SOME ASPECTS or III: EFFECTS OF HIGH
CONCENTRATIONS OF CALCIUM CARIIIONATE'
AND VARIOUS ORTHOPHOSPHATESI ON THE

ABSORPTEO-N, TRANSLOCATION. AND UTILIZATION
O? [RUIN av CHRYSAN‘FHEMUM IIImImLIuM

 

Thesis for the Degree: of Ph. D.
MICHSGAN STATE UNIVERSITY
Rufizs Burr Ruffiand

19:65

    
 
 

sumao a.“ I _ ‘

1‘3“ RY "
l‘viidfigm Stat-3 I

University ‘ , ~

’ HESIS

   

This is to certify that the

thesis entitled

SOME ASPECTS OF THE EFFECTS OF HIGH CONCENTRATIONS OF
CALCIUM CARBONATE AND VARIOUS ORTHOPHOSPHATES ON THE
ABSORPTION, TRANSLOCATION, AND UTILIZATION
OF IRON BY CHRYSANTHEMUM MORIFOLIUM

presented by
Rufus Burr Rutland

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Horticulture

M§M~V

M. J. Bukovac

Major professor

 

Date April 29, 1965

 

0-169

SOME ASPECTS OF THE EFFECTS OF HIGH CONCENTRATIONS
OF CALCIUM CARBONATE AND VARIOUS ORTHOPHOSPHATES
ON THE ABSORPTION, TRANSLOCATION, AND UTILIZATION

OF IRON BY CHRYSANTHEMUM MORIFOLIUM

 

By

Rufus Burr Rutland

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1965

ABSTRACT
SOME.ASPECTS OF THE EFFECTS OF HIGH CONCENTRATIONS OF CALCIUM
CARBONATE AND VARIOUS ORTHOPHOSPHATES ON THE ABSORPTION, TRANSLOCATION,
AND UTILIZATION OF IRON BY CHRYSANTHENUMiMORIFOLIUM

~—

‘HY Rufus B. Rntland

The total concentration of iron in chlorotic leaf tissue of
chrysanthemums grown in solution culture under conditions of high lime
was reduced to essentially that observed in chlorotic leaves resulting
from an absence of iron in solutions of low lime and low pH. There
were differences among cultivars in leaf content of iron associated
, with various stages of chlorosis, but in general levels of leaf iron
above 70 ppm were found in green leaves, and lower levels were asso-
ciated with chlorotic leaves. This reduction in leaf iron resulted
from a reduction in the amount of iron absorbed by the roots and
translocated from the roots to the leaves, apparently partially due
to internal changes.

Chlorotic leaves from either minus-iron, lowalime pre-treatment
or plus-iron, high-lime pre—treatment absorbed iron at rates equal to,
or exceeding those of green leaves. The presence of sodium bicarbonate
in foliar application solutions. or conditions of high pH (above
pH 6.0) reduced absorption. Chelation of iron with ethylenediamine
tetraacetate did not increase the rate of absorption.

Iron. boron, zinc, and manganese were all reduced in the leaves
of azaleas as a result of high bicarbonate irrigation.

High concentration of phosphates in the root.medium.induced

chlorosis. dilorosis was more severe when both phosphate and

Rufus B. Rutland

bicarbonate were high than when bicarbonate alone was high. There

was a higher iron content in severely chlorotic leaves than in slightly
chlorotic ones when phosphorus was in high supply, suggesting the
accumulation of iron that was not functional in chlorophyll synthesis.

Chlorosis was not induced in the presence of high lime and high
phosphate when iron was supplied as ethylenediamine tetraacetate.
Transport of iron from roots to leaves was increased, and a concomitant
decrease of phosphorus in the roots occurred. Furthermore, a more
efficient use of iron in chlorophyll synthesis appeared to result from
chelated iron compared to ferric ammonium citrate.

Associated with the appearance of chlorosis was a reduction in
shoot growth as indexed by leaf dry weight and dry weight percentage
and by shoot length. Root dry weight increase was not adversely
affected. especially not by high phosphate conditions. Under high
bicarbonate conditions, however, root development was abnormal as a
result of death or inactivation of the root tip and subsequent
excessive development of laterals.

These responses noted in solution culture were also noted under
similar treatment of azaleas in peat soil culture. but not with chry-
santhemnms in soil culture. It is possible that the presence of high
quantities of iron in the soil, even if insoluble, provided abundant
and continuous contact between root and iron particle so that, as long

as root development continued, an adequate supply of iron was obtained.

Acknowledgments

The author expresses sincere appreciation to Dr. M. J. Bukovac,
who served as his major professor during the preparation of this
thesis. Dr. Bukovac made an exceptionally valuable contribution
to the progress of the research through advising the author in the
planning and developing of the line of research and through teaching
techniques and methods which were essential to its completion. His
warm friendship was an invaluable assistance during difficult
periods and his constant encouragement left no doubt about the outcome.

Dr. A. L. Kenworthy was especially helpful in making available
his plant analysis laboratory and in counseling in regard to
interpretations of the analyses, tor which the author is most grateful.
His assistance in the administration of the degree program contrib-
uted greatly to the orderly progress toward its completion.

The availability of a wealth of knowledge and experience in
various aspects of the problem from the other members of kthe
guidance cOmmittee, Dr. R. F. Stinson, Dr. R. S. Lindstrom, Dr. H. C.
Beeskow, and.Dr. Henry Foth, was a constant source of encouragement.
Their willingness to devote their time in counseling and in making
many material contributions to the program is greatly appreciated.

The opportunity to serve on the faculty under Dr. H. B. Tukey
and under Dr. John Carew’was of real value in deveIOping teaching
ability. The close freindships with members of the horticulture
department and the high regard for those in positions of leadership
that developed through that association are treasured products of

that experience.

TABLE OF CONTENTS

REVIEWOFLITERATURE............

Introduction . . . . . . . . . . . . .

The Nature of Lime Induced Chlorosis .

Changes within the Plant . . .
Effect of Absorption . . . . .
Effect of Translocation . . . .
Iron Utilization . . . . . . .

SYnthBtic Chelates o o o o o 0

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

'Iron Solubility in Nutrient Solution .

Lime-Induced Chlorosis in Chrysanthemums and Azaleas

Root Absorption and Distribution of Radioiron . . . .

F0113}? Absorption Of Iron 0 o o o o o o o o o 0

High Lime and Phosphate EffGCts o o o o o o o 0

RESULTS 0 I O O O O O O O O 0 O O O O O O O O O O 0

Iron Solubility in Nutrient Solution

Lime-Induced Chlorosis Chrysanthemums and Azaleas

Root Absorption and Distribution of Radioiron . .

FoliarAbsorptionOfIronooooo oo o 00. 0

High Lime and Phosphate Effects . . . . . . . . .

DISWSSION O O O O O O O O O O O O O O O O O O O O O O

Solubility of Iron in Nutrient Solutions and Soils

LimeInChlcedQllorosis.oo...........

Phosphorus and Lime Interactions

IronTranSIOcationooooooooeooooooo

iii

\0 \n +4 s4 h‘

12
16
18
21
21
24
30
36
1+3
52
52
58
69
86
91

11L»

111+

116

119

125

~iv

SW. 0 O O O O O O O O O O O O O O O O O O O O O O O O O O 129
BIBLIOGRAPI-IY O O O O O O O O O O O O O O O O O O O O O O O O O 133 .

APPENDH O O O O O O O 0 O O O O O O O O O O O O O 0 O O O O O 151

Table

5.

7.

9.

10.

LIST OF TABLES

Effects of Ammonium Citrate on the Stability of Iron
Solutions Containing Bicarbonate . . . . . . . . .
Effects of Lime and Phosphate on the Stability of Iron
in Aerated Hoagland's Number One Nutrient Solution.
Effects of Lime on Growth, Chlorosis and Iron
Accumulation in Chrysanthemum Plants. . . . . . . .
Effects of High Lime and Iron Chelation on Chlorosis
“and Mineral Composition of Chrysanthemum Leaves . .
Effects of Bicarbonates in Irrigation water on Growth,
Chlorosis, and Leaf Mineral Composition of Hybrid
Mollis Azaleas. . . . . . . . . . . . . . . . . . .
Effects of Bicarbonatss in the Irrigation water on
Growth and Chlorosis of Hollis Azalea After the
Second Season of Treatment. . . . . . . . . . . . .
Effects of Bicarbonates in Irrigation water on Chlorosis
and Concentration of Iron and Phosphorus in Leaf
Tissue of Mollis Azalea After the Second Season
of Treatment. . . . . . . . . . . . . . . . . . . .
The Effect of Lime on Iron Absorption by Roots of
'Q.‘morifolium, cv. Legal Tender . . . . . . . . . .
The Effect of Lime on the Distribution of Root-Absorbed
Iron in Q. morifolium, cv. Legal Tender . . . . . .
The Effect of Lime on the Accumulation of Iron in

Tissue Segments of 9.:morifolium, cv. Legal Tender.

57

57

60

62

66

67

7O

72

7L1

Table

11.

12.

13.

1h.

17.

18.

The

Effect of Prior EXposure to High-Lime Conditions

on the Amount of Iron Absorbed Under LoweLime
Conditions by Q. morifolium, cv. Legal Tender . . . .
Effect of Prior Exposure to High Lime Conditions

on the Distribution of Root-Absorbed Iron to Tissues
of g, mgrifolium, cv. Legal Tender . . . . . . . . .
Effect of Prior Eprsure to High lime Conditions

on the Accumulation of Root-Absorbed Iron in Tissues
of|§.:morifolium, cv. Legal Tender . . . . . . . . .
Effects of High Lime Conditions on the Long-Term
Distribution of Root-Absorbed Iron in g. morifolium,
cv. Legal Tender . . . . . . . . . . . . . . . . . .
Effect of Age on a Leaf of'g. morifolium, cv.

Legal Tender on its Rate of Absorption of Iron . . .
Effect of Prior Ebcposure of Plants of g. morifolium,
cv. Legal Tender to High Lime Conditions on the

Rate of Fbliar Absorption of Iron . . . . . . . . . .
Effects of Solution pH and the Presence of Sodium
and/or Bicarbonate in Foliar Treatment Solution

on the Rate of Absorption of Iron by Detached Leaves
of Q, morifglium, cv. Legal Tender . . . . . . . . .
Effects of the Presence of Bicarbonate and Form

of Iron Used in the Foliar Treatment Solution on

the Stability of Iron in Solution and Rate of
Absorption of Iron by Detached Leaves of

E. morifOlium, cv. Legal Tender o o o o o o o o o o 0

vi

75

77

77

79

87

87

9O

Table
19. The Effect of Form of Iron in Foliar Treatment

Solutions at pH 3.0 on the Rate of Absorption

by Detached Leaves of g. morifolium, cv. Legal

Tender .......................92
20. The Effect of pH of Foliar Treatment Solution on

the Rate of Absorption of Iron by Leaves of

9.. morifOIium, CV. Legal Tender o o o o o o o o o o o 924'

 

21. The Effect of Phosphate Concentration in the Nutrient
Solution on Shoot Growth and the Absorption and
Accumulation of Iron and Phosphorus by _C_. morifolium,
cv.Mermaid.....................95

22. The Effects of High Concentration of Various Forms
of Orthophosphates on the Development of
Chlorosis and on Iron and Phosphorus Accumulation
in Leaves and Roots of _C_. morifolium, cvs. Oregon
andYellowDelaware.................98

23. The Effects of High Concentration of Various Forms
of Orthophosphates on the Production of Tissue
Dry Weight by g. morifolium. cv. Oregon and Yellow
Delaware ......................lOO

24. The Effects of Lime and Phosphate on Growth and the
Development of Chlorosis in _C_. morifolium. cv.

Legal Tender . . . . . . . . . . . . . . . . . . . .103

25. The Effects of Lima and PhOSphate on Iron and Phosphorus
Accumulation in Root and Leaf Tissue of Q. morifolium,

CV.L6galTender..................101+

s r

viii

Table
26. The Effects of Lime, Phosphate, and Iron Chelation
on the Development of Chlorosis and on Iron and
Phosphorus Accumulation in Root and Leaf Tissue
. of Q. morifolium. cv. Legal Tender . . . . . . . . .106
27. The Effect of Form and Concentration of Iron Applied
to the Soil on the Concentration of Iron in Leaf
Tissue of _C_. morifolium, cv. Oregon and Yellow
Delaware ......................111
28. The Effects of High Bicarbonate Irrigation and High
Soil Phosphorus on the Accumulation of Iron in
Leaf Tissue of _Q. morifolium, cv. Legal Tender,

Grown in Soil of Moderate Iron Content . . . . . . .113

Figure

2.

3.

LIST OF FIGURES

Solubility of F859(F659C13) in non-aerated, half-

strength Hoagland's number one solution adjusted

to various pH levels. Concentration of iron is

expressed as per cent of original concentration

remaining in solution at each time of observation . . 53
Solubility of Fe59(FeS9C13) in aerated half-strength

Hoagland's number one solution in presence of

high (3 mM CaCO3. pH 7.0) or low (no C3003.

pH 5.5) levels of bicarbonate . . . . . . . . . . . . 56
Autoradiogram of a young chrysanthemum.plant prepared

#8 hours after absorbing Fe59C13 from half-

strength Hoagland's number one solution adjusted

to pH 5.5 with ammonium sulfate . . . . . . . . . . . 82
Autoradiogram of a chrysanthemum plant, which had

absorbed Fe59 under identical conditions as the

plant in Figure 3, and subsequently had grown for

two weeks in half-strength Hoagland's number one

solution at pH 7 and containing 3 millimolar

calcium bicarbonate and aerated with 2% carbon

dioxide in air. A source of stable iron was

available to the roots . . . . . . . . . . . . . . . 82
Autoradiogram of a chrysanthemum plant, which had

absorbed Fe59under identical conditions as the

0‘
\_a

O
t
I
.
.
u
‘1
n!‘ '
.,'
.-
l ‘I
.\'
‘a
‘ p.
I“ ,
' i

plant in Figure 3. and subsequently had grown

for two weeks in half-strength Hoagland's

number one solution at pH 5 containing no

dissolved bicarbonates and aerated with air.

A source of stable iron was not available to

the roots . . . . . . . . . . . . . . . . . . . . . . 82
6. Autoradiogram of a chrysanthemum plant. which had

absorbed Fe59 under identical conditions as the

plant in Figure 3, and subsequently had grown

for two weeks in half-strength Hoagland's

number one solution at pH 5 containing no

dissolved bicarbonates and aerated with air.

A source of stable iron was not available to

the roots . . . . . . . . . . . . . . . . . . . . . . 82
7. Autoradiogram of a chrysanthemum lateral shoot and

the leaf from whose axillary bud it grew. The

plant had been grown for nine weeks in Heagland's

number one solution at pH 7 and containing 3

millimolar calcium bicarbonate and aerated with

2% carbon dioxide in air after having been

allowed to absorb radioiron. A source of

stable iron was available to the roots . . . . . . . 84
8. Autoradiogram of a chrysanthemum lateral shoot and

the leaf from whose axillary bud it grew. The

plant had been grown for nine weeks in Hoagland's

number one solution at pH 5, containing no

bicarbonates, and aerated with air after having

been allowed to absorb radioiron. A source of

stable iron was available to the roots . . . . . . . 84

9. Autoradiogram of a chrysanthemum lateral shoot and

the leaf from whose axillary bud it grew. The

plant had been grown for nine weeks in Hoagland's

number one solution at pH 5, except containing

no bicarbonates, and aerated with air after

having been allowed to absorb radioiron. A

source of stable iron was not available to the

roots . . . . . . . . . . . . . . . . . . . . . . . . 8h
10. Autoradiogram (above) and photograph (below) of an

azalea plant that had been grown under high-lime

59

for twelve weeks after having absorbed Fe under
optimum conditions. An external source of stable
iron, but no radioiron was available to the root
system during the treatment period. The two lateral
shoots grew from buds which were present at the time
of initial absorption of Fe59 . . . . . . . . . . . . 85
11. Photograph (left) and autoradiogram (right) of an
azalea plant which had been grown under low-lime

59

for twelve weeks after having absorbed Fe under
optimum conditions. An external source of stable
iron, but no radioiron was available to the root
system during the treatment period. The single

lateral shoot grew from a bud which was present

at the time of initial absorption of Fe59 . . . . . . 85

12. Effect of solution pH on rate of absorption of iron

by Chrysanthemum leaves. Solution pH is plotted

on the abscissa with rate of absorption expressed

as millimicromoles of iron per square centimeter

leaf area per hour . . . . . . . . . . . . . . . . . 93
13. Effect of HCOB. P04 and iron chelation on tissue

accumulations of iron and phOSphorus . . . . . . . .108

REVIEW OF LITERATURE
Introduction

Green plants are dependent on chlorophyll for photosynthesis. A
deficiency of certain mineral elements can result in an inadequate
maintenance of chlorophyll in leaf tissues. As early as 18h5 Gris
(1+8) demonstrated that an absence of iron would result in a deficiency
of chlorOphyll. Furthermore, iron must be in continuous supply (20,
21, 56).

under conditions of high pH chlorosis resulted in the presence
of iron (#3, 56, 86). Prevention or correction of such chlorosis is
possible by acidifying the soil or nutrient solution (52, 56). Though
high.pH is detrimental, so also is too low a pH, primarily because of
limited calcium supply (1, 3, he, 99). Plants have an Optimum pH which
varies among species, as is indicated by the attention of standard
texts to this point.

High soil lime appears to be the most important cause of induced
iron chlorosis as indicated by the great volume of research reports
and comprehensive reviews (9, 18, 122, l36). Those papers which di-
rectly relate to the question of altered plant ability to absorb, trans-

locate, and utilize iron will be examined here.
The Nature of Lime Induced Chlorosis

When lime or calcium carbonate is high in the soil, several soil
components may be affected. Obviously the concentration of calcium
is high. .Much.of the calcium carbonate will be present in the solid
State, but it dissolves in equilibrium with the dissolved 002 in the

l

.—. c

soil solution (1h). Gile (#3) did not believe that a high concentra-
tion of calcium in the soil was the cause of chlorosis, since he grew
plants in a soil of 15% Casoh without inducing chlorosis. More re-
cently, however, Thorne et al (122) concluded that it was possible
that calcium affected the mobility of iron in plants. Lingle, et al
(82) and Cain (31) showed that iron uptake was reduced when the cal-
cium in solution reached 8 millimolar concentration, and.McGeorge (87)
found soil calcium to be high in chlorotic citrus groves.

Bradfield (1%) pointed out that the bicarbonate concentration in
the soil solution increased as CaCO3 dissolved in the carbonic acid
resulting from dissolved 002. This could be a causal factor in lime-
induced chlorosis, and such a possibility has been supported by several
researchers (29, 1+3, 51+). It hasbeen (ha, 105, 130) shown that the
bicarbonate effect is additive to the pH effect by comparing plant re-
sponse under hithCO3 with that under the same high pH of NaOH treat-
ments.

Soil pH is high under conditions of high lime. Bradfield (1h)
demonstrated a relationship between C02 and CaCO3 as it affected soil
pH. .As the 002 concentration in the soil atmosphere increased in the
presence of insoluble CaCO3, solution pH decreased; but at 002 levels
less than hi, the solution would be neutral or alkaline. Such low
levels of 002 might exist in the zone except after recent incorpora-
tion of organic matter. High pH has been confirmed as a cause of chlo-
rosis under controlled conditions using a variety of means of con-
trolling ph (11, 36, 8h, 86, 97, 105). There is, however, at least
one reported case of a plant (sorgham) accumulating more iron and de-

veloping a greater chlorophyll content under alkaline conditions than

under neutral or acid conditions (77). The opposite is predominately
true, though, and often the effect of excess lime has been corrected
by acidifying the soil with such agents as mineral acids (52), sulfur
(107), or amonium salts (kl, 51+). The effect of pH as demonstrated
in solution cultures is not always similar to that obtained in soil
cultures (99).

The solubility of iron is reduced at high solution pH. Welcher
(1&6) gives the solubility product for ferric hydroxide as 10"36 and
Latiaer (78) as 6 x 10‘38. .At either value solubility is extremely
low. ‘Using Latimer's value, wallace (136) calculated the concentra-
tion of ferric ions to be 3 parts per billion at solution pH h, and it
would'be reduced by'a factor of 1000 at pH 7. Furthermore, it is quick-
ly oxidized in the presence of oxygen, especially in alkaline or neu-
tral solutions (136).

Brown (18) also mentioned the instability of ferrous iron in solu-
tions close to neutrality in reaction. The concentration of iron in
solution cultures was reduced by bicarbonate (26) and by hydroxide and
orthophosphate (89). .At appropriate concentrations of cation and anion,
floculation was instantaneous (86).

Somers and Shive (lit), and Oertli‘ and Jacobson (97) found that
iron accumulation in the leaf correlated with the concentration of
soluble iron in the root medium. It has been established that the use
of organic acid or humic acid forms of iron prevents chlorosis by main-
taining iron in solution (12, 56, 106). Jenny and Overstreet (80) found
that radioactive potassium was exchanged from roots to irondbentonite
suspensions but not to ferric hydroxide colloids in their studies of

that mechanism 0 1’ root absorption.

Important as soluble iron might be in the prevention of chloro-
sis, it is not always essential. Gile and Carrero (uh) grew plants
successfully using dialyzed colloidal iron in suspension. The col-
loidal form was not as effective as ferric chloride at the same con-
centration of iron; but with the concentration of metallic iron in the
collodial form five times that of the concentration of the chloride form,
equal growth was observed. Others (31;, to, 119) have used magnetite as
the source of iron in sand-solution studies and obtained healthy green
plants. The use of frits (1118, 1&9, 150, 151, 152) and other fans
(72) is based on this same principle: roots can absorb iron form solid
forms on contact either by direct exchange or by dissolution of iron in
the microenvironment surrounding the root. wynd and'Wildon (1&9, 152)
collected the nutrient solution leachate from frit-sand mixtures in
which healthy, green plants were growing and found that the leachate
did not contain enough iron to prevent chlorosis when applied to plants
in send only. Furthermore, Jenny (71) and Guest (1+9) demonstrated that
there had to be a large number of contact sites between root and iron
particle for the plant to be able to absorb sufficient iron from insolu-
ble sources. This principle might account for the findings of Twyman
(129) that one large application of ferrous sulfate was better than
several small ones. Such application probably assured good distribu-
tion of iron within the treated soil mass, thus permitting more contact
with.a restricted root system.

7 In addition to these direct effects of lime in the soil there are
indirect effects -- for example, the altered solubility of phosphorus.
Sendory (110) noted that calcium phosphate was more soluble in serum than

in saline solution and that serum contains traces of sodium bicarbonate.

This effect was confirmed in the soil solution (#6). Brown et a1. (23,
26, 91) also observed this and noted that high HCO3 with high phosphorus
reduced leaf-iron and produced chlorosis. This indirect effect of HCO3
has been studied extensively as a major factor in lime induced chlorosis.
Others (59, 108, lhO) have shown chlorosis to be more of a problem on
calcareous soil if phosphorus is also high. And still others (12, 27,
#1, 98, 106, 113) have shown that high phosphorus alone can cause chlo-
rosis. Hageman and Hartman (52) observed phosphoric acid to give poor
results as an acidifying agent for HCO3 solutions compared to sulfuric
and nitric acid. 0n the other hand, Franco (#1) observed good growth
without chlorosis with high KHQPOu maintaining a solution pH 5.5 in
Hoagland solution. Bingham and Martin (13) had similar results with
high phosphorus in three soils under conditions that did not result in

an increase of soil pH.
Changes within the Plant

In addition to the direct and indirect effects on the soil solu-
tion as previously discussed, high lime is known to induce changes in the
plant that may influence iron nutrition. Changes in mineral composition
would of course be expected to follow changes in the soil solution. Ben-
nett (9) in reviewing the early literature reported a consensus that the
ratio of potassium to calcium in chlorotic leaves was higher than that
in green leaves. In fact, many of the papers that he reviewed reported
less calcium in the leaves of plants growing on a calcareous soil than

those on a non-calcareous soil. Others (6h, 87, 138) have reported simi-

lar observations.

\,./

Baxter and Belcher (6) observed, though, that root tissue of chlo-
rotic citrus had more calcium than did that of green plants in the same
orchard. In controlled experiments, Wadleigh, et al. (130) found that
bicarbonate resulted in a decrease in leaf calcium and an increase in
leaf potassium. Heller, et a1. (55) and Wall, et al. (132) similarly
found that bicarbonate depressed the accumulation of calcium and mag-
nesium in plant tissues. Cain (31) varied the calcium concentration in
sand-cultures and found in the leaves a high K:Ca ratio at low calcium
treatments and a low K:Ca ratio at high calcium, as would be expected.
Leaf phosphorus has often been observed to be higher in chlorotic plants
than in green ones (9). This has been shown to be brought about by
high levels of bicarbonate (37, 132). Furthermore, deKock (37, 38)
pointed out that the ratio of PzFe in leaf tissue was higher in chlo-
rotic plants.

Internal pH of the plant can also be affected by lime. There are
reports that no differences exist between the pH of expressed sap of
chlorotic and green plants (101) and that the growing of plants in solu-
tions of different pH did not alter their sap pH (3). 0n the other
hand, there are reported differences. Baxter and Belcher (6) found root
pH to be higher in chlorotic citrus plants on calcareous soil than in
green plants although there were no differences between leaf pH of chlo-
rotic and green plants. Haas (51) observed a higher pH of expressed sap
from plants of clover, barley, timothy, and wheat on limed plots than
of those on non-limed plots. This was true also of the root tissue of
mustard, but no difference was found for corn or oats. The sap of chlo-
rosis-susceptible cultivars of corn were found to have a higher pH than

that of resistant cultivars (51). Higher pH of expressed sap of corn

\l \

\f/

and oats growing on limed plots than of those plants on non-limed plots
has been reported (80). wadleigh and Shive (131) and Colgrove and Rob-
erts (36), studying the effects of ammonium and nitrate forms of nitro-
gen at different solution reaction, found tissue pH to correlate with
solution pH, and also tissue pH of plants on nitrate nitrogen was high-
er than that of those on ammonium at the same solution pH.

Rogers, et a1. (50, 109) also found differences among species as
well as daily variation within a plant - lower pH during the night.
Furthermore, the latter observed that there was more iron in solution
in plant sap of low pH than in that of high pH. There was a higher con-
centration of iron in plant sap than in nutrient solution of the same pH.
Furthermore, there were differences among tissues, with the pH of xylem
being lower than the pH of phloem, root cortex, or leaf parenchyma. Iron
apparently accumulated along pH gradients between tissues (50, 65, 109).

Differences also exist in biochemical composition of plants as a
result of exposure to high lime. The organic acid content was found to
be higher in chlorotic than green plants of 17 species (63). Citric
acid increased in apple foliage before any visual manifestation of chlo-
rosis developed. Increased malic acid in some species was related to
increased calcium absorption resulting from increased supply. Su and
Miller (119) observed higher citrate in chlorotic soybean than in green
plants when the chlorosis was due either to withholding iron from the
nutrient solution or to induction by high bicarbonate, phosphate, or man-
ganese.

Clark, et a1. (35) and Bedri, et al. (8) noted that roots of some
plants could absorb and fix 002 into organic acids. Others (2A, 103,

130) have noted the absorption of bicarbonate with a subsequent increase

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in organic acids and more recently this has been confirmed with radio-
isotope labelling techniques (8, lo, 116). Jacobson (71) proposed that
the synthesis of organic acids is one way in which plants maintain ca-
tion - anion equivalence when they are absorbing more cations than
anions, as demonstrated when barley absorbs KHCO3.

The respiration of potato disks was affected by the pH of the am-
bient solution, it being optimum in neutral solutions and slightly re-
duced at both lower and higher pH (118). Miller (914) and his students
observed fairly uniform respiration rates over the pH range h.5 to 9 for
enzyme systems in vita—33. However, bicarbonate reduced the activity of
cytochrome oxidase and prior eXposure of roots to high bicarbonate caused
such reduction even when bicarbonate was not present in the substrate
during respiration measurement (93). Similarly, Brown, et al. (17, 19)
found catalase activity to be reduced in some species when grown on
calcareous soils. Both of these research teams observed the reduction
of respiration to be more pronounced in those plants susceptible to lime
induced chlorosis than in resistant ones (19, 91). Furthermore, Miller
(91) noted that high phosphorus reduced the activity of cytochrome oxi-
dase in preparations from soybean root, although some phosphorus was es-
sential for activity; and Huffaker and Wallace (62) found that high
phosphate reduced phosphoenolpyruvate carboxylase and carboxykinase pre -
parations from orange leaves and fruits.

Species of plants differ in their susceptibility to chlorosis (23,
he) as do cultivars of a species (9, 18, 23, 136), and even individuals
of a cultivar (6, 80, 87, 90, 1%, 122, th). Such differences in sus-
ceptibility could be due to differences in iron requirement, but they

any also be due to differences in the ability of the plant to absorb,

9

translocate, or utilize iron. Some evidence has been presented to re-
late plant differences to differential ability of roots to absorb (22,
2k, 135), or translocate iron (133). Soybean cultivars differed in
their absorption of bicarbonate and subsequent organic acid synthesis
(8), and respiration rates of susceptible species were reduced by bi-
carbonate; whereas, this was not true for resistant plants (9%).

There are also indirect plant effects brought about by high lime
that complicate our interpretations of causes and effects. Foremost
among these is the reduction in growth. This has been Observed in most
cases of chlorosis (9, 18, 122, 136), even when the cause is not typical
of lime induced chlorosis (77). The cause of reduced growth has been
attributed to high calcium concentration in the root environment (#3),
an adverse pH of the soil (1, 2, 3, h, 60), high bicarbonate and C02
(29, #2, SS, 130), or high phosphorus (60). Root extension is reduced
under high bicarbonate conditions (116). In addition to overall re-

duction in plant size there is reduction in dry weight of the tissues

(105. 123, 136)-
Effect on.Absorption

,Aspects of the problem so far discussed dealt with indirect evi-
dence that internal changes in the plant as brought about by exposure
to lime might possibly affect the ability of the plant to absorb and
translocate iron. Such changes, if they occur, would complicate any ef-
forts to correct or prevent chlorosis of plants growing on calcareous
soils. Direct evidence for such changes is limited, but it is being de-
veloped.at the present, and of particular importance in such studies

is the use of radioisotopes to facilitate accurate measurements of the

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small quantities of iron moved during short periods of time.

Evidence has been presented that indicates that some of the genetic
difference in susceptibility is due to a differential ability to absorb
iron. Brown, et a1. (20, 22, 25) and Bhan and Wallace (11) found that
Hawkeye soybean absorbed more iron than did PI 5h6l9-5-l, and Brown (25)
pointed out specifically that a difference in the rate of reduction of
ferric iron in the ambient solution accounted at least in part for the
difference in absorption of the iron.

Reference has already been made to the reduction in respiration
rates of plants afflicted with lime-induced chlorosis. Brenton and Ja-
cobson (15, 16) found that when root respiration was reduced by dinitro-
phenol, iron absorption and subsequent accumulation in the tops of pea
plants was reduced. This is consistent with earlier observations by
Hoagland (57) and with Kramer's discussion (76) to the effect that an
expenditure of energy obtained from metabolic processes requiring oxy-
gen is necessary for active absorption against a concentration gradient.
This is further supported by observations that aeration of nutrient sol-
utions facilitates the absorption of such minerals as calcium, magne-
sium, phosphorus, nitrogen, and potassium, and that CO2 represses such
absorption (33). Linsay and Thorne (81) showed oxygen to increase iron
absorption from solution unless that solution was high in bicarbonate,
in which case the precipitation of iron was apparently accelerated. De-
pendence on high rates of respiration for iron absorption also probably
accounts for the observations that low soil temperature and high soil
moisture (with associated low soil oxygen) increase the susceptibility

of plants to chlorosis (30, 108, 109).

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Insoluble frits (151) and magnetite (3h, 71) can be sources of
iron in the presence of lime, but Chapman (3h) observed that chlorosis
could result when the lime level was high unless the amount of finely
ground magnetite was increased in the soil. He did not resolve the ques-
tion of how this reduction was brought about, but it is likely that the
extension of the root system was reduced under these conditions, and
higher magnetite levels increased the number of iron particles to com-
pensate for root reduction and thus maintained the necessarily high root-
particle contact. Guest (#9) also noted a reduction in iron absorption
from a magnetite source under conditions of high calcium and magnesium
carbonate.

Very little information has been developed pertaining to the effects
of lime-induced chlorosis on foliar absorption of iron (133). The form
of iron applied is a factor in the foliar treatments: inorganic forms
are absorbed more readily but chelated forms are translocated more read-
ily (85). In laboratory experiments inorganic forms were more effective
than organic, but in field tests better recovery from chlorosis was ob-
tained from some of the chelated forms (85). These observations were
not related to the level of lime or condition of chlorosis; however,
Teubner, et al. (121) have provided some information about foliar ab-
sorption in general.) They found that light and warm temperatures sti-
mulated foliar absorption. The pH of the treatment solution was a factor
with different optima for different elements. Surfactants increased the
wetting of the leaf but decreased total absorption because the quantity
of treatment solution that would remain on the leaf was reduced. Wal-
lace and Bedri (13h) found more foliar absorption of iron from treatment

solutions of low pH than from those of high pH.

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There is some direct evidence that lime reduces root absorption
of iron. Steward anlereston (118) observed an effect of pH on root
absorption of potassium. Hoagland and Broyer (58) noted bicarbonate
repression of potassium absorption. Hale and Wallace (53) and Walli-
han (1&2) reported that bicarbonate reduced the root absorption of
iron. Cain (31) found a reduction of iron absorption as a result of
high calcium concentration in the rooting medium, as did Lingle, et
a1. (82) for magnesium. Hale and Wallace (53) observed that phosphorus,
in the presence of bicarbonate, reduced root absorption of iron, whereas

phosphorus without bicarbonate did not.
Effect on Translocation

The effect on translocation is not easily separated from that on
absorption, but some tentative conclusions have been drawn. Rediske
and Biddulph (106) concluded that absorption and translocation of iron
was greater at low pH that at high, and that more iron appeared to be
translocated to the tOps of beans when iron and phosphorus were approxi-
mately equimolar (2 x 10-5M) in the nutrient solution. High bicarbonate
impeded the translocation of calcium and iron from root to shoot (A5).
Using radioactive tracers, Wallihan (1&2) found no iron translocated to
the t0ps of orange plants in five hours from high bicarbonate solutions,
whereas there was some from the zero bicarbonate treatment. The data
were such as to permit the conclusion that both absorption and translo-
cation were reduced by high bicarbonate. Brown and Jones (25) concluded
that the resistant Hawkeye soybean translocated more iron than did the

susceptible PI cultivar.

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Using a split root technique, Brown, et al. (26) found that high
bicarbonate in the lower root medium did not reduce translocation of
iron absorbed by the higher portion of the root system, but when both
bicarbonate and.phosphorus were present in the lower medium there was
a reduction. Similarly, there was no reduction in iron absorption from
high calcium in the lower medium, but sufficient reduction from calcium
and.phosphorus together in the lower medium that soybean plants became '
chlorotic with concomitant reduction in leaf-iron (28). High phospho-
rus with either bicarbonate or chloride salts of sodium reduced the
translocation of iron in other experiments (91).

Olsen (98) early postulated that iron was precipitated with phos-
phorus within the plant when grown at solution pH 6 - 7 unless the source
of iron was iron hulate. Thorne (122) made reference to several such re-
ports in his review. Some evidence given for this phenomenon is that
root iron is very high compared to foliar iron (83, 87, 10%, 122). More
direct are the observations of Hoffer (59) and Loehwing (83) that iron
acmmulsted in the nodes of corn. Hoffer (59) indicated that liming and
applying phosphorus to the soil reduced this accumulation by reducing
the amount of iron absorbed; whereas, Loehwing (83) related such.preci-
pitation to a higher sap pH under the influence of high lime. Biddulph
(12) demonstrated the probable internal.precipitation of iron from high
phosphorus in the rooting medium. Brenton and Jacobson (16) also showed
accumulation of iron in root cortex and in the leaf parenchyma near vein-
let endings, although this accumulation was not studied in relation to
lime or’phosphorus induced chlorosis. High lime was found by North (93)
to reduce the translocation of iron after injection into the root; but
he, like Brown and Holmes (20), found no reduction in translocation when

iron was introduced into the stem.

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Translocation from, and mobility within the leaf has been studied
to a very limited extent. Brown (18) in his review concluded that the
mobility of iron in the leaf varied with the form of iron applied. Such
reports as that of Biddulph (12) support the commonly held position that
iron is not translocated readily from the leaf, even when introduced by
infusion through the cut leaf. Wallace and Bedri (13%), however, de-
termined that there was some translocation of foliar applied iron and
that the amount translocated was proportional to the amount absorbed,
being in the range of one to ten percent. Lunt and Kohl (85) found that
translocation varied with the form of iron applied to the leaf. Trans-
location of radioactive iron from the leaf to which it was applied was
reduced by high calcium, magnesium, phosphorus, copper, manganese, and
iron, itself, in the rooting medium (95). Growing plants under high bi-
carbonate for a few days prior to foliar application of iron increased
the amount translocated from the site of application (39). And al-
though it was translocated from the site of application to the leaf, iron
remained in the petiole and stem under the influence of prior treatment
with high bicarbonate, high pH or high phosphorus, as much as ho% of the
iron translocated out of the treated leaf remaining in the conducting
tissue, contrasted to 28% under low pH, bicarbonate, and phosphorus
treatments.

In spite of such evidence for the precipitation of iron within and
contiguous to conducting tissues, attempts to correct chlorosis by in-
fusion or injection of chelating agents have not been successful in re-
dissolving and mobilizing that iron to the extent that chlorosis was

corrected (111).

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Tiffin and Brown (126, 127) presented evidence of malic and malonic
acids acting as carriers of iron in the conducting tissues of soybeans.
Jones and Eagles (73) showed that Cluoe was fixed by leaves as products
which were translocated to other leaves. Primarily translocation of
these products was to young immature leaves of the plants and to the less
mature portions of mature leaves. To some extent it was translocated to
older leaf tissue, but only into the veins and veinlets, accumulating so
as to Show by autoradiography the veinal pattern of the leaf in a manner
closely resembling patterns observed in iron translocation studies (15).
Papers dealing with the root fixation of C02 and bicarbonate have re-
ported specifically that malic and malonic acids were increased in the
roots under the influence of high bicarbonate (35, 69, 130). Others
(29, 63, 130) have shown an increased organic acid content in chlorotic
leaves -- namely, malic, citric, oxalic, and tartaric acids. Wallace,
et al. (137) even found a higher content of citric and malic in the
chlorotic portion of variegated leaves of ivy and camellia than in green
portions. A possible expectation as a result of these observations
would be that chlorotic plants growing under high bicarbonate conditions
would have a high production of organic acids in the root system, and
these organic acids would facilitate the translocation of iron absorbed
through the roots. However, Bedri, et a1. (8) found that the C11+ fixed
in the roots after being absorbed as C02 did not move upward into the
shoots and leaves.

Iron has generally been considered not to be redistributed from
old to young tissues, since deficiency symptoms appear in the youngest
tissues first. However, plants which have been growing with an adequate

iron supply for some time often can produce considerable normal-appearing

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leaf tissue on a medium devoid of iron (100, 128). Young azalea plants
were grown in sand-solution culture involving iron-free solutions for
210 (100) and 150 days (128) before chlorosis developed. For plants to
have grown that long without showing symptoms of iron deficiency there
either had to be an internal iron reserve which could have been trans-
located to the meristematic tissues, or sufficient iron was present in

the nutrient solutions used to maintain threshold supply.
Iron Utilization

..There are numerous reports (6h, 80, 87, 90, 101, 113) of chlorotic
leaves containing as much iron as green leaves, and this has been con-
sidered evidence that some of the iron in the leaf was inactive in chlo-
rophyll synthesis.

Oserkowsky (102) developed this concept to a considerable extent
by correlating acid-extractable leaf iron with chlorophyll. By extra-
polating to zero chlorophyll, he established the amount of leaf iron
that was not functional in chlorophyll synthesis, and this fraction he
called "inactive." McGeorge (87) concurred in principle. .Acid-soluble
iron was decreased by bicarbonate, whereas total iron was not (29).
Brown (18) concluded that "active" iron was that iron soluble in nor-
mal hydrochloric acid. The form in which the "inactive" iron occurs
has not been established, although several workers (12, 26, 37, 98) have
suggested iron phosphates.

Bennett (9), on the other hand, argued that most of the iron in
the plant is not in solution but is absorbed on protein (enzymes) and as
such is not extracted by acid but is metabolically active. Not all pro-

tein-iron would be involved in chlorophyll synthesis, but a considerable

\1/

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17

portion of it would be. Therefore, he did not accept the hypothesis
that acid-extractable iron was the fraction active in chlorophyll syn-
thesis. Jacdbson (66) also stated that some of the iron that he found
to be inactive in chlorOphyll synthesis was possibly active in other
processes in the leaf cell.

Direct studies of the role of iron in chlorophyll synthesis have
Just begun. Cheberek and Martell (32) cited evidence for the role of
iron in the condensation of pyrrole groups to form urophophyrin and in
coordinating bonds in the cyclic condensation of porphobilinogen groups
as they assume the spacial configuration of chlorophyll. Magnesium dis-
places iron from the cyclic molecule, and thereafter the iron is avail-
able for further condensation of pyrrole or porphobilinogen groups.
Granick's pathway (#6) for chlorophyll synthesis from protoporphyrin -

9 indicates a similar role for iron. As these roles of iron are further
defined, our understanding of the effect of bicarbonate in the root med-
ium should be improved.

Jacobson (66) demonstrated the importance of cleaning leaves before
analysis. The iron content of leaves washed in 0.3 N hydrochloric acid
had.on1y 6% the iron content of nondwashed leaves. Others (18, 122, 120,
130) have further established this point by comparing different methods
of washing. Several later reports (66, 68, 79, 97, 111, 130, lhl) have
indicated that with careful sample preparation good correlations were
found between the total concentrations of iron and chlorophyll in the
leaf. Some (66, 97, 111, 122) have correlated iron with chlorophyll but
indicated that it was not possible to establish.a "critical“ level, since
incipient chlorosis occurred at different levels of iron indifferent

orchards, species, or clones. More consistent differences between green

l8

and chlorotic leaves have sometimes been found when the iron content was
expressed on a leaf area basis (lll). Porter and Thorne (105) on the
other hand, found that total iron within the plant was reduced by bicar-
bonate, but not the ratio of iron to leaf dry matter, since the dry
weights of plants were also reduced under high bicarbonate conditions.
In addition to careful sample preparation, it is important that
the iron supply be constant. Iron in leaves increased greatly after
treating deficient plants with available iron (68, 79, 80). This would
indicate that fluctucations in iron supply result in fluctuations in
iron concentration in the leaves, and probably leaf iron fluctuates more
rapidly than chlorophyll content. Oertli and Jacobson (97) were able
to establish a correlation between the iron concentration in the leaf
and that in the root medium by using large volumes of nutrient solution
for each root system and by changing the solutions frequently, in con-

trast to reports of no correlation (106).
Synthetic Chelates

Synthetic chelates have been studied as a means of correcting lime-
induced chlorosis, and hence offer some information about the nature of
the disorder. Early investigations established that chelation with or-
ganic acids and humates improved the stability of iron at high pH (7, 12,
38, 61, 8h), but not to the extent of making it absolutely stable at any
solution reaction (11, 37, 85). Bear (7) indicated that ferric ethy-
lenediamine tetraacetate (EDTAsFe) was stable to pH 8 or 9, and ferric
diethylenetriamine pentaacetate (DTPA-Fe) to pH ll. Jacobson (67) found
100 per cent of initial EDTA-Fe still in solution after 90 days at pH 6,

82 per cent at pH 7, 70% at pH 8, and 10 per cent at pH 9. Furthermore,

\
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19

the chelate-metal system tends to maintain a constant concentration of
free metal in solution (103).

This increased solubility of iron at high pH undoubtedly accounts
for some of the success obtained in correcting or preventing chlorosis
under high lime conditions (37, 60, 98, 99, 133, lhO, lhh), though this
would not likely be the case in the acid, sandy soils of Florida where
chelates have also been successfully used (79, 11h).

Stabilizing iron in this manner also makes possible observations
about the effects of soil conditions on absorption and translocation of
iron independent of its precipitation from the soil solution. By using
EDDHA, Bhan and Wallace (11), and Jeffreys, et a1. (70) showed that high
pH reduced root absorption of iron, as did the nitrate form of nitrogen
relative to ammonium. This was evident from the accumulation of iron in
both the root and taps of the plants. High bicarbonate reduced the ac-
cumulation of EDTAéFe in leaves and stems but not in roots, i.e., trans-
location of the absorbed iron was reduced (81).

Some evidence has been presented which indicates that synthetic
chelates increase the translocation of iron within plants, and this
might account for the improved response under high bicarbonate conditions.
Weinstein, et al. (1&3) reported higher leaf-iron and lower root-iron
in plants receiving EDTAéFe relative to those receiving ferrous sulfate.
The same authors (lhh) noted that supplying the sodium salt of EDTA to
one portion of the root system with the other portion in ferrous sulfate
prevented chlorosis under conditions that induced chlorosis when EDTA was
not supplied. Chelation with ethylenediamine di-(o-hydroxy phenylacetate) -
EDDHA - and with.DTPA increased the translocation of root-absorbed iron,

even when the chelating acid was supplied either before or after tagged

\/

20

iron sulfate.(70).

Wallace, et al. (139) measured the relative absorption and trans-
location of the EDTA molecule and the chelated iron and found essentially
equimolar accumulation. This indicated that the iron was absorbed and
translocated as part of the chelated molecule. Lunt and.Koh1 (85) also
found increased translocation of iron when it was applied to rose
leaves in a chelated form. By contrast, Neher, et al. (95) observed no
increase in the translocation of chelated iron over that of ferric chlo-
ride.

More recent work by Tiffin and Brown (12%, 125, 126, 127) has ques-
tioned the possibility of translocation of an intact, chelated iron mole-
cule. Tiffin, et al. (125) first reported that stem exudate of soybean
contained more total iron than EDDHA-chelated iron when EDDHA-iron was
supplied to the rooting medium, and concurrently free EDDHA was found to
accumulate in the nutrient solution as iron was absorbed. At the same
time EDDHA was found to be absorbed since the chelating ability of the
exudate increased when the roots were supplied the free-acid form of
EDDHA. In a later paper the same authors (127) showed that Clh labelled
chelates were absorbed, but the electrophoretic migration was not the
same as that of the intact molecule, indicating that some degradation
had taken place within the plant. Wallace and North (138) reported that
the nitrogen from Nlu-labelled chelate molecules remained in the residue
of plant tissue after extraction with water, alcohol, and acid, which
also suggested that the chelate molecule was not intact within the

plant.

MATERIALS AND METHODS
Iron Solubility in Nutrient Solution
Ezperiment I: Stability of Iron in Non-aerated Nutrient Solution

The stability of iron in nutrient solution was determined using
radioiron (Fe59). Half-strength Hoagland's number one solution
(Appendix Table l) was prepared and adjusted to pH 4, 5, 6, 7, or
8 by adding either HCl or NaOH. Aliquots of 50 ml to which were
added 0.“ uc of Fe59c13 were placed in each of three test tubes and
stoppered with sterile cotton. This gave an initial acitivity of
1000 cpm.per m1 as determined with a gas-flow'Geiger-Mueller tube
used with a Versamatic sealer.

A one-milliliter aliquot was removed from each test tube after
one-half, 1. 3, 6, and 12 hours, and after 1, 2, 3, 4, 5, 9, 11. 18,
and 25 days. It was evaporated to dryness on a stainless steel planchet
and its radioactivity determined.

The percentage of iron remaining in solution was calculated by
dividing the activity of each sample aliquot by the activity of the

initial aliquot and multiplying by 100.
The pH of each treatment was determined on an aliquot of nutrient

solution at each sampling time by means of a Beckman Zeromatic pH
meter and was not observed to change.
Three replications were provided in a completely randomized

design.

21

22

Haggariment II: Influence of Lime and Ammonium Citrate on Iron Stability

in Aerated Nutrient Solution

The influence of ammonium citrate on iron stability in aerated
nutrient solution of pH 5 and low lime concentration was contrasted
with that at pH 7 with high lime.

In this experiment, as well as in those that follow, the term
"low lime" refers to a solution to which no carbonates or bicarbonates
were added. The pH was adjusted to pH 5.5 by adding ammonium sulfate
and the solution was aerated with compressed air. A trace of bicar-
bonate was probably present since the air can be assumed to have
contained approximately 0.03% carbon dioxide. The term, "high lime",
refers to the concentration of calcium bicarbonate which resulted
from suspending finely ground calcium carbonate in the solution and
aerating with air enriched with carbon dioxide to a final carbon
dioxide concentration of 2% by volume. Calcium carbonate dissolved
in equilibrium with the dissolved carbon dioxide and resultant carbonic
acid as calcium bicarbonate. A calcium bicarbonate concentration of
approximately 3 millimolar was assumed on the basis of equilibrium
studies reported by Bradfield (14). After the system.had come to
equilibrium, solution pH was 7.0 and remained constant as long as
solid calcium carbonate was present. "High lime", therefore, refers
to the combined effects of calcium and bicarbonate at approximately
3 millimolar concentration and solution pH 7. These conditions are
indicated in all instances when the terms "low lime" or "high line" are

used in this paper.
Iron was added to each 50 ml aliquot of nutrient solution in

aerated test tubes after the pH had stabilized at the desired pH.

23

Ferric chloride was added as one form of iron in sufficient quantity
to have 3 ppm iron in solution. In the other iron treatment. a stock
solution of ferric chloride was combined with ammonium citrate at a
molar ratio of 1 iron to 6 ammonium citrate and allowed to stand for
a few minutes before being added to the nutrient solutions to give a
final concentration of 3 ppm iron. The instantaneous development of
.green color in the solution of ferric chloride and ammonium citrate
indicated that a reaction had occurred between the two compounds and
thus the ferric ammonium citrate complex was formed.

The solutions were analyzed colorimetrically after 20 hours by
the O-phenanthroline method described in Methods of Analysis (5 ).

The experiment was repeated for the two treatments containing
ferric chloride. so that observations could be made after 1. 2. 5.
and 7 hours by preparing fresh solutions of those treatments and
analyzing five-milliliter aliquots removed from each test tube at
each time of observation.

Three replications of each treatment were provided in a completely

randomized design.

ggperiment III: Influence of Lime and Phosphate on the Stability of

Iron in Aerated Nutrient Solution

The stability of iron as ferric ammonium citrate in aerated
nutrient solution containing either high bicarbonate or high phoSphate
or both was determined by sampling nutrient solutions in which Chrys-
anthemums were being grown as part of Experiment XVIII. which is

described in detail on page #3. The solution treatments are

2’4»

The solution treatments are described in brief in the table below.

 

 

 

p_H_ Hm; HLPQL; _ Aeration

5.5 o 0.5mM KHZPOL, Air

7.2 0.5mM K200 0.5mm mm. 2% 002 in air
excess CanB

7.2 0.5mM K200 0.5mM KHZPOL, 2% 002 in air
excess Cang 1.5mM KZHPOLI,

The base solution was half-strength Hoagland's number one with
Hoagland's "A" solution (Appendix Table l) for minor elements.
Ferric ammonium citrate was used to provide a concentration of
2 ppm iron.
Aliquots were removed from the crooks of aerated nutrient solution
in which young chrysanthemum plants were growing at intervals of 2,
24, and “-8 hours after fresh solutions were added to the crooks.
"Iron in solution was determined by the g-phenanthroline method.

Four replications were provided in a randomized complete block

dOSigno

lime-Inmced Chlorosis in Chrysanthemums and Azaleas
merinent IV: Effects of High Lime on Growth and Chlorosis of

Glrysanthenmms

'1 comparison was made of plant growth, development of chlorosis.
and iron content of leaves of chrysanthermms resulting from high-
lime induced iron stress with that resulting from iron stress caused
by Withholding iron from the nutrient solution. Rooted cuttings of

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the cultivar Beauregard were selected for uniform size (four expanded
leaves) and well developed roots. Tue plants were transferred to each
two-gallon. glazed crock after washing the roots to remove the rooting
medium. The crocks were filled with half-strength Hoagland's number
one solution containing Hoagland's "A" solution for minor elements
and modified as indicated below to maintain the treatment conditions.
Aeration was provided by forcing air through aquarium aerating stones.
A masonite cover supported the plants by means of polystyrene plugs
fitted in the holes through which the root system and basal stem
passed.

mess plants. as well as those of the other experiments reported
here. were grown in a greenhouse having a minimum night temperature
of 60° 1?. A double layer of cheesecloth was suspended over the
plants along the south side of the experimental area, this shade being
maintained for all experiments conducted during the months of May
through August. The natural photoperiod was extended when necessary
to 16 hours with supplemental lighting of 20 r. c. intensity to main-
tain plants in a vegetative state.

The terminal bud was pinched out of each plant after one week
of growth and three lateral shoots near the apex were allowed to
develop.

Three treatments were provided. as follows:

(a) pH 5.0. no line. 2 ppm iron as ferric ammonium citrate

(b) pH 5.0. no lime. no iron

(c) pH 6.8, high lime. 2 ppm iron as ferric ammonium citrate

Va

26

The low pH treatments were obtained by adding ammonium sulfate at

0.5 millimoles per liter to Hoagland's number one base solution. The
high pH. high lime treatment was obtained by adding 0.5 millimoles

per liter of potassium carbonate and suspending finely ground C. P.
grade calcium carbonate in the solution and aerating with air enriched
to 2% 002.

Four replications were provided in a randomized complete block
design.

The plants had attained a height of about 18 inches and produced
about 22 nodes. after six weeks under these treatments. At that time
they were rated for degree of chlorosis and necrosis. The relative
scale for chlorosis was:

1. None
2. Slight yellowing
3. Monte yellowing with green vein pattern
4. Severe yellowing with some white areas
The scale for necrosis was:
1. None
2. Slight. along margins
3. lbderate
14'. Severe, with entire leaf affected in some cases.

At, that time the experiment was terminated. The non-necrotic
leaves from all shoots of each plant in each crock were removed and
combined as one sample. They were washed in 0.1 normal hydrochloric
acid in distilled water containing 0.5 ml per liter Tween-20 as a
wetting agent and were rinsed twice in distilled water. The leaves

were then dried in a forced-draft oven at 70° C. and their dry weights

O.

27

determined as an index of growth. The roots were also removed and
dry weights determined.

The dried leaf samples were ground to pass a 20-mesh screen in
a Wiley Mill fitted with bronze blades. For total iron the tissue
was extracted by ashing at 500° C. and dissolving the ash in hydro-
chloric acid and analyzed colorimetrically by the g—phenanthroline

method.

meriment V: Effects of High Lime and Iron Cnelation on Chlorosis

and Mineral Composition of Chrysanthemums

The effects of EDTA (ethylenediamine tetraacetic acid) chelation
of iron on iron accumulation in the leaf and prevention of chlorosis
under high pH and high lime conditions were determined. Four treat-
ments were established. as follows:

(a) pH 5. 5. no lime. 2 ppm iron as ferric ammonium citrate

(b) pH 5.5. no lime. no iron

(c) pH 7.0. high lime. 2 ppm iron as ferric ammonium citrate

(6.) pH 7.0. high lime. 2 ppm iron as ferric EDTA
The methods of controlling solution pH were the same as in
Dcperiment IV.

Two plants of Chrysanthemum morifolium. cv. Legal Tender. were
grown in each two-gallon glazed crock under the same methods of
culture as in Ecperiment IV.

Three replications were provided in a randomized complete block.

The degree of chlorosis that had developed in four weeks was
I‘ated. and the leaves that had developed on all shoots of both plants

-1.»

Ci

1...-

l!

'v
L

1

28

in each crock were removed and prepared for analysis. The leaf
samples were analyzed by spectrographic methods to determine leaf
concentration of calcium. magnesium. phosphorus. iron and manganese.
This analysis was perfbrmed in the tree fruit nutrition laboratory

of the Department of Horticulture of Michigan State University under

the supervision of Dr. A. L. Kenworthy (75).

Eageriment VI: Effects of Bicarbonates in Irrigation water on Growth

Chlorosis. and Mineral Composition of Azalea

The effects of pH. bicarbonate. and associated cation in the
irrigation water on the growth and chlorosis of azaleas was determined.
The azalea was selected because of its known sensitivity to high soil
pH and bicarbonate concentration. Young seedling plants grown from
hybrid seed lot ##9, a Hollis type produced by Dr. W} J. Haney. were
grown in four-inch clay pots containing German peat moss which had
an initial pH 5.2 and was of the type used extensively by Michigan
azalea growers because of its low pH and high buffer capacity. Plants
‘were fertilized weekly by applying 200 ml of a nutrient solution that
'was twice the concentration of Twigg and Link's azalea solution (125).
They'were irrigated daily by applications of 200 ml per pot of the

appropriate solution listed below:

(a) Distilled Whter

(b) Tap Water with 0.1 ml concentrated P55q+per liter
(c) Tap Whter with 0.25 ml H3PQu per liter

(d) Tap water

(e) Distilled water with 5 me Oa(HC03)2 per liter

(1') Distilled Water with 5 me NeHoo3 per liter

29

The tap water used was that at the Plant Sciences greenhouse and had
a pH of 7.# and an electrical conductivity of 45 mho x 10‘5 due
primarily to dissolved bicarbonates of calcium, magnesium, and
sodium. The calcium bicarbonate solution was prepared by suspending
5 me CaCOB per liter in distilled water and bubbling carbon dioxide
through it until the carbonate dissolved. The sodium bicarbonate
solution was prepared from chemically pure sodium bicarbonate and
distilled water. The quantities of acid added to the tap water were
sufficient to neutralize it. I

The pH of the first 50 ml of leachate from the pot was determined
at least once every two weeks. The additions of acid to the tap water
was adjusted as necessary to maintain the same pH as occurred in the
distilled water treatment. pH 5.5. The pH of the treatments receiving
tap water. calcium.bicarbonate, and sodium bicarbonate remained within
the range pH 7.0 to 7.4 throughout the duration of the experiment.

The plants were subjected to treatment conditions from July 26
until October 7 in a randomized complete block design with three
replications. They were soft-pinched to encourage lateral shoot
development. By October the shoots had terminated in dormant buds.
The leaves that developed during the experiment were rated for chlorosis
on.the relative scale, removed, washed, and analyzed Spectrographically
for calcium, magnesium, potassium, sodium. phosphorus, iron, manganese.
boron, and zinc. Leaf dry weight was determined and used as an index
cf growth.

The cold requirement of the plants was satisfied by subjecting

them to 34° F. for a period of 12 weeks (October 7 to January 5).

30

Then they were returned to the greenhouse and grown at 60° F. minimum
night temperature. By May 5, shoot extension was complete and leaves
had matured on the new shoots. The plants were segmented into leaf
tissue. lateral stems. and primary stems. Each tissue segment was
weighed after drying and dry weight used as an index of growth. The
tissues were analyzed for phosphorus and iron by spectrographic means.

The data were analyzed by analysis of variance with emphasis on the
effects of the irrigation solutions on the accumulation of these

elements in the various tissues. and particularly to determine if

iron accumulated in stems in association with phosphorus.

Root Absorption and Distribution of Radioiron

garment VII: Effect of High Lime on the Absorption and Distribution
of Iron in Qirysanthenmm

Three groups of rooted cuttings of _c_:_. morifolium. cv. Legal
Tender, were grown for two weeks in serated Hoagland's number one
solution plus Hoagland's "A" solution under the following conditions:

(a) pH 5.5 and 2 ppm iron as ferric ammonium citrate

(b) 133 5.5. and no iron

(c) pH 7.0. high lime, and 2 ppm iron as ferric ammonium citrate
10pr conditions were maintained by adding ammonium sulfate, and high-
PH. high-lime conditions were maintained by adding calcium carbonate
and aerating with 23% carbon dioxide as described for Experiment II.

After two weeks of treatment, the plants under treatment "a"

were green. but those under "b" and "c" were slightly chlorotic.

A‘.

r}

r?‘

31

At that time the plants were moved from.the greenhouse to the labora-

tory for the second phase of the experiment. Radioiron was used to
determine the effects of these treatments on root absorption and

distribution of iron.

The same treatments were continued during the 24- and 36-hour
absorption periods, utilizing fresh solutions. Radioiron (Fe59Cl3)
stabilized with ammonium citrate was added to these solutions in the
amount of 57 uc/l. Stable iron was also included at 2 ppm iron as
ferric ammonium.citrate. Absorption was allowed to occur under con-
tinuous illumination of 800 f. c. and at a temperature of 28° C.

A split plot design of five replications was employed with
treatment as main.plot and time as sub-plot.

At the end of each absorption period. the plants were removed from
the solutions and segmented into: (a) 'upper four leaves. (b) lower
four leaves. (c) stem. and (d) roots. The lower stem sections with
roots attached were washed by repeated dipping in acidified Tween-20
solution and then in distilled water. This was followed by soaking
30 minutes in Na-EDTA solution. flushing with running tap water for
two minutes, and rinsing indistilled water. They were then blotted
dry on absorbent tissues and the roots cut from.the stem. Ehch tissue
sample was dried at 70°C. overnight and weighed.

.The dry tissue samples were crushed in the bottom of paper cups
to give a uniform distribution on the bottom surface and their radio-
activity was determined with a Geiger-Mueller tube using a Nuclear-
Chicago sealer. The relationships of radioactivity and iron

concentration of the treatment solutions were determined by counting

32

one-milliliter aliquots. Total iron absorbed by each plant was deter-
mined by assuming a constant relationship of radioactivity to iron
concentration, and the percentage of the total amount that was in each

plant segment was calculated.

Qeriment VIII: Effect of High Lime on the Absorption and Distribution
of Iron by Girysanthemum Plants after Transfer to a Low-Lime Solution

A detersdnation was made of the effect of prior treatment with
high-lime conditions. and subsequent develOpment of chlorosis, on the
absorption and translocation of iron by chrysanthermlm plants after
their transfer to a solution of low-lime conditions.

Young rooted cuttings of _Q. morifolium. cv. Legal Tender, were
treated for two weeks under the same three conditions as described in
the previous experiment. Then the plants were transferred to the
laboratory and allowed to absorb radioiron under the same conditions
of light and temerature as before. Only one solution treatment was
used during the 22-hour absorption period: pH 5.5 obtained by adding
0.5mM ammonium sulfate to half-strength Hoagland' 5 number one solution.
Iron was supplied at 2 ppm as ferric ammonium citrate and radioiron
at 57 no per liter as ferric chloride stabilized with ammonium citrate.
The absorption procedure, sampling and determination of radioactivity
was identical to that described in Duperiment VII.

A completely randomized design with five replications was used.
The data for each tissue segment were analyzed separately by analysis
01' variance and mncan's Multiple Range Test for evidence of effects

01‘ pre-treatment.

lid-

”I

Y!

.1

11

3.3

Qweriment IX: Effects of Lime on Long-Term Distribution of Iron in

Chrysanthenmm

The redistribution, or long-term distribution of root-absorbed
iron in chrysanthemums was examined in this eacperiment.

Uniform rooted cuttings of Q. morifolium. cv. Legal Tender, were
selected and allowed to absorb radioactive iron from half-strength
Hoagland's number one solution modified with 0.5mM ammonium sulfate.
After 12 hours in this solution under continuous illumination at 800 f .c
and at 30° C., the plants were removed. Their roots and basal stems
were washed by flushing under running tap water for 10 minutes, dipping
in distilled water, and draining dry on tissues.

To determine initial distribution, nine plants were selected at
random and segmented into: (a) leaves, (b) stems, and (c) roots. These
were dried at 70° C., weighed, and their radioactivity determined by
counting by the methods described earlier. Other plants were prepared
for autoradiography by cutting the stem above the roots, cutting each
leaf petiole from the stem, and pressing each severed shoot portion
between blotter papers. After drying at 70° C., each plant shoot was
mounted on fresh blotter paper with each leaf in the same position as
it had grown on the stem. (Roots were not mounted, since previous
experience had proven that radioactivity of the roots was so high,
relative to that of the stems and leaves, that the film would be
flatly overexposed in the area contacting the roots.) The mounted
Plants were placed in an exposure box with only a thin film of plastic

between the plant parts and a sheet of x-ray film. A sheet of masonite

34

and a 3/16 inch thick sheet of steel separated each specimen from the
one above it as they were stacked in a light-proof box. After two
weeks, the exposed film was removed and developed in fresh x-ray
developer for 60 seconds, fixed, washed, and dried. The image on the
x-ray film was copied on photographic film and printed using constant
exposure and development.

Long-term distribution was established by utilizing the remaining
rooted cuttings and transferring them to half-strength Hoagland' 5
number one solution containing Hoagland's "A" solution for minor
elements and modified for treatment: conditions, as indicated:

(a) pH 5.0, no line, 2 ppm iron as ferric ammonium citrate

(b) pH 5.0, no lime, no iron

(c) pH 7.0, high lime, 2 ppm iron as ferric ammonium citrate
As in similar earlier experiments, the low pH treatment was provided
by adding ammonium sulfate at 0.5mM, and the high pH/high lime treat-
ment was obtained by adding 0.75mM potassium carbonate and equilibrating
solid calcium carbonate with 2% 002.

Three plants were grown in each one-gallon crock. Each crock
was considered that unit of replication, and there were six replica-
tions in a randomized complete block design.

The plants were pinched to remove the terminal bud after two
weeks, and immediately thereafter the lower lateral buds were removed
leaving only the upper three to develop. At that time one plant was
removed from each. crock and autoradiograms prepared.

At the end of the ninth week, one plant from each crock was

renewed and the roots and lower stems were again washed in running

35

tap water for ten minutes and blotted dry. The plants were segmented
into: (a) leaves from lateral shoots grown after pinching, (b) stems
of lateral shoots, (c) leaves from the shoot below the point of origin
of laterals, (d) stem below the point of origin of laterals, and (e)
roots. These tissue samples were dried at 70° C. to constant weight
and dry weight recorded, then ashed at 500° C. The ashed samples were
dissolved in 50% hydrochloric acid, transferred to paper cups, evaporated
at room temperature, and radioactivity was determined. An indication
of long term distribution or redistribution was obtained by calculating
the per cent of the total radioactivity of each plant that was
attrihlted to each segment of that plant.

Autoradiograms were prepared using the remaining plant from each
crock. Because of the size of the plants, it was not possible to
include the entire plant on one autoradiogram film (8" x 10").

Separate autoradiograms were prepared of the lower stem with its
associated leaves and of the lateral stem with its associated leaves

plus the leaf from whose axil it had developed.

mariner“. X: Effect of Lime on Long-Tenn Distribution of Iron in
Amae ‘

Uniform rooted cuttings of the Rhododendron hybrid, Alice Mueller,
an evergreen azalea of the type grown as a florist pot plant, were
allowed to absorb Fe59 through their roots from nutrient solution.
Eleven cuttings were provided with 80 ml of solution containing
26.9 no R59 (concentration 3’40 uc/l). They remained in this solution

for 72 hours in mu sunlight on a greenhouse bench. The roots were

36

washed by dipping in a container of water and then flushing under
running tap water. The rooted cuttings were then potted in clay pots
filled.with number four quartz sand, assigned to two treatments with
five replications in a completely randomized design.

One treatment consisted of a base solution of Twigg and Link's
(128) azalea solution of pH 5.5. The other treatment received nutrient
solution with sufficient calcimm bicarbonate (obtained by aerating
a calcium carbonate suspension with carbon dioxide) to maintain pH 7.
The treatments were continued for twelve weeks.

At the termination of the experiment, the plants were segmented
into: (a) leaves of sheets which developed from.apical buds that were
present at the time of Fe59 absorption, (b) stems of the new shoots,
(c) leaves which were present on the cuttings, (d) stem.within the

zone of leaf attachment of the original cutting, (e) basal stem, and
(r) roots. These tissue segments were dried and their radioactivity

determined. Autoradiograms were prepared of representative plants of
each treatment. Photographs were made of the mounted.plants and of the

xpray film image of those plants whose autoradiograms were selected for

presentation.
Fbliar Absorption of Iron

The method of treatment. in the series of experiments on foliar
absorption was similar to that described by Jyung (7n). Absorption
was determined on detached leaves with the blade placed in.a designated
treatment solution contained in a Petri dish (50 ml) and held in a
submerged position with a glass rod support. The cut end of the

37

petiole was coated with lanolin and supported on a glass rod of large
enough diameter to keep the end of the petiole out of the treatment
solution. During the absorption period, the leaves were illuminated
on a laboratory bench with fluorescent lamps (600 f.c.). and the air
and solution temperatures were maintained at 20° c. The top of the
dish was covered with a piece of rigid plastic which had been notched
to accomnodate the protruding petiole]. This cover reduced evaporation
and consequent change in salt concentration for the solutions.

The leaves were removed from the treatment solutions at the end
of the absorption period and washed in a standardized manner. Each
leaf was washed in a separate series 'of three containers of wash
solution. The first solution was 50 ml of distilled water containing
one drop of Tween-20. no second was‘a 0.06 millimolar solution of
Na-EDTA. The third was distilled water. The washing was accomplished
by dipping the blade in the wash solution and removing it, repeating
ten times. In addition,the leaf was left to soak in the chelate '
solution for one hour. Before changing the leaf from one solution to
the next the excess solution on the leaf was drained onto absorbant
tissue paper. The leaf was blotted dry between absorbent tissues at
the end of the third wash.

To provide an area basis for expressing absorption, the leaf area
was determined by tracing its outlineand measuring the enclosed area
with a planimeter. In most cases an alternate method was used, wherein
a plug of 2.27 square centimeters in area was removed with a cork borer.
The sample was then dried at 70° C.

Radioactivity of the whole treated portion of the leaf blade was
determined by placing the dried samples in paper cups and using a

38

Geiger-meller tube with a VersaMatic Sealer. That of the sample plugs
was determined by placing the dried plugs on stainless steel planchets
under a gas-flow Geiger-Weller tube fitted with a mica membrane window
and «playing the VersaMatic Scalar.

garment XI: Effect of Age of the Leaf on Rate of Foliar Absorption

of Iron

In the development of chlorosis the first leaves to be affected
are those being formed in the terminal meristems, and when chlorosis
is corrected by re-establishing an iron supply in the nutrient medium,
the leaves expanding from those meristems develop normal chlorophyll
content. Mature, chlorotic leaves on a lower position of the stem do
not recover. If thisis due to their not receiving a sufficient amount
of the iron being translocated through the xylem, and not due to an
inability to utilize that iron in chlorophyll synthesis, then by
supplying iron by foliar application to those leaves, their recovery
could be induced. The first experiment relating to foliar absorption
was designed to eammine the question of the effect of the age of the
leaf on its ability to absorb foliar-applied iron.

Girysanthemum plants, cv. Yellow Delaware, which had been grown
in a complete nutrient solution for six weeks were used. Leaves of

different physiological age were selected as follows: (a) young newly
expanded leaves from near the growing point, (b) mature hilly eaqaanded
leaves from the central portion of the sten (six to eight nodes from
the apex), and (0) old leaves from the base of the shoot. All leaves

appeared healthy and no chlorosis was present.

39

Iron absorption from a solution containing 0.3 millimolar ferric
chloride labeled with 10 uc Fe59 as ferric chloride plus 0.01% Tween-20
at pH 3.2 was observed. No precipitation of iron from the treatment
solution occurred during the duration of the absorption period.
Furthermore, no change in pH occurred, so a buffered systan was con-
sidered unnecessary and was not used. The radioactivity of this solution
was 617 cpm/umole of iron, as determined on the same instruments used to,
determine radioactivity of the leaf samples. The absorption period
was 214» hours. 15 minutes.

A completely randomized design with five replications was used. The

absorption rate for each sample was determined in terms of mumole iron

per square centimeter per hour.

fieriment XII: Effect of Chlorotio Condition on Rate of Foliar

Absorption of Iron

ercts of pre-treatment to induce chlorosis on the capacity of
the leaves to absorb foliar-applied iron was next determined. Chrysan-
themum plants of the cultivar. Legal Tender, were grown under the following
nutritional environments:

(a) pH 5.5. no lime, 2 ppm iron, as ferric ammonium citrate

(b) pH 5.5. no lime, no iron

(c) pH 7.0, high lime, 2 ppm iron as ferric ammonium citrate
lhese conditions were maintained in aerated nutrient solution of
Hoagland's number one solution in the same manner as described in
earlier experiments. The plants under "a" were green and vigorous,

those under “b" and "c" were slightly chlorotic and slightly stunted
at the time that leaves were utilized for the foliar absorption studies.

#0

One leaf of comparable physiological age was selected from each
of five plants grown on each of the above treatments. The selected
leaves were free of mechanical injury, insect or disease infestation and
were selected from among those which had expanded while the plants were
subjected to differential nutritional conditions.

The five leaves of each treatment were allowed to absorb radioiron
according to the detachednleaf method described previously. The
treatment solution in this case was 0.3mM ferric chloride in 0.01%
Tween-20, deionized water, containing F059C13 at 20 uc/l. Forty milli-
liters of this solution was provided in each Petri dish. The absorption
period was 18 hours, 20 minutes with continuous light.

A completely randomized design with five replications was used.

moment XIII: Effects of pH, Sodium, and Bicarbonate on the Rate

of AbSorption of Iron

The following treatments were employed to determine the effects
of solution pH, sodium.concentration and bicarbonate concentration on

the rate of foliar'absorption of iron by chrysanthemum.

P“ 8223;” was?“ that
(a) 5 0 O (Tris*)
(b) 5 3 0 (sodium acetate)
(c) 7 0 0 (Tr-15)
(d) 7 3 0 (sodium acetate/Tris)
(e) 7 3 3 (sodium carbonate)

* Tris is tris-hydroxy, methoxy methane

1+1

Green leaves of the cultivar. Legal Tender, were allowed to absorb
iron in the above solutions at a concentration of 0.3mM as ferric

chloride labelled with Fe59c1 at 21» uc/liter. The period of absorption

3
was “.33 hours under conditions of continuous illumination of 600 f.c.
and temperature of 28° 0. Leaf disks were removed, dried, and their
radioactivity determined by counting in a scintillation well using a

versaMatic counter.

Five replications were provided in a completely randomized design.

ggperiment XIV: Effect of Chelation on Stability of Iron in Foliar

Treatment Solutions containing Bicarbonate

The effects of form of iron on rate of absorption by chrysanthemum
from solutions of high bicarbonate and.high pH was next determined by

observing the absorption of Fe59 from the following solutions:

pH sodium. Bicarbonate , iron pH
(mM) (mM) (form) Control
(a) 5 3.0 O FeCl3 (sodium acetate)
(b) 7 3.0 3.0 Feel3 (sodium carbonate)
(c) 7 3.2 ’ 3.2 FeAC* (sodium carbonate)
(d) 7 3.6 3.6 ' , FeEDTA** (sodium carbonate)

* FeAC is ferric ammonium citrate

** FeEDTA is ferric ethylene diamine tetraacetate
Endioiron as F659C13 was added to give a concentration of 2.h cpm/mumole
of iron, and where chelation was desired the chelating agent (ammonium

citrate or ethylene diamine tetraacetate)was added to give 1.5 moles
for each mole of iron. Tween-20 at 0.01% was included in each treatment.

1+2

Forty-milliliter aliquots of each treatment solution were allowed
to stand in a Petri dish under conditions typical of an absorption
experiment except without a leaf. At the prescribed time these aliquots
were transferred to paper cups, and, after the precipitate had settled
to the bottom, five one-milliliter aliquots were taken for determination
of radioactivity.

The treatments were replicated five times in a completely randomized

design. The absorption period was 17 hours, 15 minutes.

gperiment XV: Effect of Chelation on Rate of Foliar Absorption from

Solutions of Low pH.

This experiment was conducted to determine the absorption rate of
chelated iron by leaves at pH 3.0 and in a solution devoid of sodium or
other salts.

Radioiron was chelated with ammonium citrate and HJTA and its
absorption contrasted with that of non-chelated radioiron (FeClB).

The iron concentration was 0.3 millimolar in deionized water containing
0.01% Tween-20. The method of treatment was the same as for the other
detached-leaf experiments. The stability of iron in the treatment
Solutions was determined by removing one-milliliter aliquots from a

’40 m1 aliquot of each treatment solution that was allowed to stand in
a Petri dish without a leaf during the course of the experiment. The
radioactivity of each aliquot taken upon terminating the experiment
"33 compared to the activity of the initial aliquot removed at the
beginning of the experiment, and the percentage of iron remaining in

solution was calculated.

1+3

Leaves were selected from plants, cv. Legal Tender, which had
been grown for a few'days under low-iron conditions and were showing
slight chlorosis. Five replications were provided in a completely

randomized design. The absorption period was 10 hours.

Experiment XVI: Effect of pH on Foliar Absorption of Iron

 

Tb examine more fully the influence of pH in the absence of
bicarbonate, the absorption of Fe59 by green chrysanthemum (cv. Legal
Tender) leaves was followed from solutions at pH 3, 4, 5, 6, 7, and
7.8 using Tris (tris-hydroxy, methoxy methane). The iron was not
chelated so that differences in stability at the given pH value could
be observed.

Five replications were provided in a completely randomized
design. Absorption was allowed to proceed for 16 hours under con-

tinuous illumination at 600 foot-candles and a constant temperature

of 29° C.

High Lime and Phosphate Effects

_Experiment XVII: Effects of High PhOSphate Concentration

 

In the first experiment of this series, the effects of increasing
concentrations of soluble phosphate in the root medium on the absorption
01' iron and maintenance of leaf chlorophyll were studied. Four phos-
Phate levels were established using Hoagland's number one solution
as the base: (a) 0.5 mM, (b) 1.0 mM, (c) 2.0 mM, and (d) 4.0 mM as KHZPOQ.

Potassium chloride was added in sufficient quantities to treatments

an,

(a), (b), and (c) to provide equal concentration of potassium. Iron
was supplied in all treatments as ferric ammonium citrate at a rate of
2 ppm iron. Minor elements were supplied by adding Hoagland's "A”
solution.

We cuttings of _guysanthemum morifolium, cv. Mermaid, were grown
in each two-gallon crock with pinching and pruning to three stems for
eight weeks under a photoperiod of 16 hours. At the end of the
experiment, leaf and root samples were collected, washed, and prepared
for phosphorus and iron analysis. '

The tissue samples were analyzed for phosphorus by the ammonium
molybdate method and for iron by the g-phenanthroline method (5).

The extract for both analyses was prepared as specified for phosphorus

analysis.

kerimemt XVIII: Effects of Various Forms of Orthophosphates at High
Concentration on Growth, Chlorosis, and Tissue Accumulation of Iron and

Phosphorus by Two Cultivars of mrysanthemum.

The effects of the form of phosphate on growth, chlorosis, and
iron and phoSphorus absorption were studied.
Four treatments were provided as follows:
(a) 0.5mM phosphate as KHZPOL,
(b) lMOM! phosphate as (NMMZPOQ
(c) mom-1 phosphate as KHZPOL,
(d) 14.011114 phosphate as KZHPOL,

45

The base solution was Hoagland's number one with Hoagland's "A" solution
for minor elements. Iron was supplied at 2 ppm as ferric ammonium .
citrate.

The nutrient solutions were changed each week, and the pH of each
was checked daily.

Two cultivars of Chrysanthemum, cv. Oregon (chlorosis-susceptible)
and Yellow Delaware (resistant) were selected as test material. Two
plants of each cultivar were grown in each crock. Four replications
were provided in a split plot design with form of phosphate as main
plot and assigned in a randomized complete block design, and with
cultivar as sub-plot.

The plants were soft pinched, pruned to three stems, and grown
for seven weeks under a 16-hour photoperiod. The plants were rated
at the time of harvest on the basis of chlorosis, using the relative
scale described earlier. Leaves produced under the treatment, and roots
were removed and analyzed for phosphorus and iron. Leaf and root dry

weights were determined and used as criteria of growth.

Experiment XIX: Effects of High Lime, High Phosphate, and Their
Combined Effects on Growth,; Chlorosis and Tissue Accumulations of Iron

and Phosphorus in Chrysanthemum

The individual and combined effects of high phoSphate and high
bicarbonate on growth, the development of chlorosis, and iron uptake

and distribution were studied in this experiment by providing the

following treatments:

(a) O bicarbonate, 0.5 mM phosphate

(b) 0 bicarbonate, 2.0 mM phosphate

(c) 3 mM bicarbonate, 0.5 mM phosphate

(d) 3 mM bicarbonate, 2.0 mM phosphate
The low level of phosphate was 0.5 millimolar monobasic potassium
phosphate, which was the level of that salt in the base solution
(Beagland's number one). The high level was obtained by adding
1.5 nillimoles dibasic potassium phosphate to the base concentration
of 0.5 MM monobasic potassium phosphate. The low bicarbonate was
essentially zero, since no carbonates or bicarbonates were added and
the solutions were aerated with air. High bicarbonate was obtained
by adding potassium carbonate to yield a concentration of 0.75 millimolar
plus excess solid calcium carbonate and aerating with 2% carbon dioxide.
An equal concentration of potassium was maintained in all treatments
by adding potassium chloride as necessary. Amonium sulfate was added
to the low bicarbonate, low phosphate treatment for a concentration of
0.5 millimolar to maintain a solution pH of 5.5.

l'bur plants of the cultivar, Legal Tender, were grown in each
crock with four! replications in a randomized complete block design.
They were soft pinched after two weeks and pruned to four branches.
After eight weeks the plants were rated on the basis of chlorosis and
harvested. Stan length of the lateral shoots produced under treatment,
and leaf and root dry weights were determined and used as criteria of

gmwth. The leaf and root tissues were analyzed for iron and phosphorus.

"W

1+7

merimenj; XX: Effects of Lime, Phosphate, and Iron Chelation on

allorosis and Tissue Accumulation of Iron and Phosphorus

A comparison was made between ferric ammonium citrate and ferric
EDTA in the presence of low and/ or high levels of bicarbonate and
phosphate. Plants were subjected to the following treatments:
(a) 0 bicarbonate, 0.5 mM phosphate, ferric ammonium citrate (2 ppm Fe)
(b) 0 bicarbonate, 0.5 Phosphate, ferric EDTA (2 ppm Fe)
(c) 0 bicarbonate, 2.0 mM phosphate, ferric ammonium citrate (2 ppm Fe)
(d) 0 bicarbonate, 2.0 mM phosphate, ferric EDTA (2 ppm Fe)
(e) 3 mM H003, 0.5 mM phosphate, ferric amonium citrate (2 ppm Fe)
(1‘) 3 mM HCOB, 0.5 mM phosphate, ferric EDTA (2 ppm Fe)
(g) 3 mi! 3033, 2.0 ml! phosphate, ferric ammonium citrate (2 ppm Fe)
(h) 3 mM H003, 2.0 mM phosphate, ferric EDTA (2 ppm Fe)
A modified nutrient solution was used in this experiment so that the
pH of the low bicarbonate, low phosphate treatment would be 5.0 or
lower, while providing the same base solution for all treatments.
The salts used and their concentration in the base nutrient solution
were as follows:

0.5 mM monammonium phosphate

1.0 mM ammonium nitrate

2.0 mM calcium nitrate

1.0 mM magnesium sulfate

0.51m]. Hoagland's "A" per liter (minor element)

Potassium was supplied as the phosphate, carbonate, or choloride salts
as appropriate in establishing the treatment conditions.

#8

The low bicarbonate treatments received no carbonate or bicarbonate
salts and were aerated with compressed air. High bicarbonate treatments
received potassium carbonate at 0.75 mM and exceSs solid calcium
carbonate and were aerated with 2% carbon dioxide in air. we low
phosphate treatments had dibasic potassium phosphate added for a concen-
tration of 1.5 millimolar, which with the 0.5 mM ammonium phosphate gave
a total orthophosphate concentration of 2.0 mM. Potassium chloride was
added as necessary to equate the concentration of potassium in all
treatments at “.5 mM.

Four plants of Q. morifolium, cv. Legal Tender, were grown in each
crock. They were soft pinched after one week and pruned to three lateral
shoots, and grown under treatments for seven weeks. Four replications
were provided in a randomized complete block design.

Leaves were removed from the lateral shoots and roots from the stem
when the experiment was terminated. The tissue samples were washed and

analyzed for phosphorus and iron.

gaperiment XXI: Effects of Form and Level of Iron Applied to the Soil
on Growth, Chlorosis, and Iron Concentration in Leaves of Chrysanthemum

The effects of high soluble phosphates and high bicarbonate irri-
gations on the availability of iron of different forms from a greenhouse
soil was next determined. A soil mix of equal parts of loam, coarse
«sand, and Michigan peat moss was used. This soil mix was characterized
‘hy low fertility and a pH of 6.2.

Iron was supplied as either ferrous sulfate, glassy frit, or
EDTUh-Fe. Ferrous sulfate was used at 65, 130, 195 mg per four-inch

1+9

pot. The glassy frit (8% iron) was used at 2, u, and 6 g per fourbinch
pot. Those materials were applied by mixing weighed portions with
'batches of soil of sufficient volume to fill a pot. EDTA-Fe was applied
at rates of 5, 10, and 15 mg per four-inch pot by dissolving the appro-
priate quantity in 100 ml distilled water and applying the solution after
the plant was established in the pot of soil.

Two cultivars of Q. morifolium were used, Oregon and Yellow Delaware.

 

The plants were grown single-stem in four-inch clay pots (one plant per
'pot) under a 16-hour photoperiod in a greenhouse at 600 minimum night
temperature for 12 weeks. Four replications of each treatment and culti-
var were provided in a randomized complete block design.

The plants were fertilized with a mixture of 2.5 g of 25-0-15-
5Ca-3 Mg plus 2.5g of monobasic potassium phosphate per liter of tap
water. This solution was applied at the rate 150 ml per pot at the end
of the second and sixth weeks. In the 10th and llth weeks, half-strength
Hoagland's number one solution was applied. Tap water containing
moderately high concentrations of bicarbonates (pH 7.h and electrical
conductivity of us at 10"5 mho/cm) was used for irrigating.

Frequent checks of soil pH were made by collecting leachate after
applying distilled water and determining its pH. Soil leachate pH
greater than 7.0 was maintained in all treatments, and by the end of
the experiment it was 7.8 to 8.“. Soil pH determined by preparing a
.1:2 soil-to-water extract after the plants were removed closely agreed
With that of the leachate determined just before termination.

When the experiment was terminated, all leaves that had developed

under the treatments (20 to 21+ per plant) were removed, washed, dried,
"Bigflaed, and analyzed for iron and phosphorus.

50

gapermment.XXII: Effects of Bicarbonates and Phosphate on the

Availability of native Iron of a Greenhouse Soil

The effects of high bicarbonate and high soluble phosphate

applications on the availability of naturally occurring soil iron was
established. A single plant of Q. morifolium, cv. Legal Tender was

grown in a four-inch clay pot filled.with soil identical to that used
in the preceding experiment. This soil contained six.ppm iron and five
ppm phosphorus as indicated by the Spurway test (115).

Five treatments were established as follows:

Phosphate Najor Fertilizer Irrigation
(a) native (5 ppm) 23-0-22 distilled water
(b) superphosphate 25-0-15-5-3 distilled water
(c) superphOSPhate 25-0-15-5-3 tap water
(d) ammonium phosphate lZ-Bl-lh distilled water

monopotassium phosphate calcium nitrate

(e) ammonium phosphate 12-31-14 tap water
monopotassium phosphate calcium nitrate

Superphosphate was used at a rate of 1.5 grams per four-inch pot, which
is equivalent to five pounds per 100 square feet, 6 inches deep. The
soluble fertilizer, lZ-Bl-lh and calcium nitrate were applied at a
concentration of one-half ounce each in three gallons water. The
material 25-0-15-5-3 (5% calcium,3% magnesium) was applied at a concen-
tration of 2.5g/1 water. approximately 150 ml of fertilizer solution
'Hls applied to each pot twice a month. Soil leachate was pH 6.0 to 6.5
in treatment "a” and 7.0 to 7.5 in the remaining four treatments.
Five replications were provided in a randomized complete block

design. The experiment was conducted for a lO-week period. The plants

51

were grown single-stem under 16-hour photOperiods and at 65° F. minimum-

night temperature.
At the end of the experiment, leaves were collected from the upper

third of the shoot and were washed, dried, weighed, and analyzed for

iron and phosphorus.

RESULTS
Experiment I: Stability of Iron in Non-aerated Solution

The rapid precipitation of iron, when supplied as ferric chloride,
is indicated in Figure l. Precipitation was very rapid at pH 7 or
pH 8, with 50% of the initial concentration remaining in solution after
only five hours. Precipitation at pH 6 was alos rapid, but 50% of
initial iron was still in solution after five days and 12% remained
after 25 days. At pH 5, ferric ions were more stable and 50% of the
initial concentration was in solution after seven days, with 35%
remaining at the end of 25 days. At pH 4, an equilibrium apparently
was reached after eight days and 70% of the initial concentration

remained in solution thereafter until the 25th day.

Experiment II: Influence of Lime and Ammonium Citrate on Iron

Stability in Aerated Nutrient Solution

The rate of precipitation of iron when supplied as ferric chloride
in aerated nutrient solutions was rapid, even at pH 5 (Table 1). After
24 hours, less than 1% of the original iron, of that form, remained
in solution, there being no difference between the high-lime and Iowa
lime treatments.

In the presence of ammonium citrate, iron remained in solution
to a greater extent but was subject to more rapid precipitation in the
presence of dissolved calicum bicarbonate than in its absence.

The rate of precipitation of ferric iron in aerated Hoagland's

Inrtrient solution can be followed in Figure 2. Only traces of iron

52

ioagland's number one
Concentration of iron

renaining in solution

Frfure l. Solubility
—‘-—-

of Fe:9 (Fe59C1 ) in non-aerated, half-strength
nutrient solution adjusted to various pH levels.
is expressed as per cent of original concentration

at each time of observation.

53

FIGURE

 

 

pHB

 

 

   

o. o.
6 5

4<z_o_mo

O m
4 3

hzmomwa

m
2

IO.

TIME ‘ DAYS

5h

‘able l.-Effcct3 Cf A.imnium Citrate cm the Stalility of Iron Soluticns
Containing Bicarbnnate.

 

 

 

 

2‘- ---.;.......:;.:" :53: ".Z-. - .. I:‘?‘..‘.;.’._';.I‘:.._z:."::'.;::'°1‘:Lil';?‘:.‘::;:'”‘;:‘_‘.2—; 2......— .::7;“.
m -...'. - H n "1.! no... , A.“ '. “a
areatmcnc re uU‘ celitie ltn nioci at Lturs

_m-W- Ch”.—a-.-v opu- w... .- -9” — _ *—

T‘fi‘ C' ’ I‘.. ,V' ,.,L ' " i, '- V J: .' 1

noL3 pm lltn utnCCuLIaban rcr CCJL original

u‘w --~"-'V '4 " ”I. m”. .' - ~-.-. '.—‘ -‘-‘.'-‘ u w ”-0 w’w" -0“. —
V, :,as »~s ,
so; AC‘Joa z. ll‘la I'

3 7.0 FcCls C.CZ”a c.c5a

 

* FeAC is ferric ammonium citrate.

a . . . . . ...
Values with the sane superscrllt letter are not Signlflc cantly oliferent
at the SJ level of confidence as cetermined by Dulce an' s Ilultiple flange

‘3Ste

55

remained in solution at the end of five hours in either the lowhlime,
pH 5.0 treatment or in the high-lime, pH 7.0 treatment. Iron appeared
to be more stable in the lowhlime, pH 5 treatment, but this could
hardly be considered to be of significance in plant nutrition in view

of the short time that it did remain in solution.

Experiment III: Influence of Lime and Phosphate on the Stability of

Iron as Ferric Ammonium Citrate in Aerated Nutrient Solution

Iron as ferric ammonium citrate remained in solution in aerated
nutrient solution in which chrysanthemum.plants were growing for less
than 48 hours (Table 2). ‘In the presence of about 3 mM of calcium
bicarbonate, either with 0.5 mM or 2.0 mM of orthophosphate, only
about 50% of the supplied iron remained in solution for two hours.

At pH 6.8 with 2.0 mM potassium phosphate. as much iron remained in
solution for two hours as in the low pH treatment. At the end of

2“ hours, less than 25% of the original iron remained in solution, and
after 48 hours no iron was detected in solution in any treatment.

Examination of the precipitate accumulating on the bottom of the
crock established that considerable quantities of iron precipitated
from all the high pH treatments but not from the low pH treatment.
This suggested the possibility that absorption of iron by the plants
in solutions of low pH accounted for the removal of iron from those
Solutions; whereas precipitation was largely responsible for its
removal from solutions of high pH. Growth response and the development

0f chlorosis further substantiated these observations.

Figure 2. Solubility of F959 (F9590. ) in aerated half-strength
Hoagland's number one solution in progence of high (3 mM Ca003.
pH 7.0) or low (no Ca003. pH 5.5) levels of bicarbonate.

PERCENT ORIGINAL

56

 

 

FIGURE 2
80
LOW I‘ICO3
60
4O
20
0
HIGH HCO3 e
I 2 3

TIME " HOURS

 

57

Table 2. -T of Line and ILLSLLate on tLe Statilit; of Iron in

cts
rated Hcafland's Lumler Cne Lutrient Solutitn.

 

 

 

 

 

 

Treatment Iron Concentration After Times Indicated

 

hCC3 P ph 2 hrs 2A hrs he hrs

 

(xii) (nil) (Ignn) (pixn) (Egzn)

0 2-C 6-3 Sta c.6220

O

 

Values with the same superscript letter are not significantly dif-
ferent at the 5; level of confidence as determined by Duncan's
Fultiple Range Test.

«‘3‘

“K

l...

‘v
H‘

18‘

58

figpegiment IV: Effects of High Lime on Growth and Chlorosis of

Chrysanthemum Plants

than chrysanthemum plants were grown in aerated nutrient solution,
chlorosis resulted from both the high-lime treatment receiving iron
and the low-lime treatment receiving no iron (Table 3). Associated
with the development of chlorosis was the development of necrosis along
the margins of the leaves which in some cases resulted in death of the
entire leaf. Root growth as measured by dry weight was not affected by
any of the treatment, but leaf growth was reduced in proportion to the
severity of chlorosis. The accumulation of iron in leaf tissue was

reduced equally in both treatments resulting in chlorosis.

figperiment V} Effects of High Lime and Iron Chelation on Chlorosis

and the Mineral Composition of Chrysanthemum

Supplying iron as ferric ethylenediamine tetraacetate prevented
chlorosis in the presence of high concentration of dissolved calcium
carbonate. It resulted in an iron concentration in the leaf tissues
which was equally as high as the lowblime. pH 5.5 treatment and which
was significantly higher than the high-lime treatment receiving ferric
ammonium citrate (Table h).

The treatment which resulted in the most: severe degree of
chlorosis (high-lime with ferric ammonium citrate) resulted in the
highest concentration of manganese in the leaf tissue.

In the low-lime, pH 5.5 treatments, the absence of iron resulted
in a higher calcium concentration in leaf tissue, being as high as that

in the two high-lime, pH 7.0 treatments.

V}

59

Table 3.~Effects of Lime on Gromth, Chlorosis and Iron Accumulation
in Cinrysanthemwn Plants.

 

 

 

 

 

 

Treatment Chlorosis Kecrcsis Tissue Dry Ht. Leaf-Fe

 

 

 

7‘ v T;
hv C3 r. A A Fe IX; 0 to 1‘3 Elf dl‘y 1‘11: 0

 

(13.) (ppm) (reli’r) (rel—‘4‘) (5;) (.7) (17w)
0 5.5 2 1.3 1.0 2.15a L.1ca 59.8

3 7°C 2 3-8 '3.3 1.93a 1.540 32.5b

Values with the same superscript letter are not significantly differ-
ent at the 53 level of confidence as determined Ly Duncan's lultiple
Range Test.

* Degree of chlorosis and degree of necrosis are indicated by relative
rating scales.

6O

.poeeo .sc: oa.aeds m nausea he we :Isaenes mm oesouawzoo
we He>mm an amp pm pecaOeMHe mmpzm0H9fl2.wm pom cam neppoa paHmOmaonzm omen one mafia meSHm>

 

 

 

 

.m

.mamom wmflpmh e>flnmamm hp vmpmofivcfl ma mHmQhoI Ho Mo ash are

.e_ +mpeomcxpe no 2H1 wfleeeeasoepo oa.:om mr manlma umpmnafio mafia oi;m Oflmaem ma u<mm *
«Wm; e \+..\e_ ...\ e _. we. I...\. e .411) . e.._ N.
A)..1.\rL U m& .N\ U ngU H qu ¢l\ .mr\.a m H d _rrrI N U IL 0

mmm.u Duem.o em¢>.H adum 3mm m.m omen

(‘1
Q

0
L“
m

\u‘ . a. \ I \III)‘ .
ummvnvno, mflcweQ wfioem more Grimm vow I Q mom 0
\l\. . .II It \ .\c u \)
Swamo.u mwvc.v one . mafia mew e.m odrm . N m.m o

«mnv aw.v A».V Asweav Aeawmv AVTx.Hmr.V Axureomv Aaeme A suv

 

 

 

 

 

a 3 no a: ma no.3. HE men:
sewvflmcmaoo Hmpogflg mmmq memoacaro +:o£immme

 

.nn>meq Edtmipem :aao
mo :oflpfimonEco Hmaocfid new mfimoaoaao no Ccapmaofio coaH mum madq :uIm mo mpom%+fll.q oammm

61

Magnesium concentration in leaf tissue was significantly higher
in the high-lime, pH 7.0, ferric-ammonium—citrate treatment than in
any of the others.

In the high-lime, pH 7.0 treatments, the use of ferric ethylene-
diamine tetraacetate resulted in a significantly lower phosphorus
concentration in leaf tissue relative to that from the use of ferric
ammonium citrate. Furthermore, the highest phosphorus levels were
found in leaves with the most severe chlorosis, although not sig-
nificantly higher than in those of less severe chlorosis resulting

from the lowhlime, pH 5.5 treatments.

gaperiment VI: The effects of Bicarbonates in Irrigation water on

Growth, Chlorosis, and Mineral Composition of Azaleas

From.leaf dry weight data of Table 5, it can be inferred that
growth was not significantly reduced by any of the treatments during
the first growing season under treatment.

The plants that had been grown under low-pH irrigation were not
chlorotic, as indicated by the relative scale. When phosphoric acid
was added to neutralize tap water, chlorosis developed earlier and
more extensively than in any other treatment. However, sulfuric acid
was effective in maintaining a low soil pH and preventing chlorosis.

Iron concentration in the leaf tissue was highest in plants
receiving distilled water or tap water neutralized with sulfuric
acid. Green leaves had a higher concentration of iron than did
chlorotic ones.

The minor elements, manganese, boron, and zinc were of lower

concentration in leaves of plants under treatment with calcium or

A

.onom .QHHm o. oprmHoa A; vopmoncH mH mHmOHOHAo Ho moamoo

*

 

 

 

 

 

 

 

.omoe 3 .dm oHAHHHdA. m.:moasm he sodemoeee no oozesHHmoo
Ho He>oH 2mm EPA Hm HcmHOeHHu HHpemo iMHAMHm won one peppoH HaraoaHeA:c omen mAH ApHs mQSHe> m
flea. CH oH.am omH ea.mH eHs.H m.m m.a moose”
eomH. ohm oa.am omm om.mH omm.a a.H e.e «Amoozveo
eeeH. omen oes.ma ems ea.HH emm.H a.H o.a an.
oaam. oomm onm.ee oeH em.mH ese.s m m m s some; \ use
.V. 4
seem. emm ee.mw eon om.Hw eI.H Q.H o.m esmci \ one
peom.o mqq mm.mm qu eu.mm muH. H o.H 0.0 UoHHHumHQ
Atv AEon ©:AAV AEAAV éeAA v va 6*Hmav
a an m an on .e: use .oaao me some:
QQHHHmOAmoo HeaouHA HmoH oSmnHB mmeH possesses

 

 

 

 

Hwhmfivfi MQOH UCJ

 

E III ’33: I1 I .1; II."

.mmonNH mHHHoA UHMQHA Ho CQHeHmOAeoo
.mHmomoHA «Aesoao Go Home; COHpmoHaHH CH mopmcooamoHu we mooemmm I.m oHQwB

 

-___._._.——-_

I d C I 0 a
(I
I a a u o o '
I
J
v
.
a ' u a o n
O
U
‘
d a o v 0 I
_ 0
J o o I C I
v o I 0 I O
O
/\

63

.pmma mmzdm mHAHaHSA m.nmocdm hp vmmHSAOAOU mm mocmvamoo

mo Ho>mH um wan pm psohmmen thqmonHsmHm #0: ohm hmppmH pAHgomAOASm 02mm man Asz moSHw>

 

 

 

 

 

 

.m
ouog.m om»m.o moon. QAHH. mama m.» moomdm
nAmq. QQHQ.H ommq. HAHN. Gama m.© mAmoouvwo
gnaw. nomo.H omam. pmunm. omqw 0.5 mag
mbq\. momw. Unmm. OQQHH. mefl m.w dammm + awe
mbq». mmmv. gmww. fiomom. QAHH o.m Aommg + age
«moo.o mmmb.o ouug.o awhm.o moo 0.0 umHHHpmHa
.Ammmmmv AAA émwp A”; It, AamA .u;

coax do A AA mA AA pmgws
COHpHmOAmoo ngquA Amoq pamfipmohB

 

 

I}. -D".t“.t"i‘i."| ‘3 i--ll‘.

 

 

I..." '!!|"" til;‘.’. 1’.- "‘. I‘I ‘

‘I.l.! ‘1'... 0!

 

I’Dial-n‘ i

.m oHQma U.Acoo

sod:

v—‘l

6h

sodium bicarbonate or tap water with phosphoric acid than in those
under distilled water or tap water with sulfuric acid. Tap water
with no acid resulted in higher concentrations of manganese and
boron. due possibly to the presence of these elements in the tap
water.

Phosphorus accumulation in leaf tissue was lower in the high
bicarbonate treatments than in the elow'bicarbonate ones, regardless
of cation associated with the bicarbonate. Surprisingly; the
highest concentration in leaf tissue of that element occurred in
plants under sulfuric acid rather than phosphoric acid treatment.
The phosphoric acid irrigation treatment did result in higher leaf-
phosphorus than did the distilled water treatment.

The concentration of basic cations in leaf tissue was
affected by the cation level in irrigation water more than by
the bicarbonate ion level (Table 5 ). Sodium.accumulated to the
greatest degree in leaves of plants under the sodium bicarbonate
treatment and in the two tap water treatments which received acid
to lower soil pH. The lowest concentration resulted from distilled
water irrigation, although it was not significantly lower than that
from the tap water without acid.

Accumulation of magnesium.was not strongly affected by any
of the treatments except to be slightly reduced under calcium
bicarbonate and greatly reduced under sodium bicarbonate.

Potassium accumulation was highest in the plants under sodium
bicarbonate treatment and lowest under calcium bicarbonate. The
opposite was observed for calcium accumulation. and this resulted

in a much higher K:Ca ratio in plants under sodium bicarbonate

65

irrigations than in those under calcium bicarbonate or tap water,
with intermediate ratios resulting from distilled water or tap water
neutralized with either sulfuric or phosphoric acid.

The leaf composition of alkali cations was not consistently
affected by bicarbonate treatment.

After the second season of growth under the same irrigation
treatments. dry weight increase of both primary stem and leaf was
suppressed in those treatments that resulted in chlorosis, viz..
tap water with phosphoric acid, tap water without acid, and calcium
and sodium bicarbonate (Table 6). The dry weights of secondary
stems was not affected by these treatments. The sodium ion in
combination with bicarbonate reduced growth significantly compared
to the calcium ion or the mixture of sodium, calcium, and magnesium
ions of tap water.

Phosphorus concentration in the plant tissues examined was
not affected by these treatments. Of particular interest was the
lack of evidence that phosphorus accumulated in the stem tissues
under those treatments that resulted in chlorosis (Table 7).

Iron concentration in these tissues also was not consistently
affected by the bicarbonate level in irrigation water (Table 7).
Accumulation in the primary stem tissue was highest in the plants
under distilled water, tap water with phosphoric acid, and sodium
bicarbonate treatments. The concentration of iron in leaf tissue '
was actually highest in the tap water treatment, which resulted
in chlorotic leaves.. This agrees with many reported observations
that chlorotic leaves can have as high an iron concentration as green

ones 0

66

Table é.-Effects cf Bicarbonates in the Irrigation Water on Gronth
and Chlorosis f Lollis Azalea After the Seccnd Season of
Treatment.

MOM" ‘1... ~.-.-'.——. or .-~—I—--—— C me. O . w...- «I’m—r.- ”——~-9u~—0 -m ew---.-z-u‘-~-r— *0.-.-

 

 

 

 

 

 

Treatment Cllorcsis Plant Tissue Dry heights
Later pH Primary Secondtry Leaf
--.. w... _..__._......, .3399: __.-_. ._ Sir-v.83?”
(reLW) (3) (L) (r)
w‘ c _- _o a -, | ‘1 A 8'
bistilleu 5.5 1.0 h.13‘ 2.;6 5.3va
. . ,.. . a‘) A , .
Tap + h2oCA 5.5 1.0 3.55 ‘ ¢.c;a 5.Cca
r1 t' -, . Q r; r~ahc r) l 3. I) IV) 5
Lap + [Vii-Ch 60C go} Ao)‘ keLLL «Late/I
/

Tap 705 1140 1.1+50 1.5C8. l.CCb
C8(HC03)9 7.2 L.O 1.93tc l.Q7a 2043b

:aucc3 7.3 2.3 1.51.c 1.6L 4.3CD

 

 

*-rw*O-‘W'--fiw. m-" w- w— __ _—_ .— 'CI-

a Values with the same suferscript letter are not sivnificantly
different at the 5" level of confidence acccrding to Duncan's

/I"'

7"1'1 " .. m-
multiple flange rest.

* Degree of chlorosis is indicated by relative rating scale.

U

67

Table 7.-Effects of Bicarbonates ‘ Irrigation Later on
Concentration of Iron ard Phosplorus in Leaf Ti
Azalea After tie 3:C<nd 3eas<n of Treatrent.

 

 

 

 

 

Treatment Chlorosis Phosphorus Concentration*
Kc. hater pH Prirary Secondary Leaf
(‘3‘ F) C r f\ V1
.. -..__._....._- ‘9 .’.":’. “1......- “~-F’.-..i‘

 

 

 

(rel) (E)

(
1. Distilled 1.0 0.126a 0.123a 0.2itb

U1
0
v1

 

- w r' - a firm.)
2. Tap + hgoOL 5.; 1.0 .L9La .llZ .21c
' ' a - a a -
30 Pa i“ + fiqPO 600 3.3 01’4“.) OC’VC) 014.1840
2 A
4. Tap 7.5 L.C .C‘éa .11oa .411“
v-n . la / 4
5. Ca( c23)2 7.2 4.0 .140 .l2ca .1960
5 Ia””C " 7 ° 7 1?“ 7103 o vb
U. . lab 3 (a) a.) o.L_)..L a“ at. I
a

Values with the same 31}e1s011 t letter are not significantly
diiferent as determintd by Duncan 3 Lultiple Range T st at tie
a level of confidence.

Up
I.‘

he significant difference azcng treatzents wit}.in a plant tissu
segment; concentration of F witLin leaf was Si'niiicaltiv Ligher
than in either primary stem or secondary stem; no difference between
primary and secondary stem.

68

Table 7. (Cont'u)

 

 

 

 

 

Treatment Iron Conccn ration Total Iron Distribution
K0. Primary Secondary Leaf Shoot Leaf Per cent

L *4. .. .. ° ‘1 -m w.
ct: L Quail ln loaf“

 

‘—--‘__ ‘—

(ppm) (ppm) (ppm) (as) (at) (x)
lo Aha 25a 42a Abba Baca €0.00

» “ -a ’ a »a .
a s7“ 243 5 3c4 “15‘ 5s.52

3. 43ab 27a tea 21.2b 1c2~ 2.15

4. 260 21a so“ 1490 790 53.c2
5. 23° 233 sea 215LC 12C,b 53.¢c
é. joabc 23a A93 iaybc 110b 58.20

 

T

* fer cent of total shoot-iron that was found in the leaf tissue.

69

Since growth had been affected to such an extent, it was
considered desirable to examine thedata on t he basis of the total
amount of iron accumulated in the shoot portion of the plants and
in the leaf tissue. There was, on the basis of these data, more
iron accumulated under low~bicarbonate and lowpr conditions than
under high-bicarbonate and high-pH conditions. The lowest percentage
of total shoot-iron found in the leaf tissue was observed for
plants under treatment with phosphoric acid in tap water, and this
might be interpreted as reduced translocation to the leaves under

those conditions.

Root Absorption and Distribution of Radioiron
Experiment VII: Effect of High Lime on Absorption and Distribution

of Iron in Chrysanthemums

The data of Table 8 indicate that chlorotic plants which had
been grown two weeks in lowhlime Hoagland's solution lacking iron
absorbed several fold more iron in the first 24 hour period after
adding iron tagged with Fe59 to the root medium than did the green
plants in eitherlowhlime, lowth solutions containing iron or the
chlorotic ones in high-lime, high-pH solutions also containing
iron. During this first 2h hours there was no significant differ-
ence in the total amount of iron absorbed by the green plants which
had received iron and the chlorotic plants which had been under the
high-lime, high-pH treatment, although absorption tended to be
higher by the green plants.

During the first 36 hours of root exposure to radioiron, plants

 

70

of Line on Iron Abso*ption by Roots of

Table 8.-The Effect
9; ncrifoligg, cv. Legal Tender.

 

 

 

 

 

 

 

Treatment Total Fe Absorbed
hCC3 pH Fe Duration

 

 

 

(ppm) (hr) hajtlant)

, ..,.~
0 5.5 e 36 2.9.67“)

3 7.0 2 3e ' 1.C934

 

 

Values with the same superscript letter are not significantly
different at the Sfl level of confidence according to Duncan's
Lultiple Range Test. Comparisons were made among treatments
within each time of observation.

1 Values with thd same superscript number are not significantly
different at the 5$ level of confidence according to Duncan's
Lultiple Range Test. Comparisons were nade between times
within each treatment. -

71

under the two lowhlime treatments absorbed equally high amounts of
iron. The chlorotic plants under high-lime conditions did not absorb
any more iron during 36 hours than they did during 24 hours, as was
true also of the chlorotic plants under lowhlime, minus iron condi-
tions, although the difference between amounts absorbed was great.
The green plants under lowblime conditions with iron did absorb

more iron during 36 hours than during 2h hours.

These observations viewed as a whole indicate that the condition
of chlorosis did not predispose the plant to low capacity for iron
absorption, but prior and continuing exposure to high lime condi-
tions did.

An indication of the effects of these treatments on distri-
bution of iron is given in Table 9, in which the percentage of total
absorbed iron that was found in each tissue segment after 24 and 36
hour absorption periods are given.

The highest percentage of iron was translocated to the youngest
leaves. during either period, in the chlorotic plants exposed to con-
ditions of low lime and iron starvation. The lowest percentage was
in the chlorotic plants under high-lime conditions, this percentage
being significantly lower during the 36 hour observation period.

The same differences in translocation to the lower (physio-
logically older) leaves were also observed. There were likewise
similar differences among the percentages found in the stem tissue.

The percentage of iron remaining in the root was lowest in
the chlorotic plants under lowblime treatment and highest in those
under the high-lime conditions (although the latter was not signifi-

cantly higher than in the green plants under lowalime treatment).

72

Table 9.-Thc Effects of LL 1e on th “Dis ririuticn of Root-AbSOIbcd
Iron in Q. norifglgug, cv. Lega 1T Hen er.

 

 

 

 

 

 

Treatment Per cent Distribution

H003 pH Fe Duration Upper Leaf Lc.rer Leaf Stem Root

Cflg) — ww:_];¢mfl (hrs) (3 total abs rbed iron in each segmenty——
o 5.5 2 21 1.7431 2.6931 4.12a1 90.5cal
o 5.5 o 24 19.21b2 20.5bb2 10.51b2 A9.7Ab2
3 7-0 2 24 C 3”a3 c.55a4 2.Aca“ 97.c1ah
O 5.5 2 BC 5.?3‘1 .BAKl 3.31xl 83.62Xl
0 5.5 c 36 1,.c712 13.1233 6.9a73 61.8333
3 7'0 2 3‘ 0-3423 0.14”“ 1.423“ 97.10ZL

 

 

W _—‘.----— ——_._

a Val ues with the ea: c su1mer cri; t letter are not sirnificantlv
differer ta the 5 level of ccnfidcnc e accord1nf to Duncan'
Zultif 1e Pw ' Test. Ccmparisons were Ladc amen: treatments

fitlin each tile of observation.

sa1m3L11crscrpt nurx_er are not si nificantly
55 1e J81 of confidence according to Duncan's

.L

I
w .- '. ' ‘4
est. Co”pariscns were race between tires victim

1 Values with the
different at the
Eu ltiple Lax 3
each treatment.

73

Apparently the reduced amounts of iron found in the shoot
portions of plants under high-lime treatment was due to the accumu-
lation in the root tissue. as well as to reduced absorption.

It can be concluded from these observations with reapect to
translocation that the condition of chlorosis did not result in
reduced translocation of iron to shoot portions and young leaf
tissue, but other changes within the plant as a result of exposure
to high-lime treatment must have.

An examination of the data of Table 10 reveals that there was
a much higher accumulation of iron on a tissue dry weight basis in
roots than in other portions of the plant, regardless of treatment.
The highest accumulation in any tissue was in those plants which
were under lowblime, minus-iron treatment.

In the chlorotic plants under either lowelime, minus-iron or
high-lime. plus iron treatment, as much iron accumulated in upper
leaf tissue in 24 hours as did in 36 hours. In the green plants
under lowblime treatment more iron accumulated in.upper leaves in
36 hours than in 2h hours. In those green plants iron continued
to accumulate in all tissue segments over the 36 hour period.

Iron accumulated in the upper leaves of the chlorotic plants
under lowhlime treatment to a concentration of approximately 50 ppm
within the first 24 hours. and this concentration would be almost

sufficient to maintain green leaf tissue, according to earlier

findings.

m

a 7 rrw-‘ '79" .- .: .. 7. a 1 4 :. . o “a, .. :
.10 lC.-1ne ullGCuo cf L113 on tLG accxiu ac1cn cl 1rca 1n

7%

h°
' ‘7’“1")
Ll.-/L)K‘k‘

.1_“,_‘,,7‘.‘v'\71“' .11 cw" f‘ r I" 1“; r. 1 .tw‘w“ Vf L3f‘“l I '3‘ ~’" ‘\
QV~ 4 .vaauo .L. V O .m... 4.}. -‘-.. Iaut.’ C V O J. «Ann; :1.
g...- o ----. . _

-. .. _ A .1. . ._.- , .. A . 7. . .. . .. . .
4 “"" -.—-‘-- -'.--.'-’€-3 37: t.‘-—‘—~...-3.2.£.o -.=-.‘~..f.'2 $1’;." 3 z.-r.:‘m::t.'...'..:.: ;. LJ- aawr..:-ra-n 3.433 3.3}- 7...: .1 1'1"; 2...: I 2.32%»

Treatment Iron Acc mulaticn

 

~‘fi‘O‘O-m’ ”-W'K m "—vfi—n‘nv-mv-h W Os-H—J--uv -u-av..—o~ *“x—v—n—o

 

.‘_Y " .‘ rm; . ‘ --A-.~- ~~ '7 - '« ‘ ' "-. ‘ '- ' 'L
pn re Duration type~ Layer oten acct
Inn f Irmif
- -— .u— o- -m o o -9 b. - Ia —- H-~ .— CI-a- N «w .-—r 'u--—-.--<‘—-- ---4-‘- -—-r> -\--e -o-"o-~ ’—fl

\ 7‘..\ 7 --
(Ll/1-.) (1.3.23 ,‘ J5; ITO/lg. LLl ‘V‘ LTD.

.‘ ‘Nqfi -...‘ ~.—'7 7. 'I..'
5.5 :2 ”u 1.3;» 0.1: .214 71.141747-

} A . 1’) ‘-~ '
t' ' "I L"? "1 .2) "7" L 2'7 “I ’2 (g: 7.2,) 7r! 1) ('7‘. U.)
2.) C‘ u' 2.20.", ' )k/e/‘D .LJ.;L' 7.7.7.111

7 c 9 7 c (raw C c‘ C 1“a5 1 77’3‘1+
l. " :11. 0 ,' 7. C; e A. .re/

. , .. m“? ,4 . , 37:: t ’7
50 5 2 30 l oiiifivv C). (fl. Sets-71;” 23C. 53"“
u '3‘ I " 1"" "VJ "\ r‘ -| N L) A) ‘vziv " " i (F '3

50 5 C /U 1+1). 7K" 1.30.19 /e:}n_.','-’ jbq.l;.7°

/ \' --. ‘ ’ x r- . i r ~ . .‘\
7.C 2 30 C.05“4 L.th b.10“) 2..y

 

J.

4“

 

—— L‘- — ‘m I”" -W m—o‘m .-- ---"' v.“ -¢ u ‘- —— v H.

n1 0;-.. -\‘ r ,‘ y-‘ I ‘.'. ~-l~_ .e‘fi‘ , v o $- V‘ 1‘ v”: A r— , ‘v I
ireatment Ledho '1tn1n the Game durati on cf‘ trrnuuznt hd‘lhg Bud
sane superscri1t etter are net significantly uifferen‘

'1. "‘
u U “U
-‘ l

7 z. __ :1 1 ' 7x“. _;,_ , .4.“ - . n W. - 1,,
uetermined b5 Dxnlcan s haltiple Henge Test at tne 5N level c1
(‘0 11.14.. ICC.

Keane of the sane treatment centres ted lctxze' en the two can’tian

of treatment having the care supe‘scriyt number are not si nil ica ntly
different as deterrined o;r Duncan's ialtille nanje Test at the St
level of conficence.

}

iteraction between cicacment and time was not s1;n1fic'rt as
detern 419d bJ P tCSt bWith fifl level of cc.fidence. Treatment

‘ O ‘I

‘1ficrences averaged over both ti.es were. Grand mean for

"CC - Fe was greater tian ticse ‘or LCD? + Fe and 3 hCO3 + Fe;
other centres s we e not 8i nificantlr different as determined ty
Duncan's Lultiple flange Tee t with 5N level of confidence.

75

7 A?” .11 1 7...: - . :1- . .' _1 .,. n.
CI Ircn ntacrced Lader Lew-Lime CCnultibno b1
"IL

-33., ,1 T .. " .17..
[11.n, CV. “01ai Tenccr.

_n_---

. .‘--- -." ~.—...—.-.-
-——- ru-voo'~——. -. c—«Q ~-- .0 w- -f“~.‘-*Q-*- _-

 

Fre-Treatnent Total Abscrbei Iron

w-“ -vwm _

 

 

 

 

*. -m” ~- ”I“. .0.” “.4- -m~<~ m‘ v.— “Won—D...-

 

O

\n

o

\‘1
N
(K
0
\Q
I‘.)

C7

0
Ln
0
\J1
O
('3
o
('0
Lu

Values with the same superscript letter are not significantly
different at the 55 level cf confidence according to Duncan's
Lultiple Range Test.

P 1*- < f“:' I T“ - ..* ' p‘ ‘ .'.' I." :v‘. ~V ‘2."‘ ‘. " " '
.iect of 1111,: discs e to I.i;_1.‘"L...‘A-.e C(a.;luJ_L.LC‘-1.S en tne

76

Experiment VIII: Effect of High Lime on the Absorption and Distribution

 

of Iron by Chrysanthemum Plants after Transfer to a Low-Lime Solution

When chlorotic plants that had been grown for two weeks under
either high-lime or lowalime, minus-iron conditions to induce
chlorosis and green plants that had been grown in lOWblime solution
containing iron were allowed to absorb radioiron from solutions
of lowblime and low-pH conditions, it was found that the greatest
amount of iron was absorbed by the plants which were chlorotic as
a result of lowhlime and minus-iron pre-treatment (Table 11).

Here, as in the previous experiment, the important contrast
is between the two treatments resulting in chlorosis (lowalime,
minus-iron and high-lime, plus-iron). If iron-starvation chlorosis
predisposes a plant to higher capacity for iron absorption than
that of green plants, then it would be expected that chlorosis
resulting from high lime treatment would also. unless other effects
of high-lime exposure interfere. Since the amount of iron absorbed
in 22 hours by the high-lime treated plants was significantly less
than that of the minus-iron treated plants, than it must be con-
cluded that the high-lime treatment did have other effects on those
plants. These effects reduced the ability of the plants for some
time after they were provided with a root environment of lowalime
and optimum pH.

Those changes brought about by the high-lime treatment also
affected the translocation of iron from roots to all segments of

the shoot, as illustrated in data of Table 12.

77

Table 12.-The Effect of Prior Exposure to High Lime Conditions on tlm
Distribution of Root-Absorbed Ilon to Iissues of Q. yorifoligm,
cv. Legal Tender.

 

”'C -0” ‘- rug—fi' 5 -0" .---—.—. a..- o-n—ve . .—"-u.."-—-’ no 'IIU—nn—a. --~ -m-u\-'-—.- “Cr-..- m~- -—-'-.--‘-~ ‘ 'ub
M —--‘ a ‘. ”m --'-' ‘ 0‘ -.-—I ”-nt— -0 i o- .5.- “. ~ m"! I.- Q - .0!— -~-—-.-- -u.‘O- fl.“ anu- .- _-
- 7‘1 - .L . J. .g‘ '
Pre-Ireasmenc Per cent Distrituticn
_ -“Ct .I-n-w wc—‘u mun- — = 'w “_—

 

 

ECO3 pH Fe Upper Leaf Lower Leaf Stem Root

 

 

.uo— w “_-‘ —

(mK) (ppm) ( total absorbed iron in cache qment)

 

0 5.5 2 - 1.94 c.3ha 2.97a 94.2ca
o 5.5 o 2.63a 1.430 4.82b 91.11b

3 7.‘ 2 ' c.53b 0.533 5.930 92.72c

Table lB.—The Effect of Prior Exycsure to hi: h Lime Ccn Wit 3 on tLe
Accumulation cf Root-Abs orLel Iron in Tissues of CT ggggggggig,
cv. Legal Tender

 

 

HM‘ ~wr=::r.-:_': m‘...“*-‘:.. ":2: -mum" .7: 3:: :‘—:::_:1--*":‘ -*-'-"- "FMS-2- .....:...":.'.-:.:a-t '.:::.._'_"‘ ”ta-3::
Pre—Treatnent Iron Accumulation
KCC3 pH Fe Upper Leaf ch.'er Leaf Stem Root

 

“Cu-r“ H -.'

 

 

-‘.—‘.-- v. IOr— w-~.——o-—“~m

(1111.?) (ppm) (ut: Fe/g dry wt.)

 

C

M"!
\J‘!

2 1.74La c.532a 1.cc33 1C2.c2a

o 5.5 c 2.994b 1.043b 1.932b 1:5.1cb

‘ ”Xia- ./ ~‘L; \
3 7.0 2 0.32:C 0.234 l.c9C 79.730

 

 

me SU'EIS cript letter are not siwfn iicantly different

a Values with thee
e confide no 3 according to Duncar's I-ulti'le Range

Sfi level 0

H, o.)

78

There was a lower percentage of total absorbed iron translocated
to both upper and lower leaf segments in the plants previously exposed
to high-lime conditions than in those plants previously exposed to
iron-deficient conditions (Table 12). There was less iron translocated
to the upper leaves of these plants than in the green plants, which
tended to be intermediate in percentage of iron translocated to the
leaves.

There was a higher percentage of absorbed iron in the stems of
the high-lime treated plants. There were slight differences among
the percentages of iron remaining in the roots under these conditions
and it is possible that the reduction in translocation to the upper
leaves was the result of accumulation of iron in the conducting tis-
sues of the stem rather than in the root tissue. The percentage of
iron in the stem was highest in the high-lime treatment.

Accumulation data presented in Table 13 indicated that there
was less iron absorbed and translocated by the high-lime treated
plants. as expressed on the basis of tissue weight. than was by
either the chlorotic plants of the iron deficient treatment or the
green plants. This was particularly the case with the upperbleaf

and root tissue.

ggperiment IX: Effects of High Line on.Long-term Distribution of

Iron in Chrysanthemums

When plants were allowed to absorb radioiron and afterward
were grown under the same treatments (as in Experiment VIII) for

nine weeks, it was feund that considerable quantities of iron.

79

 

 

rv: '-, m —~. - ,n 1': __ _- ‘ '1. ‘4. - - . W ."1. ‘ .
Laple lA.-Lhe aifzcts c; LL_h Lan bona-tlons on tLe LCJL‘iUfT
° ‘ :1- -.:, *j J. n‘ . I, n ., ' fl ., ‘ rm" .° .‘
Dlptr*,ut*3n of Loch-Absurueu Iron 1n 3. :gggr;;ggfib
A .n V‘.‘ T
CV'. L'JI-Jsll iUi’l'v‘Cr.
”pr-‘u-crs-- -‘r-z?:-: O :"‘..;." -’-:‘:.:.-‘ t a-.. . -‘.~ - o .1 _n ‘90,?” “0.;- ~.' -- -I...—*'—---’ -0. --
h .,. - . 4. “' 4 v": 1.‘ .
ireatment fer cenu UlSurLVUUlCH
*m WW ‘0‘...“ ”-mcw — m“-.—~«-‘—.’-~I—O~O m..- -0 "a“... -w Q
' w. ' h‘ ". v *, .‘0 - '3 ‘ A ‘ '19 .1. 'v .‘: ‘4-
LOC-Q {h rc hen Lcaf L3H Utah Clu Leg; C;d Stun RCLV
.2

.--‘-.~-* .‘ — -.¢ -.m-~#~o ”- Q h..- . ~-.~'--—”u—~ no. --I- “—ID-‘v. 00-. a..- ".- wan -rF .- mu.“ -‘u—

 

(:13 (fpm) (yer ent of tetal in each scfmcnt)
c- 5.5 2 2.7.3a 0.Laa 3.253 4.L4a 6;.09
(Jot/21E C.

C 5.5 C 13.13” 1.71

3 " o ” 0.73“ “73 1.!"C .Qla 0° "“3

l. U‘ I Its-lo“

 

 

H-.- “.‘-“-.-uov mm -‘p r-w-o..———— v, - .. -——n.ov “”_‘~-

80

which had been absorbed and remained in the root tissue initially,
was translocated to new sheet tissue produced during this period.
Percentages of total radioiron found in tissue segments at the
termination of the experiment are recorded in Table 1”. Initial
distribution is not shown, since there was only a trace of radio-
activity in stems and leaves after 12 hours. The radioactivity in
the roots averaged 750 cpm per root.

Greater amounts of radioiron were found in the secondary
leaves and stems of chlorotic plants receiving no iron during the
nine weeks and maintained under conditions of low lime than in
comparable tissues of chlorotic plants grown under conditions of
high lime with 2 ppm iron provided or of green plants under treat-
ment with low'lime receiving stable iron. The percentage of
radioiron in the primary leaves was highest in plants under the
lowblime, minus-iron treatment also. which leaves were present as
the young leaves near the apex of the plants during the absorption
period. Differences in the percentage of iron found in the primary
leaves at the end of the nine week period was considered to be due
to differences in initial distribution rather than differences in
translocation out of those leaves.

The difference in percentage of total radioiron in the root
tissue indicates that iron was translocated from that tissue to
the growing points and incorporated in new leaf and stem tissue.
There tended to be an inverse relationship between the percentage
of radioiron found in root tissue with that found in new leaf

tissue at the termination of the experiment. There was a highly

 

 

81

significant difference between the percentage remaining in the
roots of plants under lowblime. lowbiron treatment than in roots
of the high-lime treated plants which received stable iron.

Iron distribution patterns are illustrated in the autoradio-
grams (Figure 3-9) after two and nine weeks on designated treatments.

Emposure of the xhray film by the radioactivity in the plants
and subsequent development of the film was constant for all treat-
ments and all times of observation. Therefore. the differences in
intensity among the various tissues of a mounted specimen as well
as among the plants may serve as an approximation of quantity of
radioiron.

The accumulation of iron in the growing point and young leaves
during the first #8 hours after the absorption period is illustrated
in Figure 3. Similar patterns were observed in plants under
treatment for two weeks (Figure 4, 5, and 6). There was much higher
activity in the youngest leaves with very little translocation to
the physiologically older leaves near the base of the plants.
Furthermore. there seemed to be progressively decreasing amounts
of radioiron from the youngest to the oldest leaves. an observation
not well substantiated by count data of the previous experiment when
an arbitrary division was made between the young and old leaves.
or mrther interest is the observation that essentially no differ-
ences existed among the patterns of distribution in the plants
representing each treatment under study. No doubt, considerable
quantities of iron had been translocated to the terminal growing

points during the first #8 hours and this iron was available to

Figure 3. Initial distribution of radioiron in young chrysanthemum
plant.

The autoradiogram was prepared #8 hours after absorbing FeS9Cl
from half-strength Hoagland's number one solution adjusted to pH 5.;
with ammonium sulfate.

Figure 4. Distribution of radioiron after two weeks in high lime
with a source of stable iron.

Autoradiogram of a chrysanthemum plant, which had absorbed Fe59
under identical conditions as the plant in Figure 3. and subsequently
had grown for two weeks in half-strength Hoagland's number one solution
at pH 7 and containing 3 millimolar calcium bicarbonate and aerated
with 2% carbon dioxide in air. A source of stable iron was available
to the roots.

Figure 5. Distribution of radioiron after two weeks in low lime with
no source of stable iron.

Autoradiogram of a chrysanthemum plant. which had absorbed F959
under identical conditions as the plant in Figure 3, and subsequently
had grown for two weeks in half-strength Hoagland's number one solution
at pH 5 containing no dissolved bicarbonates and aerated with air. A
source of stable iron was available to the roots.

Figure 6. Distribution of radioiron after two weeks in low lime with
a source of stable iron.

Autoradiogram of a chrysanthemum.p1ant, which had absorbed Fe59
under identical conditions as the plant in Figure 3. and subsequently
had grown for two weeks in half-strength Hoagland's number one solution
at pH 5 containing no dissolved bicarbonates and aerated with air. A
source of stable iron was not available to the roots.

   

Figure 3

   

Figure 6

83

those leaves which developed during the first two weeks after
absorption.

Some of the radioiron was translocated to new shoot tissue even
after nine weeks as illustrated in Figures 7, 8.and 9. There was
considerable difference in the amount reaching those tissues under
the various treatments. That amount of radioiron that was translo-
cated to new tissues was apparently evenly distributed within the
new shoots with no tendency to accumulate along veins of the leaves

as has been reported for other plants.

eriment X: Effect of Lime on Long-term Distribution of Iron

in Azalea

Growth of azalea plants in sand culture was poor and variable.
Root systems which were not well developed at the time of potting
were subject to injury from drying as the moisture level in the
sand near the surface flucuated. Consequently, some plants in the
lowhlime treatment made very poor growth and became chlorotic. There
was considerable variation in growth in the high-lime treatment,
also, but the development under the high-lime treatment was inferior
to that under either lowelime with iron or lowblime without iron.

count data was erratic, and because of large variance within
treatment there were no significant differences between treatments.
These data are not presented because of their erratic nature.

Two autoradiograms are presented in Figures 10 and 11. which
demonstrate. in comparison with the photograph of the mounted plants,

that there was a strong correlation between the presence of radioiron

Figgre 2. Distribution of radioiron after nine weeks in high lime
with a source of stable iron.

Autoradiogram of a chrysanthemum lateral shoot and the leaf from
whose axillary bud it grew. The plant had been grown for nine weeks
in Hoagland's number one solution at pH 7 and containing 3 millimolar
calcium bicarbonate and aerated with 2% carbon dioxide in air after
having been allowed to absorb radioiron. A source of stable iron was
available to the roots.

Figgre 8. Distribution of radioiron after nine weeks in low lime
with a source of stable iron.

Autoradiogram of a chrysanthemum lateral shoot and the leaf from
whose axillary bud it grew. The plant had been grown for nine weeks in
Hoagland's number one solution at pH 5. containing no bicarbonates. and
aerated with air after having been allowed to absorb radioiron. A
source of stable iron was available to the roots.

Figure 2. Distribution of radioiron after nine weeks in low lime
with no source of stable iron.

Autoradiogram of a chrysanthemum lateral shoot and the leaf from
whose axillary bud it grew. The plant had been grown for nine weeks
in Hoagland's number one solution at pH 5. except containing no bicar-
bonates, and aerated with air after having been allowed to absorb
radioiron. A source of stable iron was not available to the roots.

 

Figgre 10. Photograph (left) and autoradiogram (right) of an azalea
plant that had been grown under high-lime for twelve weeks after having
absorbed F659 under optimum conditions. An external source of stable
iron, but no radioiron was available to the root system during the
treatment period. The two lateral shoots grew §§om buds which were
present at the time of initial absorption of Fe .

Figgre 11. Photograph (left) and autoradiogram (right) of an azalea
plant which had been grown under lowhlime for twelve weeks after
having absorbed Fe59 under optimum conditions. An external source of
stable iron but no radioiron was available to the root system during
the treatment period. The single lateral shoot gr from a bud which
was present at the time of initial absorption of Fe 9.

85

Figure 10

Figure 11

86

and chlorophyll development. Tissues were green in those areas of
the leaves where radioiron accumulated. Radioiron was uniformly
distributed in the leaf tissue of the green leaves corresponding
with uniform green color deve10pment. Radioiron was present in the
highest concentration in tissues immediately adjacent to the veins,
and the interveinal pattern of chlorosis developed which is typical

of this species.
Foliar Absorption of Iron

eriment XI: Effect of Age of the Leaf on Rate of Foliar Absorption

of Iron

The absorption rate of physiologically old leaves, when
measured by the detached-leaf technique was found to be equal to
that of young leaves and significantly higher than that of mature
leaves of intermediate age (Table 15). No attempt to measure foliar
absorption by leaves on the intact plant was made due to the diffi-

culty of separating translocation from absorption processes.

Egperiment XII: Effect of Chlorotic Condition on Rate of Foliar

Absorption of Iron

Absorption rates of detached leaves from plants which had been
subjected to either high-lime or minus-iron conditions to induce
chlorosis were found to be equal to those of’leaves from plants
which had been grown.under lowblime conditions with iron and were

green (Table 16). This can be interpreted as evidence that no

 

 

87

cv. Legal Tender

"O
Hml-v‘

 

rm 1- m '4 1 ‘ a _. . '~ 1‘ . 4".
iaole lS.—ihe ulfCCt cf aye of a Leei oi o. Irzli ill”,
V" — ww—-~——.—.—an—.
. : ,, ‘t ' y 't' A“ ,‘ x _W L - _ V -. -_ ' .
on it” gate Cl absorptltn cl lrbn.
“'2: ._“ ". Tm ‘22.: 3 .2 '13 .2 ':....“ ‘2..?"..'. .2: 7..."- .u... ....... n...‘:..""~..: ..... _‘r .. ...%""--.....“" If..." ':-=: a ;—'.'.._.""'.a-.?
r 1,. ,. J. ' . '7“. L
It- JCT; Lal-CIL .0. be
_m w ‘Cl‘b "‘~—“.*_”

 

m1.
471 A .1-. , r, _
irea LANG; lJu
. . . .é /‘ .
id mole/cm‘/rr

wow—a-.. a.“ -. u-.--A-"-~O _
,-. 3
Lb

.M “W

 

‘

\ g' . a.
CofUU

a
TIC LILIILJ
.1. ‘ r‘v y a.
131‘?- will 6 C Q ( ‘
1 . ,o
.L. 4.21.4.

 

old
disorigt letter are not Sifnificantly
of confidence as determined by Duncan's

 

Table 16.4113 Effect of Prior Exposure of Plants of g. [zyillifil‘ijul cv.
;e Conditions on the hate cf Foliar

.«a- --..a-. — -._"- ‘o- F‘-

e;al Tender to high Lin

Absorption of Iron.

--.-n a flaw...“ .‘-- o a... o- H-.--- -~%-.‘
0

 

 

 

 

Pro—Treatment Atsorpticn hate
H003 pH Fe mu mole/cm2/hr
O 5.5 2 1.56a
a 1
O 505 L 10A3(
ea
3 7.0 2 107)
t significantly
idence as determined by Duncan's

 

q

a Values with th3 same superscript letter are no
I level of corf

88

changes within the leaf itself as a result of either iron starvation

or exposure to high lime altered the absorption capacity.

Aggperiment XIII: Effects of pH, Sodium, and Bicarbonate on the Rate

 

of Fbliar Absorption of Iron

Although prior exposure of the roots of chrysanthemums to high
lime did not affect the absorption rate of iron by detached leaves,
the presence of bicarbonate in the foliar treatment solutions did
result in reduced absorption rates (Table 17).

High concentration of sodium as sodium acetate at pH 5 did not
reduce the rate of absorption relative to that at the same pH when
tris was used for pH control. Foliar absorption rate was reduced
at pH 7 under all three means of control used here. There were no
statistical differences among the three treatments of pH 7, although
there tended to be a greater reduction under sodium bicarbonate
treatment. The rate under that treatment was only half what it was

under the sodium acetate treatment.

Experiment XIV: Effects of Chelation of Iron in Foliar Treatment

Solutions Containing Bicarbonate

When ammonium citrate and ethylenediamine tetraacetate were used
to stabilize iron in solutions containing sodium bicarbonate. it was
found that both agents prevented precipitation from solution. Howa
ever, there was no increase in absorption rate over that resulting
from high bicarbonate and iron as ferric chloride (Table 18). The
absorption rates were lower in the presence of sodium bicarbonate

than in its absence and at solution pH 5.

89

Table l7.—The Effects of Solution pH and the Presence of Sodium and/or
Eicarbcnate in Foliar Treatment Solution on the Rate of
Alscrption of Iron by Detached Le ves of Q. morifoliug,
cv. egal Tender.

 

 

 

 

_- ._ - A... -- - -..-

 

 

 

Treatment Absorption hate
HO 3 la pH Control mu mole/ch/hr
(1:11':) (Iii)
o o 5 Tris zucsa
0 3 5 peAc 3.94a
i . . b
O C / Tris O.a7
o 3 7 Tris + I aAc 0.61b
. b
3 3 7 ra20t3 0.29

 

Values with the same superscript letter are not significantly
different at the 55 level of confidence as determined by
Duncan's hultiple Range Test.

9O

Ta‘ole 18. -Tk L8 Effects cf the Presence of bicarbonate and Form of Iron
Used in t1 8 Pol iar Treatment Solution on the Stability of
Iron in Solution and Rate of Absorption of Iron by Detached
Lw

ves of C. rcrifL'iLl, cv. Legal Tender.

.l- ." ‘n‘..- -‘— .0 -\-~ - .- -.-.-- -.-——‘ M” .~»‘-M‘ -—.... "-0.. -w----- _v-n_.~.'-.W n...-4-s.-~

 

 

 

 

 

 

¢
Treatment Absorption hate Iron Stability
If" .'r , .fi ...2 , -4. ,-.. : 4. ,. '
LoC3 pn lion mu mole/om /Lr Per cent Iemalning in
solution after 1] hours
(Iii-L) (Form)?

b.) \JJ U)
K}
o
('3
’7']
' t'.‘
in
, O
O
;.\
0‘
(N)
r1
[in
O
C

.~”9‘*

 

ll

:3

FeAC is f rric aram nium citrate; FeE TA is ferric ethylenediaminc
tetra acetate
3 Eralues ‘Ut-it

different at the Si level of c<- nfi<Le
lulti ple Ear 3 Test.

} the same slp'iuc111t lo otter are not sirnificantl
no ce as {eternincd by Duncan's

91

‘Experiment XV: Effect of Chelation on Rate of Foliar Absorption

 

from Solutions of Low pH

Ammonium citrate and ethylenediamine tetraacetate, had no
effect on the stability of iron at pH 3.0 as evidenced by the absence
of precipitation when ferric chloride was used. Maximum absorption
rate occurred from ferric ammonium citrate, the minimum from the
ferric EDTA solution, and an intermediate rate from ferric chloride

solution (Table 19).

Experiment XVI: Effect of Solution pH on Rate of Foliar Absorption

of Iron

Solution pH had a marked effect on the foliar absorption rate
of iron (Table 20, Figure 12). Maximum absorption occurred at pH 5
and pH 6; absorption was slightly reduced at pH 4 and considerably
so at pH 3, 7, and 8.

In Table 20 absorption rates have been expressed on a leaf dry
weight basis as well as leaf surface basis so that the quantities of
iron absorbed can be related to the concentration reported for leaf

tissue of Chrysanthemum in earlier experiments.
High Lime and Phosphate Effects
Egperiment XVII: Effects of High Phosphate

Phosphate concentrations as high as 4 millimolar as monobasic
potassium phOSphate (eight times the concentration in half-strength

Hbagland's number one solution) did not reduce shoot growth (Table 21).

92

Table lQ.-Tie Eff C
at {h 3.C on the h.

D

J.

p..-- -0. vc. ...¢‘~-.,I
.- — -r

,.---.- -~
' - o .

oo-o-w- o. '.‘-'.

u—u—wu- “’04—‘I- --~-‘— "u.-.-~.-c— — .~ .o : o s
n..- .n ..-—. -..”- .g. _..—- ---._._. ————u--§

Treatment Absorption Rate

-- ‘ w ‘—

‘ \I ‘. . i' .. k: I?
form of ii-ozrn' mu LLClLZ/ cm. /nr

1‘3Cl3

-. ~n‘a-m-u-Iuc—

‘I
I -.,..‘L

FeAC is ferric ammonium.citrate; FeEDTA is ferric ethylenediamine

>§<

tetraacetate.

a Values with the same superscript le't r are not significantly

t e
different at the 5% level of confid‘nce as determined by
‘. i J.

Duncan's hultipie hangs fest.

Figure 12. Effect of solution pH on rate of absorption of iron by
ohrysanthenmm leaves. Solution pH is plotted on the abscissa with
rate of absorption expressed as millimicromoles of iron per square
centimeter leaf area per hour.

93

FIGURE l2

0.?

 

m: emu \ use“. 3:
zo_._.n_¢omm< no whdm

0|

SOLUTION pH

91+

m*ole HC.-’}e 3f1ect of H cf Foliar irea on z1t S::lution on tie hate of
Absor ti(n cf lron I; Lea es of C. Lfrli(lld', cv. Legal Tender.

I. CG". v’-

”n- -—~o—.—~ o-~.---v- “- .- -‘o—O"w~0- o—o CO—Q p- -w*,—'--w —.-~.'-.-— m'm-‘~ ~.-..-.--.—o—--‘..-’-..
”Q...” w- .“ w...—:.——-_.—-—-. -c- rv~ M '. Ifi'H

f1 '4‘“ 'I" ”I" 'u -. V-' 4 ‘1’ .h' '
ll‘Udonliino AUQLI‘LUiC/u 11.11138

pH mu mole/cmE/Lr ug/g dry wt./hr

b \A)
C C
o
\1‘ L1.)
«.11 (r.
C‘~ P
O {D
\ -\7
o o
C 1 N
f“ r‘
C“ 93

v
O
O
C‘
c.
‘1
O
1.4
1.4
O
\I
(‘1
£4.

 

- .1 .,oc
7 c.33t1 c.1/

a ~ac
0 (.373 (:OCL

 

 

Values with t e sane supe iscri1t lette r are rot si:nifica1tly
di1ferent at 1e 5; level of confidence as determined by Duncan's
4.1.1.149 irlC 1.1.6.119“ e TGS'C.

Ct *“

Table Bl.-Tte Effect of PLOSPLECB Concentration in the Lutrient Solution
on Shoot Growth and the Abscrpticn and Accur ‘aticn of Iron
and Pkos1hcrvs Ly g. gtrifclinn, cv. Lernaid.

a
——-- .-.ru

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Leaf Slcct Lt. Rcot-Fe Leaf—Fe Root-P Leaf—P
P ccnc. chlc 5. dry (3 (ppm) (3) (fl)
.1 ri- 29* “ 1.
1 ma .. EL}; .3 1 an .
c.5 l.L 9.9; o.c17a 54 0.479 1.5173
‘ a b ‘ 8’10 b . n Kb

2.0 1.3 1c.10a 0.5c50 913 0.669ab c.4116

1.0 1.7 3.95a -o.5ssa“ 75b 0.3A7b c.LAA°

 

 

-— -.- _— D ‘—

* Degree of chlorosis is indicated by relative rating scale.

a - .. .. . . 1
Values with the came ”up rscritt letter are not Signi icantiy

e
differeLt at the 55 level of confidence acccrcing to Duncan's
1~1ulti1cle Range T S

(L

96

Furthermore, there was no consistent effect on the concentration
of iron in the leaf tissue. The iron concentration resulting from
2 mMthosPhate was higher than that resulting from the 4 mM treat-
ment, but equal to that under the lower two treatments. There was a
tendency for the concentration of iron in the root tissue to be
somewhat less at the higher levels of phosphate than at the lowest.

The concentration of phosphorus in root tissue was smmewhat
higher under the three higher levels of phosphate than under the ,
lowest level. although the concentration at 2 mM phosphate was not
significantly higher than that at 0.5 or 1.0 mM;

Perhaps the most important observation is that slight chlorosis
did develop under the 2 mM and u mM levels (one plant under # mM
phosphate was severely chlorotic). Since this was not apparently
due to a reduction in the concentration of iron in the leaf tissue,
it.mey be concluded that the higher concentrations of phosphorus,
somehow altered the utilization of iron.

It could also be concluded from these data that the absorption
of iron hy the roots was slightly reduced hy the highest phoSphate
treatments, but the amount of iron translocated from the roots to

the leaves was not reduced appreciably under any of these conditions.

ggperiment XVIII: Effects of Various Forms of Orthophosphates at
High Concentration on Growth. Chlorosis.'and Tissue Accumulation of

Iron and Phosphorus by Two Cultivars of Chrysanthemum

Chlorosis developed in plants of the cultivar. Oregon irres-

pective of the form.or concentration of phosphate. Chlorosis was

97

slight under the low concentration of monobasic potassium and under
the high monammonium phosphate treatments and severe under the high
concentrations of mono- and dibasic phosphate treatments (Table 22).
The chlorosis developing in this cultivar under the influence of both
the low level of monobasic and the high level of dibasic potassium
phosphate could be due to the high solution pH. Ammonium.sulfate was
not used in the low phosphate treatment as it was when this solution
was used as the lowblime, lowpr treatment in other experiments, in
order that the effects of concentration and form of phosphate on
solution pH could be observed. The chlorosis that developed in
Oregon under treatment with high monoammonium and high monobasic
potassium phosphate. however, was not due to solution pH, since in
both treatments the pH was well within the optimum range for chrysan-
themums as established in earlier experiments. This was especially
noteworthy in the ammonium phosphate treatment. in which the solution
pH was 5.4 in the beginning of the experiment and decreased to 4.3 in
the latter stages of the experiment.

With the cultivar. Yellow Delaware, chlorosis was not present
with either low potassium phosphate or high ammonium phosphate treat-
ment. There was slight chlorosis as a result of the high monobasic
potassium phosphate and.moderate chlorosis under the high dibasic
potassium phosphate treatment (Table 22).

Only in the high dibasic potassium phosPhate treatment (high
solution pH) could reduced iron concentration in the leaf be a cause
of chlorosis in either cultivar. The low concentration of iron was

apparently due to reduced absorption, since there was a corresponding

98

Table 22.-The Effects of High Concentration of Various Forms of
Orthophosphates on the Development of Chlorosis and on
Iron and Phosphorus Accumulation in Leaves and Roots of
Q, morifolium cvs. Oregon and Yellow Delaware.

 

 

 

 

 

 

Oregon
Treatment Leaf Root
Conc.- Form pH* Chlo Fe Fe P
(3) (T1) (Tfiflroll (m7 (i) (ppm) T%)
0.5 11112201 6.0 7.0 1.8 5131 1.3681 1550.11 0.267all
11.0 (NHQHZPOh 5.11 11.3 2.0 6033 1.5932 226033 .385a2
11.0 KHZPOL 5.11 5.7 2.2 “£1.55“ 1850“ .373“
11.0 KZHPOL 7.11 7.2 3.8 33b5 2.32b5 330b5 1.1.58b5

 

Yellow'Delawere

 

_ri

0.5 W01. 6.0 7.0 1.0 92112 0.92K1 81.20"2 0.162"1
11.0 (NHfiszoh 5.11 11.3 1.0 66Y3 1.01x3 1570?3 .2606
11.0 10121901 5.1. 11.3 1.2 60xyh1.16x’* 11.90?“ .278“
tho xzupo,‘ 7.1. 7.2 2.8 11025 1.1.136 33025 .80076

 

a Treatment means of the same cultivar having the same superscript
letter are not significantly'different as determined by Duncan's
multiple Range Test at the 5% level of confidence.

Cultivar means of the same treatment having the same superscript
number are not significantly different as determined by Duncan's
Multiple Range Test at the 5% level of confidence.

Ti Initial Time: TH Time of Termination

e
1
4
. .
a
\ I
.
.
\
. b
..- _ ..-... ... . ----. _H
' 1 u
1 ‘ , .
e o
I
a
.
a -
-' i t‘ ~ I
_h~ K e k w x .
l
x \ s . .
‘ . . t .
t
\ . ~ \
.' 1 _

 

99

reduction in the roots (Table 22).

Associated with the reduced iron concentration in leaves was
an increase in phosphorus concentration resulting from high dibasic
potassium phosphate in Oregon. There was a slightly higher phOSphorus
concentration in leaves resulting from high monobasic potassium and
high ammonium phosphate than from the low monobasic potassium phos-
phate._but not significantly higher.

Translocation could not be judged to have been affected by any
of the treatments in either cultivar. In all cases, reduced leaf
iron was associated with reduced root iron. Furthermore, the
concentration of phosphorus in the roots under high phosphate was
higher than that under the low phosphate treatment only in the
treatment with high dibasic potassium phosphate. There was no
evidence that iron and phosphorus accumulated together in the root
tissue with a resultant decrease in transport of iron to the leaves.

Leaf growth in both cultivars varied inversely with the devel-
opment of chlorosis. This was not true of root growth. however.
Root growth was favored by the high phosphate and high pH conditions.
the greatest growth being made under the influence of the high
dibasic potassium.phosphate treatment.

Under these conditions Yellow Delaware made greater root and
leaf growth than did Oregon (Table 23). There were no statistical
differences among treatment effects on leaf dry weight of Oregon,
although it tended to be reduced under high phOSphate. especially
high dibasic potassium phosphate. This latter treatment did result

in significantly less leaf dry weight of Yellow Delaware. There was

100

’3’) r. '1 7‘1'9- :15 P '1‘} ‘. 1" , A 1.. 1.1,.--¥. .3 . y m P Y n- , ..~ ‘fi‘|Y~--1 P
Ia J-G~/.-'11.'3 -‘JiiQCuu Cl. 1-... .. bC/ILV‘:11UA Csu.~'\-1.u C‘i wrtel'iCz'nu ”911.1. Ca.

‘1I_ ~\‘. " V _f‘ 3 . .. ‘1‘ rfi:f“.*“ ‘» .V t 3'
(1U1.CllleCILIlAaUGS L21 0110 1'11"ch halal Cl in-uQ-AKE L'L: "Unint-

b.qr (‘ ---"r fi-‘-- .4. ~ ”‘1 _ g- L ‘,3 .-'. (: .l—’)1 1 (‘_._ Y} 1‘ '1' "II”‘Lfl
LJ J. ...-1_1.-\...-_.1.’ Co. I wk on aILJ J.v.1..1.~/n Ub+Cht1vo
” "‘mu¢-l“-.fi

"* “mm
'~-.--o

 

..—.- --u~..n-.-. P... -....O --0
”h..—-*. gn—u... ”-‘c—o—fl“ . -‘~ ~m-—- -~q~.-—uo MW..."” 0

 

m .2 . 1 ,—_ . “’1'1‘ ., “.-“,.-.._
1133b1..b‘ilu Li‘UL‘UIl icilbfl UUid (mil 3
____ W Avf _——— ~ ~- —- —

 

 

 

Cone. Form pH Leaf icct Leaf Reut

 

 

 

—-‘1-~ --- - .O

(11.1) ’ (1:) (14) (1)

vrvv I -a -~ . I
0.5 -u21c 7.0 1.10 .;;C 1.5ya 2.5;L

z,
1.0 (211)19101 1.3 2.33a 1.053 5.19a 2.16a

1.0 11:101+ 5.7 2.17a 1.11“ 3.71a .22L

a Values with the sane ou;erac:i1t are not Statistically d7°13re1t
a3 doom-in"J by Duncan's haltiple hange Test at the 5» protecticn
level on leai dry weight data which Lal a significant ir-te1acticn
Lethe en t1ca6mcno an‘ cultivar.

101

not a significant interaction between treatment and cultivar in root
dry weight. The average root dry weight for both cultivars was
lower under high amonium and high monobasic potassium than under
high dibasic potassium phosphate. Apparently solution pH had a
stronger effect on root growth than did phOSphate concentration.

The important differences in response to the two cultivars,
which could account for the reduced tendency of Yellow Delaware to
develop chlorosis, were that Yellow Delaware absorbed more iron and
less phosphorus than did Oregon under high solution pH and low-
phosphate. The leaf tissue acquired more iron. but probably not
related to differences in translocation. In the high ammonium phos-
phate treatment, Yellow Delaware absorbed essentially the same amount
of iron as did Oregon, with equal concentrations accumulating in the
leaf tissue. but absorbed and accumulated less phosphorus in the
leaves. Under the high monobasic potassium phosphate treatment. there
was no difference between cultivars in respect to either root or leaf
concentrations of iron or phosphorus. Under the high dibasic potassium
phosphate treatment, the differences with respect to phosphorus
absorption and accumulation in leaves were essentially the same as
under the low phosphate treatment. except Yellow Delaware did not absorb

or accumulate more iron than did Oregon.

Qgeriment XIX: Effects of High Phosphate and High Lime and Their
Combined Effects on Growth, Chlorosis, and Tissue Accumulation of Iron

and Phosphorus in Girysanthemum

Chlorosis was induced with high lime or high phosphate, but it

 

 

102

was most severe in the treatments having a high phosphate concentra-
tion (Table 24).

Growth was repressed to the greatest extent in the high-lime,
high-phosphate treatment. although this repression was not significantly
greater than either high-lime or high-phosphate alone except in respect
to stun length.

Root growth was similarly reduced as a result of either high
lime or high phosphate. Root tip activity was affected in a way that
cannot be easily described by numerical means. Roots in treatments
with high lime tended to be shorter and branched. Their color was
dark. and the tips were black. The root tips died or ceased to give
rise to new tissue and subsequently laterals developed. The laterals
also ceased to elongate after attaining only a few millimeters in
length. This process was repeated until a root system analagous to
"witches' broom” of the shoot had developed.

The concentration of iron in leaf tissue was reduced by either
high lime or high phosphate (Table 25). However. the significant
effect of high phosphate under high-lime conditions was an increase
in iron concentration. Thiswas not due to a greater reduction in
leaf dry weight. There was, in fact. a greater absolute amount of
iron in the leaves. is indicated by the development of chlorosis,
high phosphate at high lime resulted in iron acctmulation in the leaf
but apparently this iron was not effective in chlorophyll synthesis.

The reduction of iron in leaf tissue was not primarily due to
reduced translocation. The concentration of iron in the root was

reduced under high lime or high phOSphate or both.

, "{ 103

Taifile 2L.-The Effects of Line and Phosphate on Cromth and the
Development of Chlorosis in C. morifnlimr, cv. Legal Tender.

 

Il‘.‘~‘-.- .- .~__
_' u! --’-.u—-_~n-s._' . m~~--‘_- .1,

 

.O" -H---

_- —._.
.I. ‘7‘ ~‘. ~.-.- . .‘- .11 1.--..- 4—. CM ”1.-“ --——~-~-¢ ‘- o .0- -v’.‘ *-... -o .- g.’ .n—u—v~_o-.- w...'----

 

 

 

 

Treatment Chlo. Leaf height Stem Root
ECC3 Lil P fresh dry dry wt. length dry wt.
; parocnt—
"a3,

 

(KL) (1L) (rel*) (g) (g) (p) (em) (5)

("05 10013705 l7e

O
\J’c
O
\J I
C";
[—1
(‘1)
O
\J
H
[—4
O
G\
b
O
\Q
‘7‘

C 6.8 20C' [$.00 7Co3 £08 900 508 3065
3 7.2 C05 2'; 71;.02 701, 9.9 605 30/2
3 7.2 2.0 A.C 65.1 (.2 0.5 5.1 3.11

 

 

 

 

 

 

* agree of chlorosis is indicated by relative rating scale.
oi niiicant Simple affects*
Comparison Leaf Weight Stem Root Weight
fresh dry dry 3 length dry
(H003) at C.SmM P yes yes yes yes yes
(hCOB) at 2.th P no no no no no
(P) at 0 H003 yes "es yes yes yes
(P) at 3 mh H003 no no no yes no

 

 

 

*Simple effects were tested at the 5% level cf confidence by the F test.

10h

Table 2§.—Tte Effects of Linea nd Phcsghate on Iron and Phosphorus
Accumu ation in Root ani Leaf Tissue cf C. morifoliur,
Cl]. Let-"3.1 T011691”.

 

w- ---~-.--n-..- -.— ”--~ owa‘ao— m-~- :. .mw .D‘ .-
ac—noo—um-n two-*cwn~w H.”_‘* «an... era "mwo. - an.- C.-

Treatxent Iron Comccsi ion P1-cs;h rus Composition

 

 

 

 

 

 

 

 

H003 pH P leaf root leaf root
7"“ z) (”37""f ”mm—"*—
0 5.‘~ C.5 12‘ BCLB 0.6h 0.7;
C 6.8 2.C A? 931 l.hl C.93
3 7.2 C.5 #3 SCC 1.Cé C.92
3 7.2 2.W CC 3th 1.25 l.C5

 

 

-m—o—~.~“ —. —~‘~—» -—w

Congariscn Iron Connositi on Phosphorus Composition

 

 

’— -..mm-u—v __ _A d."-

 

“—

leaf root leaf root

 

 

*---A——.-‘e-u.¢ Maw-“C'-o-. ‘ ”'.~.-' ”‘“*“—~--

 

.‘~
. ‘m‘ - n ' " . V ' .fi
:u1 0.531. . zes yes yes no

ITO

E’
h.)
o
,1
L— 0
":1
:5
C
:3
O
A
1
O
O

’r ' 7' ""1 w 1‘ a. ..
(t in 0 LL tho yes yes ”as no

e s no y: 5 no

{A

(1*) in 3 n1: Inc;

m“.~M--‘u—- *Or‘noflm ~_.-.--- 4 ‘1‘-.- -.. ‘wu.< '~“0--‘ -- ‘ ‘n-u‘"m-O“- —s.--d--. ‘fl— - "av--m-"m

* Sirple effects were tested at the 5% confidence level by the F test.

~"-"— ~‘ . ‘~ 1--' n :- -. w. ,. r... . . a: : . -1
"n Interaction between cloazoonate and 1h<sllate was not significant at
.L.~_ rr' . V‘ _3 “j ,_ n _ 1,, ' ,1 _j.‘ p p 1-L -‘ _b a a L 1 .__.L 4
ohe ”a CUI.fJ.L'.Glawe lutsl. 41.1.11 efl‘BCUQ Of thaILCIIlc. (A. and }.i.L,Si.l.aeC

" _' .

Q \

greater as a result of either high bicarcona te or high ghos; hate
treatment.

fer“ 3i nilioane :.ith root—glcsp 1.1te cone ent1aticn
q. 1

105

Under conditions of low phosphate, there was significantly more
accumulation of phosphorus in leaves at high lime than at low.
However, under conditions of high phosphate, the effect of lime was
«opposite. The concentration of phosphorus in the roots was increased
under either high lime or high phosphate, but there was. not a signif-

icant interaction.

gpefiment XX: Effects of Lime, Phosphate and Iron Chelation on

Chlorosis and Tissue Accumulation of Iron and PhOSphorus

In this experiment treatment solution pH was lower than in
comparable treatments of earlier experiments. This was apparently
due to using ammonium.phosphate in the base solution in all treat-
ments. Chlorosis developed in those treatments of either high lime,
high phosphate, or both, when the form of iron was ferric ammonium
citrate, but not when the source of iron was ferric ethylenediamine
tetraacetate (Table 26). The use of the latter form resulted in a
significant increase in iron concentration of leaf tissue relative
to that obtained from ferric ammonium citrate under the treatments
having either high lime, high phosphate, or both. There was no
significant effect of chelation on the concentration of iron in leaves
under the lowblime, lowbphosphate treatment. Furthermore, neither
high lime nor high phosphate affected leaf-iron when EDTA was used;
whereas, either reduced leaf-iron accumulation when ferric ammonium
citrate was used. (Figure 13)

Greater concentrations of iron were found in roots when iron

was supplied as ferric ammonium citrate rather than ferric EDTA under

‘
e
‘t
5 .
a
_ _
0
‘ \
4

f"
.L

4‘-

 

'0‘
~

I

.
..
. . . o
‘. ‘3 .
4 . “ a n
O l
I ‘ _ ,
. t‘
. n '
.. ,
‘ v,» L
F
I I \
‘ i .
‘-,'rf '1‘ ‘: . '
u c .,
, .
>‘ ' O
. . \,. e-
< .
1 ’ .
.-‘ ‘ l
u ‘, (- vr
‘ o .A I
.' . 3‘ -
‘ \.. . t \J . 4
. 'J
’2 I ‘
. Io .J'n\ ~ ’
.‘ . ‘j
I O
'4. .a ‘ e - '1
A .- ‘ J- t ‘
. .'. -‘ a
J _, L; _
. , .
- I r r
v. r.
s. _. , :‘J .' .)
9‘ 'V' '
AOL. "' '
I ' ~ ~I
.
n r h l
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106

’T“

11031:} at oe, and Iran Che CLa ation on the
an d on Iron and 1nosrloru

Table 2C.-The Effe c s c-f Llr
f 3 sis
and eaf Tissue of Q, gyrjjfiggggg, cv.

Development 0
Accumulation in Re
Legal Iznder.

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*w Chlorosis is rated on a rel at we scale 1 - L, increasini severity.

107

Tahle 26. (ccnt'o)

 

 

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F Tests for Significant Effects

 

Effect Leaf-Fe Root-Fe LeafuP hoot—P
HCC3 no yes no yes
P no yes no ‘os
Chelate yes yes yes yes
HCCQ - P yes yes no yes

ECC3 - Chel 10 yes yes no
P - Chel DC yes no no

ECOB - P — Chol res no no no

 

— w 1 ‘—-—'——— —- m

Significant Siryle effects

 

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Leaf-Fe Root—Fe Leaf-P Rooth

 

hCCB in C.5uh P KCC3 in 0.5;? P Chel in 31L ECO} hCCg in ZmL P

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P in AC P in CLL.HCCB KCCB in CDTA
Chel in 2.CuL;P hCC3 in AC

Chel in C.5LL.P AG in Cat ECCB
at th E003

Chel in C.51L P
at 3mh hCC3

P in AC at Cub HCC3

 

Figure 1g. Effect of bicarbonate, phOSphate, and iron chelation
on tissue accumulations of iron and phosphorus. The concentration
of iron or phOSphorus in leaf or root tissue is plotted on the
ordinate of the graph against treatment bicarbonate and phosphate
on the abscissa.. The effect of iron chelation is indicated by
the use of separate lines identified as FeAC (ferric ammonium
citrate) or FeEDTA (ferric ethylenediamine tetraacetate). In each
graph the positions on the abscissa represent the following '
conditions:

1. 0 mM H003 and 0.5 mM P04
2. 0* HM 3003 and 2.0 mM P04
3. 3 mM H003 and 0.5 mM P04
4. 3 mM H003 and 2.0 mM P04

108

FIGURE l3

   

ROOT- IRON

— F0 AC
..... FoEDTA

   
  
  
  
  

LEAF - IRON

IOO‘

 

l
.....

PP" 0'" WEIe HT

Hco3 - P04 H005 - p04

LEAF-PHOSPHORUS ROOT-PHOSPHORUS

5

PER CENT DRY WEIGHT

 

109

each condition of lime and phosphate. except where both were high.
Since high leaf-iron was associated with low root-iron under EDTA
treatments, it may be concluded that the EDTA chelated form of iron
was more readily translocated from the roots to the developing leaves.

Either high lime or high phosphate reduced the iron concentration

in roots when iron was supplied as ammonium citrate. but not supplied
as MA. This reduction was most pronounced when both lime and phos-
phate were high.

High levels of phosphorus accumulated in leaf tissue when ferric
ammonium citrate was used under either high phosphate. high lime, or
both. Of particular interest was the observation that no such increase
resulted when ETA-iron was used. and in fact, there was a tendency
for the reverse to be true. Furthermore, the form of iron used had

a stronger effect on resultant phosphorus concentration in leaves than
did the concentration of phosphate in the treatment solutions.

The use of EDTA-iron also resulted in slightly reduced concen-
trations of phosphorus in the root tissue. under each condition of
lime and phosphate concentration relative to that from the use of

ferric ammonium citrate. There was a significant main effect of
chelation on such accumulation. but no significant interaction with
either lime or phosphate.

High lime combined with high phosphate resulted in less phos-
or'us in the root tissue relative to low line with high phosphate.
e concentration of lime in the treatment solution had no effect on
ot-phOSphoms concentration when phosphate was low in the treatment

1111:1011. Furthermore. the phosphate concentration in the treatment

 

 

 

110

solution had a direct effect on root-phosphorus under lowblime

conditions, but not under high-lime conditions.

Experiment XXI: Effect of Form and Level of Iron in Soil Applications
on Growth. Chlorosis. and Iron Concentration in Leaves of Chrysanthemum

Growth was not judged to be affected by any of the treatments
of this experiment. Leaf dry weight data indicated no differences,
although the samples weighed were useful only as an approximation.
since all the leaves of each plant were not weighed. Plant height and
overall appearance were also judged visualLy to be essentially equal.
Furthermore. no chlorosis developed under any of the treatments.

EBsentially'no differences existed between cultivars in terms
of iron concentration in the leaf tissue. although Yellow Delaware
tended to have a higher concentration when the highest applications of
each form was used (Table 27). There was no consistent increase in
leafeiron as the level of each form of iron was increased in the soil.
There was somewhat higher leaf-iron at highest ferrous sulfate and
EDTA treatments as compared to that at the lowest levels; however, the
opposite was observed in the case of the fritted form.

Statistical analysis of these data indicated that the soluble
:forms of iron (ferrous sulfate and Ferric-EDTA) were more effective in
producing high leaf-iron than the fritted form. EDTA was the more

effective of the soluble forms.

111

Table 27.-ifs Effect cf Form and Co centre tion cf II: nA pl Mo to the
Soil on tke Concentration of IICD in Leaf issue of
. _piiitliuq, cv., Cregcn and Yellou D: laa are

_'-‘-‘-’ -

no

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Treatment Leaf-Iron Concentration

 

 

a“-.. w _.

*.'~-‘— ”-9.- “a-”

Form Level Oregon Yellow Delanere

 

--.’~_ , ‘—

(rel) (ppm) (ppm)

FeSCh low 59 CC

Frit med 76 73
e a c I

Frlt hlgu 52 L4

EDTA low 66 72

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S‘uple Effecte of FormoLevel Interaction

A

 

Comparison Significant
SoluLle vs frit linear yes
Soluble vs frit quadratic no
Fesoh vs FeEDTA linear yes
F8504 vs FeEDTA quairatic no

 

112

_Egperiment XXII: Effects of Bicarbonate and Phosphate on the

Availability of Native Iron from a Greenhouse Soil

High applications of superphosphate -- Ca(H2P04)2 -- did not
result in a lower concentration of iron in leaf tissue, compared to
the treatment receiving no additions of phosphate, unless the soil was
irrigated with water high in bicarbonates (Table 28). When the more
soluble form of phOSphorus, monobasic potassium phOSphate, was used,
the resultant concentration of iron in leaves was lower than in the
treatment receiving native phosphate regardless of whether bicarbonates
'were high or low in the irrigation water.

The concentration of phosphorus in the leaf tissue was higher
under any of the high phosphate treatments than under conditions of
low phosphate. However, neither the form of phosphate nor the presence
of bicarbonate in the irrigation water had any significant effect on
the concentration of phosphorus in the leaves.

Chlorosis did not develop under any of the treatments, and growth

was not judged by visual observation to have been affected.

Table 28.—The Effects of high Bicarbonate Irrigation and E
the Accumulaticn of Iron in Leaf TC“‘
cv. Lekal Tender, Crown in Soil c

mm-.—.—-

.3
‘\ (N' “ln
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(:11

113

_-__ _—-. .fl'w-w..».’--fi.“

 

 

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Treatment

 

Phosphate

 

 

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Leaf Fe aid P Composition

 

 

a Values with the sane superscript letter a
different as determined by Duncan's Lulti

 

 

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DISCUSSION
Solubility of Iron in Nutrient Solutions and Soil

The precipitation of iron when supplied as ferric chloride in
nutrient solutions of high pH was so rapid as to indicate that the
presence of other ions in those solutions had no tendency to prevent
or delay its precipitation. The rapidity and extent of precipitation
was essentially in agreement with reports dealing with iron precipi-
tation from.water devoid of other ions (78, 136, 1&6). The extreme
instability of iron as ferric chloride in highly oxygenated nutrient
solutions or in nutrient solutions containing dissolved lime indicated
that such a form of iron would be completely unsuited to such situa-
tions (57).

Although ferric ammonium.citrate was a more stable form of
iron than ferric chloride. it did precipitate from.solutions of high
pH. Its rate of precipitation appeared to be affected by the presence
and activity of microorganisms which contributed to its decomposition.
And, if such decomposition was a factor under laboratory conditions.
it would be expected to be of much greater importance in a soil with
a well developed microbial population. Iron did not precipitate when
ferric othylenediamine tetraacetate was supplied at pH 7.2 in the
presence of bicarbonate, as has been reported under other conditions
(8. 67).

when the stability of ferric ammonium.citrate was studied in
the presence of rapidly growing plants. it was found that iron did

not remain in solution for more than one or two days under conditions

115

of either high or low solution pH. At high pH, iron was precipitated,
and the rate of precipitation was greater if the cause of high pH
were high lime (calcium and potassium bicarbonate) than if it were
high phosphate. However. the disappearance of soluble iron from
solutions of low lime and low'pH could only be attributed to adsorp-
tion and absorption by plants, since no iron precipitate was recovered
from the bottom of the crooks.

Growth of chrysanthemums was rapid and leaves green under all
conditions which favored the presence of soluble iron in the nutrient
solution, including that of EDTA-Fe in solutions of high soluble lime
or phosphate. Plants under conditions favoring rapid growth were
subject to a fluctuating supply of iron. though, and when solutions
were not changed each week it was necessary to add iron at weekly
intervals. even when EDTA-Fe was used. ‘Weekly additions of iron as
ferric ammonium citrate to solutions of high lime or high phosphate
(both having high pH) did not prevent the development of chlorosis.

It was obvious that a constant supply of soluble iron in aerated
nutrient solution cultures was essential to healthy growth and green
foliage. This was not so for chrysanthemums in soil cultures. Chry-
santhemums in soil under the conditions of high lime and/or high
phosphate were green and.made apparently normal growth. Azaleas,
in a peat soil, did develop chlorosis under high bicarbonate, high
phosphate, or high pH conditions and were green under low pH conditions.
Possibly the differences in responses of the two plants in soil culture
could have been due to differences in iron supply; or differences in

root growth of the two plants, or both. The chrysanthemums received

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116

fair1y*high additions of iron applied by mixing with the soil before
planting in one experiment. In a second experiment a soil with an
high iron supply (6 ppm by Spurway test) was used. The mode of
absorption might have involved direct root-iron contact phenomena
similar to that described for magnetite (3h. #0. 49) or glassy frit
(1#8, 149, 152). Root growth of azaleas appeared to have been F“
repressed more than that of chrysanthemums.

Adsorption of colloidal iron might have been the first step in

absorption of iron from aerated solutions (h#). Visual observations

 

established that suspended particles did become attached to the roots ;_
in solution culture as a result of the agitation by stirring and

aerating. Furthermore. this material continued to adhere to the roots

until the plants were harvested for analysis, and it was removed by

a washing technique used to avoid interpreting adsorbed iron as

absorbed iron. Analysis of that material established that iron, phos-

phorus and calcium were present in it.
Lime Induced Chlorosis

Chlorotic leaves of chrysanthemums grown under conditions of
high bicarbonate or high lime contained a lower concentration of total
iron within the leaf tissue than did green leaves. The concentration
under highplime was comparable to that under lowblime, minus-iron.
Iron concentration in chlorotic leaves varied among cultivars of
the species to such an extent that the concentration in severely
chlorotic leaves of plants of one cultivar can be as high as that of

green leaves of another. Such variation complicated any attempt to

117

establish a critical level of iron for chlorophyll maintenance in
leaf tissue (66, 97, 111, 122). However, the observations that
chlorotic leaves contained less total iron than green leaves indicated
that chlorosis developed as a result of a decreased supply of iron to
the leaf rather than an inactivation of iron within the leaf when lime
was the cause of chlorosis.

In all cases reported here, growth, and particularly leaf dry
weight, was reduced in those plants which became chlorotic, regardless
‘of cause of chlorosis (105, 123. 136). When leaf iron is reported
as the total amount occurring in the leaves of the plants. a greater
difference is usually noted than when the iron is expressed as a '
concentration in the dry matter. This difference between total leaf
iron and leaf iron concentration on a dry weight basis was particu-
larly evident in the case of the Mollis azalea. grown during two
seasons. At the end of the first season of growth under treatment,
only a slight reduction in leaf dry weight was noted and the depres-
sion in iron concentration was related to the severity of chlorosis.
However, during the second season. growth reduction was severe and no
difference in iron concentration in leaf dry matter was observed. The
total amount of iron in the chlorotic leaves of the second season was
less than that in the green leaves. The reduction in growth was
greater than the severity of chlorosis.

A reduction in iron available to the develoPing leaves was not
the only effect of high lime in the rooting medium. Spectrographic
analysis of the leaf tissues of the Hollis azalea established that

other important minor elements were also reduced in availability to

 

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118

the leaves as a result of high bicarbonate and high solution pH.
Manganese was reduced, and this element is known to have a function

in chlorophyll maintenance. Boron and zinc were reduced, and these
two elements have important functions in cell division and enlargement.
The sodium bicarbonate treatment resulted in leaves having higher iron
content than did the less severely chlorotic leaves produced under the
calcium or mixed bicarbonate treatments, but leaves under the sodium
bicarbonate treatment had the least concentration of zinc and the
least leaf dry weight of the high bicarbonate treatments. Further,
the visual symptoms developed in an evergreen azalea (data not
reported) under that treatment resembled those typically ascribed to
zinc more closely than those of iron deficiency. The concentration

of the major cation elements were also affected by these treatments,
but these changes apparently were due more to the effect of relative
cation concentration in the rooting medium than to the high concen-
tration of the bicarbonate anion. The high ratio of potassium to
calcium.which has been observed in many studies of the effects of high
lime (9), was noted in leaves of plants under sodium bicarbonate
treatment, but the ratio was low under calcium bicarbonate. Develop-
ment of chlorosis was not affected by these differences, however, and

this phenomenon is not considered to be a causal factor.

 

 

 

 

 

 

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119
Phosphorus and Lime Interactions

The first indications that high phosphorus could be involved
in causing chlorosis were obtained when phosphoric acid was used to
neutralize the high bicarbonate of tap water. Chlorosis developed
. in azalea plants irrigated with tap water plus phosphoric acid while
plants were green with distilled water irrigation at approximately
the same soil pH. Chlorosis was induced with phosphoric acid more
readily than with any of the high-bicarbonate treatments. Chlorosis
was most severe under that treatment in both the first and second
season of growth. During the latter season, the amount of phosphoric
acid used was greater and a lower soil pH was maintained. ‘This is
consistent with the observations of Hageman and Hartman (52) that
phosphoric acid was not effective in reducing the harmful effects
of bicarbonate in water.

High concentrations of monopotassium phosphate in nutrient
solution (four and eight times that in Hoagland's solution) induced
chlorosis at solution pH 6. When various forms of phosphates were
used at eight times the concentration of Hoagland's solution,
Chlorosis developed to the greatest extent in the treatment having
the highest solution pH. However, with the cultivar, Oregon,
chlorosis developed at solution pH less than 6.0, which pH otherwise
promoted green foliage.

Accumulation of iron that was not functional in chlorophyll
synthesis was indicated by the relatively high concentration of iron
in slightly chlorotic leaves of chrysanthemum under certain conditions

Of high phosphate.

120

Dibasic potassium phosphate, on the other hand, induced chlorosis
by reducing the accumulation of iron in the leaves.

By comparing the effects of high phosphate with those of high
lime (or bicarbonate), it was found that chlorosis developed earlier
and became more severe as a result of high phosphate. Growth
reduction was most severe when high phosphate and high lime effects
were combined.

Additional evidence of accumulation of inactive iron was obtained
in another experiment in which leaf dry matter under high phosphate
and high lime had 60 ppm iron contrasted to #9 ppm in the leaves of ‘
equally severe chlorosis and of equal dry weight'under high phosphate
alone, and contrasted to #3 ppm in the less severely chlorotic leaves
under high lime alone.

EDTApchelated iron prevented chlorosis under conditions of either
high lime, high phosphate, or both. Concomitantly higher concentra-
tions of iron and lower concentrations of phosphorus occurred in the
leaf tissue from chelated iron than from ferric ammonium citrate.

The direct effect of high concentrations of dissolved line
(i. e. bicarbonate) on absorption of iron by roots was to reduce its
stability in solution so as to reduce its availability to the roots.

Indirect effects were also noted, particularly in two experiments
in which radioiron was used to measure absorption during short periods
of time. In one such experiment, chlorotic plants which had been
grown previously under conditions of low lime and no iron absorbed
more radioiron in 24 hours than did green plants previously grown

‘under conditions of low lime with adequate soluble iron. Chlorotic

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121

plants grown under conditions of high lime with iron absorbed much
less radioiron than did the others. Absorption, in this case, was
measured under the same conditions to which the plants had been
exposed for two weeks, except that the absorption period was short
and the iron stabilized with ammonium citrate so precipitation was
not an important factor. Iron deficiency apparently increased the
capacity for iron absorption if the cause of iron deficiency was an
absence of iron in the root medium.and not the presence of bicarbonate.
Similar differences in capacity for iron absorption were noted
when plants were grown under identical conditions but allowed to
absorb radioiron under lowblime conditions. The increased capacity
for iron absorption by chlorotic plants of lowhlime, minus-iron pre-
treatment and decreased capacity of chlorotic plants of high-lime
pre-treatment was interpreted as evidence of internal changes in
the plant as a result of exposure to high bicarbonate concentrations.
Such internal changes reduced the plant's capacity for iron absorp-
tion, and such reduced capacity continued to affect the plant for
some time after transfer to favorable conditions of low bicarbonate.
These observations regarding the rapid rate of iron absorption
under conditions of low lime have further significance. When the
iron supply flucuates widely in aerated solution culture,successful
iron nutrition would depend on the ability of the plant to absorb
most of the soluble iron during its brief exposures to it. When
ferric ammonium.citrate is added to aerated solutions on a weekly
basis, it remains in solution for only one or two days even at low

pH. 'me plant then grows essentially in a minus-iron solution for

122

the next five or six days. If it is growing rapidly during this time,
a considerable amount of new tissue would.undoubtedly be formed under
iron stress. When iron is again made available in soluble form, that
plant would absorb it at a rapid rate. Such a cycle would be repeated
again and again for the plant in the lowblime solution. If, on the
other hand, the plant were growing in a high-lime solution it would
also have tissue lacking in iron, but other changes brought about by
the high-lime effect would reduce its ability to absorb iron rapidly
during the brief periods every week when iron was in solution. And
since ferric ammonium citrate would.remain.in solution in the high-
lime solution for only a few hours, the problem of availability is
intensified. As a consequence of these phenomena, the plant in a
lowhlime solution goes through periods of incipient iron deficiency
followed by periods of rapid absorption of iron and recovery from
that stress; whereas, the plant in the high-lime solution develops
incipient deficiency during the long absence of available iron and
fails to absorb enough iron during the subsequent brief exposure to
soluble iron to recover, so chlorosis develops as a permanent condition.
The tendency of plants under lowhlime treatment to recover from.incipient
chlorosis created a problem in maintaining the minus-iron treatment.
Often the iron in distilled water was sufficient to bring about recovery
when such water was added as a means of renewing the nutrient medium.
Plants supplied with EDTA-chelated iron do not go through these
cycles of availability and nonpavailability of iron. The chelated
iron remains in solution for a longer period of time so as to provide

a continuous supply for absorption and subsequent translocation to the

 

0V

123

developing leaves. This difference could account for the lower iron
concentration observed in leaves of plants grown in solutions
receiving weekly additions of EDTAriron contrasted with that in leaves
of plants grown under conditions of lowalime with weekly additions of
ferric ammonium citrate. Quantities of iron in excess of that needed
for chlorophyll synthesis probably'were absorbed by the plants receive
ing periodic additions of ferric ammonium.citrate, because of rapid
absorption when flhe iron was available. The plants receiving EDTA-
chelated iron never developed an iron deficiency and so did not go
through periods of very rapid iron absorption which could result in
excess accumulation.

Not only did bicarbonate in the rooting medium reduce iron
absorption by roots, but bicarbonate in solutions applied to the
leaves reduced absorption by leaves.

When the foliar treatment solution contained no salts other
than iron, and when its pH was favorable for foliar absorption, such
absorption proceeded at an appreciable rate even if the leaves were
chlorotic as a result of prior exposure of the plant to high-lime
conditions. This indicated that the condition of chlorosis, itself,
did not predispose the leaf to a reduced rate of foliar absorption
of iron. This was further emphasized by the fact that under certain
experimental conditions the detached chlorotic leaves both from.minus—
iron, lowhlime pre-treatment and from high-lime pre-treatment absorbed
iron at a faster rate than did the green leaves.

The age of the leaf did not apparently have an effect on its

rate of foliar absorption when it was detached from.the plant. If

12“

older leaves on the intact plant are capable of similar rates of
absorption, then foliar application provides a means for introducing
iron into those older leaves of the plant that do not receive appre-
ciable quantities of root-absorbed iron.

The quantities of iron absorbed under optimum conditions indicate
that the leaf would absorb enough iron in less than one hour to raise
the iron concentration in the leaf tissue to a level equal to that
which was found in green leaf tissue of most cultivars studied. This
could lead to recovery from chlorosis, if other conditions for chloro-
phyll synthesis were favorable. It must be kept in mind, however,
that leaf analysis data from earlier experiments showed that other
elements were in reduced concentration in the chlorotic leaf when that
leaf had been formed under high-lime conditions. Important among these
is manganese,because of its role in chlorophyll maintenance. It is
possible that the use of foliar sprays to correct chlorosis will not
prove completely successful unless all the elements lacking in the
leaves are included in the foliar spray solution and application is
made at a time when enzyme systems involved in chlorophyll synthesis
are still functional.

At solution pH 7, resulting from either sodium.acetate, sodium
bicarbonate, or Tris the rate of foliar absorption of iron was sharply
reduced. Sodium bicarbonate inhibited foliar absorption more than
did the sodium acetate at the same concentration, because of the
higher resultant solution pH, This indicates that preparing spray
solutions in tap water that is high in bicarbonates would predispose

the treatment to failure because of this inhibition.

 

 

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125

Treatment solution pH had a pronounced effect. The optimum
pH was not the lowest tested, at which stability of iron in solution
would be the greatest, but it was at the intermediate levels of
pH 5 and pH 6. At higher pH, inhibition occurred, due primarily,
of course, to precipitation of nonpchelated iron from.solution.

Both ammonium citrate and ethylenediamine tetraacetate reacted
with ferric chloride to chelate the iron and maintain its solubility
under conditions of high pH during the time periods necessary for
successful foliar treatment. Ammonium citrate increased the absorp-
tion above that of the non-chelated form.of iron at high solution
pH, but not to the point of being equal to the absorption rate of
iron at the optimum pH (pH 5). The observation that chelation with EDTA
did not increase the absorption of iron under high pH conditions agrees
with reports by Lunt (85) and Wallace (131+). Under conditions of
low pH, ammonium citrate increased the absorption relative to that of

ferric chloride, but EDTA had no favorable effect.
Iron Translocation

The effect of lime on translocation of iron was best observed
in experiments in which the distribution of radioactive iron to
various plant parts was followed. Iron.was translocated rapidly to
the young leaves of plants which were chlorotic from exposure to low»
lime, minus-iron conditions. A relatively high percentage of the
total amount of iron absorbed by each plant was translocated to the
leaves within the first 24 hours. The percentage of absorbed iron

that was translocated to the young leaves of green plants was less,

126

and it was greatly reduced in the plants under the high-lime treatment.
Count data was interpreted as evidence that essentially as much iron
was translocated to the lower four leaves as to the upper four leaves
under all treatment conditions, in terms of percentage translocated to
each. However, the resultant accumulation in the tissues on a dry
weight basis was higher in the young leaves. The interpretations

from autoradiograms did not agree with that of count data. The
youngest leaves evidently had more radioiron than did the older
leaves, and the difference was progressive with age. If the older
leaves did have as much iron as the younger ones, it was not distri-
buted throughout the leaf tissue.

The tissue in which accumulation took place was the root. A
high percentage of iron was being translocated from the roots of
plants under the lowhlime, minus-iron treatment during the first 2h
hours.‘ During the remainder of the observation period more iron
was absorbed than translocated out of roots, so the percentage in the
roots increased. And the highest percentage of iron remaining in the
roots was observed in those plants under high-lime treatment.

In 36 hours the concentration of iron in chlorotic leaf tissue
produced under lowalime conditions reached a level essentially equal
to that observed in green leaves in other experiments. This could
mean that enough iron had been abSorbed and translocated to leaves
to bring about a correction of chlorosis, provided that the iron-
requiring'mechanism.for chlorophyll synthesis had not been irrevocably
altered. Recovery was not observed in the short-term experiments, of

course, but did occur in other experiments.

127

Internal changes affected the movement of iron within the plant.
Exposure of plants to high-lime conditions reduced the distribution
of iron from roots to leaves after transferring the plants to solutions
of lowhlime and low pH. Iron accumulated in stems as much as in roots.

The concentrations of iron in root tissues were less when EDTA-
iron was used under high lime, or high phosphate, or both,than when
ferric ammonium citrate was used. This could readily be accepted as
evidence that this synthetic chelating agent prevented the accumulation
of iron in the roots and favored the translocation of higher percent-
ages of absorbed iron to the growing points.

Internal changes as a response to high lime developed rapidly.
When plants were allowed to absorb radioiron under lowblime conditions,
and were then transferred to high-lime conditions, they became
chlorotic more quickly than did similar plants transferred to lowb
lime. minus-iron conditions. Very little of the iron that had been
absorbed prior to treatment was translocated to the shoot apices under
the high-lime conditions. 0n the other hand, sufficient iron was
translocated from roots to developing leaves under the lowblime,
minus-iron conditions to meet the requirements of those leaves for
some time and thus delayed the onset of chlorosis. Such a means for
supplying iron to developing leaves under lowblime could be even more
important in a woody plant and could be the basis for the long delay
in the development of chlorosis in azaleas as observed by Orr (100)
and by Twigg and Link (128).

It was apparent from examination of autoradiograms that most

of the iron was translocated from the roots to the shoot apices, with

 

 

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128

progressively less to the more mature leaves. Much of the iron
initially incorporated in the apical tissues could have been utilized
in the leaf tissue that developed from them. Lateral buds do not
appear in the autoradiograms to have received an appreciable amount
of radioiron in the early period. Therefore, the shoots that grew
from the lateral buds after the terminal buds were removed must have
received radioiron from the roots, and possibly lower stem, to account
for the radioiron present.

The first leaves that formed on the lateral shoots contained
more radioiron than did the later ones. This observation could be
interpreted as evidence that much of the iron in the lateral buds
was present in the emerging leaves and more advanced primordia and
was supplemented by radioiron that was being redistributed from the
roots to these leaves during their enlargement. Leaves that developed
later probably did so from primordia that were not present in the
meristems during the period of initial distribution. After redis-
tribution from the roots declined, there was no source of radioiron
for those leaves.

Chlorosis developed in the leaves of the growing plants in
relation to the decrease in radioiron as revealed on examination of
the autoradiograms. There was only slight interveinal pattern of
chlorosis in chrysanthemums and little restriction of radioiron to
the veins. In azaleas, on the other hand. there was a pronounced
interveinal pattern of chlorosis with an equally pronounced pattern
of accumulation of radioiron in and near the veins as seen in the

autoradiograms.

SUMMARY

Iron when supplied as ferric chloride in aerated nutrient
solutions precipitated so rapidly that such a form of iron could not
be considered for meeting plant requirements in nutrient solutions.
When supplied in the form, ferric ammonium citrate, iron was more
stable, but was subject to the precipitating effect of high pH
associated with high bicarbonate, and particularly so when micro-
organisms were present to hasten the decomposition of the organic
molecule. As ferric ethylenediamine tetraacetate, iron was stable
under all conditions examined here. Frequent additions to nutrient
solutions were necessary to maintain an iron supply as plants
absorbed it.

High concentrations of soluble lime (3mM bicarbonate) in nutrient
solutions induced chlorosis in several cultivars of Chrysanthemums.
In all such cases, the total concentration of iron in leaf tissue
was less than it was in green leaves. This reduction was comparable
to that observed in chlorotic leaves resulting from withholding iron.

Iron concentrations in chlorotic leaves of chrysanthemums were
observed to be higher than in green leaves when chlorosis was induced
by high lime associated with high phosphate, or by high phosphate
alone, suggesting the accumulation of iron under those conditions
which was not active in chlorophyll maintenance. With the azalea
plant, lower iron concentrations were observed in chlorotic leaves
induced by high bicarbonate in the irrigation water or by high

phosphate when phosphoric acid was used to neutralize a tap water

129

130

of high bicarbonate content. However, during the second season under
the same conditions, growth of foliage was much reduced and higher
iron concentrations were found in chlorotic leaves than in green
leaves. A greater total amount of iron was accumulated by the leaves
of the green plants than by the chlorotic plants, however, and the
difference in resultant concentration of iron in the leaf tissue was
attributed to the reduction in growth rather than to an accumulation
of inactive iron. .

Conditions of high lime which induced chlorosis in chrysanthemums
reduced the capacity for root absorption of iron and reduced the
translocation of absorbed iron to the developing leaves.

The chrysanthemum plant was found by radioisotope techniques to
have an iron reserve capable of supplying iron to developing leaves
over a period of at least nine weeks. Such a reserve apparently was
in the roots, having accumulated during a brief exposure to radioiron.
This root iron continued to move into developing leaves whether or
not there was a continuing supply of iron to the root system, though
a higher proportion of initial root iron was translocated to leaves
under conditions of no iron for root absorption. Under conditions
of high lime, however, very little iron initially absorbed was trans-
located to new shoots.

Absorption of iron by leaves of chrysanthemum plants was not
adversely affected by conditions of high lime in the root medium.

On the contrary, a deficiency of iron in the leaf tissue, whether
caused by high lime or by an absence of iron, led to increased rates

of foliar absorption of iron. If bicarbonate were present in the

131

foliar treatment solution, or if that solution were of high pH
(greater than pH 6) by any cause, absorption of iron by the leaves
was greatly reduced. There was no demonstrable effect of bicarbonate
on foliar absorption other than in causing precipitation of iron from
the treatment solutions. However, when iron was stabilized in such
solutions as either ferric ammonium citrate or ferric ethylendiamine
tetraacetate, there was little or no improvement in absorption rate.
FUrthermore, iron as ferric ethylenediamine tetraacetate was not
absorbed from solutions of low pH as readily as was ferric chloride
or ferric ammonium.citrate.

Autoradiograms established that most of the iron being absorbed
by healthy plants is translocated to the youngest leaves and to
active terminal buds. It was found, however, that older leaves from
the basal portion of plants could absorb iron from foliar treatment
solution when detached from the plant as readily as could detached
young leaves.

A possible interaction between bicarbonate and phosphate as
causal factors in lime-induced chlorosis was suggested in experiments
in which both phosphate and lime rates were varied. High lime led
to increased phosphorus and decreased iron accumulation in leaves
of chrysanthemums. High phosphate supply under either lowhlime or
high-lime conditions had similar effects. Chlorosis developed under
any of these three conditions. The decrease in leaf accumulation of
iron was associated with a decrease in root accumulation of iron,
suggesting decreased absorption as the effect rather than decreased

translocation. A decrease in root accumulation of iron was observed

 

 

 

132

when the form of phosphate was ammonium phosphate and solution pH
was “.5, as well as when dibasic potassium phOSphate was used and
solution pH was 7.2, which indicates that some means other than
precipitation of iron from solution could account for the effect on
iron availability. Precipitation from solution of high pH was
observed to be a factor in inducing chlorosis.

The use of ferric ethylenediamine tetraacetate made possible
the maintenance of a supply of soluble iron under all conditions
examined, and in such cases chlorosis did not develop. The concen-
tration of iron in leaves of plants supplied with this chelated form
of iron was higher than chlorotic leaves of plants supplied with
ferric ammonium citrate under high lime or high phosphate or both.
Furthermore, there was less leaf accumulation of phosphorus when
EDTA-iron was used than when ferric ammonium citrate was used under

the same adverse conditions of lime or phosphate.

h.

‘e

-letlttnar'v

Arnon, D. I. 1937. LEA and ROB nitroge; nutrition of barley
at ciiferent seasons in relation to hi Concentration, Ln, Cu“

‘v': e '\ .1 " Ij/‘r‘. Q ' ’ (" .‘ I)
8.1m; 2 8111.1)-.3e Jail: uCle lye: ylv'll,

 

\(1
C

I?
o (2’)

 

Effect of LEA and EC3 nitrogen on tLe
mineral co position and sap characteristics of barley roots.
Qgil Sci. Ah: 235-3C7.

and C. R. Johnson. l9hl. Influence of h+ con-

 

centration on the growth of higher plants under controlled
conditions. [lent P} ";_ic1. 17: 525-539.

, a. 3. Fra Ms:e, and C. L. Johnson. 1942. ir
concentration in relation to absorption of inorganic nutrients
by 113 nor plants.£lvit.2133111. 17: 515—524.

sscciation of Cfficial Agricultural Chemists. 195(

O

'p

g1 he tlods g__ nalvsis., 8th Ed. ACAC, Washington, D. C.

--_.-a- s“-

4:1

.LJa

in
b:

xter, P. and R. Belcher. 1955. The role of the bicarbonate

ion in lime induced chlorosis. Jour. Aust. 1532, of Afrig. §g1.

fine-O

O}
’__r
}_J

Scar, F. 3., Editor. 1957. Chelates in Plazmt utrition. c'

oi. 8h: 1-97.

m

0)

hedri, A. A0, A. ldauacc, and U. A. 33108118. 1960. ASSlillilathn

of bicarbonate by roots of diffem‘ rt p ant sIeoie . Sc.1 Sci.

——---

01
\L‘
O O
(‘0
n
«J
I
I
(~ \
kc
0

e
O\
O
\O
'T’
I...)
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e

Eennett, J. P. l?45. Iron in leaves. Soil Sci

 

133

134

1C, Lhan, K. C., R. C. Ema: a 1;er, A. A. becri, H. T. Lueller, R. A.

Jeffreys, R. h. Carnack, B. I. Eiely, and A. wallacc. 19CC.

Possible relationshigs of bicarbonate or CCQ assimila tion to
cation accumulation by plant roots. §§i §31. 89: 276-2Eh.
11‘ a: A° ”allace, and E- Jo trohn. 1962. Effect Cf TH

 

and nitrogen source on ability of corn and soybean to obtain Fe
chelated wit} ct} lenedranine ul-O-}"“‘OA then; lacetaoe. A rcn.
{333. EA: 119-21.

d r

a. Eiodulph, C. 11L7. Interrelations Letween Fe a P in plant

“-1

le'e Fioio'v Colloeriu , Tutriritn:
- H- _.

' O-b a“- -‘- '~-.-.\ *r‘ 1.... ~. --_... ‘.-.—.

nutrition. (re. St. 90

-.-—-

13. Einglan, F. T., and J. P. Lartin. 105i. affects of soil phos-

f ,-, ° l."(.,‘ 11.. 1“. 1.?‘1 . P)( . ’2' h) K;
Krlo U» v. “JV; 0 J “we 4. o J- J'", o
- -- .9-“

1h. Eradfield, R. lChl. Calcimr in the soil. Physico—Cleljcal

Relations. Srfl Sci. doc. A'cr. Proc. 6: 8—

‘-
”‘V‘N 0“ -—-v . o .— M—-.——

L-)
\J‘:
O

15. Brenton, D. and L. Jacotscn. 1952. Iron tranSport in yea plants.
221i 5;“ 1 3'7: 53'2—SLF.
16. and L. Jacobson. 1?C2. Iron localization in pea

 

plmtSe Plantl“1":__ia C :0 37: Shred-5:310

l7. Eroun, J. C. 19);. fine effect of dominnice of a Letaholic sysoen

requiring Fe or Cu on developnent of line—induced chlorosis.

 

int .1. A—c
lo. . 1955. Iron chlorosis. 1;. Rev. £1a1t Pl mig1.
f: ”1-1 lo

». .—" C‘ -. A- -‘ 1W. ‘rrfi .’ r7171 .. . I 1 . . 4' 4 ".4 ‘IV‘
1?. and o. L. Hchulicuue 1&54. an“ i.aoio actiViolos as

 

135

incication Cl copy r and ircn deficiency in plants. Plant

PLVsicl. 27: 651—60.

/

26. and h. 5. Holmes. 1955. Fe, the limiting element

 

in a ctlo.: Part I availability and utilization of Fe donendent

A

uycn nut. and p1. 8100188. Plan; ILysiol. 3C: A51—57.

 

 

21. . 956. Iron supvly and interesting
factors related to line-induced chlorosis. $051. 931. C2:

 

P.)

I“)

o
‘

, and L. C. Tiffin. 1958. Iron

 

ctlcrcsis in sogbeans as related tc genctype of rootstcck. 1511

 

 

 

121. CE: 75-82.
23. , , and . 1959. 1;}ctheses
ccncernin: Fe chlorosis. £211,§§;. §:§;_mj§gp 1:33, 23: 231—1.
21. , , alu . 1§(l. Ircn chlorosis

 

 

 

in scybears as related to genotyyes rootstock. II; A relationshiy
between susceptibility to chlorosis and reductive capacity at tte
rcot. Soil Sci. 91: 127-132.

25. , and h. E. Jones. 1762. Absor1tion of Fe, Ln, Zn,

 

Ca, fit, and khostnate ions by soybean rccts tLat ciffer in their

reductive caLacity. Soil §91. 9h: 173—17§.

 

26. C. B. Lunt R. S. hcltes L. C. Tiffin. 1959. The
9 D ,

 

KCL ion as an indirect cause of Fe chlorosis. §ci1 Sci. 5a: ELL—o.

27. , and L. C. Tiffin. 1962. En deficiency and iron

 

chlorosis debendent on plant s;ecies and nutrient element balance
in Tylare clay. A run. dour. 3t: 356-6.

26. , a. 5. holmes, A. a. Specht, and J. N.

‘

 

. I \

Resnickf. 1957. Internal inactivity of Fe in soybean as affected

4"“

A
451.

\ 0
\M N
C

b.)

Kt)
b
.

37.

’3:
/U.

by root {routh Leuinm. 3*i1 Sci. ;.: cj— A
brown, J. H. and C. H. Madieigh. 1§55. Influence of LaHSC} on

grcwth and chlorosis of garden beets. got. 682. 11c: 2C1-3L9.

 

n

11111101., EL. 1'10, U. 1H". TI'AJI‘I-€, r. L). 112111.10 1,443. The effdct 0f

light, soil teuierature, and soil moisture on lime—induced

chlorosis. Sail §ci. ggg. gger. Frag. 13: 374—8.

Cain, J. C. 1352. A Comgarison of ERA. and LG3-L for LlueLerries.

 

Ciaberek, 3., and A. E. Lartell. 1959. trgan"c Secuestcring

I
.5

Agents. Proc. ALer. Soc. Hort. Sci. 75: LAL.

 

Chang, K. T. and I. E. Loomis. 1§45. Effect of CC, on absorption

of mater and nutrients by roots. Plant Physicl. 2L: 221-32,

 

Chapnan, H. D. 1939. ALscrytion of Fe from finely ground
maénetite by citrus seedlings. £93; ggi. L6: 3C9-13.

Clark, R. E., A. wallace, h. T. Lueller. 1961. Dark 0C2
fiAaticn in avocado rocts, leaves, and fruit. Proc. Amer. Soc.
5223' égi. 78: _161-8.

Colarove h. 8. Jr. and A. L. Roberts. 1956. Growth of azalea
(.4 , , ’

 

 

 

as influenced by RNA and HOB-K. Proc. Amer. Soc. Hort. Sci.

Dechk, P. C. 1955. Iron nutrition of plants at high ph.

§9il Sci. 79: 167-175.

, and E. L. Strmecki. 195A. An investigation into

 

growth promoting effects of a liquite. Physiologia ”lantaricm
7: 5C3-11.
Dovey, R. C., R. L. Smith, H. K. hiebe. 1960. Ef“ects of various

levels of bicarhcnate, phosphorus, and pH on the translocation

AC.

Al.

I)

In...

43-

L7.

137

of foliar applied iron in plants. §gil_§gi. 8?: 259-275.
Eaton, F. M. 1936. Automatically operated sand-culture equip—
ment. ggyw. figg. figs. 53: ABB-LAA.

Franco, C. K., and d. E. Loomis. 19AE. The absorgtion of P
and Fe from nutrient solutions. Plant Eggsiol. 22: 627-3h.
Gaugh, H. G., and C. H. hadleiph. 1951. Salt tolerance and
chemical composition of Rhodes and Dallis grasses grown in sand
cultures. gig. Egg. 112: 171—5.

Gile, P. L. 192C. Cause of line-induced chlorosis and availa-
bility of Fe in the soil. £333. 51;. Res. 2C: 33-Cl.

, and J. O. Carrero. 191A. Assimilation of colloidal

 

Fe by rice. £335. 5:3. ififi- 3: PCS—210.

Goss, J. A., and E. H. Romney. 1959. Effects of bicarbonate
and some other ions on the shoot content of P32, Cah5, F059,
Rbaé, Sr9o, Ruloé, 03137, Celhh in bean and barley plants.
Eleni.§ni.§9i1 10: 233—2Al.

Granick, S. 1950. The structural and functional relationships

r11) \ r
(_L. ,— ,)0

between heme and chloroyhyll. The Nerve; Lecture series AA

 

Greenwald, I. l9h5. The effect of phosphate on the solubility

of CaCO3 and of H003 on the solutility of Ca Lg phosyhates. Joug.
“_- w r“ _ /-v ~

Lioi. 2&23“ 101: 697-7CA.

Gris, E. lth. Rouvelle's experiences sur l'action des composes

ferrugineux solubles antliqucs a la vegetation et specialment en

traitnent de la chlorose et de la detilite des plantes. Cnggjr

"w

Rand. figgg. §g§. (Paris) 19: 18-19.

Guest, P. 1944. Root—contact phenomena in relation to Fe nutrition

and growth of citrus. Proc. Aner. Soc. Port. Sci. 4L: L3-h3.

 

138
in the acidits

 

 

 

 

 

173-85 0

SC. Custafscn, F. C. l).,. Diurnal clan es
E“"C“‘."Il ‘1 001.11.111.11... 211-..;- *1 :1: 3...- 7 : 719-7
51. haas, A. L. C. 192”. Stuu110 (a t. reaction of plant guices.
52. ha exar, n. 1., and E. L. Iaro an. 1939. lnjur519 produced
1:” sail! e anti alkaline meters on reexfircuse ,1aILts and the
alleviation of alkaline inj 13y I:y neutralization. £333. Agggy
Soc. “ort. Sci. 39: 375 ~33 C
53. h 1e, t. .., and A. wallace lQCC. ICC3- azd P effects on
uptake and distrinuclon in soybeans of iron che lated with
etkvloneoianiro di-o-hydroxyphenylacetate. §gil ggi. 89: 235-7.
54. Harley, C. P., andR C. Linuner. 19h5. Chservcd ponses
of aklle and pear trees to 9016 irrigation waters of north
entral uasiix ton. I: 3. A333. §33. Io o-rt. §3;. to: 35- LA.
55. Heller, V.C uo, R. h. rabouan, and E. L. hart1_an. 1940. Sand
culture stuuios of the use 01 saline and alkaline waters in
greenhouses. lrnt jigging. 15: 727-33.
56. HoaLlanu, u. n., and D. I. Alxon. 1433. The water culture
.ethod for rruin; plants Wiu.CUL soil. _Cigf. ngh. éta. Cir
347.
57. , and T. C. Eroyer. -93é. C neral nature of the
krocc s of salt accumulacion by roots with d850“1ption of
e3}e*Luenta1 m1thods. I ant P’"siq;. 11: A71~§C7.
53. , and . lQAC. I crogen ion effects anu
accurulation of salt by barley roots as influ were ed by metabolism.

A533. Jour. Pot. 27:

59. hoifer, C.I. l??3. Aceunulation of a uLiI-um aI;d iron com-
fomals in corn 1-1an‘ts and its probatly relatio n to roetrots.
gogg. 5;. 12:. 23: 3Cl—23.

6C. Holmes. R. S. 1?CC. dfigct of pIo3“htrus and CH on iron chl o-
ros is of tlueierz" in mat 3r culture. §;i_ égg. 9C: 37L-9.

Cl. Korner, C. K., D. Burk, and S. R. Foo~er. 173A. Preyaration

of IIuLate iron and other Lunate netals. §1g§t_§§;picl. 9: 663—9.

V

(Y\
(X)

. Huffa mr, R. C., and A. gallace. 195). Dark fixation of 0C9

in honofeha es from citrus leaves, fruits, and roots. Free.

3:;‘(3ro £31.90 EiC‘I‘E/o 2;. 7L: BLS‘BSZQ
63. Iljin, W. 3. 951. Ketabolism of plants affected with line-

induced chlorosis (caloiosis): II Cr:anic acid ane carboh"drates

 

64. 3* . 1952. Letabolism of plants affected with lime-

 

inducedo mlorosis (caloiosis). Plant and Soil A: 11-23.
65. Ingalls, R. A., and J. U. Shive. 1931. Relation of H—ion
concentration of tissue fluids to dis ribution of iron in plants.

Ilant PMI‘VSjCjJ 6': 103‘1250

 

CC. Jacobson, L. 1945. Iron in tIe leaves and cIloro; lasts of
some plants in relation to til Ieir chlorophyll content. Plant
’hvsitl. 2C: 233~2LS.

67. . 1951. Laintenance of iron surply in nutrient

 

solutions by a sin; 1e addition oi‘ ferric 1osassiun ethylene-

dicmine tetra-acetate. Plant Physiol. 26: All—A13

 

68. , and J. J. Certli. 1956. The relation between iron
and ohloroyhyll contents in chlorotic sunflower leaves.

Plant F‘Vsi cl. 31: 199-2C4.

 

*3

72.

N}
KL)
0

71+.

”.3
[J O

79.

140

, 8rd L. Crdin. 1956. Crganic acid and icn atsorption

 

in roots. gggggiggigg59, 29: TC.

Jciireys, R. A., V. g. Rale, and A. hallace. 1961. Uptake and
translocation in plants of labeled iron and la oeled chelating
agents. £32; :23. 9?: 2 8-73.

nanny, Hans. ‘Plant root-soil interactions" in K. K. Zarrow,
ed., C Chth_ Ln Lixgp“ S"s§en§. Eas ic Books, Inc., flew Ycrk 3.
1961. pp. 665-9Ao

Jenny, H., and R. Cverstreet. 1933. Contact effects between
roots and soil colloids. Ergo. ggt'l. fipgg. §ci. 53: BBL-332.

lh

Jone s, H., and E. H. Eagles. 1962. Translocai ion of Carl (n

ca ‘
'.L..Lo

witlin anJ betneen leaves. gnnggg a; Egtgn; 26: 5C5—,
Jyuné, N. K., and S. H. fiittver. 1963. Kinetics of foliar
abscrpticn. {lggt LL;§§£}. 38 suppl): XAVI

Kenzxorflny, A. L., and E. J. Liller. 19576. IJEutri exit-element
analysis of fruit tree leaf 53U1)1CS by several labo rator1es

11cc. 5:31;. 5333. $5.3:- §_c;. 67: 16-21.

Kraner, Paul J. 1957. Du or Space in Plants §g;. 123: (33-5.
Kurtz, E. E., Jr., and R. K. Laier. l?6 . Acid chlorosis and
iron u;ta1:e by sorgham (Double Dwarf-33) grown in solution
culture. A"rCn. £533. 52( ) LE6—Ao7.

Latiner, I. L. l?52._ C: iolt'“n Pot _ont a1 2nd Ed. Prentice-
Hall, Lew York.

Leonard, C. D., and I. Stewart. l?53. An available source of iron

for plants. Frcc. Aver. Soc. LC?

 

Lindner, R. C., and C. P. Karl y. 194v. Lutrient interrelations

in 1:"J—induccd chlorosis. £2932 Fkvsicl. 19: AZC—Eg.

A“
was.

f'\

(a

f‘ L
"J 0

E60

(5
(J90

141

(TH

. - _ . * -11 r ~.' , ‘ "r1 '_.‘ “‘.\ .,..'- ',.-. ,.
LJ.C..JC“,, H. Lo, 3114 .LJ'. .1. 1.. 1‘1‘30 1.5/1+0 LiCEiuCILouo’ leJ. «:1le

‘ ~V'r-J-‘v "-- “'1 . ' r - ‘. a'l‘tr- ‘~‘ . .r-'. ‘..' r:r rrw
CLJQUJ leVUi as leluLeu to CulutCClb. SCil 0,1. {1: C(1—V.
.1 'r m: .5“: C-,: '1 1",, -C is C.“
1:141:16, U. C., L. C. $1114.11, 841k; J. U. 4.102.110 l/U/‘o 1CD
LTI‘L , _‘L " 1 t, (*r“~‘1‘)'1~ fi '10 ‘ ”i" 13" >4 "7 f1 ’4‘! '3
.l--.3— UL J15} (.1 («f 0L}, . D‘uad C...) «Lli .LLLILCCIL‘ [91' CC; .91 Ck-‘qlC’llJ.
' 1— __ v "1 F n’
Egan-Lat TI.“"»‘.._;.. 3k): (l—‘Fm

Loehwiny, X. F. 1?:.. alciuu, octarsiuu, and iron balance

in certain crop plants in relation to their metatolisna Elgfit
Lucas, R. 3., and J. F. Davis. 1961. elationshiys between ;H
values of o‘ga.ic scils and availabilities of twelve plant
nutrients. Soil §3i. 92: 1'7-1C2.

Lunt, C. R., and K. C. Kohl, Jr. 1956. Abscrytien £.nd tr his
,; . . .- ; ; 1 ; A ;
location Cf iron from various Sources by rose leaves. arm. Cn
ti: E;e o; Leta; Chela43§_ 32L§_a. Lutr;tiop, A. uallace ,Editcr.
LcCall, A. 0., and J. R. Haag. 1921. TLe relation of H+ CCn—
rentraticn of nutrient solutions to ”rcntn and chlorosis of

wheat plants. Soil Sci. 2: 67-77.

”—‘M

\' "1 v~-

LCr“Cr€G, w. T. 1919. A study Cf lih:e -iriuced cthrosis in
Arizona orchards. £512. §;. gig. §ta. _gci. £311. 117.

Karen, R. P., and J. H. Shive. 1925. Adjustment of iron sugply

to requirements of soybean in solution culture. 2C3. Caz. 79: 1-27.

Eattson, S. 193C. The laws of soil colloidal behavior: III

ISO-electric precipitates. Soi Sci. 36: h59-h9)

 

lilad, Y. 1921. Tne distribution of ilcn in chlorotic pear

trees. Prcc. Aver. Soc. Hrrt. Sci. 21: 93-98.

 

 

 

killer, G. W., J. C. Brown, R. 5. Holmes. 196C. Cnlorcs is in

so; teen as related to Fe, P, HCC3, ad cytochrome cxiuase activity.

92. , and H. J. Evans. 1996. Inhibition of cytochrome
oxiuase Ly bicarbonate. Lgtgrg 178: 974—976.

93. , and . l95o. The influence of salts on

 

 

activity of particulate cytochrome oxiuase from roots of higher
plants. 41:.1t 1.7:;ci. 31: 57-CL.
9A. , and D. M. Throne. 195$. Eli'ect of L-icazLonate on

t1le respiration of C; cised roots. Plant thsigl. 31: 151—5.

 

95. 1.81181”, Do Do, R. L. 81111:“, alld 11. 11. 1dieL o 756). EffGCt 0f

plant micronutrient balance on the translocation of foliar

applied chelated and non-chelated iron. S'n. on use E£.L2;Q1

£3
E.
H
c...
C“
0)
IE
'3
p a

intrititn. A. wallace, Elitor.

—. J' 0.... -

 

96. Lorth, C. P. 956. Translocation of chelated and non-chelated

iron applied to avocado seeulings. S"Rh on use cfi_1etal 91913392.

"U

lent fiutritiCn. A. Wallace, Editor.

, --—. --__v .5.-

1.11

" Oertli, J. J., and L. Jacobson. 196C. Some quantitative con—

\0
O

siderations '1 iron nutrition of ligle }lants. Plan lvsiol.

w—o—u—o—h—
35: 6”3~ 8.
9o. Olsen, C. 1‘93 9. Iron atscrpti on and chlorosis in green I,W1ant3

I

Corpt. rend. Eran. lay. CarlsterL, ser. chin 21: 35—50. Abstract

only .

79. . 1953. Iron u;take in different plant Species or a

 

function of pH value of the nutrient solution. Physiologia

antarum 11: 869-9C5.

mwcm- -

1C0. Orr, H. P., Tokuji Furuta, and Charles H. Bell. 19‘57. Azalea

fertilization. Ala. EXo. §t_. Cir 118.

 

1C1. Cserko;sLy, J. 1931.1;Cro en ion concentration and i; on content

103.

lCA.

1C5.

1C3.

1C9.

11C.

11.3

'1

of tracheal sa:\ fr 1 green and chlorotic pear trees. Eigrt
Bl? iFI-l. 7: 33:53.7.

H...»

. 1733. Quantitative relation between chlorophyll

 

"+
IT!
,3.

and iron in green and chlorotic pear leaves. 41am,
Lh9~€8.

Overstreet, R., S. Ruben, and T. C. froyer. 1VAC. The absorytion
of ti ar onate b3' bar ley plants as indicated by stud es Lith
radioactive carton. 239g. {31. gape. §_hi 26: 638-95.
Parry, R. U. Che Mfi and the theory of Leterocyclic ring
formation involving metal ions. QE§§° cg Eggrginatign o;
Pailar, Euitor.

For or, L. K., and D. W. TLorne. 1y,=. Interrelation of carton

dioxide and bicarbonate ions in cauoinr plant chlorosis. §; 1

it).

Sci. 79: 373—32.

_‘u'

Rediske, J. H., and C. Biddulph. 125 . TLe aisorltion al'd
translocation of iron. £;;€£ §p"§;;l, sg- fié-,}.

Reuther, 1., and C. L. Crawford. 194$. Effect of certain soil
and irrigation treatments on citrus chlorosis in a calcareous
soil: I Plant Responses. 3o1. "l §:i. '2: A77-91.

, and . 19A7. 31 f ects of certain soil

 

 

and irrigation treatments in citrus chlorosis in a calcareous
soil: lI soil atmosphere studies. §gil_§gi. 63: 227—52.
Rogers, C. h., and J. h. Shive. 1932. Factors aifecting th
distx luticn of iron in plants. Plant pvsigl. 7: 227—52.
Scndroy, J., Jr., and A. B. beatings. 1925. The solubility
product ofs secondary and tert_a1y calcium pho ospnate under various

CbflditiOflL. Joug. giol. GL3;. 6h: 5C9-

I
. I
a 0 9 a no
- ~
) a a
O
0
U
‘ I O k.
A
' .
) . O .0
I
>
a O 0 q
4
. ‘ q
a
v
Q I I t
9.
\
I 4 Q. '{

v.

111.

112.

113.

115.

116.

117.

118.

119.

120.

144

Shannon, L. M. 1956. Some chelate studies concerning the
behavior of iron chlorosis. SEQ. gg_thg use gg’Metal Chelates
in 2132: Nutrition. A. Wallace, Editor.

Smith, P. F., W. Reuther, and A. W. Specht. 1950. Mineral
composition of chlorotic orange leaves and some observations
on the relation of sample preparation technique to interpre-
tation of results. {lent Physiol. 25: h96-506.

Sims, W. H., and W. H. Gabelman. 1956. Iron chlorosis in
spinach induced by phosphorus. 2593. Amer. §gg.{§gg§. §gi.
67: ALS-ASO.

Somers, I. 1., and J. w. Shive. Iron-manganese relation in
plant metabolism. Plant Phx§;9_. 17: 582-602.

Spurway, C. H. 1933. Soil Testing, A Practical System of
Soil Diagnosis. high. Ag. £5925. Sta. 23911,. M. #132.
Stalwijk, J. A., and K. V. Thiamann. 1959. On the uptake of
carbon dioxide and bicarbonate by roots and its influence on
growth. Plant Rhysigl. 32: 513-20.

Stewart, I., and C. D. Leonard. 1952. Chelates as sources of
iron for plants growing in the field. §gi. 116: 56h-566.
Stewart, F. C., and C. Preston. 1941. Effects of pH, and
components of bicarbonate and phosphate-buffered solutions on
the metabolism.of potato discs and their ability to absorb ions.
Elan; Physiol. 16: 481-519.

Sn, L. Y., and G. W. Miller. 1961. Chlorosis in higher plants
as related to organic content. Elan; Physiol. 36: LlS-hlB.
Taylor, G. A. 1956. The effectiveness of five cleaning pro-

cedures in the preparation of apple leaf samples for analysis.

:3

K- .1...

193.

l‘:o1* .

126.

1'?

13C.

r. ‘\‘_ _ c_'. vfig“. ,«I 4 r7 :- —)
rroc. a1,r. soc. Lxluo 03.. 6;: 5-7.

Teutner, F. C., A. A. Hittuar ,L. C. Lon;_, aid h. E. Tuisy.

Dcting ahsorg ion and trinsport of

}_.J
\
\ '1
i
(J)
a
O
F I,
Q)
0
’1
‘1
Q
Q.)
‘J
H:

foliar-ark] ied nutrients as revealed ty radi‘ ctive isotof:es.

'. full. 39: 39C-L13.

' ' ”W ‘N
‘ 1"“ A,“ -'~—-wA. \o-’
I fin-‘tl. . Q4“; . s’l.
r-ho- o-Q-- - .—

'\rcne, D. U., F. L. Lann, and H. Robinson. 195C. Errotheses

concerning lixhe induced crmlcrc;1.3. Soil Sci. Soc. A er. Pro

 

, and A. Wallace. 1941. Some factors affecting

‘ fl

chlorosis on hign line soil: I ferrous and ferric iron. DC

11

Tiffin, L. C., and J. C. Erown. 1959. Alsorption of iron from

 

 

 

 

iron-chelate by sun fl er rrcts. Sci. 130: 274-5.

, and . 1961. Selective absormtion of iron
fr m iron-chelates by'soyhean plarts. Plart Elgggagl. 16: 710—1

, and __ . 1961. Iron chelates in s<:"1ean
exudate. S31. 135: 311-313

, and , and R. U. Krauss. 1960. Differ-

 

 

ent 1al absorption of netal chelates components by plant roots.

Elgnt Fljsigl. 35: 362-7.

Ttifg, h. C., and C. B. Link. 951. Futricnt deficient syr

gl‘tCLZS

and leaf analysis of azaleas grown in sand culture. Pros. £333.

Taynan, E. 3. 1959. The effect Cf iron supp‘“ on tls "191d anc
co*no"1t‘ou of lw es of torato plants. Plant and Soil 1C: STE-CC.

.7 ~15 ‘1‘.' Y ‘1‘ v-T '7‘“ V
“8.111.151, Co Lo, 8.11.»; J. to. TAG-“Til.

fl
.,

bean plants airlicted with bicartcrate induced chlorosis.

1’ .)

1.1
k-)
b.)
O

A I
1"“.10

y"!
\
C\
o

79‘
*JKJ.

14C.

146

_..._._.........--..J mid U. H. ~111in l/3(;o L338
as influe.eed Ff 1X 01 ou13trate and form c
“8.3-1., i1. 1‘ o ’ 831C} 3. L. 1.5.1.”: V ‘ T“. JT‘ZIA. 38.“.

. 'r. 1 fi ' ' /1
2L; ‘1’... 3’: L‘. a_’._:\ . x) :--. O+C . L!‘}_L-/ .
“'fl J -'.~v‘ ' “.w ‘11-!" 'I ”I“ K
II 8.1- .L.L (29.2, A. ‘J‘ _I_ ’(‘z' A../ :T_J.)3 (.L L‘(\ [_r Cjab )3
\ ' - .‘ 1. ‘. 't 1 1 .‘
licarcrna.L3. *7. CJ 1.1 L 111 c.a e3
mu- ..0 ”—9 —r'-'. .-‘~-'- Oh. ' '0 _I _-

- -" ‘I‘\§_-_o_ qrr/
A. ..8._.___(133, 44(“..._-“;I:/lfi O J., "L O
,J
, €J1-.L A. A.

133(3 ),

\0
U1
0
H
'1
O
:5

h ,3 ‘-
113111 1. 1

(J.

 

05.71?" ,‘..1
«.L....J.o a‘L 101'030
—-.o -‘—ru ‘

 

, R. A. JV fir‘u 1'3, and V0 .. Alta-.13
alllitg c1 taO 5c; Lgan va'ie Lie” to take ug

 

ccrLanc cf ccrn ylants

t‘ " n». 3 v 1‘ ‘ 3 .'
1 1. C71.J_--).z-:'. or 11 vL.Lo
,3 J. n \

u cul:u“e snuaies cf

: - r . .L. . ,
1r: c1 t1:auc. Y1rc.
m. D g .
111 jI‘EmBLCO C1 ..)L {.1731
in f: ('1 it T 11 ‘12.”. ’1 \ '1': ,

to a nutrient soluticn. Soil Sci. 9L: 111-111.
: and C. R. Lunt. 19(C. rcn chlorosis in horticultural
11‘1ts, A I3vie". nge. 1133. 459. Lcrt. Q31. (5: $19-111.

 

 

 

 

, :L. To 1.1161161, D.VCI1 IL‘Ol‘t (Jul (J P. luCI‘tlxo lrk/Co
Lari Caxbcn diexide fi.a;i cn cf v1ru3-dieee:e6 , ircn—cllc: chic,
and genetic-ch1c1cpi leaveu. Efiéx- Amer. §Lg, 1:5}. §gi.
76“: (:79-Ct31.

, and C. F. Hersh. 1953. ime ~1n¢ueed enlcrcsis.
ggggg. £93. I 3): 1C.

, , 11. T. Lueller, L. 1-. 01311111., 8.111
C. Keaiiean. 1955. Behav1er c chelatinr enbs in ylal s.

fiallace, T. 1723. Investigations on c130;

I‘ n‘._ °_'. .1_.‘,,
esis C1 I1dlu t1ec3.

 

153.

'7
.- o

’3
(J.

149.

151.

-
r‘l~‘.-. F“, r
byg»L. -l k

' L.)
J

hallihan, 3. F. lTEJ. R'f eticn Ci cLiclcsi. to concentratian

‘0 7'1" .' ' 3 ~ ‘- L .-,-. 7.-....“ A n *3. J I) ~ 2 .
CL .e in CltEUQ ieaves. A ut- .uui. Li _LL. L_; iLl—LLA.
“ID-0“- WD-u— -~

{-11 3““..- I_ an «.u.. ‘ -‘u n .. I ‘
lzti. niiect c1 seeiim Li: ~Ltnu. cn lFLn

 

‘. A '- ‘ 'l' . i ‘ ‘N‘w fi~~ ‘ _"u‘. « ; . u. v I —y!- r.
l/f '0 “hi?“ ItLlCI; 11). t1 al.u...CCt-uJL11 Ci '1“. LJ' ondl;.l.b..b'; f'ldl.uoo

‘~ «m4 e ,2 “1.» e 6 er
1i 0 (5...“ rUCu U.-1:-i... LL: AKl-A~_).

 

' '3“ 7-x... ' M 1.: r! L '1 . ‘ ‘ .
, No 1.1. RVIJL'LLQ’ 2.11;]. II. F. PUl‘AtJLSo l‘P/H’. Cl-(3.1_atl_né}

 

aeeits and plant nquition. Sci. 120: 51-53.
heiss, K. G. 19L}. Inreritance and f“*°“oio v of efficiency in
iron utilizaticn in soybean. Eggptics 28: 253-63. Abstract cnly.

delcher, F. J. TZe Ar2"ti ;; ngg E“ 3025. Von Iostrand, Inc.,

.-

‘i- C" V? ‘ ‘y;
Luau}, ADI 1's.

hhite, K. E. 1354. Lesponse of rose and gardenia to treatment

4"- . r— 1..- -'- — ‘~ .1 '1'..--: “a" ”a v)“~ v» .. o - 7“.-
with irun enclate and Chem L it a cnt. ice. A cz. v03. Leit.

_-

ci. Ch: #23’430’

’Jildon, C. E. 1957. Croxth cf rose, alfalfa ald tobacco plants

as affected by different sources of Fe. ligfi. 5;. gge.

Ga .
C .5.

1 U;

5355. 39(4): 628—634.

. 195$. Tie effect of prolonged leaching and of

 

‘ I O

successive generations of plants on the solutilit y cf iron and

manganese in glassy frits. L 5:3. 5.;;e3;.__ gee. filer-t. 91:3. 72

 

P “'1 av 4-._ v Tl”. 7. -' $\".I‘f W I‘ 31h ~ "
CJ. .L..C.'.l ibl _ “6.64.4.5 J.LL1.I.LJQ C11 C<

55-63.

h-u-g

 

for Lyi

» *\~: 1‘ J' ‘s‘ .0 ‘t‘- ~n‘
rcpcnic ciltuie Ci LLLQLO.

M“: C *3 ,4 f
, Chin I. .4. n..-.L~J.L/.l. .L'

lie.

“-5‘.

Q

'9 ‘4
\....‘-a
a—“u

ll‘

- ’7 ‘7‘

5’
.LUs)

0 149

APPENDIX TABLE 1. Nutrient Solutions

I.

II.

III.

Hoagland's Number One Solution

Compound Stock
Concentration
KH2POA 1 molar
KNO3 1 molar
Ca(NO3)2 1 molar
Mg 805 1 molar

Twigg and Link's Solution

Compound Stock
Concentration
KH2POu 1 molar
(NHh)2SOh 1.75 molar
Ca(NO3)2 1 molar
Mg 805 1 molar

Hoagland's "A" Solution

Compound Stock
Concentration (g/l)
H B0 2.86
3 3
Zn SOu-YHQO 0.22
Ca SOu°5H20 0.08

H2MoOu-H20 0.02

Dilution

(ml/l)
l

5

Dilution

(ml/l)
1

l