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This is to certify that the

thesis entitled
THE ‘mrLUENCE or SIMAZINE
ON THE QUALITY AND COMPOSITION
OF POME FRUIT
presented by

Donald Bernard Carlson

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Horticulture

éfiflz

Major professor

Date October 0 l 6

 

0-169

 

 

 

 

 

 

 

 

ABSTRACT

THE INFLUENCE OF SIMAZINE
ON THE QUALiTY AND COMPOSiTION

0F POME FRUiT

by Donald Bernard Carlson

The successive annual application of simazine is a common practice
in many Michigan fruit orchards. it was therefore postulated that simazine
may have an obscure influence on fruit quality. The influence could be
due to the direct effect of the chemical, or the well documented indirect
effect of increasing the foliage nitrogen; which may be a critical
factor in the quality of pome fruit.

Simazine applications increased the foliage nitrogen in only one
pear and one apple orchard, out of a totai of nine experiments conducted
during l965 and 1966. The increase in nitrogen level of apple foliage
was not reflected in the protein content of the buds, fruit firmness,
total pectic material, soluble solids or titratable acidity. In
addition, protein content and the respiration rate of whole apple fruit
were not altered.

in one experiment one pound of supplemental nitrogen per tree, as
compared with non and one=haif poundg increased the apple foliage

nitrogen.

 

Donald Bernard Carlson

Simazine slightly reduced the level of soluble solids in one apple
and one pear orchard in l965. No other effects of simazine were observed
for fruit from these orchards, and a similar reponse was not observed
for fruit from the apple orchard in 1966, negating the practical
significance of this observation. The application of simazine to the
remaining orchards had no influence on either the nitrogen metabolism,
or any of the aforementioned quality or composition parameters.

Under the conditions of these experiments simazine had no obscure

effects on the quality of apple or pear fruit.

 

THE INFLUENCE OF SIMAZINE
ON THE QUALITY AND COMPOSITION

OF POME FRUIT

By

Donald Bernard Carlson

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1967

cq§%3q

5,

wwtg

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to
Dr. S. K. Ries for his strong, but timely, criticism and encourage-
ment during the period of this research. Further appreciation is
expressed to Drs. H. Davidson, D. R. Dilley, R. A. Mecklenburg,
C. J. Pollard, B. G. Ellis and A. R. Wolcott for serving on the
guidance committee and editing the manuscript.

Acknowledgement is also given to Dr. F. M. Morrison,
Dr. A. R. Putnam and G. R. Stephenson for aid in the establishment
of field experiments, and constructive discussion.

This research was supported by the Public Health Research
Grant CC 00246 from the National Communicable Disease Center,

Atlanta, Gerogia.

 

 

ACKNOWLEDGMENTS . . . . .
LIST OF TABLES. . . . . .
INTRODUCTION. . . . . . .
REVIEW OF LITERATURE . .
Physical Properties of
Mode of Action . . . .

Metabolism of Simazine

TABLE OF CONTENTS

0

0 O O O C

Simazine. .

O O O I O

O O O O .

Simazine and Nitrogen Metabolism . .

Cold Hardiness and Nitrogen Compounds.

Orchard Weed Control. . . . . . . . .

Fruit Quality and Composition . . . .

Pesticides-Crop Quality and Composition.

Summary of Literature Review . . . . .

MATERIALS AND METHODS. . . . . . . .

Field Studies. . . . . . . . . . .

Tests Conducted on Tree Portions .

Nitrogen Analysis . . . . . . .

Nitrate Reductase-Leaf and Root

Water Soluble Protein

Determination-Apple

0

Tissue.

Buds.

Tests Conducted on Fruit a o o o o I o o o o a o a a

FirmneSS. . o o o O o o o I o o a o O o o o o 0 O

SOIUb‘e Solids O O O O O I 0 O o a o o o O I O 0

2l
22
22

23

 

Total Pectic Material

Determination of Titratable Acidity of Fruit .

Water

0 O U . . O O 0 O O I O .

Respiration Studies . . . . . . . .

Statistical Analysis

RESULTS AND DISCUSSION. . . . . . .

Tests Conducted on Tree Portions

Nitrogen Analysis . . . . .

Nitrate Reductase . . . . .

Protein Determination-Apple

Tests Conducted on Fruit
Quality of Fruit at
Finnness .... . . . .

Soluble Solids. . . .
Total Pectic Material
Titratable Acidity .
Water Soluble Protein
Respiration Studies .
SUMMARY . . . . . . . . . .
LITERATURE CITED. . . . . .

APPENDIX

0

O

O

Buds.

0

Harvest

Soluble Protein Determination of Fruit.,

Determination-Fruit . .

O O o o o 0 o e

o o o a O O

o o o o o o

o o o 0 o O

23
25
25
26
26
27
27
27
36
37
37
37
37
39
no
no
1.3
LII-l
50
52
59

 

Table

LIST OF TABLES

Pear tree leaf nitrogen content and qualitative
characteristics of fruit in relation to treat-
mentsinorchardA........o......

Apple tree leaf nitrogen
characteristics of fruit
ments in orchard B . . .

Apple tree leaf nitrogen

content and qualitative
in relation to treat-

I I I I I I I I I I I I

content and qualitative

characteristics of fruit in relation to treat-
ments in orchard C . . . . . . . . . . . . . . .

Apple tree leaf nitrogen content and qualitative
characteristics of fruit in relation to
treatments in orchard G . . . . . . . . . . . .

The nitrogen content of the foliage from trees
in orchards E and J in relation to weed control
t rea tmen t s I O O O O O O O 0 O O O I I O D D O 0

Apple tree leaf nitrogen content and qualitative
characteristics of fruit in relation to treat-
ments in orchard D . . . . . . . . . . . . . . .

The nitrogen content of the foliage from trees
in orchards H and F in relation to herbicide and

nitrogen treatments. . . . . . . . . . . . . . . .

Protein content of lateral buds of twigs from
McIntosh apple trees in relation to treatments
in orchard G . . . . . . . . . . . . . . . . . .

Protein and total pectic material content of the
flesh of McIntosh apples in relation to treat-
ments in orcha rd G O O O O O D O O D O O O O O 0

Protein and total pectic material content of the
flesh of McIntosh apples from orchard D as
influenced by herbicides and their area of

application. I I. I I I I I I I I I I I I I I I I I

28

29

30

3]

32

33

34

38

Al

42

 

Table

Carbon dioxide evolved and oxygen utilized by
apple fruit from orchard C at the time of the
preclimacteric minimum and climacteric

maximum of reSpiration . . . . . . . . . . . .

Carbon dioxide evolved and oxygen utilized by
apple fruit from orchard D at the time of the
preclimacteric minimum and climacteric

maximum of respiration . . . . . . . . . . . .

Carbon dioxide evolved and oxygen utilized by
apple fruit from orchard F at the time of the
preclimacteric minimum and climacteric

maximum of respiration . . . . . . . . . . . .

Carbon dioxide evolved and oxygen utilized by
apple fruit from orchard G at the time of the
preclimacteric minimum and climacteric

maximum of respiration . . . . . . . . . . . .

Carbon dioxide evolved and oxygen utilized by
pear fruit from orchard A at the time of the
preclimacteric minimum and climacteric
maximum of respiration . . . . . . . . . . . .

#5

1+7

#8

1+9

 

INTRODUCTION

The controversial book ”Silent Spring”, by Rachel Carson (16),
called attention to the widespread use of pesticides in our environ-
ment. The publicity and reviews following the appearance of this
publication acted as a major stimulus to numerous investigations.

In 1950, twelve years prior to the printing of this text, the 8lst
Congress of the United States passed a resolution to create a committee
to conduct an investigation of “the nature, extent, and effect of the
use of chemicals, compounds and synthetics on the production, processing,
preparation and packaging of food products.“ (86). The purpose was to
determine the effect of the use of these materials. This was in the
interests of the health and welfare of the nation, and the stability
and well-being of the agricultural economy.

Unfortunately, little attention has been given to the possibility
that the pesticides we use may not only have detrimental effects upon
the organisms exposed to them, but may have harmful effects on the
crops they are often used to protect. A deleterious effect on the
quality or composition of a foodstuff could outweigh the advantages

gained through control of a plant pest.

Simazine (2-chloro—h,6-bis(ethylamino)-S-triazine), introduced n

1955 (90), has become a widely uSed selective herbicide for the control

 

of broadleaf and grassy weeds in numerous horticultural crops. This
chemical has been found applicable to weed problems in deciduous
orchards.

The objective of this research was to determine the effects of
simazine on the quality and composition of deciduous tree fruit,
and the composition of selected fruit tree portions, with primary

emphasis being placed on apples of the McIntosh cultivar.

 

 

REVIEW OF LITERATURE

Physical Properties of Simazine

Simazine, first reported to have herbicidal activity in l956
(34), is a white, crystalline material, with a molecular weight of
20l.7 and a melting point of 225-227°C (90). It is nonflammable
and noncorrosive under normal use conditions. A low solubility in
water, (varying from 2.0 ppm at 0°C, and 5.0 ppm at 20°C to 8% ppm
at 85°C), and the adsorption to certain soil constituents, limits
the leaching of simazine. Tests have shown that after application
the greatest portion of the compound is found in the top two inches
of the soil (is, 6). This gives a soil placement selectivity which

is important in the case of some deep-rooting perennial crops.

Mode of Action -- The low solubility of simazine in water is
accompanied by a low lipoid solubility, and as a result its toxicity,
when applied to plant foliage is negligible (40). Davis 2; 21- (2])
found that unless the cuticle was disrupted in some manner, the foliar
absorption of Inc-labeled simazine, was limited, indicating that
the intact cuticle is a rather effective barrier to the absorption
of this compound. Simazine is readily taken up by the roots, and
moves to the upper portions of the plant (21, 72). Sheets (72),

using lLIC-labeled simazine, determined that uptake by the roots of

seedling oats (Avena sativa, L. Cv. Kanota), and distribution

 

 

throughout the plant occurred within three hours. Absorption and
translocation of the herbicide were greater at 37°C than at 26°C. In
addition, with a constant temperature, absorption and translocation
were greater in an environment of low relative humidity. This
indicated that the amount of material in the plant is dependent on
the transpiration rate.

‘Absorption involves entrance into the stele of the root, migra-
tion to the apoplast, and movement in the xylem (20). Once it
reaches the leaf there is a marked tendency for marginal accumulation
in wide leaves, accumulation at the tip of grass leaves, and a general
distribution in the leaves of the tolerant corn species (20, 2i).
Following translocation it enters the living cells of the chlorenchyma,
and, in the presence of light, is believed to block the photolysis of
water and the evolution of oxygen. (Hill reaction). (27, 40, 59, 60).‘
Photosynthesis is very likely the physiological system most sensitive
to the activity of triazines. A 50 percent inhibition of the Hill
reaction in isolated chloroplasts from corn (Egg may; L.) and spinach
(Spinacia oleracea L.) was reported by Exer (27), using simazine at a
concentration of 7 x 10'”7 M. Moreland 33 a1. (59), working with
isolated barley (Hordeum vulgare L.) chloroplasts, and Moreland and
Hill (60) using chloroplasts isolated from turnip greens (Brassica spp),
feund a 50 percent reduction of photochemical activity at simazine con-
=6

centrations of 4.6 x lo M and 5.9 x i0"'6 M, respectively.

 

IFurther proof of simazine's action on the photosynthetic process
is its prevention of the accumulation of starch (32). However, plants
may be kept alive and growing, in the presence of otherwise lethal
concentrations of simazine, by supplying glucose through severed
leaf tips (59).

Various investigations have demonstrated that simazine inhibits
02 release in Elodea (68), C02 utilization by intact plants (87), and
l4c02 fixation by illuminated bean leaves (5). Clearly, many of the
toxic effects of triazine herbicides are related to photosynthesis.

One exception to this mode of action is that suggested by Ries _£ _1.
(68). They suggest that simazine toxicity may be the result of
excess production of nitrite or ammonia, or both.

Ashton 2; gl.(h), found that the fine structure of chloroplasts
in Phaseolus vulgaris L., was greatly altered by treatment with
atrazine (2-chloro—h=ethylamino-6-isopropylaminaus-triazine), a triazine
with a similar mode of action to simazine. These changes occurred when
the plant was kept in the light, but the chemical had no effect in the
dark. These workers proposed that the alterations were brought about
by the formation of a toxic substance, or substances, involving the
interaction of atrazine and light in the presence of chlorophyll.

In addition to the effect on photosynthesis, there are reports in

the literature on the influence of simazine and triazines in general,

 

on other physiological processes in plants. Increases (l, 63) and
decreases (2h, 3i) in re5piration have been detected. Differences
under light and dark conditions in the distribution of ‘TC in

compounds, following ll*Cm

sucrose uptake in atrazine treated plants,
indicated not only interference of photosnythesis, but also of other

metabolic processes. (3)

Metabolism of Simazine -- Roth (68) was the first to observe that
simazine was degraded by expressed corn sap, whereas juice from a
susceptible plant Such as wheat did not degrade the herbicide.

Further studies disclosed that simazine was converted, both in vitro
and in vivo, to the nonephytotoxic Substance 2ahydroxy-h,6-bis(ethylamino)-
S-triazine. (l7, #3 58). Metabolites other than hydroxysimazine have

been found in barley (Hordeum vulgare L.), oats (Avena sativa L.) and

 

sorghum (Sorghum vulgare) by Hamilton (42) opening the possibility
of other degradative mechanisms. A dealkylated product of atrazine
was reported by Shimabukuro (73) in both the roots and shoots of yOung
pea plants. Funderburk and Davis (3]) found ‘hcoz released from
plants treated with chain—labeled simazine, suggesting the possibility
of dealkylation of this herbicide.

Though the detoxification to hydroxy-simazine accounts for
resistance in some crops, such as corn, little is known of the toler—

ance of fruit trees to herbicides such as simazine. in a study

employing reciprocal grafting of peach and apricot seedlings (81), it
appeared that the greater tolerance of young peach trees over apricot
trees to the triazines simazine and prometryne (2,h-bis(isopropylamino)-
6-methylmercapto-5-triazlne), was the result of a physiological
detoxification occuring in the scion. (8]). Although simazine is
readily absorbed and translocated by the plant, sensitive residue
determinations (0.02 — 0.06 ppm), of apple and pear fruit from field
trial plots and treated commercial crops, have not disclosed

detectable residues of simazine in any edible parts when the chemical

was used at the recommended rate and time of application (30).

t al. (67)

Simazine and Nitrogen Metabolism -- In l963, Ries
reported that peach and apple trees growing in soil sprayed with
simazine had higher leaf nitrogen and more terminal growth than
trees where the weeds were controlled by hand=hoeing or black plastic
mulch. Similar observations on fruit trees were made by Karnatz (48).

A study of the effect of simazine on Monterey Pine (Ejggg radiata)
and corn, as influenced by lime, bases and aluminum Sulfate, disclosed
that Simazine always increased topnroot ratios of corn, but generally
decreased those of pine seedlings (22). Simazine significantly
increased the uptake of nitrogen by corn in all soils, of magnesium

and phosphorus in limed and of potassium in acidified soils.

 

Simazine applied at 0.6 ppm in solution culture increased the
yield of corn tops and the uptake of nitrogen, phosphorus, magnesium
and potassium (29). Applications of l.5 ppm simazine to soil in pots
increased dry matter yields and uptake of nitrogen in corn only when
additional nitrogen was applied to the soil. It was also found that
inoculation of soil with simazine did not increase mineralization of
the soil nitrogen, nor did it have any effect on immobilization of
nitrogen. It was therefore suggested that the herbicide increased
plant growth by a direct effect on plant metabolism and not through
any interaction with the soil°

Ries and Cast (64) disclosed that the addition of simazine to
nutrient solutions, in which corn was being grown, increased the
nitrogen content of corn. In a second test, conducted by the same
workers, under more favorable environmental conditions for growth
(higher temperatures, longer days and higher light intensities), the
total quantity of nitrogen per plant was not increased, but the
percent nitrogen was increased in the shoots of corn grown at a low
nitrogen level.

The nitrogen content of corn forage was found to increase as
nitrogen fertilizer, simazine, and atrazine rates were increased (28).
The percent nitrogen rose more sharply with increasing herbicide rate
on plots not receiving nitrogen fertilizer than on plots receiving
nitrogen. Grain nitrogen content increased as the fertilizer level

increased, but was unaffected by herbicide treatments.

 

Similar descriptions of higher nitrogen percentages in corn due
to atrazine treatment have been presented by Gramlich and Davis (37).
Enhanced growth and total leaf nitrogen in citrus trees, growing in
soils receiving simazine treatments, were reported by Goren and
MonseliSe (36).

Tweedy (80) found that simazine increased the nitrogen content of
corn plants grown in nutrient culture solutions under conditions of
low temperature and low nitrate level. If, however, the ammonium ion
was substituted for the nitrate ion, or if the nitrate levels or
temperature were optimum for growth, no increase in nitrogen was
observed with simazine applications. Further studies disclosed that
this increase in nitrogen content of corn plants, grown under sub-
optimal conditions (low temperature, low nitrate) was due to an
effect on nitrate reductase (82). A ten=fold increase was found in
the nitrate reductase activity of corn leaf extracts from plants grown
in low nitrate levels, and at low temperatures, in the presence of
simazine. Though this phenomenon occurred only under sub=optimal
growing conditions, the authors emphasize that low nitrate and low
temperature conditions are not unusual during the growth of plants.

Current reSearch has shown that the protein content of plants
may be increased by treatement with simazine (63). Rye plants
receiving 0,5 too.8 micromolar simazine contained up to 45 percent more
water extractable protein per plant than controls. Pea plants, grown

to maturity, contained more seed protein when treated with simazine.

 

Cold Hardiness and Nitrogen Compounds -- Cold hardiness is of

great interest to fruit growers. Some researchers have suggested that
various nitrogen compounds may be related to this phenomenon. AS
early as l926, Hildreth (44), in a study on the determination of
hardiness in apples, suggested there was some indication that high
reserves of carbohydrates and organic nitrogen might be correlated
with greater hardiness. In another study (79), protein nitrogen was
found to be highest during the dormant season in apple trees, and
decreased as the active growing season approached, reaching a minimum
in June. More recently, Siminovitch and Briggs (74, 75) studied the
changes which occurred in the water-insoluble protein nitrogen, the
water-soluble protein nitrogen, water—soluble non-protein nitrogen,
and the reducing and nonreducing sugars of the living bark of Black

Locust (Robinia pseudo-acacia L.) in relation to the season variations

 

in its frost hardiness. Of the nitrogen fractions, only the waterw
soluble proteins were found to increase in concentration in the fall,
along with the development of frost hardiness. This constituent also
decreased in concentration in the spring, with the disappearance of
hardiness. The authors stated that the correlation observed between
water-soluble protein and hardiness, suggested that this constituent
bears some causal relationship to the mechanism of development of frost
hardiness. Studies on the changes in metabolites of Red‘Osier Dogwood

(Cornus stolonifera Mich) by Li, t al. (53) disclosed that protein

nitrogen increased in the early fall, decreased somewhat at the outset

 

of rapid cold acclimation, and then increased more slowly in the late

fall. Since it has been established that the plasma membranes of plant

cells become more permeable to water as they acclimate to cold, and

that sugars increase during cold acclimation, the authors Suggest the

possible involvement of proteins in these phenomena, either as membrane

components or specific enzymes.
The possibility that high nitrogen levels could cause excessive

succulence, reducing the hardiness of the plant, shOuid also be

considered. .
The increase in nitrogen, due to the use of simazine, would be

of obvious importance in relation to the previous suggestions.

Orchard Weed Control —— In 1960, Larsen and Ries (51) reported
commercially acceptable weed control under bearing and nonbearing fruit
trees by employing simazine. A combination of either simazine plus
dalapon (2,2—dichloropropionic acid), or simazine plus amitrole
(3-amino-l,2,4~triazole), was reported to give excellent weed control
and resulted in no injury to young apple trees (18). Benson and
Degman (9), however, found that 5 lb/A of simazine alone did not provide
adequate weed control around non-bearing apple and pear trees. The
use of 10 lb/A of simazine resulted in tree injury, leading the 7
authors to the conclusion that more testing of this herbicide was needed.

More recently, Saidak and Rutherford (70), and Ries t §l° (65) have

reported that combinations of simazine plus amitrole-T (amitrole +

 

12

ammonium thiocyanate) gave satisfactory weed control on apples, non-bearing
peaches and sour cherries. The former workers suggest that annual
applications of simazine should not exceed 6 lb/A in order to avoid

injury to young trees. The standard recommendation for apples in

Michigan at the present time is 2-4 lb/A simazine (depending on whether

the trees are non-bearing or bearing), plus 2 lb/A amitrole-T (62).

Apple Fruit Quality and Composition -- Smock and Neubert (77)
use the term quality to describe those attributes of the apple that
pertain to edibility. These factors affecting eating quality may be
classified as follows, chemical factors, which includes acids, sugars
and astringent materials (eg. true tannins, tannin derivatives, and
coloring materials, such as flavonols); physiological-anatomical
factors, including changes in protopectin, which cements cells
together, to soluble pectin; and physical factors which would include
size and color.

Malic acid is the primary acid in the fruit of apple. Citric may
also be present, and lesser amounts of ascorbic. lactic, glyoxyiic,
m-tartaric, oxalacetic and an uronic acid have been reported (50, 77).
Carboxyi groups of pectic substances are usually Sufficiently esterified
or engaged in salt formation to make their contribution to the total
acidity insignificant. Acidity may be expressed as the total titra-

table acids (a representation of organic acids) or as hydrogen ion

 

concentration. Haller and Magness (41) found that the acidity of the
apple varieties Ben Davis and Delicious, increased as the number of
leaves per fruit increased.

Simple Sugars constitute the major portion of the carbohydrates in
apples, and of the soluble solid material in the flesh of the apple.
Since the fruit is primarily dependent on the leaves as the source of
sugar, high leaf/fruit ratios usually result in a greater percentage
of sugars in the fruit, up to a certain point, beyond which more
leaves per fruit does not result in Sugar increases. According to
Smock and Neubert (77), the amount of sugar present is an important
consideration, however, the acid=sugar ratio is probably more critical.
The authors point to the situation where on variety may be slightly
higher in sugars than a second, but more sour to the taste; this
greater sourness is probably due to the presence of a greater amount
of acid.

The term pectic substances refers to complex carbohydrate
derivatives which contain a large proportion of anhydrogalacturonic acid
units in a chain-like combination (77). The carboxyl groups of these
polygalacturonic acids may be partly esterified by methyl groups.

Three principal types of pectic substances may be found in fruit:
pectic acid, pectin, and protopectin (57). Pectic acid appears to be

a long straight-chain molecule built up by the condension of a large

 

number ofoé-galacturonic acid molecules. In pectin, many of the carboxyi
groups of the pectic acid have been esterified with methyl groups, and
the chain length is greater. Protopectin applies to the water-insoluble
parent pectic substances. The cells of the apple are cemented together
with protopectin, and during the ripening of fruit, the insoluble
pectin (protopectin) is hydrolyzed to soluble pectin, and the cells
of the tissue are no longer held firmly together. Thus, it is this
conversion from protopectin to pectin that is reSponsible for the
softening of fruit. When apples become mealy or over=mature, there
is a breakdown of the soluble pectic components into nonpectic
substances. The amount of total pectic substances remains fairly
constant until the fruit becomes overripe and mealy. Following this,
there is a decrease in the quantity of these components.

in relation to previous sections of the literature review, it
is important to consider that, ”nitrogen exerts more influence on
apple quality and storage life than any other element.II (83)
investigations have shown an inverse correlation between leaf nitrogen
and fruit firmness (ll, 14, 26, 55, 76, 88). Though the decrease in
firmness may not be critical if the fruits are marketed during the
normal marketing season, it is generally agreed that higher nitrogen

levels reduce the potential storage life (10, 55, 88).

 

Weeks _£ 21. (88) found that the amount of red color development
was associated with both nitrogen and potassium. Increases in leaf N
were associated with reduction of fruit color, whereas increases in
leaf K were associated with increased color. Hill _3 pl. (46) also
found that a widening of the N/K ratio was associated with a decrease
in fruit quality. A leaf nitrogen level of approximately 2.1 percent

has been reported to be associated with good quality fruit (25, 46).

Pesticides —- Crop Quality and Composition - An extensive review

of the literature has disclosed a minimum number of references to

 

the effect of pesticides on crop quality and composition. Studies by
Garman _£ pl. (32) showed that sprays (insecticides and fungicides)

can affect the quality of apples. The fungicide glyodin (2-heptadecyl~
glyoxylidine acetate) was found to significantly increase total sugars
when included in the spray mixture. No significant effect of insecti-
cides on sugars was detected. A complete spray schedule employing

the fungicide phygon (2,3-dichloro-i,4=naphthoquinone) plus lead
arsenate reduced acid content.

Palmiter and Smock (67) found that glyodin tended to advance the
maturity of McIntosh apples. This may relate to the increased sugars
noted by German (32) due to the same chemical, since sugars increase
with maturity, up to a certain point.

The storage and ripening of Delicious apples and Anjou pears as

influenced by DOT (dichlorodiphenylntrichloroethane) and parathion

 

(0,0-diethy1-O-p-nitrophenyl phosphorothioate) has been studied (35).
No significant difference was found in the storage behavior of sprayed
and unsprayed fruit. Neither spray influenced the appearance, rate of
ripening or the dessert quality of the fruit.

Studies on a number of herbicides in relation to taints in jams
indicated that there is normally little risk of tainting from the use
of a wide range of herbicides (88). This was expected since herbicides
rarely come into direct contact with the fruit itself.

Arthey (2), using the herbicides chloroxuron (N-4-(4-chlorophenoxy)
phenyl-N,N-dimethylurea),dalapon, diphenamid (N,N-dimethyl-2,2-diphenyl~
acetamide) and lenacil (3-chlorohexy1-5,6-trimethyleneuracil) to
determine if there was an effect on the flavor of canned and quick-frozen
fruits and vegetables, found that none of the above listed materials
left a taint in processed fruit. The author stresses the point that
the tests had been conducted for one year only, and therefore must be
interpreted with care. (According to Arthey, a minimum of three years
is necessary to provide reliable information on the effect of an
agricultural chemical on the flavor of a processed product).

Amiben (3-amino-2,5-dichlorobenzoic acid), dichlobenil (2,6—
dichlorobenzonitrile), diphenamid and dalapon were found to have no
adverse effects on the flavor, internal color, storage, or yield of
sweet potatoes (89). NAP (N-l-napthylphthalamic acid), on the other

hand, caused Severe and objectionable roughening of the surface and a

 

marked enlargement of the Surface veins of sweet potatoes. Cooke (19)
found that monuron (3-(4—chlorophenyl)-l,l-dimethylurea) caused a
large decrease in the sugar content of several legumes, and, at the
same time, caused a considerable increase in the soluble nitrogen

fraction of the plant.

Summary of Literature Review
Simazine is a triazine herbicide which is widely used in deciduous

orchards. Several investigations indicate that simazine's mode of

 

action is inhibition of photosynthesis. Recent studies open the
possibility of nitrite or ammonia excesses being responsible for this
chemical toxicity. Some resistant plants degrade simazine to the non=
phytotoxic hydroxysimazine. However, the possibility of additional
modes of degradation, such as dealkylation, have been suggested in
recent investigations.

Simazine applications may also result in increased growth, nitrogen
level, nitrate reductase activity and protein content of plants.

High nitrogen levels in pome fruit trees have been found to have
adverse effects on fruit quality.

Reports of pesticide effects on quality and composition of edible
products are limited. Some chemicals have a definite effect on the
quality and/or composition of an edible product; however, the majority

of pesticides have been used without any deleterious results.

 

MATERIALS AND METHODS

Field Studies

in 1965 and 1966 trials were conducted on pears (EILEE communis, L.
"Bartlett"), and apples (Mglgg sylvestris, Mill. ”McIntosh”).

Following the completion of fruit quality studies in 1965, plans
were made to concentrate on apples and the number of experimental sites
was increased in 1966. A summary of orchard locations and other pertinent
data is found in the Appendix - Table 1. Future reference to an orchard
will be by use of alphabetical listings from this table.

Herbicide treatments, using commercial formulations, were applied
with a carbon dioxide-pressured small plot sprayer (66). The rate of
chemical used is expressed as lb/A of active ingredient unless otherwise
noted.

Supplemental nitrogen was applied as evenly as possible, with a
perforated coffee can, around the tree out to the drip line. Herbicide
and supplemental nitrogen treatments were applied in the early spring.
The treatments listed by orchard may be found by reference to the
Appendix-Table 2. It should be noted that simazine was applied at
4 lb/A alone or in combination with amitroleuT except in orchards B

and C where an additional 8 lb/A rate was utilized.

 

 

The experiments in orchards B, D and E were single factor experi-
ments. Those in orchards A, C, F, G, H, and J were factorial
experiments. A randomized complete block design was utilized in all
cases. All treatments were replicated three times, except in orchards
F and H where four replications were utilized.

Fruit were harvested at approximately the same time the grower was
harvesting. In 1965 orchard A was harvested on August 18. Orchards

B and C were harvested during the week of September 13th. All 1966

 

harvests were made during the week of September 25-30. Random samples
were obtained by picking at various heights, and depths within the

tree crown. The fruit were held in cold storage at approximately l-2°C.

Tests Conducted on Tree Portions

 

Nitrogen Analysis -- Leaf samples for nitrogen analysis were
collected between July I and August 15 as recommended by Kenworthy
and Larsen (49). After washing and rinsing, the plant tissue was
dried in a forced air oven at 80—90°C. The dried tissues were then
ground through a 20 mesh screen using a Wiley Mill. In 1965, samples
were analyzed for nitrogen using macro-Kjeldahl procedures.I In 1966
samples were collected, dried and ground in the previously Stated
manner; nitrogen determinations, however, were made using the following

micro-Kjeldahl procedure. A 30 mg sample was weighed in cigarette

 

IPlant Analysis Laboratory, Horticulture Dept., Michigan State
University

 

20

paper, using a Mettler analytical balance. The sample, wrapped in
the cigarette paper, was digested in a 50 ml Taylor digestion tube
using the following reagents: 3 ml concentrated sulfuric acid,

0.5 g K250“, and 0.3 ml 1M CuSOh solution. Two glass beads were

used in each tube to promote even boiling. A standard containing

1 ml of (NH4)2504 solution (4.717 g (NH4)2504/L) and a blank were
included with each set. All samples were digested for approximately
two hours, cooled, and then 1-2 ml of deionized water was added to 1
each tube to dissolve any solids. The digest was then transferred to
a one—piece distillation apparatus, rinsing the tube thoroughly four
or five times. After adding 8-10 ml of 13 M NaOH, the sample was
distilled, collecting the distillate in 8 ml of a 4 percent boric
acid solution containing methyl red-methylene blue indicator. After
collection, the sample was titrated to a gray end point using

0.01 M HCL. The percent nitrogen was calculated using the standard

formula (7).

Nitrate-Reggctase - Leaf and Rpot Tisggg =- Both leaf and root
ti55ue from Orchard C were assayed for enzyme activity. Crude enzyme
preparations from leaf tissue were made in the following manner:
one 9 of fresh tissue was ground, in a cold room with a mortar and
pestle previously cooled in a freezer, in 5 ml of 0.1 M tris buffer
plus 0.001 M cysteine pH 7.5. The homogenates were centrifuged, using
an international High Speed Refrigerated Centrifuge at 10,000 R.P.M.

for 20 minutes.

 

 

The crude enzyme preparations were assayed as follows: Fiventenths
ml of 0.1M potassium phosphate pH 7.5, 0.1 m1 df 0.15 KNO3, 0.1 ml of
distilled, deionized water, 0.1 ml of 0.001 M NADH2,.and 0.2 ml of the
enzyme extract were added to a 12 x 100 mm test tube. The solutions
were incubated in a water bath at 25°C. for 20 minutes. Triplicate
samples of each extract were run. A fourth sample was stopped at
zero time, serving as a blank for determining the quantity of nitrite
produced during the incubation period. The reactions were stopped by
adding 1 ml of 1 percent Sulfanilamide in 3 M HCL. One ml of 0.02
percent N-l-napthylethylenediaminedihydrocholoride was added which
forms a diazo derivative of nitrite having a reddish purple color.
After allowing 15 to 20 minutes for color development, the optical
density was measured with a Beckman DB-G grating spectrophotometer
at a wavelength of 540 millimicrons. A standard curve was run, and
0.013 0.D. units equaled 1 millimicromole nitrite.

Root samples were prepared and assayed in the same manner except,
prior to grinding they were rinsed for 15-20 minutes in a 5 percent
sodium hypochlorite bleach solution. Following rinsing, the outer,
easily removed layer, was stripped off the roots to reduce the chance

of nitrate bacterial contamination.

Water Soluble Protein Determination u Apple Buds -- Lateral buds

from trees in Orchard G were removed from twigs shortly after their

collection in February, by making an incision at the base of the

 

22

leaf scar, and then carefully slicing the bud off the twig. A 250 mg
sample of tissue was weighed, using an analytical balance, as rapidly
as possible to avoid excess dehydration. The tissue was then ground
with a mortar and pestle in 5 ml of tris buffer (tris-(hydroxymethyl)-
aminomethane). After centrifugation at 10,000 x g for twenty minutes,
a 2.5 ml aliquot of the supernatent was precipitated with 2.5 ml of

20 percent trichloroacetic acid. The precipitate was rinsed twice
with 10 ml of 10 percent trichloroacetic acid and centrifuged at
2,000 R.P.M. in a clinical centrifuge for at least ten minutes. After
decanting, and discarding, the supernatent, the pellet was dried

in an air stream, and then redissolved in 4 ml of 1M NaOH using a
Vortex mixer. Protein assay was made using the method of Lowry, 3; pl.
(54). The optical density of the samples was determined at a wave-
length of 700 millimicroms using a Beckman DB-G grating spectro-
photometer.

Standards were prepared using 50, 100, 150 and 200 micrograms of
bovine serum albumen. The actual protein content of the bovine serum
albumen was determined by multiplying the micro-Kjeldahl nitrogen
value by 6.25 The final results were expressed as mg protein/g fresh

weight.

Tests Conducted on Fruit

 

Firmness -- Firmness of the fruit was determined using a plunger

type pressure tester. Orhcards A, G, and D were tested prior to

 

 

23

storage and B and C after approximately four months storage. Ten
fruit were randomly chosen from each treatment. A small slice was
made with a paring knife on opposite sides of the fruit, exposing the
flesh. The pressure in pounds required to force a plunger point

ll mm in diameter into the flesh of the fruit to a depth of approxi-
mately 7 mm was measured and recorded. The average of the two tests
was computed, and an overall mean for that treatment was computed

from these ten averages.

Soluble Solids -- Fruit juice collected during the firmness
determinations was used for the soluble solids measurement, employing
a hand refractometer. The results were expressed as percent soluble

solids.

Total Pectic Material -- The total pectic material was determined,
on fruit which had been stored for eight to nine months, using a method
similar to that developed by McCready and McComb (56). Twentwaive
grams of fresh fruit were macerated in a Waring blender for 5 minutes
with 125 ml of 95 percent ethyl alcohol. The ethyl alcohol, containing
the sugars, was then filtered and discarded. After washing twice with
75 percent ethyl alcohol, the moist pulp was transferred to a 250 ml
beaker. The pectin was deesterified by mixing the pulp with 200 ml
of a 0.5 percent EDTA (tetrasodium salt of ethylenediaminetetra~
acetic acid) solution at pH ll.5 for 30 minutes. The mixture was

acidified to pH 5.0 to 5.5 with acetic acid. One—tenth gram of pectinase

 

2h

was added to the solution, which was transferred to a 250 ml
Erlenmeyer flash and vigorously shaken for one hour on a Eberbach
variable speed shaker, Following agitation the solutions were diluted
to 250 ml and filtered, discarding the first few ml of filtrate. Two
ml of the filtrate were diluted to l00 ml, and three 2 ml aliquots
were used for analysis.

The assaying procedure was as follows: Twelve ml of concentrated
sulfuric acid were measured into a 25 x 170 mm test tube. The tube and
contents were cooled to about 3°C in an ice bath, and a 2 ml aliquot
of the pectic material was added to the solution, and mixed. The tube
was replaced in the ice bath and the contents cooled below 5°C. After
heating for ID minutes in a boiling water bath, cooling to approximately _
20°C, l.0 ml of 0.15 percent carbozole reagent (reagent grade carbazole
in lOO percent ethyl alcohol) was added to the sample. The color was
allowed to develop for 20 to 30 minutes, at room temperature. The
optical density of the samples was determined using a Beckman DU
spectrophotometer at a wavelength of 520 millimicrons. Samples were
analyzed in sequence so that the-time and temperature between addition
of carbozole and color development were the same. A standard curve was
run on samples containing .0], .05, .l0 and 1.5 mg/lOO ml of Dualpha-
galacturonic acid. The results were expressed as mg galacturOnic acid

per g of fruit.

 

 

25

Determination of Titrgggbie Acidity of Frglt -- Twenty wedges were
removed from the same fruit, used for determining firmness, including
the peel and the flesh, but not the core. Two lOO gm samples were
weighed, placed in appropriately labeled polyethylene bags, and rapidly
frozen to avoid excessive browning of the tissue. For analysis each
tissue sample was placed in a #00 ml beaker. Two hundred ml of
distilled deionized water were added, and boiled for three minutes.
After cooling, the sample was macerated in a Waring blender for three
minutes. The suspension was transferred to a Buchner funnel
containing a milk filter pad and filtered under suction from a water
aspirator. Two 50 ml aliquots of the filtrate were titrated to a
pH of 8.] with 0.l0 fl NaOH. The titratable acidity was expressed as

meq per 100 g of fruit.

.flgter Soluble Protein Determination in Fruit -- Twentyafive g of
tissue from fruit harvested at orchards D and G and stored for eight
to nine months, were ground with 20 ml of l.0'fl phosphate buffer at
pH 9.0 in an electric mortar grinder. After centrifugation in a
Sorvall GLC-l at 6000 R.P.M. for 20 minutes, a 3 ml sample of the
supernatent solution was precipitated with one mi of no percent
trichloroacetic acid. The precipitate was washed twice with l0 ml
of 10 percent trichloroacetic acid, centrifuged, and the supernatent
solution discarded. The pellet was dried in an air stream, and
solubilized in 2 ml of [Q NaOH. Protein assay was made in the same

manner as described under protein determination on apple buds.

 

26

Respiration Studies -- Respiration rates of whole fruit samples
harvested in l965 and l966 were monitored utilizing an open, or gas
flow, system. Fruit from each treatment were monitored in seperate
containers, in a room held at 20°C. Respiratory rates were measured
for the following time periods. Orchard A, 8/2l/65 to 9/8/65, B and
C ll/h/65 to ll/l5/65. Orchards D and G, from ll/ih/66 to ll/Zl/66.
The analysis of carbon dioxide was achieved with a Beckman lR=llS
infrared analyzer. Oxygen levels were determined with a Beckman Model
G-2 oxygen analyzer. The respiratiOn rate was expressed as ml of O2
consumed, or C02 produced per kilogram of fruit per hour at standard

conditions (23).

Statistical Analysis

Data were subjected to analysis of variance. Where mean comparisons

were necessary, Duncan's Multiple Range Test was employed. Respiration
data were analyzed using the Control Data Corporation 3600 Computer,

installed in the Computer Laboratory at Michigan State University.

An analysis of variance routine was programmed to analyze the differences

in the respiratory rate for each 12 hours cycle during the monitoring

process.

RESULTS AND DISCUSSION

Tests Conducted on Tree Portions

Nitrogen Analysis -- Simazine applications to the soil, around
the base of fruit trees, caused no increase in the foliage nitrogen
level in any orchard treated in 1965 (Tables 1, 2, 3). In 1966, the
simazine treated trees in orchards G (Table A) and J (Table 5), had
higher nitrogen levels than those where no herbicide was applied. A
significant increase in leaf nitrogen, not related to herbicide
application, occurred at the highest nitrogen level in orchard F.
Similar effects were not evident in other orchards treated that year
Table 5, 6, 7).

The lack of increase in nitrogen content, due to simazine
applications, in the majority of the orchards may be related to
several factors. Examination of this data, and prior weed control
studies where simazine was applied, indicate a trend for increased
nitrogen content appearing after two or three consecutive years
application of herbicide. This may be related to the low solubility
of simazine and associated minimal leaching to the root zone where
absorption takes place. In both orchards G and J simazine was applied
by the grower, or weed control researchers, prior to the year of the
increased nitrogen. Climatological conditions in 1966 add support to

this postulate. The temperature for the five months from April through

27

 

28

Table 1. Pear tree leaf nitrogen content and qualitative characteristics
of fruit in relation to treatments‘in orchard A.

 

 

 

 

Weed Leaf N Firmness Soluble Titratable

Control %) lbs) Solids Acidity
(%) (meq)

None 1.1191/ 19.81/ 12.23/ 6.641/
Plastic mulch 1.35 18.6 11.1 6.16
Simazine 1.63 20.8 11.0 6.67
Simazine +

amitrole-T 1.5h 18.6 11.5 5.78
1/

— F value for treatments not significant at 5% level.

3/ F value for comparison of no weed control vs weed control significant

at 5% level.

 

29

Table 2. Apple tree leaf nitrogen content and qualitative characteristics
of fruit in relation to treatments in orchard B.

 

 

 

Treatment Soluble Titratable
Weed Area Leaf N Firmness solids acidity
Control (sq ft) (%) (1b) (%) (meq)

' None 2.041/ 9.31/ 12.33/ 6.51/
Simazine 16 2.04 9.1 11.5 6.0
Simazine 64 2.05 8.9 11.8 6.0
Simazine 144 2.08 9.1 11.5 5.3

 

Simazine +

 

amitrole-T 16 1.87 9.3 11.3 6.1
Simazine +

amitrole-T 64 2.15 9.0 11.5 5.6
Simazine +

amitrole-T 144 2.01 8.8 11.7 5.6
1/

— F value for treatments not significant at 5% level.

3/ F value for comparison no weed control vs weed control significant
at 1% level.

 

30

Table 3. Apple tree leaf nitrogen content and qualitative characteristics
of fruit in relation to treatment in orchard C.—

 

 

 

 

 

Treatment '-. ‘_—
Suppl. Soluble Titratable

Weed Rate nitrogen Leaf N Firmness solids acidity
control (lb/A) (lb/tree) (%) (1b) (%) (meq)
None 0 0 1.82 9.8 11.7 5.0
Simazine +

amitrole-T 4+2 0 1.89 10.0 11 0 5.2
Simazine +

amitrole-T 8+2 0 1.53 10.2 11.9 5.7
None 4 1.91 10.3 11.3 4.0
Simazine +

amitrole-T 4+2 4 2.39 10.3 11.5 4.6
Simazine +

amitrole-T 8+2 4 1.93 10.7 11.8 6.0

 

1/

- F value for treatments not significant

at 5% level.

 

Table 4. Apple tree leaf nitrogen content and qualitativelgharacteristics
of fruit in relation to treatments in orchard G.-

 

 

 

Treatment Soluble Titratable
Weed Nitrogen Leaf N Firmness solids acidity
Control (lb/trees) Source (%) (1b) (%) (meq)
None 0 1.74c 16.4 12.5 8.3
Simazine +
amitrole-T 0 2.04b 16.3 12.2 10.1
None 1 Ca(N03)2 1.73C 16.5 12.0 10.0

 

Simazine +

 

amitrole-T 1 Ca(N03)2 2.01b 16.0 11.7 9.8
None 0 1.57c 16.3 12.0 11.4
Simazine +

amitrole-T 0 1.94b 16.3 11.7 8.5
None 1 (NH4)2504 1.74c 16.1 12.2 10.2
Simazine +

amitrole-T l (NH4)250u 2.21a 15.9 11.5 8.9
1/

— Means followed by unlike letters significantly different at 1 percent level.

l/ Means not followed by letters not significantly different at 5 percent level.

 

 

32

Table 5. The nitrogen content of the foliage from trees in orchards
E and J in relation to weed control treatments.

 

 

 

 

A 1 '

Weed Control Ordflafa E Oréhglg J
None 1.591/ 1.386/
Plastic mulch -- 1.55
Simazine 1.68 1.72
Simazine +

amitrole-T 1.65 --
1/ F value for treatments not significant at 5% level.
2/

— F value for comparison of weed control vs weed control significant
at 5% level.

 

 

 

33

Table 6. Apple tree leaf nitrogen content and qualitative characteristics
of fruit in relation to treatments in orchard D.—

 

 

 

 

 

Treatment ' Soluble Titratable

Weed Area Leaf N Firmness solids acidity
Control (sq ft) (%) (lb) (%) (meq)
None 1.66 15.3 11.2 8.2
Simazine 16 1.74 15.1 11.7 8.7
Simazine 64 1.77 15.1 11.0 9.2
Simazine 144 1.66 15.7, 11.5 9.6
Simazine +

amitrole-T 16 1.67 15.1 11.3 9.2
Simazine +

amitrole-T 64 1.79 14.8 11.2 9.1
Simazine +

amitrole-T 144 1.80 15.1 11.2 8.5
1/

— F value for treatments not significant at 5% level.

 

34

Table 7. The nitrogen content of the foliage from trees in orchard H,
and F in relation to herbicide and nitrogen treatments.

 

 

 

Nitrogen levels

 

 

 

Treatment Apple Apple
Need Nitrogen Orchard F Orchard H
Control (lb/tree) Source (%0 (%0
None 0 1,913/ 1.731/
Simazine +
amitroleeT 0 1.92 1.76
None 1/2 (Ca(NO3)2 2.07 1.94
Simazine +
amitroleaT 1/2 (Ca(N03)2 1.96 1.75
None 1 (Ca(N03)2 1.98 1.72
Simazine +
amitroleeT l (Ca(N03)2 2.06 2.07
None 0 1.62 1.74
Simazine +
amitroleeT 0 1.71 1.76
None 1/2 (NHh)2304 1.99 1.75
Simazine +
amitroleeT 1/2 (Nthzsoh 1.90 1.87
None 1 (141114,)2501, 2.04 1.92
Simazine +
amitroleeT 1 (NHA)2504 2.21 1.87
1]

significant at 5% level.

.3/ F value for average 1 l/btree of N vs average of others

significant at 5% level.

F value for herbicide treatment within fertilizer level not

 

35

August did not vary appreciably between experimental sites. The orchard
G area, however, received approximately two and four inches more rain
than the area around orchards H and F, reSpectively. The greatest
difference occurred in the critical growth month of June, when the G,
H, and F areas received 1.23, .66 and .87 inches, respectively (84,
85). The orchard J area received considerably more rain than either
of these three, with the month of June accounting for a large part of
the difference.

The optimum time of fertilizer application, in relation to maximum
availability, absorption and utilization of nitrogen by perennial
fruit trees, is not agreed upon among research workers. Much of the
literature relating to this topic is reviewed by Boynton and Oberly
(12), who believe apple trees take up appreciable amounts of nitrogen
during the winter months. However, little of this is translocated
upward until the spring. The presence of sub-optimal temperatures
and a low level of nitrate, conditions under which simazine appears
to increase either nitrate absorption, nitrate assimilation, or both
(82), could exist at this time. The necessity of having simazine in
the root absorption zone at this time, or in the late fall or early
Spring, when the previously listed conditions could exist, would be of
obvious importance in relation to consecutive herbicide applications.

Summarizing, it appears that successive annual applications of

simazine, adequate rainfall to leach the chemical into the root zone,

36

and the time of nitrogen absorption in relation to- environmental
factors (i.e. nitrogen availability and temperature), are important
in accounting for the lack of nitrogen response in the majority of
the treated orchards.

Nitrogen analyses were used as a guide to Select apple orchards
for the quality and composition studies. Two orchards were chosen:
G, where simazine caused a marked increase in the percent leaf
nitrogen; and F because it did not show an increase in nitrogen due
to simazine. However, orchard F had a history of several years of

herbicide application on the same plots.

Nitrate Reductase -- Assay of leaf and root tissue from orchard G
disclosed no nitrate reductase activity in either plant portions.
Attempts by other workers to detect this enzyme in apple tissue resulted
in similar findings (71). Recent studies by Gasmanis and Nicholas (39),
disclosed the presence of inhibitor(s) of nitrate reductase in extracts
of apple roots. By dialzying the extracts against phosphate buffer for
42 hours, or by passing them through Sephadex G-25, the inhibitor(s)
were removed and nitrate reductase was detected. Since neither of
these latter two procedures were used in the method employed in this
study, this may account for the lack of detection of nitrate reductase

activity in root extracts.

 

37

Nitrate nitrogen normally is not found in apple leaves or other
tissue of the tree above the fibrous roots (12). Since nitrate
reductase is an inducible enzyme requiring the presence of nitrate
(8), this could account for the lack of detection of enzyme activity
in apple leaves. The possibility also exists that the best conditions

for extraction and detection of enzyme activity were not utilized.

Protein Determination - Apple Buds -- The marked increase in leaf

nitrogen found in orchard G was not reflected by a higher protein
level in the lateral buds (Table 8). This may be due to the incor-
poration of this nitrogen in other nitrogenous compounds. Arginine,
asparagine, aspartic and glutamic acids have been found to account
for up to 90 percent of the nitrogen reserves of apple twig, bark,

bud, root bark, and thin root tissue (13).

Tests Conducted on Fruit

Quality of Fruit at Harvest —- Fruit harvested in both 1965 and

1966 had an exterior appearance of high quality, with no evident

differences in color.

Firmness -- Neither simazine, nor the increase in foliage nitrogen
due to simazine, affected apple or pear fruit firmness (Table i=5).
No detectable residues of the herbicide have been found in the fruits
(30), which could directly affect respiration and alter ripening and

softening processes.

 

 

38

Table 8. Protein content of lateral buds of twigs from McIntosh apple
trees in relation to treatments in orhcard G.l

 

 

 

 

 

Treatment Protein

Weed ' Nitrogen (mg/g
Control (lb/tree) Source fr wt)
None 0 25.8
Simazine +

amitrole-T O 23.4
None 1 Ca(N03)2 26.8
Simazine +

amitrole-T l Ca(N03)2 30.5
None 0 24.2
Simazine +

amitrole-T 0 2h°5
None 1 (NH4)ZSO4 23.9
Simazine +

amitrole-T 1 (NH4)2504 25.9
1/

‘ F value for treatments not significant at 5% level.

 

 

39

Positive correlations between nitrogen, cell volume and higher
internal breakdown have been found by Letham (52) and Martin 5; al.
(55). The possibility exists that if a critical threshold value for
nitrogen were exceeded due to simazine, it could result in a greater
cell volume and its associated effect on firmness as reported by these

workers.

Soluble Solids =- Except for orchards A and B (Tables 1, 2),
simazine did not alter the soluble solids (Tables 3, 4, and 6). The
average decrease in these two orchards of approximately 6 percent
would not be of practical importance. To attribute this effect to
the herbicide without further research seems questionable, since there
was no apparent effect on the other fruit quality parameters, or on
the nitrogen content of the foliage.

Large applications of nitrogen have been found to cause a trend of
decreasing soluble solids (76), however, the increase in nitrogen
content of orchard G was not accompanied by any alteration of the
soluble solids content. Since more leaves per fruit usually results
in a greater percent sugar, up to a certain point, the possibility
exists that increased nitrogen content, causing a more vigorous tree,

could result in greater leaf/fruit ratios, and thereby increase sugars.

 

 

40

[12391 Pectic Material -- The total pectic material of apples was
not altered by herbicide treatment in any of the plots (Tables 9, 10).
The amount of total pectic substances remains fairly constant following
harvest until the fruit becomes overripe, at which time a decrease
occurs. During this time, soluble pectic materials are being trans=
formed into nonpectic materials (77). Since this test was conducted
after considerable storage, any hastening of breakdown, to the point
where there was a loss in pectic material should have been detected.
Further contemplation discloses several factors that could be considered
in relation to firmness, pectic materials and nitrogen. SharplesI
expresses the opinion that the effect of excess nitrogen may in that
of causing an imbalance of nutrient levels that are necessary for
quality fruit. Calcium would be an important element in this relation»
ship, since it is involved in binding the middle lamella in the form
of calcium pectates. An increase in nitrogen, due to simazine,
without a readily available source of calcium could result in growth
and cell enlargement without normal strengthening, possibly related to

the aforementioned calcium pectates.

Titratable Acidity -- As stated in the literature review, acidity

has been shown to increase as the number of leaves per fruit increased.

 

IR. Sharples. Personal Communication. East Malling Research
Station. East Malling, England

 

41

Table 9. Protein and total pectic material content of the flesh f
McIntosh apples in relation to treatments in orchard G.—

 

 

 

 

 

Treatment Protein Total pectic

Weed Nitrogen (mg/g material
Control (lb/tree) Source fr wt) (mg/g)
None 0 .123 7.07
Simazine +

amitrole-T 0 .121 6.83
None 1 Ca(N03)2 .126 6.07
Simazine +

amitrole-T 1 Ca(N03)2 .145 6.33
None 0 .126 6.37
Simazine +

amitrole-T 0 .124 6.08
None 1 (NHp)250p .098 6.23
Simazine +

amitrole-T l (NH4)ZSO4 .146 5.83
1/

‘ F value for treatments not significant at 5% level.

 

42

Table 10. Protein and total pectic material content of the flesh of
McIntosh apples from orchard D as influenced by herbicides
and their area of application.—

 

 

 

Treatment
Weed Area Protein Total pectic
Control (sq ft) (mg/g material
fr wt) (mg/g)

None .173 6.37
Simazine 16 .131 6.22
Simazine 64 .109 6.30
Simazine 144 .126 6.78
Simazine +

amitrole-T 16 .149 6.42
Simazine +

amitrole-T 64 .119 6.28
Simazine +

amitrole-T 144 .139 6,45

 

l/ F value for treatments not significant at 5% level.

 

 

 

43

The longer terminal growth reported by Ries, 25 al. (67), associated
with increases in nitrogen due to simazine application, would be
reflected by higher leaf to fruit ratios. No effect was noted on
titratable acidity among the fruit tested, including those harvested
from orchard G, having an increased leaf nitrogen (Tables 1-5). It
should be noted that no terminal growth measurements were made to
determine if the increase in nitrogen was reflected by longer terminal

growth in this orchard.

Water Soluble Protein Detennination - Fruit -- There was no

 

difference in water extractable protein of apple flesh due to simazine
or its effect on nitrogen content of foliage (Tables 9, 10). Recent
work by ChmielI in assaying for protein, has disclosed that the most
dramatic simazine induced protein increases occur in plants that
normally synthesize high levels of protein. Apples are characteris=
tically low in protein.

In a study carried out on Jonathan apples, it was found that only
a diminishing preportion of a noted increase in nitrogen was incorporated
into the protein fraction of the fruit (55). Similarly, Hill (45)
found that a 19 percent increase in leaf nitrogen was accompanied by
176 percent increase in soluble nitrogen of the fruit, but only a

16 percent increase occurred in fruit protein.

IH. Chmiel. Personal communication. Dept. of Horticulture, Michigan
State University. East Lansing, Michigan

 

The relationship between fruit respiration rates and proteins,
or vice versa, has resulted in many hypotheses attempting to relate
protein to fruit climacterics and senescence. The necessity for
formation of high energy compounds utilized in DNA replication,
transcription and RNA biosynthesis, and translation, all steps involved
in protein formation, make respiration of obvious importance in the
overall process of protein biosynthesis. Though respiration is
necessary for protein synthesis to occur, synthesis may not be strictly
dependent on the respiratory rate. To relate the lack of increase in
proteins to the fact that there was no significant increase in the
reSpiration rate of fruit from either orchard D or G (to be discussed

in next section), would be extremely presumptuous.

Respiration Studies

Quantitative analysis of carbon dioxide, and oxygen utilized,
at each separate cycle of the respiratory monitoring process, indicated
that there was no significant differences in respiration of apples
from orchards C, D, F, and G or pears from orchard A, due to any of
the treatments. The quantity of carbon dioxide evolved and oxygen
utilized at the time of the pre- and post climacteric mimimums, and
the climacteric peak in respiration are presented in

Tables 11-15.

 

45

 

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46

 

 

 

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47

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48

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SUMMARY

The possibility of a detrimental effect on pome fruit orchards
from consecutive annual applications of herbicides suggested a study
of the influence of simazine on fruit quality. The triazine herbicide,
simazine, is widely employed for orchard weed control. Simazine
increases the nitrogen content of several crops including fruit trees.
This is considered to be of critical importance, since there is an
accepted correlation between nitrogen content and fruit quality.

Simazine was applied, either alone or in combination with amitroleeT,
to four pome fruit orchards in l965, and five in l966, throughout
Michigan. Supplemental nitrogen applications up to h lb/tree were
also applied at six experimental sites.

Kjeldahl analysis of foliage detected an increase in nitrogen,
associated with herbicide, in only one pear and one apple orchard out
of the total number treated. This foliar increase was not reflected
by an increment in water soluble protein in apple tree buds. Enzyme
inhibitor(s) may have interfered with the detection of nitrate
reductase activity, which has been associated with the effect of
simazine on increasing nitrogen content. This increase in foliar
nitrogen of apples had no effect on the firmness, total pectic material,
soluble solids, titratable acidity, protein content or the respiration

rate of the fruit.

50

 

5i

One pound of supplemental nitrogen per tree, increased the leaf
nitrogen, as compared with none and one-half pound in a single
experiment.

Where no increment in foliage nitrogen occurred, herbicide
application had no influence on any of the quality characteristics,
except for a 6 percent decrease in soluble solids in pear and apple
fruit from individual orchards treated in 1965. Because of the lack
of effect on any other quality parameter, or recurrence of the con=
dition in l966, this decrease was not considered significant.

This research indicates that there are no direct effects of
simazine on pome fruit quality. Furthermore, simazine's indirect
effect of increasing foliar nitrogen should not alter quality, provided

the grower annually considers the nitrogen status of the fruit trees.

 

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and . l967. Effect of simazine on nitrate

 

reductase activity in corn. Plant Physiol. 42:280m282.

 

83.

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85.

86.

87.

88.

89.

90.

58

United States Department of Agriculture, Agricultural Research
Service. 1965. A review of literature on harvesting, handling,
storage and transportation of apples. ARA 51-4, 2l5 pps.

United States Dept. of Commerce, Environmental Science Services
Administration. 1966. Climatological Data-Michigan. 8l:49-128.

, . l965. Climatological
Data-Michigan, Annual Summary-l965. 80—203-213.

 

U. 5. Government Printing Office. I952. Hearings before the
house select committee to investigate the use of chemicals in
food products. Chemicals in food products. H. Res. 323. Blst
Cong. 2d sess. pp l-2.

Van Oorschot, J. L. P. and M. Belksma. l96l. An assembly for the
continuous recording of C0 exchange and transpiration of whole
plants. Weed Res. l:245-2 7.

Weeks, W. D., F. W. Southwick, Mack Drake and J. E. Steckel. l952.
The effect of rates and sources of nitrogen phosphorus and po—
tassium on the mineral composition of Mclntosh foliage and fruit
color. Proc. Amer. Soc. Hort Sci. 60:ll-Zl.

Welker, William V. Jr. l967. Effect of herbicides on quality and
yield of sweet potatoes. Weeds. 15:112-114.

W. F. Humphrey Press, Inc. l967. Herbicide Handbook of the Weed
Society of America. W. F. Humphrey Press, Inc. Geneva, N. Y.
42-47.

 

 

APPENDIX

 

6O

 

 

 

 

Orchards A and J, B and D, and C and E: same orchards receiving
treatments in consecutive years.

Table 1. Sites of experiments and additional pertinent information for
the years 1965 and l966.l
Year of Approx.
herbicide age of
Orchard Fruit Type Cultivar Location Treatments trees (yrs)
A Pears Bartlett Pearl Grange, Mich l964, I965 lO
B Apples Mclntosh Belding, Mich. 1964, I965 l2
C Apples Mclntosh Sparta, Mich. l965 20
D Apples Mclntosh Belding, Mich. l965, l966 l3
E Apples Mclntosh Sparta, Mich. l965, l966 2]
F Apples Mclntosh Traverse City,Mich. 1966 9
G Apples Mclntosh Ludington, Mich. I965, l966 20
H Apples Red Delicious Shelby, Mich. 1966 9~l2
J Pears Bartlett Pearl Grange, Mich. l964, 1965,
1966 ll
1/

 

Table 1.

60

Sites of experiments and additional pertinent information for
the years I965 and 1966.1

 

 

 

Year of Approx.
herbicide age of
Orchard Fruit Type Cultivar Location Treatments trees (yrs)
A Pears Bartlett Pearl Grange, Mich 1964, 1965 10
B Apples Mclntosh Belding, Mich. 1964, 1965 12
C Apples Mclntosh Sparta, Mich. 1965 20
D Apples Mclntosh Belding, Mich. 1965, 1966 13
E Apples Mclntosh Sparta, Mich. 1965, 1966 21
F Apples Mclntosh Traverse City,Mich. 1966 9
G Apples Mclntosh Ludington, Mich. 1965, 1966 20
H Apples Red Delicious Shelby, Mich. 1966 9=12
J Pears Bartlett Pearl Grange, Mich. 1964, 1965,
1966 11

 

Orchards A and J, B and D,

treatments in consecutive years.

and C and E:

same orchards receiving

 

61

 

 

 

Table 2. Chemical and cultural treatments utilized at experimental sites
Fertilizer
Weed Rate Supplemental applied by
Orchard Control (lb/A) nitrogen grower
A None —- None
Black plastic mulch =— None
Simazine + amitrolewT 4+2 None
Simazine 4 None
None -- 1 lb NHANO3/tree None
Applied
Black plastic mulch -= None
Oi
Simazine + amitrole-T 4+2 None
None ~— 2 lb NH4NO3/tree
Black plastic mulch == None
Simazine + amitrole-T 4+2 None
Simazine 4 None
C None =- None
Simazine + amitrole=T 4+2 None
Simazine + amitrole-T 8+2 None None
Applied
None =- 4 lb NHhNO3/tree
Simazine + amitrole—T 4+2 None
Simazine + amitrole=T 8+2 None

 

 

 

 

 

 

 

 

62
Table 2, continued
Trt. Fertilizer
Weed area Rate Supplemental applied by
Orchard Control (sq.ft) (lb/A) nitrogen grower
B None -~ —= None

Simazine +

amitroleaT 16 4+2 None

Simazine +

amitrole-T 64 4+2 None Approx. lei/2
lb. Ntho /

Simazine + tree on a l

amitrole-T 144 4+2 None plots

Simazine l6 8 None

Simazine 64 ‘8 None

Simazine 144 8 None

D None =~ -- None

Simazine +

amitrolenT 16 4+2 None

Simazine +

amitrole-T 64 4+2 None
1=l/2 1b

Simazine + NHhNO /tree

amitrolewT 144 4+2 None on all
plots

Simazine 16 4 None

Simazine 64 4 None

Simazine 144 4 None

 

 

 

 

 

 

63
Table 2, continued
Fertilizer
Weed Rate Supplemental applied by
Orchard Control (lb/A) nitrogen grower
E None -- None Approx. 191/2
1b. NHhNO /
Simazine + tree on all
amitrole-T 4+2 None plots
Simazine 4 None
F None -- None
Simazine +
amitrole—T 4+2 None
None -- 1/2 lb ca(N03)2/tree
Simazine +
amitrole-T 4+2 1/2 lb ca(NO3)2/tree None
Applied
None -= 1 lb Ca(N03)2/tree
Simazine +
amitrole-T 4+2 1 lb Ca(N03)2/tree
None =~ None
Simazine +
amitrole-T 4+2 None
None == 1/2 lb (NHh)ZSOA/tree
Simazine +
amitrole=T 4+2 1/2 lb (NH4)2504/tree
None —- 1 1b (NHu)2504/tree
Simazine +
amitrole-T 4+2 I Ib (NH4)2504/tree

 

 

 

Table 2, continued

64

 

 

 

 

Fertilizer
Weed Rate Supplemental applied by
Orchard Control (lb/A) nitrogen grower
G None -- None None
Simazine +
amitrole—T 4+2 None None
None -- 1 lb Ca(NO3)2/tree None
Simazine +
amitrole-T 4+2 1 1b Ca(NO3)2/tree None
None -- None None
Simazine +
amitrole-T 4+2 None None
None -- 1 1b (NH4)2/tree None
Simazine +
amitroleaT 4+2 1 1b (NH4)2/tree None
J None -- None
Black plastic
mulch —- None Approx.
2 lbs.
Simazine + 15=5=15/
amitrole-T 4+2 None tree on
all plots
H All treatments, rates, supplemental nitrogen similar to

orchard F