo citrange grew extremely well. The roots appeared whiter,

healthier, firmer, and straighter than those in 0.1 mg Al L-1










More new roots and lateral roots grew and, near the end of the

experiment, the roots of Rough lemon grew fastest among the five

rootstocks. When the Al concentration was 8.3 mg L-1 or higher, the

growth of roots was retarded. Fewer new roots and lateral roots grew

and root tips became thickened (Fig. 4-4). At the 28.4 and 44.6 mg
-1
Al L- levels, the root system as a whole appeared coralloid, with

stubby new-growth roots (Fig. 4-5). At the 44.6 mg Al L-1 level,

some older roots rotted, with Rough lemon roots deteriorating most

seriously. At 8.3 mg Al L- or higher, some root tips were covered

by a root cap with black gelatinous material (Fig. 4-6). The number

of blackened root caps increased with increased Al concentration in

solution. Among the five rootstocks, Cleopatra mandarin had the

greatest number of blackened root caps. It appeared that the

rootstock which was more tolerant to Al had more of this kind of root

cap. Therefore, the black gelatinous material on the root cap might

be related to avoidance of Al toxicity. What the black gelatinous

material was, how it formed, and what its function was, however, were

not known. The black gelatinous material was probably the excreta of

roots or complexes of the excreta with some components in the

solution, such as Al.
-1
Shoots of the seedlings grew faster in the 2.7 mg Al L- treat-
-1
ment than in the 0.1 mg Al L- treatment. When Al concentration was

8.3 mg L-1 or higher, growth of shoots was retarded. Shoots of five

rootstock seedlings were shorter and leaves were fewer and smaller

(Fig. 4-7) on plants grown of the higher Al concentrations. However,

no Al toxicity symptoms or other elemental toxicity or deficient

symptoms were observed during the first 50 days. Near the end of the





































Figure 4-1.


Figure 4-5.


Thickened root tips of Sour orange seedlings
grown in solution with 24.4 mg Al L'.


Stubby new-growth roots of Rough lemon seedlings
grown in nutrient solution with 24.4 mg Al L






























































Figure 4-6. Root tip covered by a root cap with black
gelatinous material for Cleopatra mandarin
seedlings grown in solution at 24.4 mg Al L




















%t%


Figure 4-7.


Young leaves of Swingle citrumelo seedlings
grown in nutrient solutions with various
concentrations of Al. From left o right:
0.1, 2.7, 24.4, and 28.4 mg Al L



F:.


j -


Figure 4-8. Shoot with yellow, mottled, and withered young
leaves and aborted terminal of Swingle citrumelo
seedling grown in nutrient solution with 44.6 mg
Al L for 60 days.










experiment, at 24.4 mg Al L-1 or higher, young leaves of Swingle

citrumelo were yellow, mottled and withered. Furthermore, the

terminal shoot was aborted (Fig. 4-8). Rough lemon had similar

symptoms but the symptoms were much less pronounced. These symptoms

were different from the symptoms of elemental deficiency or excess

for citrus, as listed by Chapman (1968). The symptoms were probably

caused by Al toxicity.

Growth Responses of Citrus Seedlings to Al Concentrations in Nutrient
Solution

Growth of five rootstock seedlings for 60 days in the nutrient

solution with various concentrations of Al is shown in Figs. 4-9

through 4-13. It was obvious that the growth of roots and shoots

was different among the Al treatments. The differences between the

2.7 mg Al L-1 and 44.6 mg Al L-1 treatments were particularly evident.

The initial, final, and new-growth of three parameters for five

rootstock seedlings were listed in Appendix (Table A-i). The effects

of Al concentration on new-growth root length of five rootstock

seedlings are shown in Fig. 4-14 and the linear regression equations

are given in Table 1. Aluminum concentration in the first treatment

was so low (0.1 mg Al L-, i.e., (Al) = 0.32) that this concen-

tration would not produce any beneficial or toxic effect on citrus

root growth (Liebig et al., 1942). Therefore, the new-

growth root length of this treatment was taken as a control. The

Al concentration at which the new-growth root length was equal to

that of a control was considered as the critical Al concentration.

Concentrations below or above the critical Al levels would cause

beneficial or toxic effects, respectively. In order to get the

critical values, regression equations were calculated. For all





























Figure 4-9.


Figure 4-10.


Effects of increasing Al concentrations in the
nutrient solution on root and shoot growth of
8-month-old Carrizo citrange seedlings.
From left to right: 0.1 2.7, 4.8, 8.3, 24.4,
28.4, and 44.6 mg Al L


(


Effects of increasing Al concentrations in the
nutrient solution on root and shoot growth of
8-month-old Cleopatra mandarin seedlings.
From left to right: 0.1 2.7, 4.8, 8.3, 24.4,
28.4, and 44.6 mg Al L


t.-
tY \rYV
x ,~c~,





































igur --- Effcc:- ncreasing A! concen--acicns in :he
nu-rien: sol:icin f n rooz and shoo: rcw'th of
-mcnth-oh d 0c.,r change seedlings. Fr3n ef_
to r -gL : -.L, 3.3, .. -. and
i-.q3 7?2 ;


Figure I--. Effects of increasing Al concentrations in :he
nutrient sol tion on rooc and shooc rc;: c :
8-month-old Rough lemon seedlings. Fro e
o right: .3, n
-s. mg Ai L



















































Fiura a--3._


Effects of increasing Ak ccncenzra-3s in :he
nutrient solution on root and shect zrcwth of
8-month-cld Swingle citrumelo eedlings.
From lef: :o right: Q0.1 2.7, -.., S.. 2.a.
28.4, and -.6 ng Al L

















7.0


6.5


6.0


5.5


5.0


4.5


4.0


3.5
0.0Th
0


----A.Corrizo citronge
- --- C. Cleopatro mandarin
--O Sour orange
--- R. Rough lemon
--- S. Swingle citrumelo


0
o^"


N
. \
\-.0


A


4 5 6 7


Figure 4-14. Effects of Al concentrations (Al, mg L-1) in
nutrient solution on new-growth root length
(L, cm plant ) of 8-month-old citrus seedlings
grown for 60 days.


0
\ 0


I 2 3

(Al)'2


I I i I A I










Table 4-1.


Linear regression equations for prediction of new-growth
root length (L, cm plant ), new-growth shoot height (,
cm plant ), and new-growth fresh weight (W, g plan )
of citrus seedlings from Al concentration (Al, mg L ) in
nutrient solution. (A = Carrizo citrange; C = Cleopatra
mandarin; 0 = Sour orange; R = Rough lemon; and S =
Swingle citrumelo).


Regression equations r2 Critical Al concentration


- 0.17(Al)
- 0.28(Al)
- 0.38(Al)1
- 0.45(Al)
- 0.23(Al)


- 0.60(Al)
- 0.40(Al)
- 0.47(Al)
- 0.60(A1)1
- 0.45(Al)


- 0.24(Al)
- 0.24(Al)
- 0.27(Al)
- 0.35(Al)
- 0.17(A1)


tAll the values of r2 were significant at P < 0.001.


(LA)
(LC)
(LO)
(LR)
(LS)


(HA)
(Hc)
(H0)
(HR)
(HS)


(WA)

(Wc)
(WR)
(Ws)


= 6.44
= 6.08
= 6.40
= 7.29
= 6.57


= 3.93
= 3.34
= 3.46
= 3.93
= 3.18


= 2.57
= 2.20
= 2.61
= 3.34
= 2.53


<2.7
7.2
3.9
4.6
4.5


4.0
8.8
6.0
3.6
1.8


0.86t
0.81
0.89
0.90
0.86


0.92
0.87
0.93
0.81
0.81


0.91
0.79
0.89
0.85
0.72


1.8
12.2
5.1
5.1
4.5










rootstocks except Carrizo citrange, new-growth root length increased

between the control and the treatment which had highest amount of

new-growth root length, but the curve was uncertain because there was

no treatment between them in most cases. There were 3 or 4 Al

treatments between the treatment which had the largest new-growth

root length and the treatment with the highest Al concentration in

solution, however, and the new-growth root length gradually decreased

between these two treatments. Therefore, the regression equation was

developed for these 5 or 6 Al treatments. Carrizo citrange was an

exception, because new-growth root length gradually decreased from

the control to the treatment with the highest Al concentration in

solution. According to the trend of the other four rootstocks,

it was possible that there might be some Al concentrations lower than

the second treatment (2.7 mg Al L- ) which might still have had a

beneficial effect on root growth. Therefore, the regression equation

was calculated for five treatments from the second treatment (2.7 mg

Al L-1) to the last treatment (44.6 mg Al L-1). The same procedure

was followed for the other parameters shown in Figs. 4-15 to 4-19,

and in Tables 4-1 and 4-2.

The critical concentrations obtained from the regression equa-

tions are shown in Table 4-1. The higher critical Al concentrations

indicated greater Al tolerance. According to these critical values,

the Al tolerance for root growth was as follows (from most tolerant

to least tolerant): Cleopatra mandarin > Rough lemon > Swingle

citrumelo > Sour orange > Carrizo citrange. The effects of Al

concentrations on relative new-growth root length are shown in Fig.

4-15. The root length for the first treatment (0.1 mg Al L ) was















- A. Corrizo citronge
--- C.Cleopatra mandarin
--& O.Sour orange
---C- R. Rough lemon
-- S. Single citrumelo


SI I I I


2 3 4 5 6 7


Figure 4-15.


Effects of Al concentrations (Al, mg L-1) in
nutrient solution on relative new-growth root
length (RL, Z) of 8-month-old citrus seedlings
grown for 60 days.


5.0


0 o


4.5-


4.0k


N


3.0



2.5
0


@\\


0 \
\ 0











taken as 100%. According to the predicted highest percentage values

shown in Table 4-2, the beneficial effects of low Al concentrations

on root growth of five rootstocks were as follows (from most to least

beneficial): Rough lemon > Cleopatra mandarin > Sour orange >

Swingle citrumelo > Carrizo citrange. This order was somewhat

different from the tolerance order. Such difference indicated that

the degree of beneficial effect on root growth at low Al concentra-

tions did not correspond well with the sequential order of tolerance.

According to the predicted lowest percentage values shown in Table

4-2, the toxic effects of high Al concentrations on root growth of

five rootstocks were as follows (from most to least toxic): Rough

lemon > Sour orange > Cleopatra mandarin > Carrizo citrange > Swingle

citrumelo. This order was different from that of the tolerance order

or that of beneficial effect. This difference indicated that the

degree of toxic effect on root growth at high Al concentrations did

not correspond with the tolerance or the degree of beneficial

effects. Rough lemon was in the second position in the tolerance

order, for example, and had the highest beneficial effect from low

Al concentrations, but suffered most from high Al concentrations

among the five rootstocks.

The effects of Al concentrations on new-growth shoot height of

five rootstock seedlings are shown in Figs. 4-16 to 4-17, with the

regression equations being given in Tables 4-1 and 4-2. The effect

of Al concentrations on shoot growth showed a different trend from

root growth. The orders of tolerance, beneficial effects, and toxic

effects for shoot height also were different from those for root

growth. Carrizo citrange and Swingle citrumelo are good examples for










Table 4-2.


Linear regression equations for prediction of relative
new-growth root length (RL, Z), relative new-growth shoot
height (RH, %), and relative new-growth shoot weight (RY,
%) of citrus seedlings from Al concentration (Al, mg L )
in nutrient solution. (A = Carrizo citrange; C =
Cleopatra mandarin; 0 = Sour orange; R = Rough lemon; and
S = Swingle citrumelo.)


Predicted
Predicted lowest percentage
highest at 44.6 mg Al L
Regression equations r2 percentage+ treatment


In (RLA) = 4.62 0.17(Al) 0.86t 76.8 32.6
In (RLC) = 5.35 0.28(A1) 0.81 114.0 32.5
In (RLO) = 5.35 0.38(Al)2 0.89 112.8 16.6
In (RLR) = 5.56 0.45(A1) 0.90 124.0 12.9
In (RLS) = 5.09 0.23(Al) 0.86 111.3 35.0


In (RHA) = 5.81 0.60(Al) 0.92 124.5 6.1
In (RHC) = 5.80 0.40(Al) 0.87 171.2 22.8
In (RHO) = 5.75 0.47(Al) 0.93 145.1 13.6
In (RHR) = 5.73 0.60(A1) 0.81 114.9 5.6
In (RHS) = 5.20 0.45(Al) 0.81 86.5 9.0


In (RWA) = 4.92 0.24(Al) 0.89 92.4 27.6
In (RWC) = 5.42 0.24(A1) 0.79 135.5 45.5
In (RWO) = 5.21 0.27(A1) 0.89 117.5 30.2
In (RWR) = 5.38 0.35(Al) 0.85 122.1 21.0
In (RWS) = 4.95 0.17(Al) 0.72 106.8 45.4
S


tAll the values of r2 were significant at P < 0.001.
+Predicted highest percentage in the treatment (2.7 or
for which the sample percentage was highest.


4.8 mg Al L-1)











3.0


2.5


2.0


1.5


1.0


(Al )/2


Figure 4-16.


-1
Effects of Al concentrations (Al, mg L ) in
nutrient solution on new-growth shoot height
(H, cm plant ) of 8-month-old citrus seedlings
grown for 60 days.


---- A. Carrizo citrange
--o C.Cleopatra mandarin
--n- Q. Sour orange
----1 R.Rough lemon
D ---- S. Swingle citrumelo









0



v\



\o
*


0.5


0.0












--- A.Corrizo citrange
--oC C. n pnntr mnndrrin


N .- 0 Sour orange
---- R. Rough lemo
S'A\ --*-- S. Swingle citru
\\ \o







\, \ -o
0











0
\ \


n
imelo


5.0-


45-


4.0-










2.5-


2.0


1.5
00T
0


I 2 3 4 5 6 7

( AI)1/2


Figure 4-17.


Effects of Al concentrations (Al, mg L ) in
nutrient solution on relative new-growth shoot
height (RH, %) of 8-month-old citrus seedlings
grown for 60 days.


\\


\










showing such differences. For root growth, Carrizo citrange was

least tolerant and did not show a beneficial effect from the 2.7 mg
-1
Al L- treatment. For shoot growth, however, Carrizo citrange had

the third position in the tolerance list and showed a beneficial

effect at 2.7 mg Al L 1. Swingle citrumelo had the third position in
-1
tolerance list and showed a beneficial effect at 2.7 mg Al L- treat-

ment for root growth. For shoot growth, however, Swingle citrumelo

was least tolerant and the predicted relative new-growth shoot height

was less than 100%. Haas (1936) and Liebig et al. (1942) also found

that low concentrations stimulated root growth but depressed top

growth for some citrus species. Several questions need answers: Are

those roots developed by stimulation of low Al concentrations normal

in their absorption of nutrients from the matrix? Are there any

stimulation effects of low Al concentrations on the function of

nutrient absorption by roots instead of on the development of root

length? Are there different physiological effects of absorbed Al on

shoot growth of different citrus rootstocks?

The different growth responses of roots and shoots indicated

that neither of these alone was a good indicator for evaluation of

the Al tolerance of citrus rootstocks. Because fresh-weight values

were the sum for roots and shoots, the fresh-weight response combined

the responses of root length and shoot height. In general, new-growth

fresh weight of whole plants should be a better indicator for evalua-

tion of Al tolerance than the other two parameters. The effect of Al

concentrations on new-growth fresh weight and relative new-growth

fresh weight of five rootstock seedlings are shown in Figs. 4-18

and 4-19, with the regression equations being given in Tables 4-1





59






3.0- -- A.Carrizo citrange
--o C.Cleopotra mandarin
3 ---- 0. Sour orange
----CR. Rough lemon
2.5- s ----S. Swingle citrumeio


2.0- `II

"- o \. A'^ -"
o13

1.5 -

~ I. 4- +.. -





^^-1
05--
O I 2 3 4 5 6 7
( Al)1/2



Figure 4-18. Effects of Al concentrations (Al, mg L-) in
nutrient solution on new-growth fresh weight
(W, g plant ) of 8-month-old citrus seedlings
grown for 60 days.











5.25

5.00

4.75


4.50

4.25

4.00

3.75

3.50

3.25


3.O01
0.o00


( A )1/2


Figure 4-19.


Effects of Al concentrations (Al, mg L-1) in
nutrient solution on relative new-growth fresh
weight (RW, X) of 8-month-old citrus seedlings
grown for 60 days.


--* A.Carrizo citrange
--o C. Cleopatra mandarin
----, O.Sour orange
---OR. Rough lemon
---GS. Swingle citrumelo




\

,\ ,


I f I L (










and 4-2. According to the critical Al concentration, the Al tolerance

for fresh weight increase was as follows (from most tolerant to least

tolerant): Cleopatra mandarin > Rough lemon = Sour orange > Swingle

citrumelo > Carrizo citrange. The benefical-effect order was (from

most to least beneficial : Cleopatra mandarin > Rough lemon > Sour

orange > Swingle citrumelo > Carrizo citrange. This order was

similar to the tolerance order. The toxic-effect order was (from

most to least toxic): Rough lemon > Carrizo citrange > Sour orange >

Swingle citrumelo > Cleopatra mandarin. This order was different

from both the tolerance order and the beneficial-effect order.

Elemental Concentrations in Roots and Shoots as Affected by Al
Concentrations in Growth Solution.

The elemental concentrations and their standard deviations for

roots and shoots of five rootstocks grown in various Al concentra-

tions in solution are listed in the Appendix (Tables A-2a and A-2b).

The standard deviations were small, and the coefficients of variation

were normally less than 5% for each Al treatment and each element

(4 replications). The elemental concentrations in roots and shoots

as affected by Al concentrations in the growth solution are shown in

Figs. 4-20 to 4-28.

Aluminum

At the 0.1 mg Al L-I level, the Al concentrations of roots of

all five rootstocks were similar (about 45 mg Al kg- ). When Al

concentrations in solution increased, furthermore, the Al concentra-

tions of roots of five rootstocks were increased (Fig. 4-20). When
-i
Al concentrations were 2.7, 4.8, and 8.3 mg Al L- in solution, Rough

lemon and Cleopatra mandarin had higher Al concentrations than the

others, while Carrizo citrange had the lowest concentration among the















SHOOTS


--

--
-a
'I --~
d


I I


I I I I


S ROOTS
/"






-F-









) 10 20 30 40 5C

Al Concentration In Growth Solution (mg L'1)


Figure 4-20.


Aluminum concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)


0.3k


02-


C,

C
0.0

E 6
c)
U
C
6 5

<:7 ,










five rootstocks. This trend was similar to those for tolerance and

for beneficial effects (Table 4-1 and 4-2). It might be concluded

that the root Al concentrations were higher for Al-tolerant than for

Al-sensitive rootstocks when Al concentrations in solution were 2.7
-1
mg L1 or higher. It might be also concluded that those roots which

accumulated more Al had greater new-root growth. These relationships

imply that Al accumulation in roots was a characteristic associated

with Al beneficial effects and tolerance of roots. When the Al

concentrations in roots were high, such as 6165 mg Al kg-1 for Rough

lemon at the 44.6 mg Al L-1 level, the accumulation of Al in roots

apparently damaged the roots. The Al concentrations in shoots also

increased with increased Al concentration in solution. However, the

increases in Al concentration of the shoots were much less than those

for the roots. Comparison of Fig. 4-20 and Fig. 4-17, led to the

conclusion that there was no certain relation between Al concentra-

tion in shoots and Al-beneficial effects and Al-tolerance of root-

stocks. The relation between Al concentrations in roots and shoots

and Al-tolerance of citrus seedlings did not belong to any of the

three groups described by Foy (1984).

Calcium

The Ca concentrations in the roots of the five rootstocks

decreased with increased Al concentration in solution up to 8.3 mg
-1
L (Fig. 4-21). However, when Al concentrations in solution were

higher than 8.3 mg L-1, the Ca concentrations in the roots of all

rootstocks underwent little further change; i.e., they all remained

similar, with the shoots having higher Ca concentrations than the

roots.




















SHOOTS


OR
Es

---- ----------


ROOTS


I I I I I I


I Ij


O 10 20 30 40 5(
Al Concentration In Growth Solution (mg L')


Figure 4-21.


Calcium concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)










The Al concentrations in the shoots showed the same trend as those

in the roots. Al-induced Ca deficiency has been associated with Al

toxicity effects (Lance and Pearson, 1969; Lund, 1970). In the

present study, the Ca concentrations in roots decreased when Al had a

beneficial effect, while such concentrations remained the same when

Al had a toxic effect. Therefore, Ca concentrations in citrus plants

might not be the main factor related to toxic-Al effects. This con-

clusion could be applied to Zn, Mn, Cu, and Fe as well in the follow-

ing discussion. There was no certain relationship between Ca concen-

trations in roots or shoots and Al-tolerance of citrus rootstocks.

Magnesium

The Mg concentrations in roots of five rootstock seedlings

increased when Al concentrations in solution increased to 2.7, 4.8,

or 8.3 mg L- and then decreased and remained the same when the Al

concentrations were higher than 28.4 mg L- in solution. In

contrast, the Mg concentrations in shoots decreased when Al concen-
-1
tration in solution increased up to 8.3 mg L- Above that Al

concentration, the shoots maintained their Mg concentrations.

Swingle citrumelo accumulated more Mg both in roots and shoots than

did the others. There was no certain relation between Mg concentra-

tions in roots or shoots and Al-beneficial effects or Al-tolerance of

citrus rootstocks.

Potassium and phosphorus

The K and P concentrations in both roots and shoots increased

when Al concentrations in solution increased up to 2.7 or 4.8 mg L-

(Figs. 4-23 and 4-24), with the P concentrations increasing rapidly.

When the Al concentrations were higher than 4.8 mg L1, the K and P
When the Al concentrations were higher than 4.8 mg L the K and P















SHOOTS


N9
-


-









0
C
o_


o
t-

t-)


Al Concentration In Growth Solution (mgL-' )


Figure 4-22.


Magnesium concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)


I II I I I I


I I


ROOTS


-4-


10 20 30 40


2F r,
~
I -------












SHOOTS


20


Al





II I I


ROOTS


0 10 20 30 40

Al Concentration In Growth Solution (mg L-')


Figure 4-23.


Potassium concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)












SHOOTS


S- -


Al Concentration in Growth Solution (mg L-l)


Figure 4-24.


Phosphorus concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)










concentrations then decreased. When Al concentrations were higher

than 8.3 mg L-1, the K and P concentrations decreased only slightly,
-1
however. At an Al concentration of 44.6 mg L P concentrations in

both roots and shoots were still higher than those in the 0.1 mg Al
-1
L treatment, while K concentrations were slightly lower. The K and

P concentrations of roots and shoots were not related to Al-tolerance

of the citrus rootstocks.

The mechanisms of beneficial effects of low Al concentration on

plant growth generally have been associated with promoting P uptake

(Mullette, 1975) or with correcting or preventing P toxicity (Clark,

1977). In the present study, there was no confounding effect of P on

Al, because all treatments had nearly the same (adequate) P concentra-

tion in solution. The beneficial effects of Al were not caused by

promotion of P uptake because Carrizo citrange, which roots had not

been affected beneficially by Al at the 2.7 mg Al L1 level, also

evidenced increased P uptake. The beneficial effects were not caused

by correcting or preventing P toxicity either, because there was no

toxic level of P in the solution. Furthermore, the P concentration

in roots of Carrizo citrange was lowest among the five rootstocks.

The mechanism of toxic effects of high Al concentration on plant

growth have been ascribed to Al-induced P accumulation (McCormick and

Borde, 1972) or deficiency (James et al., 1978). In this study, the

highest P concentrations in roots or shoots at low Al treatments did

not depress root or shoot growth. When root or shoot growth continu-

ously decreased with increasing Al concentrations in solutions more

concentrated than 8.3 mg L-1, the P concentrations in the roots or

shoots basically did not change. These data suggest that toxic











effects of Al were not caused by P accumulation. When plant growth

continuously decreased, the P concentrations in the roots or shoots
.-I
were still higher than those in the 0.1 mg Al L treatment. There-

fore, the toxic effects were not caused by P deficiency.

The fact that there was no certain relation between P concentra-

tions in roots or shoots and Al-tolerance of citrus rootstocks also

supports these explanations. It might be concluded that neither

beneficial nor toxic effects of Al on growth of citrus seedlings were

directly caused by P accumulation or deficiency induced by Al supply,

although increased Al concentrations in the matrix caused an increase

in P concentration of the plant tissues. The conclusions about P

which have been made here could be applied to the cases of Mg and K

in this study as well.

Zinc and manganese

The Zn and Mn concentrations in roots of the five rootstocks

greatly decreased with increasing Al concentrations in solution up to
-1
8.3 mg L- (Figs. 4-25 and 4-26). When Al concentrations in solution

were higher than 8.3 mg L-1, Zn and Mn concentrations in roots were

maintained at the same levels or slightly decreased. However, the Zn

and Mn concentrations in shoots basically did not change when Al

concentrations in solution increased continuously from 0.1 to 44.6 mg
-1
L No certain relation was found between Zn and Mn concentrations

in roots or shoots and Al-beneficial effects and Al-tolerance of

citrus rootstocks.

Copper

In the 0.1 mg Al L-I treatment, Carrizo citrange had the lowest

Cu concentration in its roots among the five rootstocks. It seemed
















SHOOTS


200C


0IC-


I I I I-----


300 ROOTS





1 -



-L -- ----------------


10 20 30


Al Concentration In Growth Solution (mg L- )


Figure 4-25.


Zinc concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)


U1----


I I I


l l 1 l


SI I I


/~\











































S- ------------ -


10 20


30 40


Al Concentration In Growth Solution (mg L- )


Figure 4-26.


Manganese concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)


SHOOTS


ICO



iO .__ -<-


RCOTS


2



100


I


[















SHOOTS


30k


20-


---., _

I I I I S I







--

-------- -

.... .. --: .


I I I I I I I
10 20 30 40 5(

Al Concentration In Growth Solution (mg L-I)


Figure 4-27.


Copper concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)


'O





S30
20
(-1








20










that the Al-sensitive rootstock had lower Cu concentrations in their

roots than did the Al-tolerant rootstocks. The Cu concentrations in

roots of all rootstocks except Carrizo citrange greatly decreased

with increased Al concentrations in solution up to 8.3 mg L-1

(Fig. 4-27). Beyond 8.3 mg Al L-1, the Cu concentrations in the

roots changed only slightly. The Cu concentrations of Carrizo

citrange changed only a little with increased Al concentrations in

solution. The Cu concentrations in shoots decreased with increased

Al concentrations in solution up to 2.7 or 4.8 mg L- after those

all rootstocks except Rough lemon changed little. The Cu concen-

trations in shoots of Rough lemon increased dramatically when Al
-I1
concentrations in solution were higher than 4.8 mg L At the 44.6

mg Al L-1 treatment, the shoot Cu concentration of Rough lemon went

up to 41 mg kg- while the corresponding level for other rootstocks

was only about 7 mg kg- At this treatment, Al-toxic effect on

shoot growth was largest for Rough lemon among the five rootstocks

(Table 4-2). There seemed to be certain relation between Cu concen-

trations in roots and Al-tolerance of citrus rootstocks at 0.1 mg Al
-1
L level in solution. Also, there seemed to be a relationship

between Cu and the degrees of toxic effects of Al concentrations.

Iron

The Fe concentrations in roots greatly decreased with increased

Al concentrations in solution up to 8.3 mg L-I (Fig. 4-28). Beyond

that Al concentration, the Fe concentrations in roots changed

relatively little. When Al concentrations were lower than 8.3 mg
-1
L Rough lemon and Cleopatra mandarin had higher Fe concentrations

than did the others, while Carrizo citrange had the lowest values















SHOTS


>- --- ------ -

01 _

O -- -
J ** .- I i-- "- "-


2
b.,.
0--- i----j -
.3- -


) 10 20 30 40 50

Al Concentration In Growth Solution (mg '


Figure 4-28.


Iron concentration of 8-month-old citrus
seedlings grown for 60 days in nutrient solution
with various concentrations of Al. (A = Carrizo
citrange; C = Cleopatra mandarin; 0 = Sour orange;
R = Rough lemon; and S = Swingle citrumelo)


i


i I










among the five rootstocks. By comparing this order and the Al-

tolerance order of roots, suggestion might be made that the more

tolerant rootstocks had the higher Fe concentrations in their roots

when Al concentrations in solution were lower than 8.3 mg L-1

The Fe concentrations in shoots decreased when Al concentrations

in solution increased to 2.7 or 4.8 mg L-1, however. Beyond this

concentration the Fe concentrations in shoots increased only

slightly. No relation between Fe concentrations in shoots and

Al-beneficial effects and Al-tolerance of citrus rootstocks could

be found.



Summary and Conclusions

Very few systematic studies have been conducted on the effects

of Al on the growth and mineral nutrition of citrus. The objectives

of this study were to investigate growth response of the most common

citrus rootstocks in Florida to Al levels, and relations between the

Al effects and elemental concentrations in plant tissue. Five

6-month-old citrus rootstock seedlings were grown in supernatant

solutions which contained 7 levels of Al ranging from 0.1 to 44.6 mg

Al L- and P concentration of 1 mg P L- for 60 days. The temperature

of the growth solution was maintained at 2510C in the greenhouse

during the summer. Before the seedlings were grown in solution,

shoot height and fresh weight of whole plants were measured and the

root length was measured by taking photographs of the roots for the

purpose of later obtaining new-growth parameters. Results showed

that, at high Al treatment levels, plants had thickened root tips and

root caps covered with black gelatinous material. Unique Al injury










symptoms were observed in new leaves and terminals of some seedlings.

The new-growth root length and shoot height had different trends with

respect to response to Al concentrations in the growth solution.

New-growth fresh weight of whole plants might be a better indicator

for Al tolerance than the other two parameters mentioned above.

According to the response of fresh weight to Al concentrations,

relative aluminum tolerance of the rootstocks were Cleopatra mandarin

> Rough lemon = Sour orange > Swingle citrumelo > Carrizo citrange.

The critical Al concentrations in solution with respect to toxic

effects were 12.2, 5.1, 5.1, 4.5 and 1.8 mg Al L-1, respectively.

Concentrations below or above the critical Al levels caused either

beneficial or toxic effects, respectively. Aluminum concentrations

of roots and shoots increased with increased Al concentration in the

growth solution. Aluminum-tolerant rootstocks accumulated more Al in

their roots than did the Al-sensitive rootstocks. When Al concentra-

tions in nutrient solution increased from 0.1 to 4.8 mg Al L-1, K,

Mg, and P concentrations in roots and K and P levels in shoots

increased; whereas Ca, Zn, Cu, Mn, and Fe in roots and Ca, Mg, Cu,

and Fe in shoots decreased. It seemed that Al-sensitive rootstocks

had lower Cu concentrations in their roots than did Al-tolerant

rootstocks at low Al concentration (0.1 mg Al L-1) in solution. The

more tolerant rootstocks contained higher Fe concentrations in their

roots than did the less tolerant ones when Al concentrations in

solution were lower than 8.3 mg Al L-1. Concentrations of the other

elements (Ca, K, P, Mg, Zn, and Mn) in roots or shoots appeared to

have no certain relationship with the beneficial or toxic effects of

Al in nutrient solution, or with Al-tolerance of the rootstocks.















CHAPTER V

GROWTH OF CITRUS ROOTS AS AFFECTED BY ALUMINUM LEVEL
IN SOILS UNDER FIELD CONDITIONS



Introduction

Research on the effects of Al on citrus rootstocks has been

essentially limited to nutrient-solution studies. Haas (1936) used

leafy-twig cuttings of lemon, Lisbon and Valencia oranges in solution

cultures. He found that, when Al was present, roots were healthy and

more extensive and root caps were numerous, but tops usually were

retarded in growth. He concluded that "concentration of 15 to 20 ppm

of aluminum was rather high for the production of the greatest

growth" (of tops and roots). His data showed that the addition of Al

to the culture solution also increased the P concentration in root

tissue. Liebig et al. (1942) found that the addition of 2.5 to 5 mg

Al L-1 to base nutrient solutions greatly stimulated root development

but depressed top growth of Valencia orange and lemon cuttings.

Lower concentrations (i.e., 0.1 and 0.5 mg L-1) did not produce this

effect. These researchers also found an antagonistic effect of Al on

Cu uptake. Yokomizo and Ishihara (1973) concluded that root growth

of Natsudaidai (C. Natsudaidai Hayata) seedlings in solution culture

improved at low concentrations of Al but began to decrease when Al

addition was 20 mg Al L-1. Growth was extremely depressed at 100 mg

SL-1
A LL










Worku et al. (1982) found that high levels of Al and Mn were

toxic to Troyer citrange [Pencirus trifoliata Raf. x C. sinensis (L)

Osbeck], Trifoliate orange (P. trifoliata Raf.), and Cleopatra

mandarin (C. reshni Hort. ex. Tan.) grown in highly weathered

Oxisols. However, the growth-inhibiting effects of Al and Mn were

considered jointly. Other researchers (Sekiya and Aoba, 1975; Huang,

1983) have linked low pH and high Al concentrations to poor citrus

growth and shortened lifespan of the tree. However, no experimental

evidence exists to evaluate the effects of different Al levels in

soils on fibrous citrus-root growth under field conditions. Few data

have been reported as well about possible effects of Al on the

mineral nutrition of citrus.

The objective of this study was to use an implanted soil-mass

technique (Garner and Telefair, 1954; Lund et al., 1970) to investi-

gate the effects of different Al levels in soil on growth and mineral

content of citrus fibrous roots under field conditions.



Materials and Methods

The implanted soil-mass technique allows one to study root

development in a natural environment with minimal disturbance and

minimal spatial and genetic variability.

This experiment was conducted using the implanted soil-mass

technique in a commercial citrus grove.

Collection and Characterization of Soil

In order to get effects of high Al concentrations in soil

solution when a certain amount of Al was added to the soil, the soil

used for implanting must have low pH (pH < 5). The implanted soil










must also have low exchangeable Al content in order to obtain low Al

concentration (<0.5 mg Al L-1) in soil solution, so that non-treated

soil could be taken as a control to get critical Al concentration in

soil solution for phytotoxicity.

Soil used for implanting was obtained from the E horizon of an

Immokalee fine sand (an Arenic Haplaquod) from a citrus grove in the

"flatwoods" area of Charlotte County, Florida. The overlying Ap

horizon was first removed by hand-shoveling before collecting the

bulk sample of E horizon. The soil was air-dried and passed through

a 2-mm sieve.

Soil pH was measured with a 1:1 water:soil ratio. Particle-

size analysis was conducted using a pipette sampling method (Soil

Conservation Service, 1972). Soil organic C was determined by a

modified Walkley-Black procedure (Nelson and Sommers, 1982).

Potassium-chloride extractable acidity, exchangeable Al, and exchange-

able H were determined in 1 M KC1 extracts (Thomas, 1982). Effective

CEC of soil was calculated from the sum of exchangeable bases by 1 M

NH 0Ac (pH 7.0) (Chapman, 1965) and exchangeable Al (1 M KC1).

Calcium, Mg, K, Na, P, Al, Zn, Fe, Cu, and Mn were extracted with

double-acid reagent, 0.05 M HC1 and 0.025 M H2SO4 (Mehlich, 1955),

and determined by inductively-coupled argon plasma (ICAP) emission

spectroscopy. Relevant characteristics of the soil are listed in

Table 5-1. It had a high sand content; was strongly acidic; and had

a low exchangeable-Al content.

Addition of Lime, Al, and Fertilizers

There were five treatments (addition of lime and addition of 0,

15, 18, or 24 mg Al kg-I of soil). A loosely woven mesh-saran bag











Table 5-1. Relevant characteristics of the E horizon of the
Immokalee fine sand used for implants.


Soil property


pH

Organic C, g kg-

Particle size
Sand, %
Silt
Clay

ECEC, mmol c kg-
c


1 M KC1 extractable acidity
Total, mmol kg-
.1 C


0.05 M
Ca,
Mg
K


HC1 and

mg kg-I


4.20

0.6



97.5
1.2
1.3

1.31



0.62
0.11
0.51


0.025 M H2SO4 extractable elements
27.9
3.4
3.0
0.4
1.2
1.5
2.4
0.1
0.3
0.1










(hole size 3 x 2 mm) was used to hold 4.5 kg of E-horizon soil. The

limed soil was amended with 125 mg chemically pure CaCO3 kg -, the

amount of lime required to bring soil pH to 6.5 as specified by the

Adams-Evans method (McLean, 1982). The Al was added as solutions of

AIC13 6H20.

Blanket fertilizer additions in solution form also were made to

the soil in each bag. The fertilizer program recommended by Koo et

al. (1984) was taken as a reference. Fertilizer rates (mg kg- ) and

forms were as follows: 5.93 N as Ca(NO 3)24H20; 0.89 P as Ca(H2P04)2

H20; 9.10 Ca as Ca(NO3) 24H20 + Ca(H2PO4)2H20; 5.35 K as KC1; 3.88

Zn as ZnSO 47H20; 1.20 Mg as MgC12 6H20; 0.13 Fe as FeSO 47H 0; 0.22

Mn as MnSO4 H20; 0.13 Cu as CuSO4*5H20; and 0.01 B as H3BO3. After

the lime or Al solution and fertilizer solution had been added, the

soil in each bag was mixed thoroughly and the moisture level was

adjusted to 12%. The soil then was allowed to equilibrate with the

amendments for 18 d at room temperature in the laboratory.

Placement and Collection of Implanted Soil-Mass Bags

A typical commercial citrus grove (with 30-yr-old trees of C.

sinensis, cv. Hamlin/C. aurantium L. sour orange rootstock) in De

Soto County, Florida was selected for the study. The experiment was

conducted according to a randomized complete-block design, with five

treatments assigned randomly in each of 15 blocks. Fifteen healthy-

appearing trees were marked as blocks. Five holes (20-cm deep and

16-cm diameter) were dug at the dripline of each tree (about 3 m from

the tree trunk) with a post-hole digger. The exact location of each

hole used for implant was determined by first digging a hole, screen-

ing out the roots on-site, and comparing the quantity of roots to











prescribed limits (about 8 roots). A bag containing the implant soil

then was placed firmly against the face of the hole on the side

toward the tree trunk. Some original top soil was tamped firmly

around each bag, with the bag then being left open at the soil

surface to approximate the same field conditions as the surrounding

surface soil. All of the holes were dug, and all of the bags were

installed in the holes, on 2 August 1988. The areas under the trees

and within 2 m of the holes were kept free of understory vegetation,

to minimize invasion of the bags by non-citrus roots.

All bags were removed 46 d after their insertion. The roots

around the outside of the bags were cut off with a long knife and

then the bags were removed from the holes. Each bag was trimmed of

protruding roots and placed in separate plastic bags to prevent soil

and moisture loss during transport to the laboratory in Gainesville,

Florida.

Measurement of Roots and Analysis of Implanted Soils

After removal from the bag, the roots separated from the soils

were put on a screen and then washed thoroughly with tap water and

rinsed with deionized water. Root morphology was assessed visually

and root length was measured directly. Root length is a better

indicator of Al effects than is root weight, since shortening and

thickening are common results of Al treatment (Munn and McCollum,

1976). Roots which entered the bag from its surroundings were

classified as primary roots. All branches produced from these

primary roots were classified as secondary.

All roots were dried at 700C and weighed. For tissue analysis,

because of the small quantities recovered per bag, the roots of the











15 replications of a given treatment were randomly combined into 3

samples. The roots were ground in a mortar to pass a 20-mesh sieve.

Tissue samples of 0.2 g were dry-ashed at 500"C in a muffle furnace

for 4 h; the ash was then dissolved in 10 mL of concentrated HC1,

evaporated to dryness, redissolved, and evaporated to dryness again.

This residue was dissolved in 10 mL of 0.1 M HCI and filtered.

Elemental contents in the solution were determined using ICAP

emission spectroscopy.

Just before the amended soil was placed in the grove, and again

after collection, the concentrations of Al and of other elements in

saturation soil extracts (Rhoades, 1982) were determined using ICAP

emission spectroscopy. Electrical conductivity (ECe) and pH were

measured immediately after extraction. Values before implanting and

after collection were averaged to represent the implanted period

(46 d).



Results and Discussion

Selected characteristics of soil saturation extracts are shown

in Table 5-2. When 1.0 kg soil was treated with 15 mg Al, Al concen-

tration and EC sharply increased and pH decreased relative to the

control. As the amount of added Al increased from 15 to 24 mg Al
-1
kg Al concentration increased greatly while pH decreased and EC
e
increased only slightly. Aluminum concentration decreased and pH

increased in lime-amended soil.

At the time of bag removal, we observed some roots which had

grown downward in soil outside but adjacent to the bag's outer

surface, apparently avoiding entry into the soil inside the bag.










Table 5-2. Relevant characteristics of soils from five treatments.


Saturation extract of soilst
Al added Al
Treatment to soil Before After Averaget EC t pHt
implant implant e

mg kg- -----mg L-1---------- dS m-

Al-0 0 0.21 0.05 0.13 d+ 0.30 c 5.1 b

Al-1 15 15.94 2.34 9.14 c 0.71 b 3.7 c

Al-2 18 37.72 5.46 21.59 b 0.83 ab 3.5 c

Al-3 24 59.63 9.57 34.60 a 0.99 a 3.4 c

Lime 0 0.03 0.02 0.03 e 0.32 c 6.4 a


t Each value is the average of 2 means, i.e., mean values just
before implanting or after collection for 15 replications.

+ Values followed by the same letter in a column are not significantly
different at P = 0.05 by Duncan's multiple-range test.










Roots in the soils of the Al-I and Al-2 treatments appeared

healthier, coarser and firmer than those in the control soil.

There were also more secondary roots. These roots were white in

color, whereas roots in the Al-3 treatment displayed abnormal root

symptoms; i.e. they were retarded, stubby and brittle. There were

also fewer secondary roots, the root tips became thickened, and some

of them turned brown.

Average root-length densities (cm root length per dm3 soil) are

given in Table 5-3. Root-length density for the Al-1 treatment (9.14

mg Al L-) was significantly higher than that for the control (0.13

mg Al L-). Root-length density for treatment Al-2 (21.59 mg Al L-1)

was lower than that for treatment Al-I but showed a tendency to be

higher than that for the control. Root-length density for the Al-3

treatment (34.60 mg Al L1) was significantly lower than that for the

control. Factors known to affect Al phytotoxicity include tempera-

ture, pH, organic matter, and soil solution concentrations of Al, Ca,

Mg, and P (Rhue and Grogan, 1976). All treatments were in the same

thermal environment, with air temperatures during the 46 d ranging

from 21 to 370C, and averaging 270C. The pH values of Al-amended

soils were lower than that of the control (pH 5.1), being less than

pH 4 (Table 5-2). Since a large portion of the added Al was in

soluble form, the effect of Al should be much larger than that of pH.

Also, the pH values were similar (3.7, 3.5 and 3.4) across the three

Al treatments. Therefore, pH of the Al-treated soils was probably

not the main cause of the significant differences in root densities.

The EC values ranged from 0.30 to 0.99 dS m-I (Table 5-2).
Citrus root growth should not be significantly affected by EC levels
Citrus root growth should not be significantly affected by EC levels
e






87





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