CHLORIDE. TOXICITY 05 FLOWERlNG STOCK
{MNTHIGLA aNCANg-g. R. BR)

AND SWEET PEA
(LATHYRUfi QDCEATUR L)

Thesis 50:!- i‘gm Degree of M. 5.
MICHEGAN STATE UNIVERSITY

Samuel Wigdor
1957

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L I B R A F3. it,
Michigan State
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CHLORIDE TOXICITY OF F LOWERING STOCK (MATTHIOLA

 

INCANA, R. BR) AND SWEET PEA (LATHYRUS ODORATUS, L.)

 

By

SAMUEL WIGDOR

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1957

Approved WM fl; @‘fld‘;

THKF‘S

SAMUEL WIGDOR ABSTRA CT

Greenhouse crops in the State of Michigan may be subjected to substan-
tial amounts of the chloride ion which may come from the fertilizers and tap
water used in their culture. The present work was designed to study the speci-

fic effects of the chloride ion on the growth of sweet pea (Lathyrus odoratus, L.)

 

and flowering stock (Matthiola incana, R. Br. ).

 

Six levels of chloride were used in different nutrient solutions, the
basis constituents being those outlined by Hoagland and Arnon. The plants were
grown on two different soil types and each treatment was replicated four times.
Growth was measured as fresh weight, dry weight, and number of leaves
for each plant species. In addition, measurements were made on the number
of branches and flower buds of sweet pea, and the number of open flowers, stem
diameter, and mean internode length of flowering stock. Chloride determina—
tions were made on both the soils and the above-ground plant tissue.
The results indicated that there was no reduction in growth attributed
to the chloride ion within the concentration range studied. F oliar necrosis was
noted on both sweet peas and flowering stock. In the latter instance, the inci-
dence of necrotic leaves was related to the amount of chloride in the solution applied.
Granby sandy loam was found to inhibit the branching of sweet pea. This

may have been due to adverse conditions of drainage and aeration, and pH.
Accumulation of chloride ion in the soil was found to be an exponential

function of solution concentration, where the exponent is greater than one, while

plant accumulation was found to be exponential as well, the exponent being less than one.

nu‘s's

' CHLORIDE TOXICITY OF F LOWERING STOCK (MATTHIOLA

 

INCANA, R. BR) AND SWEET PEA (LATHYRUS ODORATUS, L.)

 

By

SAMUEL WIGDOR

A THESIS

Submitted to the School for Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

I957

THFWS

 

.«Y'P‘ '7

.r.-- 7-5:. ti

ACKNOWLEDGMENTS

The author extends grateful appreciation to Dr. Richard F.
Stinson for his suggestions and criticisms throughout the duration of
this investigation and for his constant encouragement during the pro-
gram.

Further acknowledgment is extended to Dr. Wade W. McCall
and Professor Paul R. Krone for their many helpful suggestions in the
planning of the study, and for their invaluable criticism in the prepara-
tion of the thesis. Special thanks are extended to Dr. Robert L. Carolus
for his criticisms and suggestions toward the improvement of the manus-
cript.

The author also wishes to acknowledge the devotion and sympa-
thetic understanding of his wife, without which the present investigation

would not have been undertaken.

THFF‘E

 

TABLE OF CONT ENTS

INTRODUCTION ....................
LITERATURE REVIEW . ................
Ion Absorption . . . ................
Salt Tolerance ...................
Chloride Accumulation '. . . . ...........
Chloride Toxicity ..................

Chlorine - An Essential Element .........
MATERIALS AND METHODS ...............
RESULTS . . . . ....................

General Observations ................

Sweet Peas . .I ...................

Flowering Stock . . . . . . . . . .....

Chloride Accumulation . . ..............
DISCUSSION .. .....................
SUMMARY .......................
BIBLIOGRAPHY. . . . .................

APPENDIX .......................

Page

11
l4
18
21
21
24
27
30
36
40
42

52

INTRODUCTION

The chloride ion is a minor constituent of soils, and greenhouse soils
may contain substantial additional amounts from fertilizer and tap water
sources. In the first instance, greenhouse operators generally maintain ade-
quate potassium levels in the soil by the application of potassium chloride.

In the second case, sources of irrigation water may contain as much as 700
parts per million of chloride. 1

Ground water high in chloride ion content is found in some areas in
Michigan as a result of large salt deposits. In some locales, formerly pro-
ductive salt mines have been abandoned, permitting the deterioration of
equipment, especially piping, through which large quantities of salt may
enter the water supply. These levels may result in suboptimal crop yields.

Although much research has been conducted on soluble salt toxicity,
some of which has been concerned with the effects of specific salts, only a
limited amount of research has been done on the toxic effects of the chloride
ion itself. The present work was designed, insofar as possible, to isolate
the specific effect of the chloride ion from those of increasing osmotic pres-
sure, electrical conductivity, or the specific effects of another ion or mixture
of ions. The plants used in this study were grown in soil and in environmental

surroundings similar to commercial growing conditions.

I
See Appendix Table A.

LITERATURE REVIEW

The subject of chloride nutrition is one which has been investigated
for many years. Observations have been made on the tolerances of different
crops to the presence of various amounts of chloride in the solution associated
with the growing medium, toxicity symptoms and levels, and the possibility of
chlorine being an essential element for the growth of higher plants. To date,
much is known about crop tolerances, especially in regard to the genus Bela
(23, 25, 85, 97, 99). It is only within recent years that the physiological prob-
lems of uptake, translocation, and distribution of chloride within the plant have

received serious attention.

Ion Absorption

 

The mechanisms of ion absorption and accumulation are nOt thoroughly
understood at the present time. Several different systems have been proposed,
and, although there is no complete agreement among the workers, differences
in points of view are not as wide as may appear upon superficial examination.

The rate and extent of solute absorption are dependent on aeration,
temperature, water and solute supply, etc. These are well known, and will
not be elaborated here.

By using a technique which exposed narrow regions of an excised barley

root to radioactive phosphorus , Kramer (61) has shown that most of the salts

translocated upwards are absorbed in the root hair zone. The apical region
of the root accumulated salts but did not release them to the shoot. In this
region the xylem is not differentiated, hence there is no pathway for rapid
upward translocation. The region of salt accumulation coincides with the
region of greatest water entry.

Cell walls and cytoplasm occupy about fifteen percent of the volume
of a wheat root cell (18). The water in the cell walls and cytoplasm may be
regarded as continuous except for the Casparian strips (15). The vacuolar
sap is discontinuous. Thus, ion transport to any individual cell may merely
follow the transpiration stream.

Further evidence substantiates this View (52, 53, 79). By the use of
respiratory inhibitors, it was demonstrated that some, but not all, ion uptake
was stopped (53). Varying the rate of water transport by any of a number of
means alters the amount of ion accumulation (IS, 52, 79). Thus, one phase of
ion uptake is transpiration-dependent, and another phase respiration—dependent.

To quote Hylmo (53): _'_'The fact that the ion uptake component which is
unaffected by inhibitors is directly proportional to the rate of water flow con-
stitutes conclusive evidence that Cl' ions are passively drawn with the transpira-
tion stream from the medium through the root to the shoot, independently of the
metabolism of the root. It does not support the theory that the ionsare trans-

ported through an endodermal barrier and actively secreted to the xylem, unless

we are willing to assume that the ion transport here is mediated by a metabolism
which is not repressed by the inhibitors employed or by anaerobic medium. "

Kinetic studies (25, 26, 27, 52, 65) have shown an initial rapid ion uptake
followed by a slower, steady accumulation. The first phase involved an equili-
brium between root and external medium which was established within sixty
minutes (27). At equilibrium there is no competition among ions, and pH bears
no influence. This may be regarded as diffusion into "outer" or "free space".
The second process is irreversible and involves active transport.

Several theories exist concerning the mechanism of active ion transport.
Most authors agree that transport is linear with time, it requires the expendi-
ture of energy, it is selective with respect to ions, and the presence of some
ions affects the uptake of other ions.

Lundegfirdh's scheme (17) involves four assumptions. The absorption of
anions is independent of the absorption of cations so that separate mechanisms
exist for each. The absorption of cations takes place in two steps, the latter
involving excretion into the vacuole and constituting real accumulation. Anion
absorption involves a portion of total respiration referred to as anion respira-
tion. It is distinct from ground respiration.

In essence, Lundeghrdh proposed that anions are carried across the
cytoplasm and into the vacuole by means of bridges of cytochrome--cytochrome

oxidases which are not necessarily continuous. Ferric iron is formed by

oxidation at the outside, accepts an anion, and reaches the tonoplast. Here
the iron is reduced by accepting an electron and thus releasing the anion. Cations
are bound to the cytoplasm by potential differences.

The data of Overstreet and his group (81, 82) and Epstein (24-27) cast
some doubt on this theory. Dinitrophenol inhibits respiration via the phosphate
system. This inhibits accumulation of ions and is not in accordance with
Lundegfirdh's theory (81). The relation between respiration and salt accumu-
lation is confused (37). Lundegardh's proposal does not explain why ions of
similar charges would not compete with each other (e. g. 25).

Epstein's theory (25) involves a different mechanism. The cytoplasm
constitutes part of the "outer space" to which ions are freely accessible. At
the outer surface of the membrane (presumably the tonoplast) which is largely
impermeable to free ions, the ions combine with the carriers; the carrier-ion
complex traverses the membrane; and at the inner surface the ions are released
in a rate-limiting, essentially irreversible step. The carrier is presumed to
be enzymatic in nature.

The rate of ion absorption has been found to follow classical enzyme
kinetics in its dependence on external ionic concentration and its response to
interfering ions (26). The ion-binding sites exhibit high degrees of selectivity.
Rb+ competes with K+ and Cs+, but not with Na+ or Li+ (26). Br' competes

with Cl- and 1-, but not with N03“ (24). Sr'H competes with Ca“ and Ba“,

but not with Mg++ (27). 504" competes with Se04", but not with H2P04- or
N03' (65). At high concentrations, normally noncompeting ions may compete
for the same binding sites.

Epstein has left a few issues unexplained. Polyvalent cations, especi-
ally Ca++ stimulate K+ uptake at certain K+ levels (55, 82, 101) and depress it
at others (82). The stimulatory effect is dependent upon respiration (101).
Although Br' uptake is independent of pH, within the range pH 4 to pr, K+
uptake decreases with increasing pH (77). These bits of data should be con-
sistent with any theory of ion uptake.

Salt Tolerance

 

One of the greatest obstacles hampering experimenters in their evalu-
ation of the role of chlorine in plant growth is the inability to separate the
effects of the chloride ion, per se, from those of increased osmotic pressure
or electrical conductivity of the aqueous part of the growing medium. Elec-
trical conductivity bears an empirical relationship to osmotic pressure (19, 60).
Hence, these may be reduced to one variable.

Osmotic pressure of the soil solution is an important factor in the
availability of water to a plant. At osmotic pressure values characteristic
of non-saline soils, water is almost equally available between field capacity
and permanent wilting percentage. As the osmotic pressure of the soil solu-

tion rises, the availability of water diminishes, and at high osmotic pressure

values a linear relationship between soil moisture tension and soil moisture
percentage exists (104). In some instances the stress may exceed fifteen at-
mospheres, a tension at which growth of most plants ceases (90).

The rate of water intake by a root is inversely proportional to the os-
motic pressure of the medium regardless of what type of compound is used to
raise the osmotic pressure (45, 70). Water uptake ceases when the osmotic
pressure reaches 6. 8 atmospheres (45). If an inorganic substance is used to
raise the osmotic pressure, nutrient uptake may diminish, whereas an organic
solute may not Show this effect (70).

Salt tolerance of a plant may be defined as the capacity of that plant to
produce a certain yield at a given level of salinity. An alternate definition of
the tolerance of a plant is its performance at a certain level of salinity as com-
pared to its performance on a non-saline substrate (47). The latter definition
is considered more acceptable from the viewpoint of agriculture.

The United States Regional Salinity Laboratory has established stand-
ards to evaluate the tolerance of crops to saline conditions (44). Electrical
conductivity of the soil solution is regarded as the most suitable criterion to
judge soil salinity. Those crops which tolerate electrical conductivity values
between 4 millimhos x10-5/cm.- and 8 millimhos XIO'S/cm. are moderately
tolerant of saline conditions. Crops of poor tolerance do not grow well at

electrical conductivity values in this range, and those crops which grow

satisfactorily at values in excess of the upper limit are tolerant.

The tolerance of a plant to saline conditions is not necessarily uniform
throughout the life history of the plant. While barley and corn may germinate
better than alfalfa or beet at high osmotic pressures (5, 92), the latter two
crops are more tolerant at advanced stages of growth (72). Beans may ger-
minate well at high osmotic pressure, but their growth is severely restricted
under these conditions (31, 72). Alfalfa, on the other hand, becomes more
tolerant with age (56).

Crop tolerances vary widely from family to family (63, 73, 83, 109),
genus to genus (e. g. 23, 3, 10, 68), species to species (3, 73, 83, 108), and var-
iety to variety (6, 10, ll, 42, 47, 108). Thus, it may be stated that although
growth of a plant may be reduced as the electrical conductivity of the soil solu-
tion is increased (10, 12, 23, 42, 43, 46, 59, 71, 73, 108, 109), the extent of the re-
duction in growth must be determined for any given plant.

Crop tolerances to saline conditions may be modified by some environ-
mental factors. As the environmental temperature rises, plant tolerancede-
creases (l, 78), and growth may be severely restricted during the warmest
part of the growing season (23). On stone fruits it has been observed that
chloride toxicity is more severe on the exposed parts of the trees (14). A
highly humid environment reduces salt uptake by plants (51). This evidence
would seem to indicate that the greater the uptake of water, the greater the

toxic effects of saline conditions.

Further evidence substantiates this hypothesis. The chloride ion is
freely mobile in the soil (74). Accumulation studies (18, 29, 69) show that
chloride absorption is a function of time and concentration in the substrate.
and thus the ion, in the main, is passively absorbed by the roots. Hence.
chloride toxicity, being a function of ion uptake, is related to water uptake,
and thus to transpiration. It is interesting to note that in the selection of
lettuce varieties for salt tolerance studies (6), heat resistant varieties were
used. In the light of what has been previously stated, it must be recognized
that if heat resistance is related to water economy, the conclusions reached

may not be applicable to all varieties of lettuce.

Chloride Accumulation

 

Any factor which affects the mobility of the chloride ion or the avail—
ability of water is important in a study of chloride toxicity. This statement
holds if the ion is in solution and is not associated with the colloidal fraction
of the soil.

Reisenauer and Colwell (89) have shown that the colloidal fraction of a
soil influences chloride toxicity. Applications of equal amounts of chloride to
virgin and cultivated soils yielded different percentages of chloride in subse-
quent leaf analysis. On theicultivated soil considerably more chloride appeared
in the leachate, hence less remained in the rhizosphere to be absorbed by the

tobacco plant. On the virgin soil, which differed from the cultivated soil in

that it contained more organic matter, less chloride appeared in the leachate -
More was retained in the soil and more was taken up by the plants during the
growing season.

Chloride accumulation may be related to the pH of the medium. The
same workers found a highly significant correlation coefficient of -0. 801
existed between accumulation and pH. A decrease of accumulation below a
pH value of 4 and above 6 has been noted in another study (80). It has been
postulated that a competing anion, HC03' may inhibit C1- uptake in the pH
range of 6 to 8.

Recent studies with radioactive Cl ‘36 (62, 96) have shed much light on
the mobility of chloride within the plant and the loci of maximal concentration.
It was found that €1-36 was translocated both upward and downward when ap-
plied to the leaves of a plant. The ion was found to accumulate in conducting
tissues and in those tissues having large cells. It was higher in the pedicels
of tomato than in attached immature fruit, but these differences decreased as
the fruit matured. The workers state that absorption of chloride is a linear
function of its content in the substrate. However, the amount accumulated in
the pulvini of lima beans appeared to be more in the nature of an exponential
function of substrate concentration. The greatest amount of the ion accumu-
lated there, followed by the mature flowers, and then the main veins.

Accumulation of €136 in alfalfa is mainly in the stems, petioles, and

ll

pulvini. In tomato the major concentrations are found in stems, leaf blades,
and petioles below the fruit, especially below the lowest clusters. It may be
noted that there is no particular concentration at the growing points. This

finding has been supported elsewhere (14).

Chloride Toxicity

 

One of the most important toxic effects of saline soils is the reduction
in growth and yield of the crops grown (6, 10, ll, 12, 59, 71). Any chloride salt,
under certain conditions, may be found to bring this about. Specifically, the
chloride ion is reported to cause a reduction in root growth (106). The con-
dition is more severe if CaCl2 is added rather than NaCl. Eaton, on the other
hand, could find no reduction in root growth which could not be attributed solely
to the increase of osmotic pressure of the substrate (22).

Foliar symptoms of chloride toxicity are clearly defined (4, 8, 14, 31, 39,
48, 94, 110). The tip of the leaf becomes necrotic and the "firing" may then ex-
tend to leaf margins. Older leaves are the first to be affected (31, 109). Oc-
casionally, necrosis may proceed down the entire leaf blade (4). Eventually,
the entire leaf blade may become necrotic and abscise (34, 39). On stone fruit
trees twig dieback may accompany foliar signs (14).

Chloride toxicity may result in a thickened, flaccid leaf, accompanied
by partial to complete cellular plasmolysis (8, 28, 41). On the other hand, a

decrease in the succulence of barley has been reported (16). The turgidity

TH F!

 

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12

of a leaf may depend on the length of time the leaf has been subjected to toxic
levels of chloride (109).

Healthy leaves generally contain somewhat less than 0. 5% chloride
(34, 87), while in injured leaves the concentration may vary between 0. 4% and
3. 5%, depending on the particular plant (4, 14, 34, 87). This may be the result
of 150 to 200 parts per million or more of chloride in the irrigation water
(57, 58, 88) or the application of as little as 150 pounds per acre of KCl (54).

The amount of chloride absorbed by any particular plant depends upon
the source of the chloride. More is absorbed by the bean plant when it is
supplied as NaCl rather than CaCl2 (32). On the other hand, more chloride
is absorbed by citrus and walnut seedlings from a substrate of CaC12 than
from KCI, NaCl, or MgCl2 (36). Stone fruit trees and orchard grass are
more sensitive to CaCl than to NaCl (14, 107, 108).

2

Chloride, especially in the form of CaCl , reduces the uptake of ni-

2
trate nitrogen (30, 36, 62, 106). NaCl causes a greater reduction in plant
protein concentration than does CaCl2 (106).

Potassium uptake by plants in the presence of chloride is variable.
Increases have been noted when NaCl was added to the substrate and the re-
verse when CaClz was added (20, 30). Conversely, the uptake of chloride is

independent of potassium uptake (18, 39). Excess chloride accumulation may

be accompanied by increased calcium absorption (75) or a depression in

13

uptake (20). Magnesium uptake may be increased or unaffected (30). Hence,
the balance between K, Ca, and Mg may be altered. This may be one cause of
the injurious effects of high chloride levels (75).

The similarity of chloride toxicity symptoms to potassium deficiency
symptoms may be due to insufficient amounts of potassium available to com-
bine with all the chloride present (13). The addition of potassium alleviates
chloride toxicity symptoms on buckwheat, but not on flax (76).

A high level of chloride accumulation in tobacco leaves is associated
with a pronounced dissipation of the malic acid content of the leaves (28).
Since the organic acids are the major components of the buffer system of
plant cells, conditions affecting dissipation of organic acids significantly
affect the pH control of the cell.

The chloride ion affects the normal amylolytic activity of the plant.
The starch level rises markedly in the leaves (12, 28,29). The sucrose level
may decrease (12), although total sugars increase (29, 66, 102). The change
in reducing sugars is not clear (102, 105). The enzyme systems responsible
for carbohydrate metabolism are intact, as hourly fluctuations in carbohydrate
levels still occur (29). There is no clue in carbohydrate metabolism as to
the mechanism of chloride injury to plants (29).

Chlorides have been found to exert a stimulatory effect on respiration

(91). ‘With chloride salts of monovalent cations, high respiration persists

14

although chloride accumulation may decline. With divalent cations an initially
high respiratory rate falls to a level comparable to one in which no chloride
has been added. Handley and Overstreet (37) postulated that respiration is

affected by ions which are both rapidly absorbed and important in metabolism.

Chlorine - An Essential Element

 

A question which has appeared in the literature is the possibility of
chlorine being an essential element. T ottingham (97) summarized early ex-
perimental work in which Nobbe and Siegert observed physiological distur-
bances of plants grown in chloride-free media. They reported that the need
for chloride appeared in the fruiting stage, and concluded that this ion was
essential in carbohydrate translocation. Subsequent experiments of Wagner
and Aschoff, and Beyer led to conflicting results. Shulov found that chlorine
was not essential for buckwheat nutrition. Tottingham criticized all the
experiments on the basis that not enough was known about balanced nutrient
solutions to permit the drawing of any valid conclusions. The purity of the
chemicals used may be questioned as well.

The first more critical experiment was conducted on peas and buck-
wheat by Lipman (67). These plants were grown on chloride-free and chloride-
added media. With buckwheat more seed production, better seed germination

and subsequent development in the F 1 generation was noted. With peas no

15

differences were observed until the seeds of the third generation were exam-
ined. Those on the chloride-free medium produced fewer, lighter and more

shriveled seeds. These, and many other experiments, merely serve to indi-
cate the possible beneficial effects of chlorine to some plants.

It was not until 1954 that experiments were conducted which proved the
essentiality of chlorine according to the currently accepted criteria of essen-
tiality. Broyer _e_t_ _al: (16) have shown that tomato exhibits nutritional defi-
ciency symptoms when grown on a halide-free substrate. The deficiency
symptoms are characterized by wilting of the leaflet blade tips, followed by
progressive chlorosis, bronzing, and necrosis. Severely diseased plants bear
no fruit. The workers found that growth was correlated with the amount of
chloride in the culture. Adequate chloride additions to the culture prevented
the trouble; severely deficient plants resumed good growth after chloride was
added to the culture solution or through stem injection. Bromide may com-
plement chloride when added at ten times the required chloride levels. They
concluded that chlorine is an essential element, and certainly the naturally
occurring essential halide.

It has since been shown that chlorine is essential to the nutrition of
sugar beets (99). The authors reported that symptoms of chloride deficiency
appeared first as a chlorosis on the leaf blades of the younger leaves. The

main veins and even the minor veins remained green, while the interveinal

16

areas became light green to yellow. They observed a netted mosaic pattern to
transmitted light. The roots of the chloride-deficient plants became very
stubby, the stubs having arisen from secondary roots to form a distinctive and
abnormal root structure. Plant growth was retarded. The addition of chloride
as CaCl2 or NaCl to the culture solution resulted in significant increases in
fresh and dry weights of both tops and roots. Under conditions of high potas-
sium concentrations, chloride deficiency caused a decrease in sucrose content
of the root. When potassium was low, a chloride deficiency caused an increased
sucrose content in the root. The authors concluded that chlorine is associated
with sugar formation rather than utilization. For plants deficient in both potas-
sium and chlorine, added chlorine removes the stress on newly formed center
leaves, and deficiency symptoms disappear. Once this stress is removed.

the young leaves can utilize potassium from the older leaves, and growth is
resumed normally.

Stout _e_t_ _a_l_. (95) reported that lettuce and cabbage may display chlor-
ide deficiency symptoms as well. A lack of chloride caused a pattern of
chlorosis followed by a necrosis of the leaf tissue. Added chlorine reduced
the deficiency symptoms, and very small additions caused a delay in the
appearance of these symptoms. Added bromine may delay some, but not

all of the symptoms. However, a much greater amount of bromine must be

added to the solution in order to substitute for the added chlorine.

17

The function of the chloride ion in plant tissue has not definitely been
established. Aside from its postulated role in sugar metabolism, another
possible function has been assigned to it. Arnon and Whatley (2) isolated
chloroplasts from plant tissue and added cytoplasmic fluid to the suspension.
In the presence of light the chloroplasts were inactivated, but the addition
of chloride to the suspension prevented this inactivation. The authors re-
ported that Warburg interpreted this as a possible function of chloride as
a coenzyme of photosynthesis, but noted that the anions Br', I’, and No-3
may serve this function as well. This effect bears similarity to salt respir-
ation of washed root discs, and starch hydrolysis by dialyzed x-amylases (33).
The stimulatory effects have a common in vitro basis, but it is a matter of

conjecture as to whether this happens in vivo.

18

MATERIALS AND METHODS

In order to determine whether the soil type alters the toxic effects of
the chloride ion, two different soils were used. Brookston clay loam and Granby
sandy loam were selected, as these soils are comparable to those used by com-
mercial greenhouse establishments. The soil was placed into eight-inch clay
pots, which had previously been coated on the interior surfaces with asphalt
paint. The soil and pots were steam pasteurized on january 29, 1957. After
pasteurization was completed, the soil was thoroughly leached with distilled

water each day for the next four days.

Seeds of Ball non-branching Number 1 Lilac-Lavender Improved flower-
ing stock (Matthiola incana, R. Br) were sown directly into half of the pots on
February 4, 1957. Seeds of spring-flowering Cuthbertson strain Lois variety1
of sweet pea (Lathyrus odo‘ratus, L) were sown into the remainder of the pots
on February 16, after twenty-four hours' soaking in distilled water. The pots
were watered as required with distilled water. The greenhouse was maintained
at a minimum night temperature of 50 degrees Fahrenheit.

On March 10 the stock were thinned to six plants per pot, and the sweet
peas to two plants per pot, on the basis of uniformity of length. The sweet
pea pots were staked by placing three rose stakes in each pot and joining them

at the top. From this date the plants were treated with half- strength nutrient

1Courtesy of George 1. Ball and Company, Chicago, Illinois.

19

solutions, every second or third day, depending on the rate of water loss.

Six levels of the chloride ion were used, namely, 0, l, 2, 3, 4 and 5
mini-equivalents per liter of solution. 1 Each replicate consisted, then, of
the six levels of chloride and the two soil types, or twelve pots for each of
the two species. Each treatment was replicated four times, making a total
of forty-eight pots per species. The pots were randomized as to treatment
using a set of random number tables. Thus, a "random block" experimental
design was employed.

On April I, the plants were thinned to one plant per pot, selection
again based on uniformity of length. Full-strength nutrient solutions were
added as of this date. Solutions were added almost every day due to the high
amount of insolation during the month of April and the beginning of May. Each
addition consisted of one-half liter of solution.

The experiment was terminated on May 11, at which time the first of
the stocks would have been ready for harvest had the plants been grown on a
commercial basis. At this time the sweet peas were just beginning to bloom.
The stocks were cut at the cotyledonary node, and the sweet peas at soil
level. All visual abnormalities were recorded at this time. Measurements
were made as to fresh weight, total length, number of leaves, number of
flowers, both mature and immature, for both species. In addition, stem

1 . . . . .
Composrtion and characteristics of the nutrient solutlons may be seen in
Appendix B.

20

diameter at the cotyledonary node was recorded for the stocks. The plants
were then placed in a 70 degree Centigrade forced-draft oven for forty-eight
hours. Dry weights were recorded, and the plant tops were ground to pass a
ZO-mesh screen in a Wiley mill. The percentage chloride was measured
for both the dried plant tissue and the air-dry soil1 by a modification of the
technique outlined by Samson (93).

Electrical conductivities and pH values were made at the conclusion
of the experiment using the Industrial Instruments Inc. "Solu-Bridge Soil

Tester" model RDlS, and a Beckman HZ model pH meter, respectively.

1
See Appendix C.

2]

RESULTS

General Observations

Brookston clay loam had the appearance of a well—aggregated, well-
drained soil. Granby sandy loam appeared to be poorly aggregated. Subse-
quent steam pasteurization seemed to promote the deflocculation of soil aggre-
gates. Consequently, drainage and aeration were impeded.

No differences were noted as to speed or percentage of germination
of the seeds germinated in the two soil types. Uniformity of stand prevailed
until the final selection of plant material on April 1.

About two weeks after this date differences in growth of the sweet peas
became apparent. Those plants grown in Granby sandy loam exhibited less
branching and linear growth. Flowering stocks exhibited no such differences.

On April 21 spots were observed in interveinal areas on the lower
leaves of some of the sweet peas and flowering stocks. The original appear-
ance was that of water-soaked areas, but these rapidly became necrotic. The
size of each spot did not change with time, but the number of such areas did
increase. The leaves on the left in Figure 1 show the symptoms observed.
The occurrence of injured leaves was sporadic on the sweet peas. There
was a significantly greater percentage of injured leaves of flowering stock'

appearing on the three higher chloride levels than on the three lower levels.

FIGURE 1

Appearance of leaves of stock (above) and sweet pea, those
on the left showing interveinal necrosis, and the stock leaf on the
right showing tip chlorosis.

22

 

23

The other injured leaf which appears to the right of the three stock
leaves in the figure is representative of a tip necrosis observed on the lower
leaves of some of the plants. This injury is significantly correlated with the
chloride level of the applied solution, the correlation coefficient being +0. 863.
Its occurrence was more prevalent on Brookston clay loam than on Granby
sandy loam.

Judging on the basis of the characteristics measured and the size and
placement of the flowers of stock, these plants were of good quality. The
sweet peas grown on Brookston clay loam were of satisfactory quality, while

those grown on Granby sandy loam would be judged inferior.

24

Sweet Peas

 

Figure 2 illustrates the growth of sweet peas on 1, 3 and 5 milliequival-
ents per liter of added chloride on Brookston clay loam and Granby sandy loam.
The growth of the plants appeared to be similar regardless of the chloride
level applied, however, those plants grown on Brookston clay loam appeared
more dense than those grown on Grnaby sandy loam. The difference in appear-
ance was due to the greater number of branches that developed on the plants
grown in the Brookston soil.

Table 1 indicates that there were no differences in any growth char-
acteristic of sweet pea which may be attributed to the chloride level of the
solution applied. Differences in fresh weight, dry weight, number of branches,
number of flower buds, number of leaves, total length, and internode length
were significant at the 1% level. Growth on the Brookston soil was signifi-
cantly greater than that on the Granby soil.

The most important single difference appeared to lie in the number of
branches per plant. A recalculation of the data on a "per branch" basis showed
that the differences due to soil type vanished. Thus, the extent of branching
is dependent on soil type, and other characteristics are dependent upon the
number of branches produced. Granby sandy loam inhibited branching in some
manner, while the individual branches manifested the same growth habits re-

gardless of soil type.

FIGURE 2

Appearance of sweet pea at the termination of the experiment.
Left to right-~l, 3 and 5 milliequivalents per liter of added chloride.
Top- -plants grown on Brookston clay loam. Bottom--plants grown
on Granby sandy loam.

25

 

 

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27

Flowering Stock

 

Figure 3 depicts the growth of flowering stock at l, 3 and 5 milli-
equivalents of added chloride on Brookston clay loam and Granby sandy loam.
From the photograph no differences are apparent in the growth of these plants
under the conditions of the experiment.

Table 2 indicates that the only differences found were between fresh
weight, dry weight, and stem diameter of the plants grown on the two soil
types. The difference in dry weight is accounted for by the difference in
stem diameter, and not in terms of leaf number or stem length.

Flowering stock is harvested when there are between eight and ten
open flowers per stem. No treatment resulted in a significantly earlier
date of harvest, as there were no differences in the number of open flowers
at the termination of the experiment.

Chloride addition had no significant effect on fresh weight, dry weight.
leaf number, stem diameter, length to the top leaf, or the mean internode

length within the range of concentration studied.

FIGURE 3

Appearance of flowering stock at the termination of the experi-
ment. Left to right - l, 3 and 5 milliequivalents per liter of added
chloride. Top--plants grown on Brookston clay loam. Bottom--
plants grown on Granby sandy loam.

 

28

 

TABLE 2

29

Growth of Flowering Stock as Related to Chloride Concentration of Growing Medium.

 

 

 

 

 

 

 

 

Measurement
Fresh Dry Length No. of No. of Stem Mean
Weight Weight of Top Open Leaves Diam. Internode
(gm 3) (gm 3) Leaf (cm) Flowers (cm) Length
Brookston ClaLLoam
_(ME Cl‘ / Liter)
0 102. 3 12. 8 42. 7 9. 5 24. 3 9. 5 l. 9
1 96. 9 12.4 39.3 11.3 24.8 10.0 1.6
2 105. 3 12. 9 44. 5 9. 3 24. 5 9. 3 1. 8
3 94.9 12.3 43.7 11.3 24.0 10.1 1.8
4 98. 4 12.1 42. 6 10. 3 25. 3 9. 6 1. 7
5 111.6 13.4 43.4 10.8 24.5 10.4 1.8
Granby Sandy Loam
(ME Cl'/Liter)
O 87.8 11.6 42. 7 10.0 25.0 9. 4 1.7
1 93.0 11.9 42.5 9.5 25.5 9.4 1.7
2 76.8 10.9 45.7 10.5 25.8 9.0 1.8
3 80.6 10.3 39. 9 10.0 23.8 8.7 1. 7
4 74.3 10.1 38.4 10.3 26.0 8.8 1.5
5 82. 5 11.1 42. l 9. 0 25. 8 9. 1 1. 6
Significant Differences
Soil 1% 5% N. S. N. S. N. S. 1% N. S.
Solutions N. S. * N. S. N. S. N. S. N. S. N. S. N. S.
Interaction N. S. N. S. N. S. N. S. N. S. N. S. N. S.

1
l.—

‘Not significant

30

Chloride Accumulation

 

The chloride content of the plants and the two soil types at the end of

the experiment may be seen in Table 3. As the solution chloride increased
from 5 parts per million to 182. 5 parts per million, the amount of chloride
in Brookston clay loam increased from 3 parts per million to 115 parts per
million, and from a trace to 180 parts per million in Granby sandy loam.
The chloride content of flowering stock increased from 0. 193 percent to 1. 108
percent on the Brookston soil, and from 0. 237 percent to 0. 966 percent on the
Granby soil, while the chloride content of sweet peaincreased from 0. 213
percent to 1. 180 percent on Brookston clay loam, and from O. 193 percent to
0. 859 percent on Granby sandy loam.

The amount of chloride in the plant tissue is closely related to the

amount of chloride added in' solution. The correlation coefficients are given:

 

 

Corggarison Correlation Coefficient
Solution chloride and stock chloride +0. 895
Solution chloride and sweet pea chloride . 800
Solution chloride and plant chloride on Brookston soil . 903
Solution chloride and plant chloride on Granby soil . 865

All correlation coefficients are significant at the 1% level.

The Relation Between Solution Chloride and Chloride Accumulation in Soil and Plant.

TABLE 3

31

 

 

 

 

 

Solution Ppm Chloride in % Dry Weight Chloride in
Number 501131011" 5011 Stock Sweet Pea
Brookston Clay Loam
l 5 3 0. 1 93 0 . 213
2 40. 5 11 . 698 . 809
3 76 30 . 749 . 770
4 111.5 59 .948 1.060
5 147 89 . 956 1. 090
6 182.5 115 l. 108 1.180
Granby Sandy Loam
1 5 tr. 0. 237 0. 193
2 40. 5 15 . 730 . 412
3 76 61 . 677 . 521
4 111.5 75 .833 .543
5 147 150 . 908 . 745
6 182. 5 180 . 966 . 859
L. S. D. soils 21. 1 N. S. 0. 081
1% levels 67. 4 0. 172 0. 219
interaction (soils x levels) N. S. , N. S. 0. 219
M 1

 

 

 

 

’Includes 5 ppm in distilled water

lNot significant

32

The correlation coefficients between solution chloride and soil chloride
at the end of the experiment were +0. 988 for Brookston clay loam and . 979 for
Granby sandy loam. Both correlation coefficients are significant at the 1%
level.

On the basis of the experimental evidence, chloride retention in both
soil and plant is directly related to the amount of chloride applied in the irri-
gation water, at least within the concentration range studied.

Mean chloride retention by the two plant species may be seen in the fol-

 

 

 

 

 

 

lowing table:
Flowering Stock Sweet Pea
Brookston clay Granby sandy Brookston clay Granby sandy
loam loam loam loam
0. 775% 0. 725% 0. 853% 0. 545%

L. S. D. = 0. 175%

Thus it is seen that retention by sweet pea is significantly less on Granby
sandy loam than on Brookston clay loam. These plants also contained less chlor-
ide than flowering stock grown on either soil type. Combining the results, there
is no difference between species uptake, but retention was greater on Brookston
clay loam than on Granby sandy loam.

Figures 4, 5, and 6 illustrate the relationship between the amount of
chloride applied and the amount of chloride retained in soil and plant. Soil re-
tention appears to be an exponential function of solution concentration where the

exponent is greater than one, while plant accumulation is exponential as well,

the exponent being less than one.

FIGURE 4

Graph of Chloride in Solution Versus Accumulated Chloride in
Brookston Clay Loam and Granby Sandy Loam.

33

ppm soil Chloride
180 i Granby Sandy Loam

160 1

140 ‘

Brookston Clay loam
120 i

100 l

801

40

20 n

 

 

S 40. 5 76 111. 5 147 182. 5
ppm chloride in solution

FIGURE 5

Graph of Chloride in Solution Versus Chloride in Tops of Sweet
Peas Grown in Brookston Clay Loam and Granby Sandy Loam.

34

% Chloride in plant tissue

Brookston Clay loam
1.201

1.05 ‘

.901

Granby Sandy loam

.75

.60‘

.451

.30‘

-IS‘

 

 

O r ' T

I ‘V
5 40.5 76 111.5 147 182.5

ppm chloride in solution

FIGURE 6

Graph of Chloride in Solution Versus Chloride in Tops of Flower-
ing Stock Grown in Brookston Clay Loam and Granby Sandy Loam.

35

% Chloride in plant tissue

1.20‘

Brookston Clay loam

1.05‘

Granby Sandy loam

.75‘

.60‘

.45‘

.15‘

 

 

0 ' ‘ ' '

5 40.5 76 111.5 147 182.5
ppm chloride in solution

36

DISCUSSION

This study has indicated that the spring flowering Cuthbertson strain
of sweet pea and the non-branching lilac-lavender variety of flowering stock did
not manifest any reduction in growth due to additions of the chloride ion up to
a concentration of 182 parts per million when grown in Brookston clay loam
and Granby sandy loam. Visual signs which appeared on the leaves of both
plant species appeared more frequently on the leaves of flowering stock grown
at higher chloride levels, and appeared sporadically on the leaves of sweet
pea. Chloride ion accumulation in soil and plant were directly related to the
concentration in the solution.

Within the concentration range of chloride studied, neither plant mani-
fested any reduction in growth. The species, evidently, are tolerant of the
chloride ion at least up to a concentration of 182 parts per million. Flower-
ing stock is reported to be moderately tolerant of high salt concentrations (71).
Although the present study is of a different nature and can neither confirm
nor deny this evidence, perhaps tolerance of saline conditions and to the chlor-
ide ion are related. Tolerance to the chloride ion in the substrate is not a
blocldng mechanism as plant and solution concentrations are directly related.

The more important difference found in plant growth was a reduction ,

of branching of sweet peas grown in Granby sandy loam. Two major differences

37

exist between the soil types, namely pH and aeration. Post (85) states that
the best pH range for sweet pea growth is between pH 6. 0 and pH 7. 5. The
pH of Granby sandy loam exceeds the upper limit of the favorable range. All
sources consulted (9, 64, 85, 100) agree that sweet pea soil should be well
drained to a considerable depth. This condition did not exist in the Granby
soil. The inhibition of branching could not have been due to a low level of
potassium, as this ion is in greater abundance in the Granby soil. Aeration
and/or pH or Other conditions may have caused the observed reduction in
branching.

Both fresh weight and dry weight of flowering stock were significantly
less in Granby sandy loam. This was found to be due, in part, to a reduction
in stem diameter. Fresh weight differences were greater and were not
wholly dependent on stem diameter. From this evidence it is concluded that
water uptake by flowering stock grown in Granby sandy loam was inhibited.
This may have resulted in a reduction in cell enlargement. The restriction
of aeration in Granby sandy loam may have been the causal factor.

Visual signs which appeared on some lower leaves of both plant
species were not similar to those reported by most authors (4, 14, 31, 39, 48,
94, 110) but resembled those found by Dilley (21) on grape and peach when
irrigated with 315 parts per million of chloride, and 624 parts per million

of sulfate. Leaf injury found in the leaf of flowering stock at the right of

38

Figure 1 is similar to that described by Baker _et 31.“ (14) who noted tiny
yellow-brown necrotic spots at the leaf tip caused by a local high concen-
tration of soluble salts at the hydathodes. This may be an early condition
of the signs reported by the other workers. Although growth of plants was
not reduced, it is possible that leaf signs were due to toxic levels of chlor-
ide as chloride may concentrate in the leaf blades (14,62, 96).

In this study, correlation coefficients between substrate concentra—
tion and plant accumulation were highly significant. This finding substantiates
those of other workers (18, 29, 84). Toth and Kretschmer (96) and Kretschmer
gt 31; (62) stated that chloride absorption is a linear function of its content
in the substrate. However, accumulation in the pulvini of lima beans (96)
appeared to be more in the nature of an exponential function. This study
supports the latter finding.

Chloride accumulation is mainly a function of transpiration (79), the
transpiration stream accounting for up to 60 percent of the total chloride
accumulated. There is no logical basis to assume that there are transpira-
tional differences between plants grown on different chloride levels, hence
the differences must be attributed to the different substrate concentrations.

Significantly greater amounts of chloride were found in both Brookston
clay loam and Granby sandy loam with additions of larger amounts of chlor- '

ide. The chloride ion does not accumulate in the soil (103) possibly due to

39

its great mobility (74). However, the type of soil colloid bears a slight in-
fluence on the amount of chloride retained (111). Present evidence would
indicate that substrate concentration is as important a factor in chloride re-
tention as is the soil type.

On an overall basis, chloride retention by plant tissue was greater
on Brookston clay loam than on Granby sandy loam. Conversely, soil chloride
retention was greater on the Granby soil. Total chloride applied was the same
for any treatment level, irrespective of species or soil type, and might be
represented thus: Total C1 = plant C1 + soil Cl. Plant chloride is a function
of the dynamic factors of growth. Hence, soil chloride differences may be
regarded as a residual effect.

Differences in chloride accumulation in plants due to soil type are
significant at the 1% level for sweet pea. Accumulation is higher in plants
grown in Brookston clay loam, the soil with the larger percentage of colloids.
Reisenauer and Colwell (89) have shown that the colloidal fraction is influen-
tial in chloride accumulation by tobacco, the plants having accumulated more
on the soil with more colloidal matter. It is interesting to note that this
effect was not observed with flowering stock in this experiment. The reason

is unknown.

40

SUMMARY

The specific influence of the chloride ion on the growth of sweet pea

(Lathjrus odoratus, L.) and flowering stock (Matthiola incana, R. Br.) was

 

 

studied. The plants were grown on Brookston clay loam and Granby sandy
loam, soils similar to those used in commercial greenhouses. A random
block design was employed, using six levels of chloride each replicated
four times. The solution composition, in other respects, was similar to
that outlined by Hoagland and Arnon (50).

Growth was measured as fresh weight, dry weight and number of
leaves for both plants. Additional measurements were made as to the number
of branches and flower buds on sweet pea, and number of open flowers, stem
diameter, and mean internode length of flowering stock. Chloride was deter-
mined as percent dry weight of the above- ground portion of the plants and
as parts per million of air-dry soil.

No significant reductions in growth were noted on either plant which
were due to the application of chloride.

Interveinal necrotic areas were found to be more prevalent on the
leaves of flowering stock grown at the three higher levels of chloride than
at the three lower levels. This injury appeared sporadically on some sweet

pea leaves. A foliar tip necrosis was displayed by flowering stock. Its

41

incidence correlated very significantly with the amount of chloride applied in
the nutrient solution.

Granby sandy loam severely inhibited the branching of sweet pea. The
cause is unknown, although it may have been due to restricted drainage and
aeration, too high a pH, or a. combination of both factors.

The chloride ion accumulated readily in both soil and plant. Significant
positive correlation coefficients were found between solution chloride and chlor-
ide accumulation. Accumulation in the soil appeared to be an exponential func-
tion of solution concentration where the exponent is greater than one, while
plant accumulation appeared to be exponential as well, the exponent being

less than one.

1°

0.)

42

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44

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45

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51

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APPENDD( - TABLE A

Amount of Chloride in the Tap Water Used by Greenhouses in Some Michigan

 

 

Communities
Community Number of Growersl PPM Chloride in Water2
Adrian 6 8
Almont l 700
Ann Arbor 6 10
Battle Creek 7 4
Bay City 8 30
Detroit 21 8
Flint 6 25
Grand Rapids 16 5
Jackson 9 55-295
Kalamazoo 10 8
Lansing 6 1 1
Monroe 5 18
Mount Clemens 8 7.
Muskegon 6 4
Plymouth 6 280
Saginaw l 1 210
Ypsilanti 4 27

 

 

1Michigan State Florist Ass'n Membership Directory, P. R. Krone, Managing
Editor, May, 1957.

2Chemical Analyses of Public Water Supplies in Michigan. Michigan Depart-
ment of Health Engineering Bulletin no. 4, 1948.

53

APPENDIX - TABLE B

Constituents and Characteristics of Nutrient Solutions

 

Millieguivalentsper Liter

 

 

 

Chemical Solution Number

1 2 3 4 5 6
KHZPO4 1 1 1
NH4H2PO4 l 1 l
KN03 5 4 4 3 2
KCI 1 2 3 4 5
NH 4NO3 0. 5 0. 5 1 2. 5
Ca(NO3)2 5 5 5 5 5 5
MgSO4 2 2 2 2 2 2

Electrical Conductivity (millimhos x 10-5)
265 270 260 290 270 300
pH

5.1

CA

5. l 5. 4 5. 4 5.

U1
0

 

Iron was supplied as "Na Fe Sequestrene" at the rate of 1 milliliter of stock
solution per liter. The stock solution contained 42 grams chelated iron per
liter. All minor elements were supplied as prescribed by Hoagland and

Arnon (50).

 

#—
_—

Soll Type

Brookston

Granby :

BIOO ks

Granb3

Soil Properties at the Termination of the Experiment

APPENDD€ - TABLE C

m

5011 Type

Solution Number

5

Egctrical Conductiv-
Cl(ppm) ity (Millimhos x 10-5) pH

 

Brookston Clay Loam

Granby Sandy Loam

Brookston Clay Loam

Granby Sandy Loam

5
6

% Sand
35. 6

62. 0

% Silt
30. 6

25. 2

ll
30
59
89
115
tr.
15
61
75
150

180

% Clay
33. 8

12. 8

lb/A available

24

18

25

22

16

18

10

10

15

16

24

24

 

P205

132

126

K20
148

165

8.

7

l

9

C. E. C.

54

(M. E. /100g)
22. 0

18. 5

 

 

 

 

c '1 \f" I“ 1"

CT:
(,1 .1
rr‘i

l

L‘ .’ 'I
a. ' . .'

l ‘ ‘ .i J u 1' ‘

Date Due

Demco-293