Title: Tolerances of certain citrus seedlings to free water in soil
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Title: Tolerances of certain citrus seedlings to free water in soil
Physical Description: x, 143 leaves : ill. ; 28 cm.
Language: English
Creator: Prevatt, Rubert Waldemar, 1925-
Publication Date: 1959
Copyright Date: 1959
 Subjects
Subject: Citrus fruits -- Florida   ( lcsh )
Seedlings   ( lcsh )
Fruit Crops thesis Ph. D
Dissertations, Academic -- Fruit Crops -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1959.
Bibliography: Includes bibliographical references (leaves 136-143).
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Rubert Waldemar Prevatt.
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Bibliographic ID: UF00098419
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000414610
oclc - 36794804
notis - ACG1790

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TOLERANCES OF CERTAIN CITRUS

SEEDLINGS TO FREE WATER IN SOIL












By
RUBERT W. PREVATT


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












UNIVERSITY OF FLORIDA
June, 1959











ACKNOWLEDGMENTS


The author is sincerely appreciative of the

guidance and for the helpful suggestions and criticisms

of Dr. J. W. Sites, and Dr. H. W. Ford, Citrus Experi-

ment Station, Lake Alfred, Florida, under whose super-

vision this study was conducted. He is also grateful to

Dr. H. J. Teas, Dr. T. W. Stearns, Dr. L. W. Ziegler,

Dr. H. S. Wolfe, and Dr. L. C. Hammond for their advice,

suggestions and criticisms of this work. He wishes to

thank Dr. W. C. Price, Citrus Experiment Station, Lake

Afred, Florida, for his invaluable assistance in taking

the pictures and with the statistical analysis. To

Dr. S. V. Ting, Dr. Roger Patrick, and Mr. R. W. Wolford,

Citrus Experiment Station, Lake Alfred, Florida, go the

sincere thanks of the author. Appreciation is also

expressed to Dr. H. J. Reitz and his staff at tne

University of Florida Citrus Experiment Station, Lake

Alfred, Florida, for facilities used throughout these

studies.

The author is especially indebted to his wife,

Edna, and parents, Mr. and Mrs. W. A. Prevatt, for their

help and encouragement in pursuing his graduate work.


ii
L11/ 6












TABLE OF CONTENTS


Page

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

II. REVIEW OF LITERATURE . . . . . 2

Flood Injury . . . . . . 2
Soil Toxins . . . . . . 9
Detection of Viable Tissues . . . 17

III. TOLERANCES OF CITRUS SEEDLINGS TO
FREE WATER IN LEON FINE SAND . . 25

Methods and Results . . . . . 25
Experiment 1. Flooding citrus
seedlings in a tank of water. . 26
Experiment 2. Citrus seedlings
flooded in January, 1958 . . 28
Experiment 3. Citrus seedlings
flooded in April, June, July
and August, 1958 . . . . 29
Experiment 4. Effect of lime on
citrus seedlings in flooded soil. 41
Experiment 5. Effect of carbon
dioxide . . . . . .. 46
Discussion . . . . . . 56

IV. DEMONSTRATION AND EVALUATION OF TOXINS AS
A FACTOR ASSOCIATED WITH WATER DAMAGE 72

Methods and Results . . . .. 73
Demonstration of the production
of a toxin . . . . . 75
Experiment I. Effect of quantity
of roots . . . . . 74
Experiment II. Effect of parts
and kinds of citrus roots 76
Experiment III. Effect of
temperature . . . . 77
Experiment IV. Effect of pH . 80
Experiment V(a). Effect of
microorganisms . . . . 81
(b) Effect of surface washing. 86


iii






Page


Experiment VI. Effect of stagnant
water from flooded Leon soil 88
Evaluation of a toxin in root
solutions . . . . . . 90
Wilting test . . . . . 90
Preparation of solutions contain-
ing the toxin for evaluation 90
Activated carbon adsorption . 91
Exchange resin adsorption . . 91
Nutrients and pH . . . . 92
Thermostability . . .. 94
Volatility . . . . . .. 95
Distillation . . . . .. 97
Ether extraction . . . .. 103
Acetone and ethanol
precipitates . . . . . 107
Paper chromatography . . 109
Spectroscopy . . . . .. 110
Discussion . . . . . . . 113

V. DETECTION OF VIABILITY OF CITRUS
SEEDLING ROOTS . . . . 119

Methods and Results . . . . 119
Experiment 1. Preliminary testing
of TTC for staining citrus
roots . . . . . . 120
Experiment 2. Determining the
viability of citrus roots using
a TTC solution . . .. 120
Experiment 3. Effect of deoxygen-
ated water on the viability of
roots of sour orange seedlings 124
Experiment 4. Effect of hydrogen
sulfide solution on citrus
seedlings . . . .. . 125
Experiment 5. Effect of acetic
acid on citrus seedlings . 126
Experiment 6. Tetrazolium as a test
for viable tissue in roots of
citrus seedlings that had been
in flooded soils . . . . 126
Discussion . . . . . . . 129

VI. SUMMARY . . . . . . . . 135

VII. LIST OF REFERENCES . . . . . 136












LIST OF TABLES


Table Page

1. Index rating of injury to Rough lemon, sour
orange, sweet orange and Cleopatra
mandarin seedlings when flooded in Leon
subsoil during April, 1958 . . . . 34

2. Index rating of injury to citrus seedlings
when flooded in Leon topsoil and subsoil
during June, July and August, 1958 . . 39

3. Index rating of injury to Rough lemon, sour
orange, sweet orange, Cleopatra mandarin,
Troyer, Carrizo and Rusk seedlings when
flooded in Leon subsoil with and without
dolomitic limestone . . . . . 43

4. Index rating of injury to Rough lemon, sour
orange, sweet orange and Cleopatra
mandarin seedlings in Leon subsoil with
and without added carbon dioxide . ... 53

5. The influence of temperatures at which
citrus roots were incubated for seven
days on the wilting of citrus seedlings 78

6. The influence of temperatures at which
citrus roots were incubated for fourteen
days on the wilting of citrus seedlings 79

7. The influence of pH of the solution in
which citrus roots were incubated on
the wilting of citrus seedlings . . . 82

8. The pH changes of the solutions following
incubation and following the wilting
of the seedlings.. . .. . .. 83

9. The influence of nutrients and pH of the
root solution on the subsequent wilting
effect of the treated solution on
citrus seedlings . . . . . . 93











10. Response of single sweet orange seedlings
in incubated root solutions and the
distilled fractions therefrom . . . 99

11. Response of single sweet orange seedlings
in soil water extracts and in the
distilled fractions therefrom after
seven and fourteen days . . . . 102

12. Response of sweet orange seedlings in the
resulting root solutions from ether
extraction after seven days . . . 108

13. Percentage transmission of distilled
fractions of soil water extracts and
root solutions at 365 mp . . . . 112

14. Specific Extinction Coefficients for flooded
root systems of citrus seedlings treated
with a triphenyltetrazolium chloride
solution and the formazan extracted
with acetone. ...... . . 128


Table


Page













LIST OF FIGUniG


Figure Page

1. Total index rating of injury to citrus
seedlings in Leon topsoil and subsoil
during ten weeks of continual flooding
from April 1 . . . . . ... 32

2. Index rating of injury to Rough lemon, sour
orange, sweet orange and Cleopatra manda-
rin seedlings in Leon soil during ten
weeks of continual flooding from
April 1 .. .. . . . . . 33

3. Total index rating of injury to citrus
seedlings in Leon soil during four weeks
of continual flooding in the months of
June, July and August . . . . . 37

4. Index rating of injury to Rough lemon, sour
orange, sweet orange and Cleopatra manda-
rin seedlings in Leon soil during four weeks
of continual flooding in June, July
and August . . . . . 38

5. Rough lemon seedlings in Leon subsoil with
(right) and without (left) dolomite
following four weeks of continual
flooding . . . . . . 47

6. Index rating of Hough lemon seedlings in
Leon subsoil with ana without dolomite
during six weeks of continual flooding .. 47

7. Sour orange seedlings in Leon subsoil with
(right) and without (left) dolomite
following four weeks of continual
flooding . . . . . . . 48

8. Index rating of injury to sour orange seec-
lings in Leon subsoil with and without
dolomite during six weeKs of continual
flooding . .. . . . . . . 48


vii









9. Sweet orange seedlings in Leon subsoil
with (right) and without (left) dolomite
following four weeks of continual
flooding . . . .... . . 49

10. Index rating of injury to sweet orange
seedlings in Leon subsoil with and with-
out aolomite during six weeks of
continual flooding. . . . . . 49

11. Cleopatra mandarin seedlings in Leon
subqoil with (right) and without (left)
dolomite following four weeks of
continual flooding .. . . . 50

12. Index rating of injury to Cleopatra manda-
rin seedlings in Leon subsoil with and
without dolomite during six weeks of
continual flooding . . . . .. 50

13. Index rating of injury to Husk, Troyer and
Carrizo citran!es in Leon subsoil with
and without dolomite during six weeks
of continual flooding . . . 51

14. Total index rating of injury to citrus
seedlings in Leon subsoil with and
without added carbon dioxide . . .. 55

15. Comparative index ratings of injury to
Hough lemon, sour orange, sweet orange
and Cleopatra mandarin seedlings in
flooded Leon subsoil without added
carbon dioxide . . . . .. 57

16. Typical yellow-veined pattern (left) in
leaves of a sour orange seedling in-
dicating early injury symptoms follow-
ing extended flooding compared with a
healthy green seedling (right) . . 58

17. Typical Rough lemon root systems follow-
ing six weeks of continual flooding
in Leon subsoil with (left) and without
(right) dolomite . .... . .. . 64


viii


Page


Figure











18. Typical sweet orange root systems follow-
ing six weeks of continual flooding in
Leon subcoil with (left) and without
(right) dolomite . . . . . . 65

19. Typical Cleopatra rindarin root systems
following six weeks of continual flood-
ing in Leon subsoil with (left) and
without (right) dolomite . . . .. 66

20. Typical sour orange root systems following
six weeks of continual flooding in Leon
subsoil with (left) and without (right)
dolomite . . . .... . . 67

21. Typical Troyer citrange root systems
following six weeks of continual flood-
ing in Leon subsoil with (left) and
without (right) dolomite . . . . 68

22. Response of sweet orange seedlings after
twenty days in a citrus feeder root
solution (400 grams per gallon), in
distilled fractions, the resuspended
residue and water. Left to right:
resuspended residue, root solution,
fifth fraction, tenth fraction and
water . ... . . . . 100

23. response of sweet orange seedlings in nine
of the ten distilled fractions of a
lateral root solution (400 grams per
gallon) and in water. Left to right:
fractions one through ten (fraction two
is omitted) and water . . . . 101

24. Response of sweet orange seedlings in
distilled fractions and in original soil
water extract from flooded Leon topsoil
in which Rough lemon seedlings had wilted.
Left to right: fourth fraction, tenth
fraction, soil water extract, demonized
water . o . . . . . . 104


Page


Figure








Figure


Page


25. Response of sweet orange seedlings in
distilled fractions and in original soil
water extract from flooded Leon subsoil
in which Rough lemon seedlings had wilt-
ed. Left to right: fourth fraction,
tenth fraction, soil water extract,
demonized water . . . . . . 105

26. Procedure used in the separation of the
toxin from the root solution with ethyl
ethnr . . . . . . . . 106












I. INTRODUCTION


Citrus is being planted on poorly drained flat-

woods soil in Florida because of the increasing demand

for more citrus land. Observations rather than experi-

mental data have been reported on the water tolerance

of citrus plants used as rootstocks. Experimental data

for the tolerance of different rootstocks in relation

to a specific soil type would provide basic information

that should be of value in determining the suitability

of a site for citrus.

This study was made to ascertain under laboratory

and greenhouse conditions the length of time Rough lemon,

sour orange, sweet orange and Cleopatra mandarin seedlings

will tolerate free water in Leon fine sand, a flatwoods

soil type of vast acreage in Florida on which plantings

of citrus are being made.

In addition to the investigations of the water

tolerance of the different citrus seedlings, experiments

were made to test the hypothesis that toxic substances

cause injury to the seedlings when the soil in which the

seedlings are grown is flooded. A study was also made of

the possibility of determining when a root is dead in

advance of injury to the top of the plant.

1











II. REVIEW OF LITERATURE


Flood Injury

Saturating the soil with water causes injury to

or death of many species of plants. The injury or death

of the root system has been attributed to deficient

aeration accompanying flooding. This does not explain

why the shoots are injured rather quickly in some species

of plants and more slowly in others. Also, this does

not account for the type of injury which occurs in all

susceptible plants. The injury of plants in such

saturated soils is usually attributed to desiccation,

caused by decreased water absorption through the injured

roots. Kramer (50) did not consider this an adequate

explanation. Aerial portions of plants have been shown

to live for some days after the root systems were killed

if the soil was kept saturated (47) or if the roots were

placed in fresh water (84). The injury of shoots cannot

be caused entirely by injury to the roots as absorbing

systems, because reduced absorption of water or of

minerals cannot explain all of the symptoms observed in

the shoots of flooded plants (50). Wilting of leaves

is often observed after flooding but this is not the only

or even the most characteristic symptom of injury.

2











Among the conspicuous symptoms of flooding injury

is yellowing and death of the leaves, beginning with the

lower ones and progressing up the stem (2,38,50). This

chlorosis superficially resembles nitrogen deficiency

but often develops within four to six days after flood-

ing, much too soon to be caused by nitrogen deficiency.

The middle leaves of tomato showed epinastic curvature

within twenty-four to forty-eight hours after the soil

was flooded. This epinasty was almost as severe on

tomato plants which were in circulating tap water as

on plants in oxygen-free water (38). Jackson (38) points

out that epinasty is induced by a slight oxygen defi-

ciency and, presumably, by light injury to the roots.

Lumps of callus tissue develop along the stem, partic-

ularly at the water level or in many species at the soil

surface or just below the water surface where the water

level is above the soil surface. Jackson (37) also found

that adventitious roots did not prevent injury to shoots

of Marglobe tomato plants when the original roots were

flooded, but leaf epinasty was less and shoot growth was

greater than the flooded plants without adventitious roots.

Except for aquatically adapted species, plants

which are growing in soil saturated with water soon

have injured root systems which cause the leaves to

yellow, reduce growth and eventually die (50). This









injury of the root systems has been attributed to the

lack of oxygen and possibly to the accumulation of

carbon dioxide, rather than to the direct effects of

water. The reason most often given to support this

gaseous concept of injury is that most species of

plants make satisfactory growth in well-aerated water

cultures.

Attempts (9,28) have been made to measure the

oxygen and carbon dioxide content in the gaseous phase

of the soil while saturated with water and as the soil

drained. Scott and Evans (80) point out that a measure-

ment of the dissolved gases in the liquid phase of the

soil should be of considerable value in characterizing

the aeration of a soil. Furr (27) concluded there was

no relation of the root rot of citrus and avocado to

low oxygen or high carbon dioxide under field conditions.

Respiration of soil organisms and of roots continually

depletes the oxygen and adds to the carbon dioxide of

the soil atmosphere and of the water film in equilibrium

with it. The activity of soil organisms varies with

temperature, moisture, and supply of organic matter that

they can use as food.

Karsten (40) points out that if the oxygen content

of the soil is reduced to such an extent that the rate of

basal respiration is radically lowered, the roots of the











plant will die and death of the tops will ensue. If

the reduction is net sufficient to cause death it is

sufficient to impair the growth of the roots, and this

condition will be reflected in reduced growth and re-

duced productivity of the aerial portion of the plants.

Floyd (24) made no gaseous measurements in his experi-

mentbut the root system of the citrus plants was

impaired by the water level in the soil and the tops of

the citrus trees reflected this injury. Conway (17)

points out that indirect effects of an oxygen deficit

are important to keep in mind when investigating the

supply of oxygen to aquatics, besides the measurements

of oxygen in the soil atmosphere or in solution. Hedox

potentials may be of importance in root respiration

apart from their use as indicating oxygen concentrations.

Black (4) proposed four different hypotheses that might

account for the failure in measuring the composition of

soil air which reflects the apparent aeration condition

of tne soil: (1) technique used; (2) the variation between

the oxygen and carbon dioxide concentration near the roots

and that in the bulk sample; (3) the difference in the

diffusion of dissolved gases through the surrounding water

film; (4) when the oxygen concentration in the soil air

decreases there may be an associated increase in certain

unfavorable effects that do not occur in solution culture










experiments. The rate of transpiration, the oxygen

supply and the temperature are closely related to the

injury of plants. Hcinicke (34) found that flooding

the soil containing apple roots during the winter caused

considerable loss of small roots but produced no serious

injury if the soil was drained before leaves began to

appear. Flooding in the summer soon caused injury,

particularly if the transpiration was rapid. Kramer (48)

found that when the oxygen content was lowered and the

carbon dioxide increased by the addition of it there

was a marked reduction in the transpiration of the tomato

plants. However, when the oxygen was removed with oxygen-

free nitrogen there was only a small decrease in the water

intake. The nitrogen gas would remove not only the oxygen

surrounding the roots, but also the carbon dioxide which

otherwise might accumulate and retard respiration and

active absorption. Kramer also pointed out that if a high

concentration of carbon dioxide or low concentration of

oxygen was maintained for many hours or days, other factors

became important. Root growth was usually stopped (12,16,

31,57) and many or all of the roots might be killed, result-

ing in a greatly decreased absorbing surface. There is

also the possibility of injury to the shoot by toxic sub-

stances escaping from the dead cells, and plugging of the

water conducting elements may occur. One or more of these










factors may cause the death of plants with roots in

poorly aerated soils. The specific effects of carbon

dioxide on permeability of the roots and hence on water

intake are probably most important during the early part

of a period of poor aeration such as occurs in waterlogged

soils. Both Chang and Loomis (13) and Kramer (48) con-

cluded that the effect of carbon dioxide seemed to be

on the water-absorption mechanism rather than on transpi-

ration. Another effect of carbon dioxide was on the

protoplasm and caused an increase in viscosity and a

decrease in permeability (25,81).

The theory that oxygen made available by nitrate

reduction would be beneficial to plants under waterlogged

conditions was investigated by Bain and Chapman (2).

heavy applications of nitrates aggravated the waterlogged

injury tc avocados and grapefruit plants. All of the

waterlogged grapefruit plants began to develop vein-

chlorotic leaves after about ten days and the severity

increased with time. This condition developed when

serious root rotting occurred. There was no significant

difference between nitrate-treated and non-nitrate-treated

plants. Klotz and Sokoloff (44) reported that flooded

sour orange and sweet orange seedlings which had received

nitrate only and those which had no organic matter or

nitrate added were not wilted after four weeks of flooding;











whereas those seedlings that were given organic matter

ana nitrate nitrogen were in a state of collapse. They

showed that initial injury to the roots could occur

several months prior to the appearance of collapse.

The toxic substance, nitrite, may disappear from the

root zone long before injury to roots or to the leaves

may become visible. In water cultures the toxic nitrite

ion very rapidly injures the roots, initially increases

and later decreases their respiration, makes them more

permeable, and permits exosmosis of materials from them.

In order for nitrites to accumulate in soil it has been

proposed (14,8) that certain conditions must be met:

the pH of soil must be above 7.0 for nitrate-reducing

bacteria, a high proportion of organic matter must be

available for the reduction of nitrate, the soil must

be well aerated, and the soil temperature must be

favorable. Chapman and Liebig (14) concluded that the

reduction reaction was more likely to take place in

deeper layers, where there was a static water table or

impeded drainage which led to saturated and anaerobic

conditions, than in surface layers. Zentmeyer and Bingham

(95) could not demonstrate that the nitrite-nitrogen

accelerated root rot by Phytophthora nor that root injury

caused by nitrite increased the rapidity or severity of

root attack by the fungus. fne growth of sweet orange










seedlings was retarded because of the amount of root rot

caused by Fhytophthora sap. (45). Environmental factors

favoring the parasitism of the fungi are excess water

and organic matter in the soil.


Soil Toxins

Soil toxins are probably related to deficient

aeration and to anaerobic conditions (79). Clements

(16) stated that this could be shown by the fact that

they were readily oxidized and soon disappeared under

proper tillage. The toxins appeared to be due to

essentially the same conditions and processes as

obtained in bogs. Livingston (56) and Dachnowski (19)

showed that bog water contained toxic substances and

that these substances were stable to boiling for ten

minutes (56) but could be removed by shaking with carbon

black or calcium carbonate (19). The toxicity of these

waters is not due to acidity nor to lack of oxygen (19).

In contrast Clements (16) states that the primary causes

of toxicity are the direct lack of oxygen and its in-

direct effect in permitting the accumulation of carbon

dioxide in harmful amounts which stimulate the production

of injurious organic acids and other compounds. In many

cases probably the first two alone are concerned, but in

sour soils and muck soils all of them must have a part

though the lack of oxygen plays the primary role.










Clements (16) concluded that organic toxins are excreted

by roots or produced in soils only as a consequence

of the anaerobic respiration of plant roots and of

microorganisms, and that the inorganic toxins may arise

as a result of chemical processes of adsorption. Russell

(79) concluded that toxins may occur on sour soils poorly

aerated and lacking in calcium carbonate, or in other

exhausted soils, whereas there is no evidence of soluble

toxins in normally aerated soils sufficiently supplied

with mineral nutrients and with calcium carbonate.

Finely divided material has a marked inhibitory

action on the toxicity of many solutions (88). The

beneficial effect has generally been ascribed to the

physical phenomenon of adsorption. In soils there are

large surface exposures and adsorption may play a large

part in inhibiting the action of plant toxins. The

great complexity of soil constituents suggests the

possibility that plant toxins may combine chemically

with certain soil constituents and thus be removed at

least partly from the soil solution, resulting in a

greatly lessened toxic action to plants. Chemical re-

actions are probably important in lessening the harmful

effects of plant toxins in soils. For example, calcium

carbonate inhibits the toxicity of copper salts and kaolin

or an acid clay soil inhibits the toxic effects of the










strong base guanidlne. The latter inhibition is

attributed to the reaction of the acid nature and

base. Undoubtedly the chemical reactions play fully

as important a role as physical phenomena such as

adsorption and possibly the former have the greater

effect.

Inorganic constituents can be toxic to plants.

Robinson (75) gives evidence that submerged soils con-

tain abnormally high concentrations of manganous and

ferrous ions which render the soil solution toxic to

most species. The substances are kept in solution as

bicarbonates because of the high concentrations of

carbon dioxide which result from the submergence of

soils, and the presence of ferrous ions is a symptom

of highly reducing conditions. Sulfides are produced

in submerged soils and are very poisonous to plants,

even in low concentrations.

In studies on the biochemistry of waterlogged

soils (85) there was a distinct increase in the free

and saline ammonia content and this was present mostly

in the soil sediment. There was no release of any

soluble reducing matter capable of absorbing dissolved

oxygen nor was there any appreciable production of

carbon dioxide. The fluctuations of dissolved oxygen

in the waterlogged soil were attributed to variations










in external conditions with time and not to any soil

factor. Waterlogging resulted in an increase in

alkalinity which was associated with the corresponding

increase in ammonia. Robinson (75) did not find that

submergence affected the pH values very markedly; if

anything, the pH fell slightly, which he attributed to

the higher carbon dioxide concentration.

Substances have been extracted from cultivated

soils which have proven to be growth inhibitors for

succeeding crops of the same plants. It was found that

toxic substances from the culture media of guayule

inhibited growth of guayule under certain limited

environmental conditions (7). These toxic agents,

although they were not isolated in pure form, were

characterized chemically as being ether-soluble acidic

compounds. Cinnamic acid was one of the toxic agents

isolated from water in which guayule roots had been

briefly allowed to steep and is a normal constituent of

the guayule plant. There was a considerable reduction

in growth in both height and dry weight of guayule

plants when one grarm of cinnamic acid had been added per

pot filled with 1500 grams of Hanford sandy loam (8).

When leaves of Encelia farinosa were applied to tomato and

other plants in sand cultures a striking growth inhibition

occurred (33). Water and ether extract of the Encelia










leaves when supplied to tomato seedlings in solution

culture may cause death of the plants within one day.

Weekly watering of fresh sand cultures of orange

seedlings with the leachate from old sand cultures re-

duced growth by approximately one-third (60). When this

sand previously cropped to citrus was leached with

sulfuric acid followed by distilled water it produced

growth comparable to that in the fresh sand. After sour

or sweet orange seedlings had been grown in a medium

quartz sand for eighteen months in the greenhouse the

growth of a second crop of sweet orange seedlings in

this sand was greatly retarded. In addition to detri-

mental organisms an organic toxic material apparently

builds up gradually in soils cropped to citrus plants

and is not readily leached from a normal soil by water

but may be partly removed from a very sandy soil by

leaching. This hypothetical toxic material could

originate by slow excretion from the citrus roots or

could be produced by microorganisms growing on root

surfaces or dead root material. Because of the gradual

build up and persistence in citrus soils as reflected

by the reduced plant growth, toxic material was probably

resistant to decay by soil organisms. The acid leach-

ing suggests that it was either soluble in or destroyed

by sufficiently strong acid. This toxin was found to be










specific to citrus in its toxic effect and not injurious

to tomatoes or avocados. Wander (89) found that a methyl

alcohol extract of grove soil when placed on virgin soil

after the alcohol was removed caused the growth of grape-

fruit seedlings to be depressed as compared to seedlings

grown in untreated soil. Ignition of the soil treated

with methyl alcohol extract destroyed most of the

inhibiting effect of the substance as reflected by the

resultant growth of seedlings in the soil after ignition.

A toxic material was extracted from the roots and leaves

of diseased citrus trees that caused wilting of citrus

and tomato cuttings in twenty-four hours after they

were placed in the solution (86). This solution was

made by covering 200 grams of wood from trees showing

decline with water in a beaker and allowing it to steep

for twenty-four hours at 450 F.

When peach roots were added to virgin soil the

growth of peach seedlings was inhibited (70). In sand

cultures the bark, but not the wood, of the roots was

found to be toxic. The alcohol extract of bark also was

toxic to peach seedlings. The injury was more severe to

the root systems of the peach than to the tops when root

bark was added. When leaching of the soil was slow, as

when peat moss was present, the injury was greatest.

Microbial decomposition of peach root residues produced a










toxic substance which is believed to be a factor

involved in the difficult re-establishment of peach

trees in old peach orchards (67).

Dried roots of brome grass were inhibitory to the

growth of the same species (3). Benedict (3) suggested

that the thinning out of brome grass stands may be the

result of the accumulation of a toxic substance from

the roots. It was also suggested that its living roots

may also excrete substances toxic to the plant.

Rovira (76) showed that the roots of pea and oat

plants when grown under aseptic conditions excreted

arino acids, fructose, glucose and compounds which

absorb and fluoresce ultraviolet. Katznelson et al.

(43) were able to recover significant amounts of amino-

nitrogen from the leachates of sand which had been dried

until the wheat plants had begun to wilt and then the

sand remoistened. Also detectable reducing compounds

were liberated in these dried and remoistened pots.

More total amino-nitrogen was found in the leachate from

pots with tomato, soybean, barley and oats which were

allowed to wilt then reiioistened and leached than was

found in leached water from the pots which were kept

wet (42). They believe that in field soil, subjected

to frequent drying and moistening, this phenomenon also

occurs, thus providing the rhizosphere microflora with










a food supply and especially with amino-nitrogen.

Anaerobic bacteria in general were consistently stimu-

lated in the rhizosphere of plants in both fertilized

and unfertilized soil and were always present in greater

numbers on the roots in the latter (41). The treat-

ment of soil with the solution of root exudate from pea

and oat plants resulted in increased numbers of gram-

negative bacteria (78). Fungal counts in the treated

soil showed no stimulation by the root exudate,

indicating an action similar to that in the root

environment in which bacteria are stimulated to a

greater extent than fungi.

Many researchers have worked with toxins which

are related to wilting in plants. Some have been con-

cerned with the physiology of toxin formation in micro-

organisms (23,56), the production of toxic material by

specific organisms (10,15,21,29,35,69,87), defense

reaction of plants to the presence of toxins (5), and

with the basis for toxic wilting (29). Gadmann (29)

points out the difference between physiological wilting

and toxic wilting. The former is caused by a lack of

water, therefore reversible. The toxic wilting is

caused by a colloid-chemical disturbance of the osmotic

mechanism through the destruction of the osmotic pre-

requisites for turgor and because of this, toxic wilting










is irreversible. Wilting plants removed from a toxin

solution to water or a nutrient solution continue to

wilt and do not recover. The toxin exerts a coagulating

effect on the plasma which leads to two pathological

phenomena: (1) damage to the water-retaining capacity

of the plasma resulting in pathological water loss;

(2) damage to the semi-permeability of the plasma

membrane which leads to a loss of turgor.

The injury, whether it is chlorosis or wilting,

and death of leaves may be caused at least in part by

toxic substances moving up from the dead roots or even

from the solution in the surrounding soil (51). It

is still unknown whether the injury is physiological

or pathological or a combination. Many factors enter

into the injury of shoots of flooded plants which make

it complex in origin.


Detection of Viable Tissues

Reasons believed to cause plants to become injured

in waterlogged and poorly aerated soils have been reviewed.

The next question to be encountered is whether the injured

tissues of the root system can be detected before visible

symptoms appear on the top of the plant. Roots in ad-

vanced stages of injury are soft and spongy, and the

cortical portion slips rather easily.










McPherson (65) immersed thick longitudinal

sections of roots for one hour in various concentrations

of different chemicals, then removed and tested them

for the presence or absence of living cortical cells.

Of the three vital stains tried-- congo red, methylene

blue and neutral red-- the neutral red was the most

satisfactory. The dead cells in the epidermis of Allium

cepa took an intense orange color while the living cells

became a cerise red color. In the cortical cells of

corn roots, the living cells took on a bright red hue

while the dead cells were colorless. The three criteria

used to distinguish the living from the dead cells were

vital staining, streaming and plasmolysis. The living

cells stained, and in many cases streaming could be seen

in the cytoplasm and plasmolysis took place readily when

they were placed in a strong sucrose solution.

It was shown by Kuhn and Jerchel (52) that dilute

solutions of 5-methyl and 5-hendecyl 2,3 diphenyl salts,

as well as the 2,3,5-triphenyltetrazolium chloride, were

capable of staining certain living cells such as bacteria,

yeasts and garden cress. Such staining was brought about

by a physiological reduction of the colorless triphenyl-

tetrazolium salt to form the highly colored and insoluble

triphenyl formazan.










Tetrazolium differs from the majority of physio-

logical indicators (52) since, in the reduced state,

it forms an insoluble formazan and the reaction is

therefore non-reversible. This is advantageous in

plant tissues. It is easily visible in minute quanti-

ties and the reaction is very sensitive. It ,wa also

possible to test the penetration of the indicator in

plant tissues by reducing the tetrazolium with sodium

hydrosulfite. Tetrazolium readily penetrates the

majority of plant tissues and it is not absorbed. The

formazan is insoluble and is neither diffused from the

cell in which it was formed nor oxidized back to a

colorless state on standing.

Tetrazolium has been used to predict the

germinability of seeds (54). The seeds which germi-

nated after soaking in a dilute solution of tetrazolium

for several hours had deep red embryos.

iaugh (90) used a 1 per cent aqueous solution

of tetrazolium to test the difference in response of

tips of twigs, both heated in a test tube suspended in a

boiling bath for fifteen minutes and unheated. All of

the unheated sections which responded to the tetrazolium

treatment developed a red coloration in the cambium layer.

The heated sections of all varieties tested exhibited no

coloration. The formazan appeared first in the cambium











but it took about four hours for the development of

the red color. Exceptions to the length of time for

the development of the red color were willow sections

and rose cuttings. The cambium of the willow sections

stained within 1-2 minutes, followed by slow development

of color throughout the phloem. Rose cuttings required

nearly twenty-four hours for the tetrazolium reduction.

Roberts (73) conducted a survey of tissues which

reduce tetrazolium in vascular plants. Observations

were made on the reduction zones in stem tissues of

vascular plants. Reduction zones in root tissue were

also observed. Actively growing root tips of all the

plants surveyed showed some degree of reducing activity.

Inner and outer cortical reduction regions were observed

in meristematic root tip tissue. Plants possessing a

tetrarch root system reduced tetrazolium in a tetra-

polar pattern which corresponds to the pattern of

secondary root formation.

Dufrenoy and Pratt (22) immersed freshly cut basal

surfaces of culms of sugar cane in a 0.5 per cent aqueous

solution of tetrazolium. The uncolored salt was rapidly

transported to the upper node and reduced there to the

insoluble red formazan. Microscopic examination of

longitudinal freehand sections showed that the precipi-

tates of formazan were localized at the sites of the










plasmodesmata and in lipidic parts of the cytoplasm.

Lakon (54) states that the staining of embryos

must be completed within twenty-four hours as micro-

organisms will appear and obscure the reaction.

Bacteria, stained by tetrazolium, may stimulate stain-

ing of cereal embryos or the cut surface of maize

kernels. In this case the tetrazolium solution itself

is stained red.

Apparently tetrazolium is reduced by several

reducing substances. Jensen et al. (39) found that

several dehydrogenase enzyme systems, prepared from

corn embryos, in the presence of diphosphopyridine

nucleotide were able to reduce tetrazolium. The

presence of succinic dehydrogenase in tissue homogenates

reduced the tetrazolium (53). This formazan was easily

dissolved in acetone for colorimetric measurement.

Reducing sugars in an alkaline medium are capable of

reducing tetrazolium and the quantity of formazan is

proportional to the quantity of reducing sugar present

(62). The tetrazolium solution is reduced immediately

on contact with a reductase system, hence the time of

staining depends on the rate of diffusion of the solution

(18). The reduction of tetrazolium could be inhibited

by many compounds (26). From the lack of specificity

for inhibitors it would seem that a number of reducing











enzymes acting on material inside the cell can reduce

the dye. Aeration by shaking retarded the reduction,

possibly because it raised the redox potential too

high (over -0.08 volt) or because oxygen competed with

the indicator. Tetrazolium can be reduced to the red

formazan by ultraviolet light, alpha rays and x-rays

(30). The formazan in visible light ( 480 ap) turns

from red to yellow. If the solution was subsequently

placed in the dark, the formazan turned from yellow to

red. This reversible conversion was caused by cie-trans

isomerism. Roberts (74) and Brown (11) concur that the

marked sensitivity of the reduction reaction to both

temperature and light may well be due to an effect of

the active sulfhydryl groups of the reducing enzymes.

Brown (11) found that split root tips or embryos

exposed one-half to one hour in a 0.5-1.0 per cent

aqueous solution of tetrazolium at room temperature

and bright diffuse laboratory light were well stained.

The root tips must be cut to stain quickly. The

epidermis and outer layers of the cortex apparently

prevent the rapid penetration of tetrazolium. The

cells of these layers do not stain well even when the

tip is split.

It was noted (11) in higher plants that











tetrazolium reduction occurred most obviously and

quickly in meristematic cells. In the time required

to stain the root tips older cells of the root were

not stained at all. This could be due in part to

the higher concentration of reducing enzyme in

mitotic cells but certainly older cells must also

contain these enzymes. Much longer treatment does

produce staining of older cells, but no study was

made of these.

Higher temperatures and higher light intensities

gave quicker and more intense staining of root tips.

Bright light (direct sunlight) and higher temperatures

(37-400 C.) affect the reduction strongly so that the

material stains almost immediately. Daylight from

blue sky was superior to laboratory light. Root tips

heated at 600 C. did not stain at all. When the pH

of a tetrazolium solution was increased from 5 to 8

or 9 with potassium hydroxide the reaction was very

quick and comparable to the reaction in direct sun-

light.

Brown (11) stated that it is doubtful that

tetrazolium reduction should be considered a specific

test for reducing enzymes. Mattson et al. (61) con-

cluded that in all probability the reduction of tetra-

zolium compounds by enzymes of living cells cannot be










considered a general test for life. Nevertheless,

the unusual properties of these reagents suggest that

they might be utilized in many types of biological

research involving differences in tissue viability.












III. TOLERANCES OF CITRUS SEEDLINGS TO

FREE WATER IN LEON FINE SAND


Nc research data are available on the behavior

of citrus seedlings in flooded soil of any specific

type. Because of tnis and the present interest in the

planting of citrus on soils which are classified as

poorly drained, experiments were conducted to study

the response of citrus seedlings, used as rootstocks

in Florida, to standing free water in soil. Citrus

seedlings were flooded during different months of the

year in the greenhouse. Leon fine sand was the soil

type used because it is the predominant flatwoods

soil type available for citrus.
Methods and Results

The soil used in this study came from a native

pasture covered with palmetto, gallberry and wire grass

in Polk County, Florida, and was Leon fine sand, which

is a somewhat poorly drained soil with a hardpan. The

description of the soil has been given elsewhere (56).

The profile was arbitrarily divided into two horizons,

topsoil 0-8" and subsoil 8-17" (leached layer on top of

the hardpan), both of which were used for growing the

transplanted seedlings in metal cans prior to flooding.
26









The kinds of citrus used in the flooding

experiments were: Rough lemon (RL), Citrus limon;

(variety unknown) sour orange (SO), C. aurantium;

Pineapple sweet orange (S5O), C. sinends; Cleopatra

mandarin (Cleo), C. reticulata; Troyer citrange (Troyer)

Poncirus trifoliata x C. alnerasii; husk citrange (Rusk)

F. trifoliata x C. sinenols; and Carrizo citrange

(Larrizo), P. trifoliata x C. sinensis. The seeds were

germinated in a sana-peat medium in flats in the green-

house in 1957 and 1956 at the University of Floriaa

Citrus Lxperiment Station. The seealings were fertilized

with a complete nutrient solution every month ana watered

once a week.

Experiment 1.- Flooding citrus seedlings in a tank of

water.

Twenty5one-yexr-old sweet orange seedlings and 8

one-year-old Rough lemon seedlings were transplanted

into 46-ounce metal cans which were filled with Leon

topsoil in July, 1957. Holes were punched in the cans at

the bottom to facilitate drainage. Two months after these

seedlings were transplanted they were placed in a galva-

nized tank in the greenhouse and the tank was filled with

water so that the water level was above the top of each

can. During tne flooding period the temperature of the

water fluctuated between 75o i'. and 950 F.










After 3 weeks in the flood tank only 1 sweet

orange seedling was wilting. All of the seedlings

were then removed and the cans with the sweet orange

seedlings were divided into 3 groups. One group of

6 cans was placed in the greenhouse, the second group

of 6 cans was placed in shade outside the greenhouse,

and the third group of 5 cans was placed in the open

sunlight outside the greenhouse. The other 3 seedlings

had been removed for observation. Six cans with Rough

lemon seedlings were left in the greenhouse after they

were removed from the flood tank. No water was added

to the cans for 3 weeks at which time only the seedlings

in the open sunlight had wilted. The soil in these cans

was dry. There were new growth and new roots on all of

the non-wilted seedlings.

Six one-year-old sweet orange seedlings in Leon

topsoil in 46-ounce metal cans were individually placed

in steel containers in a tank of water which was thermo-

statically controlled at 720 F. The cans and containers

were filled with water and the surface of the soil was

covered with aluminum foil. The cans containing the

seedlings had drain holes in the bottom, necessitating

a second container which was surrounded by the water in

the tank. For 6 weeks during October and November the

seedlings remained green and turgid.









Experiment 2.- Citrus seedlings flooded in January, 1958.

Forty,46-ounce metal cans were filled with topsoil

(pH 4.5). These cans were divided into 4 groups of 10

each and 4 seedlings of 1 variety were transplanted

into each of the 10 cans. The 4 varieties transplanted

were Rough lemon, sour orange, sweet orange and Cleopatra

mandarin; they remained in the greenhouse. Nine of the

10 cans of each variety were flooded in January, 2 months

after the seedlings had been transplanted. The remaining

cans were watered once a week as a control. There were

no drain holes in the cans, therefore the water in the

cans became stagnant, but fresh water was added when

needed to maintain the water level above the soil

surface. Four flooded cans, 1 each with Rough lemon,

sour orange, sweet orange and Cleopatra mandarin seed-

lings, were emptied and the root systems of all the

seedlings were examined after 2, 4, 6, 9, 14, 21 and 39

days of flooding. The other flooded cans, 2 of each

variety, remained flooded for 1 year.

None of the seedlings, regardless of the length

of time in the flooded soil, showed any wilting symptoms.

There were no sloughed roots on any of the varieties

after 14 days of flooding. Less than one-tenth of the

root tips of each seedling were sloughed after 21 days










of flooding. The lower half of the root system and

approximately 100 per cent of the root tips on all

of the seedlings were rotted after 39 days of flood-

ing. There were 1 or 2 new roots on each of the

Rough lemon, sour orange and Cleopatra mandarin

seedlings which had been flooded for 59 days. These

new roots were from the tap root near the base of

the stem just below the soil surface. The yellowing

of the leaves was slightly more pronounced on the

flooded seedlings after 1 year than on the non-flooded

seedlings. There were well-distributed new root

systems on all of the seedlings that were flooded for

1 year. None of the original feeder roots was present,

but about 2 inches of the tap root did remain and it

was from this portion that the new roots originated.

Experiment 3.- Citrus seedlings flooded in April, June,

July and August, 1958.

Thirty 46-ounce metal cans were filled with top-

soil and 30 cans were filled with subsoil. Four seed-

lings, 1 each of Rough lemon, sour orange, Cleopatra

mandarin and sweet orange, were transplanted into each

can, and watered with 100 ml of water once or twice

weekly as needed. Ten of the cans with the topsoil and

seedlings and 10 cans with the subsoil and seedlings









were flooded on April 4, five months after the seedlings

were transplanted.

Also, on each of the following dates, June 1,

July 1, and August 7, six of the cans with topsoil and

6 with subsoil were flooded. The water remained

standing in each can following its flooding for the

duration of the flooding periods. Additional water was

added when needed to keep the water level above the

soil surface.

The first visible symptom of injury on any of

the seedlings that were flooded in April occurred 5

weeks later. A numerical rating was given to the

various symptoms observed during this and the other

periods of prolonged flooding:

0 Leaves green and turgid.

1 Leaves yellow-veined and turgid.

2 Leaves wilted (either drooped or rolled).

3 Leaves defoliated or desiccated.

In order to compare the tolerance of the different

citrus seedlings to standing free water, the weekly index

rating for each variety in each soil was calculated by

multiplying the number of seedlings in each category by

their respective numerical rating (numbers from 0 to 3),

adding the products, and then dividing the summed

products by the total number of seedlings. The results









for the seedlings flooded in April are presented in

Figures 1 and 2 and Table 1.

Analysis of variance showed that there was a

highly significant difference between varieties, weeks

and soils (subsoil and topsoil). There was no signifi-

cance between the interactions of any of these components.

The seedlings in the flooded subsoil were injured to a

significantly higher degree than those in the flooded

topsoil (Figure 1). The Rough lemon seedlings were more

tolerant of the water than the sour orange, sweet orange

or Cleopatra mandarin seedlings (Figure 2). The sweet

orange and Cleopatra mandarin seedlings were more

tolerant of the free water than the sour orange. There

was no significant difference between the sweet orange

and Cleopatra mandarin seedlings in their tolerance of

the free water. Therefore, the order of tolerance of

the citrus seedlings to free water in Leon soil when

flooded in April in cans was Rough lemon, sweet orange,

Cleopatra mandarin and sour orange. As was expected,

there was a highly significant difference between the

weeks (5-10) although there was no difference in the

appearance of the seedlings until 5 weeks after flooding.

The citrus seedlings which were flooded in June, July

and August showed water injury symptoms within 2 weeks

after the cans were flooded. The weekly index ratings

















10
9-
e 8- SUBSOIL
7-

S6-
I 5-
4-
M ,TOPSOIL
0
0
E 1-



WEEKS




Figure 1.- Total index rating of injury to citrus
seedlings in Leon topsoil and subsoil during ten weeks
of continual flooding from April 1.
















TOPSOIL


4


SI I I I
5 6 7 8 9 10


SUBSOIL
SO


4 5
4 5


I
10


7
WEEK S


Figure 2.- Index rating of injury to Rough lemon,
sour orange, sweet orange and Cleopatra mandarin seed-
lings in Leon soil during ten weeks of continual flood-
ing from April 1.


1-1


B















r* 0 r-

Hf HHc1-


o 0 0 0 C\ 0




0C to 0 I0

r..


0 C -4' ~0
i \ O;


O'd



0)0
0


hOC
$4 =








0 r
o d
4 r-t







o
Q) 0











HO


a

Sa)


0 r
0 (D









C
0 r-4 ^








r-
0










r-





C C


0 CO 0 CO O
HS0C0H CO



0 0 0 0 0







0000 CO


CQ r.O O-O
000 0
HQ.C~HH


0 0 C0 0 0-

OHOO C'


Or-1 O OC
S0d t'0 C\2
. * *



V ) 0 \0 0
O8 r; 8


0
o
5. F i


* 0

ca C



Q 0
00









44
cs





0


H -
00
01 03

0 3

I I
E-4 to


CO0 0
* 0 0
c c0


0
1-1


0 C
a)


4.,
0
4-'
05
>





















1C. r-I to r a- V) tO -4
o0 0'0 0)








o% to wE0- loe)
a--


H 0
C- Va a) 00 0




a a a a a a








0l f ,-I 000










o i a) co co ,n H
1 -H x


(0o0 00 0



m4 *
0 He a
o








0 00
1a a a a a a3 a




&< ctf
H



00
'M O~O~r-K tO-X
0..) OH -





oa -l o s00 2










for the flooded citrus seedlings are presented in

Figures 3 and 4 and in Table 2.

Analysis of variance showed that there was a

highly significant difference between varieties in the

over-all flooding of the soils. By the t-test the Rough

lemon seedlings were significantly more tolerant of the

free water than the sweet orange seedlings; the Cleopatra

mandarin seedlings were significantly more tolerant of

free water than the sour orange and sweet orange seed-

lings. There was no difference between varieties in the

subsoil. However, both the Rough lemon and Cleopatra

mandarin seedlings were significantly more tolerant of

the flooded topsoil than the sour orange or sweet orange

seedlings, with no significant difference between the

Rough lemon and Cleopatra mandarin seedlings.

There was a highly significant difference in the

water tolerance of the citrus seedlings in the topsoil

and in the subsoil, between the weeks (1-4) and between

the months (June, July and August). This is illustrated

in Figure 3. Other highly significant differences were

between the interactions of varieties and soils, weeks

and soils, and weeks and months. After 2 weeks of

flooding the seedlings in the subsoil were much more

severely injured than those in the topsoil, which is

shown by the highly significant difference of the
















S8-
8-- AUGUST
E-

6-
~ X

S4- JUNE
JULY
2


0 T
1 2 3 4

12-
SUBSOIL

10-

a //AUGUST


a-
6- JUNE

I JULY
4-


2-


0I I I
1 2 3 4
WEEKS

Figure 3.- Total index rating of injury to citrus
seedlings in Leon soil during four weeks of continual
flooding in the months of June, July and August.













10 --
TOPSOIL

8--








21


0.



10-
SUBSOIL

8-
g6- w^ -









Cleo





2-



1 2 3 4

WEEKS


Figure 4.- Index rating of injury to Rough lemon,
sour orange, sweet orange and Cleopatra mandarin seedlings
in Leon soil during four weeks of continual flooding in
June, July and August.












TABLE 2.- Index rating of injury to citrus seedlings when flooded
in Leon topsoil ana subsoil during June, July and
August, 1958.


Variety: weekss

1 2 3 4


: onths

:June July Aug. June July Aug. ,ure July ug. June July


Soil*

:T 3 T 3 T S T S T T ST T T S T S T



RL 0 0 0.66 0.66 0 0 0 0.63 0.50 1.83 0 1.. 1.00 1.83 0.86 3.00 1..3 3.0 1.00 2.50 1.3 3.00 i.y

30 0 0 .50 0.66 0 0 0.53 0.66 1.66 .00 1.16 1.16 0. 1.16 1.83 3.00 1.83 2. 8 1.16 .. 2.3 3.00 2.3

Sw0 0 0 0.83 .16 0 0 0.16 0.50 1.83 2.00 0.83 1.00 1.83 1.50 2.00 2.66 l.t 2.33 1.83 .66 .16 3.00 2.3

Cleo 0 0 0 0.16 0 0 0 0.50 0.50 ...13 0.33 1.83 '.50 ..' u 1.00 2.33 1.. 2.50 .b0 .00 1.50 6.00 1.8


0 0 0.49 1.99 4.49 7.98 9.' .9.FA 4-.1 6-4Q 5.9 1fif .Qq


Total 0 0 1.66 1.61


4.4P P.fi 7.. 12.nn00










TABLE 2-Continued


Analysis of Variance


Source of d.f. S.S. M.S. F. Required F
Variation .05 .01


Total 95 103.53
Variety (V) 3 1.59 0.53 10.16 3.16 5.09
Weeks (W) 3 63.40 21.14 404.86 3.16 5.09
Soil (3) 1 12.15 12.15 232.72 4.41 8.28
Months (M) 2 9.74 4.87 93.27 3.55 6.01
V x W 9 0.73 0.08 1.56 2.46 3.60
V x S 3 2.86 0.95 18.25 3.16 5.09
V x M 6 0.76 0.13 2.44 2.66 4.01
W x S 3 5.05 1.68 32.23 3.16 5.09
W x M 6 2.89 0.48 9.21 2.66 4.01
S x M 2 0.28 0.14 2.74 3.55 6.01
V x W x S 9 0.62 0.07 1.33 2.46 3.60
V x W x M 18 1.27 0.07 1.35 2.19 3.07
W x x M 6 0.70 0.12 2.23 2.66 4.01
V x S x M 6 0.55 0.09 1.75 2.66 4.01
V x W x M x S 18 0.94 0.05










interaction between the soils and weeks. This is

graphically shown in figure 4. After 2 weeks of

flooding the seedlings flooded in July had a higher

index rating than those flooded for the same period

of time in June or August. At the end of 3 and 4

weeks of flooding the index rating for the seedlings

flooded in July and August were approximately the

same and higher than the index rating for the seed-

lings flooded in June for the same length of time.

This offers an explanation for the highly significant

difference of the interaction between weeks and months.

The average monthly maximum and minimum temper-

atures recorded at the United States Weather Climatological

Station 4707, Lake Alfred, Florida, for the months in

which the seedlings were flooded are as follows:

(maximum, minimum respectively) January 65,44; February

65,41; March 74,54; April 82,60; iay 85,65; June

91,71; July 92,73; August 92,73; September 92,72.

Experiment 4.- Effect of lime on citrus seedlings in

flooded soil.

Seven-month-old seedlings of Rough lemon, sour

orange, sweet orange, Cleopatra mandarin and Troyer citrange

were transplanted into 46-ounce metal cans which were

filled with Leon subsoil. The subsoil was used because of

the results from the April flooding. There was 1 seedling










per can and 20 cans of each variety in the virgin soil.

Also, 20 seedlings of each variety were transplanted

into cans filled with subsoil to which dolomitic lime-

stone had been added at the rate of 6000 pounds per acre

at the time of transplanting. There were 12 Carrizo

citrange seedlings in each of the limed and unlimed

soils, 10 Rusk citrange seedlings in the limed soil, and

7 in the unlimed soil. The Carrizo and Rusk citrange

seedlings were 18 months old at the time they were trans-

planted. The transplanted seedlings were grown in the

greenhouse for 6 weeks before they were flooded. During

this period the cans were watered once a week with

approximately 150 ml of demonized water. The pH of the

soil was determined at the time the seedlings were trans-

planted, before they were flooded, and after 4 weeks of

continual flooding.

The seedlings were flooded on July 22, 1958, in the

greenhouse. Water was added to the cans when needed to

keep a water level above the soil surface. A weekly

numerical rating of various symptoms was made for 6

weeks of continual flooding for each seedling. The

weekly index ratings were made in a similar manner as in

Experiment 2 using the same rating system for all seed-

lings of each variety in both limed and unlimed soil.

The results are presented in Table 3. An analysis of










TABLE 3.- Index rating of injury to Rough lemon, sour orange, sweet orange, Cleopatra mandarin,
Carrizo and "uEs- seedlings when flooded in Leon subsoil with and without dolomitic



Variety oeeks

1 2 3 4 5 6 :


Lime Treatment


:lime no
: lime


lime no
lime


lime no
li me


lime no
lime


lime


no
lime


lime no
lime


RL
SO
Swo
Cleo
Troyer
Carrizo
Rusk


0.45
0.60
0.80
1.20
0
0
0


1.3
1.2
1.75
2.00
0.15
0.50
2.00


0
0
0
0
0
0
0.42


1.6
1.0
1.85
2.15
0.75
1.00
2.80


lin


0.05
0
1.40
0.55
0
0
1.43


2.10
1.20
2.45
2. 55
0.90
1.16
2.80


0.10
0.15
1.30
1.05
0
0
1.70


2.3
1.4
2.7
2.75
0.85
1.41
2.90


0.55
0.85
1.60
1.20
0
0
1.86


2.45
1.25
2.75
2.80
0.95
1.41
2.90


_ ___


_ __ __










TABLE 3-Continued


Analysis of Variance


Source of d.f. S.S. MS. F Required F
Variation .05 .01




Total 83 77.80

Time (T) 5 15.66 3.13 41.87 2.53 3.70

Lime (L) 1 30.81 30.81 411.85 4.17 7.56

Varieties (V) 6 17.97 3.00 40.04 2.42 3.47

V x L 6 3.06 0.51 6.82 2.42 3.47

T x L 5 2.76 0.55 7.38 2.53 3.70

V x T 30 5.30 0.18 2.36 1.84 2.38

V x T x L 30 2.24 0.07










variance was made to determine any significant differences.

The pH of the soil at the time the seedlings were

transplanted was 4.6. Six weeks after the dolomitic

limestone had been added to the soil and just prior to

flooding the pH of the limed soil had increased to 7.5.

The pH of the limed soil with citrus seedlings was 7.0

after 4 weeks of flooding, but was not changed without

seedlings. The unlimed flooded soil with citrus seed-

lingn had a pH of 5.0 after 4 weeks of flooding and a

pH of 4.0 without citrus seedlings.

There was a highly significant difference between

the degree of injury to the seedlings of each variety

in the limed soil and the unlimed soil for the 6 weeks

of flooding. The injury to the seedlings in the unlimed

soil was progressively more than the injury to the seed-

lings in the limed soil. There was no difference between

the rating of the seedlings of the 7 varieties in the

limed soil after 3 weeks of flooding. In the unlimed

flooded soil during the first 3 weeks of flooding the

least injured varieties to the most injured were: Troyer

citrange, Carrizo citrange, sour orange, Rough lemon,

sweet orange, Cleopatra mandarin and Rusk citrange. After

4 weeks of continual flooding the Rough lemon, sour

orange, sweet orange and Cleopatra mandarin seedlings

were photographed to illustrate the difference between the









condition of the tops of the plants in the unlimed and

limed soil (see Figures 5, 7, 9, 11). Figures 6, 8, 10,

12 and 13 illustrate the change in the condition of the

seedlings (in the limed and unlimed soil) during the 6

weeks of continual flooding. It is possible for one to

look at the graph accompanying each picture and compare

the index rating of all the seedlings after 4 weeks of

continual flooding with the representative seedlings

in the photograph.

After 6 weeks of continual flooding there were

significant differences in the injury index rating

between the Rough lemon, sweet orange, Cleopatra manda-

rin, and Rusk citrange seedlings; the sour orange, sweet

orange, and Rusk citrange seedlings; and the Cleopatra

mandarin and Rusk citrange seedlings in the limed soil.

In the unlimed soil there was no significant difference

between the sweet orange, Cleopatra mandarin and Rusk

citrange seedlings, and the sour orange and Carrizo

citrange seedlings, whereas there were significant differ-

ences among all of the other seedlings.

Experiment 5.- Effect of carbon dioxide.

One-gallon metal cans were filled to within 2

inches of the top with Leon subsoil. A glass tube with a

fine mesh wire over the bottom was placed in the center of

each can at the time the cans were filled with the soil































Figure 5.- Rough lemon seedlings in Leon subsoil
with (right) and without (left) dolomite following four
weeks of continual flooding.


5-

SNO DOLOMITE





3.

DOLOMITE

0 -
1 2 3 4 5 6
WEEK S


Figure 6.- Index rating of Rough lemon seedlings
in Leon subsoil with and without dolomite during six weeks
of continual flooding.


~ ~_















I-.


Figure 7.- Sour orange seedlings in Leon subsoil
with (right) and without (left) dolomite following four
weeks of continual flooding.


3-



S2-
SNO DOLOMITE
x


WEEKS


Figure 8.- Index rating of injury to sour orange
seedlings in Leon subsoil with and without dolomite
during six weeks of continual flooding.
















4
/


Figure 9.- Sweet orange seedlings in Leon subsoil
with (right) and without (left) dolomite following four
weeks of continual flooding.






% NO DOLOMITE


x
w*


DOLOMITE




1 2 3 4 5 6
WEEKS


Figure 10.- Index rating of injury to sweet orange
seedlings in Leon subsoil with and without dolomite
during six weeks of continual flooding.















/N
ell10


Figure 11.- Cleopatra mandarin seedlings in Leon
subsoil with (right) and without (left) dolomite follow-
ing four weeks of continual flooding.




NO DOLOMITE

1 2-




DOLOMITE



1 2 3 4 5 6
WEEKS

Figure 12.- Index rating of injury to Cleopatra
mandarin seedlings in Leon subsoil with and without
dolomite during six weeks of continual flooding.
















2-



1- NO DOLOMITE


DOLOMITE
-4 6
CARRIZO


E-4
NO DOLOMITE








RUSK x

SNO DOLOMITE
-F4 2- DOLOMITE
O DO LOIT


-14


0
1 2 3 4 5
WEEKS


Figure 13.- Index rating ot injury to Rusk, Troyer
and Carrizo citranges in Leon subsoil with and without
dolomite during six weeks of continual flooding.











and 6 seedlings of each variety were planted per can.

The varieties of seedlings used were Rough lemon, cour

orange, Cleopatra mandarin and sweet orange. There were

3 cans of each variety. Two cans of each variety were

flooded with deionized water in June, 1958, three months

after they were transplanted. Compressed carbon dioxide

was slowly released into one flooded can and one unflooded

can of each variety through the glass tube for the first

24 hours the cans were flooded. During the succeeding

8 days carbon dioxide was released into the cans for

15 hours each day. The injury symptoms of each plant

were rated using the rating system of Experiment 2 and

an index rating for all the seedlings of each variety

with and without carbon dioxide was made the same as

that in the above experiment.

The index rating of the seedlings in the flooded

soil with and without added carbon dioxide is presented

in Table 4. After 1 week of flooding there was no

difference between any of the seedlings in the flooded

or non-flooded cans with or without additional carbon

dioxide. After 2 weeks of flooding the flooded seedlings

with additional carbon dioxide had a higher index rating

of injury than the flooded seedlings without additional

carbon dioxide. This relation continued for 5 weeks.

There was a highly significant difference between the














TABLE 4.- Index rating of injury to Rough lemon, sour orange, a eet orange and Cleopat:
in Leon subsoil with and without added carbon dioxide.


Variety:


Weeks


: Tot


G0o


Tr t.tment
CO. No
CO,
2 r


CO
4-


No
CO2


CO2


No
2


C02


0 0 1.00

0 0 0.66

0 0 0.66

0 0 1.00


0

0

1.0,


1.83



3.00

2.00


0 3.00

0 3.00

0.60 3.00

2.50 3.00


1.00

1.00

1.20

2.50


3.00 2.33 8.83

3.00 1.50 8.32

3.00 2.00 9.66

3.00 2.50 9.00


1.00 8.49 3.10 12.00 5.70


1J0
CO
CO


RL


CS'

Cleo


- --


- --- I- -- ---~-- ---~ --


- ---- -I ----


12.00 8.33 35.81


Total 0 0 3.32











TABLE 4-Continued


Analysis of Variance


Source of d.f. S.S. M.S. F Required F
Variation .05 .01



Total (T) 39 56.46

Varieties (V) 3 2.50 0.83 5.88 3.49 5.95

Weeks (W) 4 37.21 9.30 65.65 3.26 5.41

CO2 (C) 1 7.81 7.81 55.12 4.75 9.33

V x W 12 2.04 0.17 1.20 2.69 4.16

V x C 3 2.06 0.69 4.85 3.49 5.95

W x C 4 3.14 0.79 5.54 3.26 5.41

V x W x C 12 1.70 0.14






55









12-

CARBON DIOXIDE
10

E--
8


6 -


4-

2_


I I I I I
NO CARBON DIOXIDE


1 2 3 4 5
WEEKS


Figure 14.- Total index rating of injury to
citrus seedlings in Leon subsoil with and without
added carbon dioxide.










flooded seedlings with and without carbon dioxide added

(see Figure 14). The only significant difference between

varieties was between the Cleopatra mandarin seedlings

ana the Rough lemon, sour orange, and sweet orange seed-

lings in the flooded soil without additional carbon

dioxide. This is shown in Figure 15. There was no

significant difference between any of the varieties in

the flooded soil in which carbon dioxide was added.

The seedlings in the unflooded cans to which

carbon dioxide was added never wilted. Two weeks after

the carbon dioxide was first introduced there was new

growth present on 80 per cent of the seedlings.

Discussion

VThen the root system of a plant has been injured

in water-saturated soils the top will eventually show

injury. The first visible sign on citrus seedlings in

these experiments was a yellowing of the leaf veins.

The leaf pattern as shown in Figure 16 was more outstand-

ing on sour orange and sweet orange. The symptom first

appeared on the lower leaves of the seedlings. Bain and

Chapman (2) observed this same condition on grapefruit

plants in water-saturatea soils. The next symptom of

injury was wilting of the leaves. The young tender leaves

wilted first. The leaves when wilted were either drooped

or curled. After the seedlings wilted the leaves sometimes

























2- Cleo



1- Sw




1 2 3 4 5
WEEKS



Figure 15.- Comparative index ratings of injury
to Rough lemon, sour orange, sweet orange and Cleopatra
mandarin seedlings in flooded Leon subsoil without
added carbon dioxide.








































Figure 16.- Typical yellow-veined pattern
(left) in leaves of a sour orange seedling indicating
early injury symptoms following extended flooding
compared with a healthy green seedling (right).










defoliated before desiccating or they appeared desiccated

and remained tightly on the stems. These symptoms were

a result of injury to the root system which may vary in

a seedling from the loss of many of the fibrous feeding

roots to the death of the tap root. In larger plants

having a more branched root system, death of both small

and large woody roots, in addition to the loss of the

fibrous roots, and rotting of the root crowns may be

manifested in the top of the tree by reduced growth.

Smallness, sparseness, and yellowing of the foliage and

more or less complete defoliation will ensue.

Sweet orange and Rough lemon seedlings in water-

saturated soil in a water tank survived flooding for

3 weeks during the month of September. More than

75 per cent of the sweet orange seedlings that were

flooded in cans in July and August but not in a water

bath were wilted after 2 weeks. The temperature range

(750 F. to 950 F. each day) was comparable. There was

a prolific growth of algae in the water in the tank in

which the seedlings were flooded which undoubtedly in-

creased the oxygen content of the water. Since the cans

had holes in them there was an interchange of water in

the cans with the water in the tank, whereas there was no

such interchange of water in the flooded cans outside of









the water tank. The water in the cans not in the water

tank was stagnant and had an odor of putrefaction. In

the flooded cans accumulation of toxic substances as

proposed by Kramer (50) or Jackson (38) would be far

greater than in the cans in the water tank. If toxic

substances had been formed in the water tank they could

have become diluted to such an extent that they were

not harmful to the plant or they could have become

oxidized as a result of increased oxygen due to algal

growth. The sweet orange seedlings, in a water-saturated

soil at a constant temperature with the soil-water

surface covered to eliminate light, were tolerant of

the water for six weeks. In this case the dilution

factor was the most probable reason for the elimination

of high concentrations of any toxin which might have

formed.

Four varieties of citrus seedlings tolerated

water-saturated soil for one year when flooded in

January. No extensive root injury was observed during

the first 39 days of flooding. There was a complete new

root system on all seedlings in cans which were flooded

for one year and the old root system had completely

decayed. Apparently the new root system was formed during

the time which the plants were under minimum transpi-

rational stress. When the plants became more active they









were dependent upon the new root system which was

adapted to the flooded environment.

When seedlings were flooded in the summer months,

plants were in a more active stage of growth and their

leaves displayed injury symptoms within 2 weeks.

During the spring and summer of 1958, the seedlings

flooded in July were damaged sooner as evidenced by the

leaf symptoms. Injury symptoms appeared sooner during

June, July and August than in April. This could be

due to variances in temperature resulting in a differ-

ential rate of transpiration. Heinicke (34) found that

flooding the soil containing apple roots during the

winter months caused considerable loss of small roots

but produced no serious injury if the soil was drained

before leaves began to appear. Flooding in the summer

soon caused injury, particularly if transpiration was

rapid.

There was a striking difference between the degree

of injury to the citrus seedlings in flooded subsoil and

flooded topsoil. Soil from both depths had a comparable

pH of 4.5. The higher organic matter content in the top-

soil could influence the water tolerance of these seed-

lings, either by a physical or chemical reaction with

substances that caused injury, be they organic or in-

organic. The subsoil was almost devoid of organic matter.










When a soil is flooded in the field and the water is

removed only by natural factors the subsoil retains the

water longer than the topsoil. Citrus trees may suffer

from decided rises of the water table during the rainy

season of the year, especially when the occurrence of

one or more fairly dry years nas induced the root systems

to extend themselves fairly deeply into the subsoil. It

is in this horizon where the root injury is most likely

to occur first and most severely. If water becomes stag-

nant more injury is likely to be shown by the tops of the

plants than if water is moving. Moving water will remove

or dilute toxic accumulations from the root areas. Water

from the surface of the soil can be removed by evapo-

ration or run off, ana sooner than water in the subsoil,

thus allowing subsoil water to become more stagnant and

more harmful to the roots and to the entire plant. Reitz

and Long (71) found tnat in poorly drained areas approxi-

mately 76 per cent of the root system of the citrus trees

was located in the surface 12-inch level.

Plants in the limed soil tolerated the waterlogged

condition for a longer time than the plants in the acid

soil. There was sufficient time for the reaction of the

lime with the soil before flooding to raise the pH of

the soil to near neutral. deslaes the pH change

additional cations were present. The reason for the










influence this change had on the water tolerance of

the citrus plants is uncertain. If roots of citrus

secrete a substance with or without the aid of

microorganisms when flooded and that substance can

be changed chemically by the presence of cations at

the higher pH, the seedlings might tolerate excess

water for a longer period of time instead of collapsing

within a short time as was the case in the acid medium.

Truog (88) proposed that an equilibrium condition,

chemically and physically, exists between toxic sub-

stances in the soil solution and the solid soil

constituents. They may combine directly or react by

double decomposition with these constituents. Thus

the equilibrium concentration is disturbed.

New roots were present on Rough lemon, sweet

orange and Cleopatra mandarin seedlings in the limed

soil but not on the seedlings in the unlimed soil. These

differences are shown in Figures 17-19 inclusive. The

new roots are white and near the base of the stem. New

roots were formed on sour orange and Troyer citrange

seedlings in both the limed and unlimed soil (Figures 20

and 21). The rapid collapse of the root systems in the

acid soil was reflected in a relatively short time in the

tops and this damage to the tops probably accounts for

the absence of new roots. Even though the old root







































Figure 17.- Typical Rough Icmon root systems
following six weeks of continual flooding in Leon
subsoil with (left) and without (right) dolomite.









































Figure 18.- Typical sweet orange root systems
following six weeks of continual flooding in Leon sub-
soil with (left) and without (right) dolomite.









































Figure 19.- Typical Cleopatra mandarin root
systems following six weeks of continual flooding in
Leon subsoil with (left) and without (right) dolomite.








































Figure 20.- Typical sour orange root systems
following six weeks of continual flooding in Leon
subsoil with (left) and without (right) dolomite.







































Figure 21.- Typical Troyer citrange root systems
following six weeks of continual flooding in Leon subsoil
with (left) and without (right) dolomite.









systems on the plants in the neutral soil had been

injured it could have been a more gradual injury and

new roots began to form which aided in the support of

the tops. Rough lemon seedlings in the limed soil had

the largest new root system with the Troyer citrange seed-

lings next. The root systems on the Carrizo citrange seed-

lings were comparable to those on the Troyer citrange seed-

lings. Both regenerated new roots in the acid and limed

flooded soil. In all of the experiments where seedlings

were flooded for extended periods of time any seedlings

which survived after 6 weeks of flooding had new roots

near the base of the stem just below the soil surface.

These newly formed roots undoubtedly become a factor in

the survival of the plants under flooded conditions. A

soil medium conducive to better root function seems to

be conducive to greater water tolerances.

Carbon dioxide, when added to the flooded soil

with citrus seedlings, caused earlier water injury

symptoms to appear in the leaves than where no carbon

dioxide was added. Also these seedlings that were

treated with carbon dioxide desiccated earlier. The

carbon dioxide apparently caused earlier root injury

which was reflected by the leaves. It is believed (25)

that the specific effect could be on the protoplasm,

causing an increase in viscosity and decrease in









permeability, and this would affect the water absorption

mechanism rather than affect transpiration. Cannon (12)

found that citrus roots could tolerate high concentrations

of carbon dioxide in a porous medium not waterlogged. No

apparent damage was done to the seedlings in the unflooded

soil to which carbon dioxide was added; indeed, new growth

appeared 1 week after the carbon dioxide treatment ceased.

It seems to the author that carbon dioxide probably

injures the roots but with water present other factors are

involved which are stimulated by additional carbon dioxide.

In flooded acid soil with one seedling in each can,

the order of water tolerance was: Troyer citrange, Carrizo

citrange, sour orange, Rough lemon, sweet orange, Cleopatra

mandarin and Rusk citrange. There was no difference in

the water tolerance of the seedlings in the limed soil

after 3 weeks of continual flooding. After 6 weeks of

continual flooding the order of decreasing water tolerance

of the seedlings was: Troyer citrange, Carrizo citrange,

sour orange, Rough lemon, sweet orange, Cleopatra manda-

rin and Rusk citrange. In the flooded cans where there

was more than one seedling, the order of decreasing water

tolerance during the summer months was: Rough lemon,

Cleopatra mandarin, sour orange, and sweet orange.

Sour orange rootstock has been classified as

being more resistant to water injury than either sweet










orange or Rough lemon rootstocks. This was attributed

to the more shallow rooting tendency of the sour orange

rootstock by Rhoads (72). Others (45,46) have attributed

these difference to the greater resistance of sour

orange rootstock to root-rotting fungi. If the soil

environment is conducive to growing vigorous plants with

good root systems the difference between the water

tolerances of the various rootstocks would be due to

the length of time the waterlogged condition was present

throughout the soil profile in which the roots were

concentrated. The rate and degree to which new roots

are formed under prolonged waterlogging is also a

contributing factor in the water tolerances of citrus

seedlings.












IV. D1EON3TRATION AND EVALUATION OF TOXINS3 AS A

FACTOR ASSOCIATED RITH VATER DAMAGE


Very little is known about why some plants may

become injured so quickly when the soil in which they

are grown becomes flooded. Lack of oxygen, increase

of carbon dioxide, and the production of toxic

substances have been suggested as the causes of flood

injury. Toxins were considered to be a possible

causal agent and therefore preliminary experiments

were designed to determine whether a soil kept under

stagnant conditions would produce toxic substances

which would be detrimental to citrus plants. It was

further realized that this would not be the same

condition as found in citrus grove soil when water-

logged. Therefore, fresh citrus roots were incorporated

in soil and all of it submerged in water. It was found

that citrus seedlings wilted in the soil water where

citrus roots had been but remained unwilted in the soil

water where there had been no citrus roots.

The purpose of these experiments was to investi-

gate the production and properties of a toxin in citrus

root solutions which causes seedlings to wilt. The

effects of temperature, pH, and microorganisms on the

72










production of the toxin were investigated. The influence

on citrus seedlings of water extracts from stagnant water-

logged soils with and without citrus roots and the sub-

sequent effect on healthy seedlings of planting them in

these soils and reflooding were determined.

Methods and Results

Various methods were used to study the properties

of the toxin in the root water. Filtering, activated

carbon, heat, vacuum and atmospheric distillation,

exchange resins, varied pH values and nutrient precipi-

tations, alcohol and acetone precipitation, and ether

extractions were all employed to remove the toxin from

the root solution. Paper chromatography and fluorimetry

were also used to gain further information on the toxin.

Demonstration of the production of a toxin.

Citrus feeder roots and small lateral roots from

healthy trees were selected for incubation in water in

sealed glass jars. This incubated root water was tested

on Rough lemon, sour orange, sweet orange, and Cleopatra

mandarin seedlings for the presence of toxic substances

which induce wilting. The jars were incubated at

different temperatures, the pH was adjusted prior to

incubating, different quantities of roots were incubated

in soil and water, and citrus roots from trees on

Cleopatra mandarin, Rough lemon, sour orange and sweet









orange stock were incubated.

Experiment I. Effect of quantity of roots.- Two

pint jars each with 10 grams of fresh citrus feeder

roots were filled with water. 1ix Rough lemon seed-

lings were placed in 1 jar and 6 sour orange seedlings

were placed in the other. Two pint Jars filled with

demonized water had 6 Rough lemon seedlings in one and

6 sour orange seedlings in tne other. These seedlings

were held upright by a circular waxed perforated

cardboard and a metal ring cap, and the jars were

placed in the greenhouse. All of the seedlings in the

jars with the added citrus roots were found to have

wilted after 2 weeks. The seedlings in the jars with

no additional roots remained turgid.

Six one-quart Mason jars were partly filled

with 600 grams of Lakeland fine sand from an old citrus

grove. To each of these jars was added respectively

0, 1, 5, 10, 25, and 35 grams of fresh citrus feeder

roots and these were incorporated in the soil. Another

6 one-quart Macon jars were partly filled with 600 grams

of Leon fine sand from a pasture with native cover. To

each of these jars was also added respectively 0, 1, 5,

10, 25 and 35 grams of fresh citrus feeder roots and

these were incorporated in the soil. All of these jars

were filled with deionized water, sealed and incubated









in the laboratory at room temperature for 2 weeks.

A one-quart jar with 10 grams of citrus roots was

filled with water only and incubated alongside the

other jars. Each jar was shaken at the end of the

incubated period and 8 citrus seedlings (2 each of

the four varieties of citrus seedlings) were placed

in each jar. Another 8 seedlings were supported

in a quart jar of delonized water as control. The

jars were covered with aluminum foil and placed in

the greenhouse.

After 7 days only the 2 Rough lemon seedlings

in the jar which had the Lakeland soil and 25 grams

of citrus roots were wilted. However, all of the seed-

lings in the jars which had the Leon soil with 10, 25,

and 35 grams of citrus roots were wilted after 7 days.

Those in the jar with roots and water without soil were

also wilted. After 2 months the seedlings in the jars

with both types of soil containing no roots, and in the

jars with Lakeland soil and 1, 5, and 10 grams of citrus

roots were still turgid and healthy except for the sour

orange seedlings, which developed yellow-veined leaves.

Less than one-fourth of the water remained in these jars

after a two-month growing period. The seedlings in the

remaining jars were all wilted at the end of the two-

month period with approximately 90 per cent of the water









still in each jar. The seedlings in the demonized water

remained turgid for 2 months with only yellow-vein

symptoms on 1 of the sour orange seedlings.

Experiment II. Effect of parts and kinds of citrus

roots.- One hundred grams each of fresh root cortex

(from xylem outward), root xylem (wood), and feeder roots

from older trees of Rough lemon, sweet orange, sour orange

and Cleopatra mandarin rootstocks were incubated sepa-

rately in sealed gallon jars filled with water for 2

weeks in the greenhouse. Six each of Rough lemon, sour

orange, sweet orange and Cleopatra mandarin seedlings

were placed in 480-ml aliquots of root solutions from

each of the incubated jars. For controls, 6 each of

Rough lemon, sour orange and sweet orange seedlings were

placed in 480-ml quantities of deionized water.

Twenty-five grams of small lateral and feeder

roots from trees which had been subjected to high water

and were in a wilted condition were incubated in 480 ml

of deionized water. Both the feeder and lateral roots

were badly sloughed. The water table at the time the

wilted trees were examined was 4 feet below the surface.

Twenty-five grams of roots from an apparently healthy

tree in close proximity were also incubated in 480 ml

of demonized water. After 2 weeks of incubation 3 sour

orange seedlings were placed in each root solution.









After 7 days all of the seedlings in all of the

root solutions were wilted, and they were desiccated

after 10 days. The seedlings in the deionized water

remained green and turgid. The stems of the sour orange

seedlings in the root solution from the apparent water-

damaged roots and non-water-damaged roots after 7 days

were bleached in addition to being wilted.

Experiment III. Effect of temperature.- One

hundred grams of feeder roots from old citrus trees were

incubated in sealed gallon jars filled with water at

o2 F., 400 F., 600 '., 7o F., 800 F., and greenhouse

temperature for 7 and 14 days. Also, 2 one-gallon jars

each with 100 grams of feeder roots were filled with

water, and placed in the greenhouse, and compressed air

was bubbled continuously into one of them for 7 days

and into the other for 14 days. Six each of JRough

lemon, sour orange, and sweet orange seedlings were

placed in 480-ml aliquots of the root solution from

each of the incubated jars and in 480 ml of delonized

water.

The influence of temperatures at which citrus

roots were incubated on the wilting of citrus seedlings

are presented in Tables 5 and 6. The greatest differ-

ence between the 7-day and 14-day incubations was the

subsequent wilting effect of the aerated solutions on









TABLE 5.- The influence of temperatures at which
citrus roots were incubated for seven
days on the wilting of citrus seedlings.


(a)
Incubation Seedlings() umber wilted after
temperature days in solution
7 16


600 F.



720 F.



800 F.



Greenhouse


Greenhouse

air

Deionized
water


RL
30
SwO

RL
SwO

RL
SO
SwO


SO
SwO

RL
SO
SwO

RL
so
SO


(a) Six seedlings
solution.


per variety per 480-ml aliquots of


- -- -- ---










TABLE 6.- The influence of temperatures at which
citrus roots were incubated for fourteen
days on the wilting of citrus seedlings.



Incubation Seedlings(a) Ntumber wilted after
temperature days in solution
4 11


600 F.



720 F.



800 F.



Greenhouse


Greenhouse

air

Deionized
water


RL
SO
SwO

RL




SwO
RL
s0

Swo


SwO

RL
so
SO
SwO


RL
SOW


(a) Six seedlings per variety per 480 ml of solution.










the citrus seedlings. The seedlings in the 14-day

incubated solution were all wilted on the eleventh day

whereas none of the seedlings had wilted in the 7-day

incubated solution.

No seedlings were wilted after 1 month in the

root solutions which had been incubated either at

32o i'. or at 400 F. Following the removal of the

aliquots from the jar which had been incubated at 400

F., the jar was resealed and incubated for an additional

2 weeks in the greenhouse. Four Rough lemon seedlings

were then placed in 480 ml of the root solution. All

of these Rough lemon seedlings were wilted after 7 days

in the root solution.

Experiment IV. Effect of DH.- Forty grams of

dried citrus feeder roots were incubated in sealed

gallon Jars filled with water for 11 days in the green-

house after the pH was adjusted to 4.0, 6.0 and 7.5

with hydrochloric acid or sodium hydroxide. One jar

was incubated without any pH adjustment and its initial

pH was 5.6. All pH measurements were made using a glass

electrode and a Beckman pH meter. The pH of the solution

in each jar, and of the solutions in the jars in which

the citrus seedlings had been for 11 days, was measured

before and after incubation. Five each of Rough lemon,

sour orange, sweet orange and Cleopatra mandarin









seedlings were placed in 480-mi aliquots of the incubated

root solutions.

The influence of pH of the solution in which citrus

roots were incubated on the wilting of citrus seedlings is

presented in Table 7. After 5 days, of the citrus seed-

lings in the solutions which had an initial pH of 4.0,

less than one-half of the seedlings were wilted. Those

seedlings that were wilted were sour orange, sweet orange

and Cleopatra mandarin.

The changes in the pH of the incubated solution

and of the solutions In which the different seedlings

had been for 11 days are recorded in Table 8. The pH

after incubation of the solutions with initial pH

values of 5.6, 6.0, and 7.5 wan lowered to 4.6. The

solution with an initial pH of 4.0 had a pH of 5.1

following incubation. The pH of the solutions in which

ceediings had been for 11 days rnged from 5.1 to 7.5.

The pH of the solutions which had an initial pHi of 4.0

was higher after the seedlings hd been in them than

that of any other solutions.

E.oerlment V (a). Effect of microorg:inisms.-

Twenty-five grams of citrus feeder roots from old citrus

trees were incubated in sealed one-quart jars with 100

grams of Leon topsoil (0-7'), 100 grams of Leon subsoil










TABLE 7.- 'he influence of ph of the solution in
which citrus roots were incubated on the
wilting of citrus -eeullngs.



Initial(a) Seedlings(b) Number wilted after
pH days in solution
2 5 4 5



4.0 RL 0 2 0 0
SO 2 2 2 2
SwO 2 3 5 2
Cleo 2 4 3 3

5.6 RL 4 4 5 5
8s 4 5 5 5
Swo 4 5 5 5
Cleo 5 3 5 5

6.0 RL 5 5 5 5
0S 4 5 5 5
SwO 5 5 5 5
Cleo 5 6 5 5

7.5 kL 4 5 5 5
SO 4 4 5 5
SwO 5 5 5 5
Cleo 5 5 5 5



(a) Incubated for 11 days.

(o) Five seealings per variety per 480-ml aliquots
of solution.









TABLE 8.- The pH changes of the solutions following
incubation and following the wilting of
the seedlings.



Initial pH after pH after 11 days with seedlings
pH 2 weeks HL SO SwO Cleo
incubation



4.0 6.1 7.2 7.3 7.5 7.4
5.6 4.8 6.7 6.6 6.7 6.7
6.0 4.8 6.7 6.2 6.3 6.5
7.0 4.8 6.7 7.0 6.1 6.8


(7-17") and no soil. The jars were all filled with

water. After 2 days of incubation in the greenhouse

1 ml of a 1:1000 dilution of the water from each jar was

placed on petri dishes which contained 4 agar media:

potato dextrose, pH 3.0 (20); dextrose, pH 7.3 (20);

orange serum, pH 6.8 (20); and mycological. The

mycological agar medium had the following composition

(grams): glucose, 10; peptone, 5; KH2P04, 1; MgSO4-7H20,

0.5; agar, 30 in 1000 ml distilled water, and the pH

was adjusted with H2804 to 4.0.

Duplicate petri dishes of each agar medium were

inoculated. One-half of the petri dishes were incubated

at 320 0. and the other half were incubated at room

temperature in the laboratory desk for 1 week. The









dishes which had prolific growth within 2 days were

removed from their respective incubators and stored

in the 0 C. room.

Twenty-five grams of citrus roots in 1000 ml

of water were sterilized in an autoclave at 15 pounds

pressure for 50 minutes in each of 9 two-liter flasks.

These flasks were checked for sterility by streaking

orange-serum plates with water from each flask using

a sterile loop.

Tne flasks with the autoclave-sterilized roots

were inoculated with the growth on the different agar

media in the following manner:

Ilask iNo. Agar medium Source of inoculum

F-1 Dextrose Roots and subsoil
F-2 Orange serum Roots and subsoil
F-1 dycolobical Roots and subsoil
F-4 Mycological Roots only
F-5 Orange serum Roots only
F-6 Potato dextrose Roots only
F-7 Potato dextrose Roots and topsoil
F-8 Potato dextrose Roots and topsoil


Two milliliters of sterile water were poured on each

plate and a sterile loop was used to loosen the growth.

Each mixture was poured into a bottle with sterile water.

Cell counts were made on the solution using the direct

microscopic method. The quantity of solution added to

each flask contained approximately 100,000 cells. These

flasks were incubated in the laboratory desks at room









temperature for 10 days after each was inoculated. Also

1 flask containing 25 grams of unsterilized roots in

1000 ml of water was incubated for 10 days.

All flasks which had been autoclaved were sterile

according to the streaked orange-serum dishes.

The bacterial colonies on the inoculated

dextrose and orange-serum media were too numerous to

count after 24 hours in the 32o C. incubator. However,

the media to which water had been added from the jar

with citrus roots and topsoil had a greater number

than those plates to which water was added from the

jar with citrus roots and subsoil. The smallest

number of colonies on these media was from the water

in the jar that contained just citrus roots.

There were colonies of fungi in all mycological

agar dishes after 72 hours. The different fungal

colonies were grown together. There were from 1 to 6

colonies of fungi on the potato-dextrose-agar plates

after 3 days of incubation under both incubating

conditions.

Following the incubation period of the inocu-

lated flasks, seedlings were placed in two 250-ml

aliquots of solution from each flask. One aliquot

contained 5 sweet orange and 3 Rough lemon seedlings

and the second aliquot contained 3 Rough lemon and









5 sour orange seedlings. These containers were

placed in the greenhouse.

None of the seedlings in any of the solutions

was wilted after 7 days. These seedlings remained green

for 2 weeks at which time less than one-half of the

solution remained in the containers.

Experiment V (b). Effect of surface washing.-

Citrus roots were washed with an intense spray of water.

Fifty grams of these roots were placed in one-liter

Erlenmeyer flasks. Three flasks were treated for 3, 5,

and 8 minutes with a 3.5 per cent sodium hypochlorite

solution to which 11 ml of glacial acetic acid per liter

had been added. Two flasks were treated with a 0.25

per cent sodium hypochlorite solution to which 3 ml

of glacial acetic acid per liter had been added. One

flask v;hich was untreated and filled with water served

as a check. The roots were rinsed four times with

sterile water by covering the roots and decanting

(after the hypochlorite solutions were poured off).

After the roots were rinsed the flasks were filled with

sterile water and sealed with a sterile rubber stopper.

These flasks were incubated in the laboratory desks.

Duplicate petri dishes with orange serum and potato

dextrose agar were streaked with water from each flask

after 3 days of incubation. One set was incubated at









320 C. and the other set at room temperature.

After the flasks were incubated for 14 days,

3 sweet orange seedlings were placed in 250-ml aliquots

from each flask.

Thirty grams of washed citrus roots were placed

in 1000 ml Erlenmeyer flasks. Two of these flasks

were filled with a 800 ppm Roccal solution (active

ingredient: alkyl benzyl ammonium chlorida) for 30

minutes and 2 flasks for 60 minutes. This solution

was decanted and the flasks were filled for 10 minutes

with a 0.25 per cent sodium hypochlorite solution to

which 3 ml of glacial acetic acid per liter had been

added. The roots were rinsed three times with sterile

water and then each flask was filled with sterile water

and sealed with a sterile rubber stopper. One flask

with untreated roots was filled with sterile water and

sealed with a rubber stopper. The stoppers in each

flask were covered with aluminum foil and the flasks

were incubated in the greenhouse. Duplicate petri

dishes with orange-serum and potato-dextrose agar were

streaked with water from each flask after 3 days of

incubation. One set of the dishes was incubated at

320 C. and the other set at room temperature in

laboratory desks.









After the roots were incubated for 2 weeks,

6 sour orange seedlings were placed in 480-ml aliquots

of the root solution from each flask.

Lone of these flasks was sterile. On the orange

serum medium which had been streaked with the solution

from the flask with untreated roots both bacteria and

molds were present. Only bacterial colonies appeared

on the orange serum medium -which had been streaked with

water from the Roccal- and hypochlorite-treated citrus

roots. Fungal colonies appeared only on the potato

dextrose which had been streaked with water from the

hypochlorite-treated and untreated roots. The Roccal

solution apparently inhibited the fungal colonies.

After 7 days in the root solutions all of

the sweet orange seedlings were wilted. The sweet

orange seedlings in the solution from the flask with

the untreated roots were desiccated after 5 days.

More than one-half of the sour orange seedlings in

the solution from the flask with the untreated roots

were desiccated after 5 days. bore than one-half of

the sour orange seedlings in each solution were wilted

after 7 days. All of them were wilted after 14 days.

Experiment VI. Effect of stagnant water from

flooded Leon soil.- One-gallon metal cans containing










Leon subsoil and topsoil from the native pasture were

flooded with deionized water in the greenhouse. One

group of cans had Rough lemon seedlings growing in the

topsoil. A second group of cans had Rough lemon seed-

lings growing in the subsoil. A third group had 70

grams of roots from old citrus trees incorporated in

each gallon of subsoil (prior to flooding). A fourth

group of cans contained the virgin subsoil. After 2

weeks of flooding the seedlings were wilted and

desiccated. The water from each group of cans was

removed with the aid of a water aspirator and a piece

of glass tubing with a fine mesh wire covering the end.

The soil water from each group of cans was passed

through chopped filter paper using suction. The

filtered soil water was a clear tan solution. Sweet

orange seedlings were put in each filtered soil extract.

There was one seedling per 50 ml of solution.

Rough lemon and sweet orange seedlings were plant-

ed in these ans after the water was extracted, and the

cans were reflooded with deionized water.

The sweet orange seedlings in the soil water

extract from the cans which had the wilted seedlings

and the incorporated citrus roots were all wilted after

7 days. However, the sweet orange seedlings in the soil

water extract from the virgin soil were not wilted in




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