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mass {@YRUS cowwsess L.) Abii‘.‘ seas 3mm
{gamma mvmvem, sum)

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MSCHSGAN HATE 'UNNEEISETY
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$66

 

 

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This is to certify that the

thesis entitled
STUDIES ON THE NUTRITION OF BARTLETT PEAR TREES

(PYRUS COWUNIS L.) AND FIRE BLIGHT (ERWINIA
AMYLOVORA, BURR.)
muted by

LOWELL N. LEWIS

has been accepted towards fulfillment
of the requirements for

degree in

DOCTOR OF PHILOSOPHY

Department of Horticulture
, A ,__>

Major professor /

Date November 11L 1960

0-169

   
     

  

LIBRARY

Michigan State
University

 

STUDIES ON THE NUTRITION OF BARTLETT PEAR TREES

(PYRUS COMMUNIS L.) AND FIRE BLIGHT (ERWINIA

 

AMYLOVORA, B URR . )

 

By

LOWELL N. LEWIS

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1960

Approved // f K Vim/LIZ}?
7

 

LOWELL N. LEWIS AETRACT

Bartlett pear trees were grown in a nutrient solution sand culture at
various levels of nitrogen, phosphorus, potassium, calcium, magnesium, iron,
zinc, boron, manganese, copper, and molybdenum during the 1958, 1959 and
1960 seasons. These trees were used to study the effect of nutrition on the
mineral composition of the leaves, on the free amino acid levels in the grow-
ing tips and older leaves, and on the susceptibility of pear trees to fire
blight.

As an auxiliary study, the causal organism of fire blight, Erwinia
amylovora, Burr. was grown in synthetic media to ascertain its ability to
utilize natural and synthetic amino acids and amides as a source of nitrogen.
Various other synthetic amides were tested for their bactericidal effect.

The studies on the effect of nutrition on the mineral composition of the
leaves showed that decreasing the amount of an element in the nutrient solu-
tion resulted in a corresponding decrease in the leaf concentration of that ele-
ment in all cases except iron, zinc, copper, and molybdenum. Increasing the
amount of an element in the nutrient solution increased leaf concentration ex-
cept for calcium, zinc, and molybdenum. In a few instances the concentration
of one element seemed to be dependent on the concentration of another. Mag-
nesium, calcium, and pctassium concentrations seemed to be closely inter-
related. Potassium and phosphorus showed an indirectly proportional relation-

ship. There were other instances of low magnitude elemental interactions.

LOWELL N. LEWIS ABSTRACT - 2

The free amino acid composition of the growing tip of a pear tree also
seemed to be related to the nutrient element composition of the leaves. In-
creasing or decreasing total leaf nitrogen resulted in a similar change in the
free amino acid concentration. High levels of phosphorus or potassium in the
leaf were associated with an increase in total free amino acids similar to that
found for trees high in nitrogen. Either increasing leaf boron or decreasing
leaf magnesium resulted in amino acid levels equally as low as those in the
minus nitrogen trees.

The free amino acid levels were also studied in the mature leaves from
non- growing shoots of trees grown in solutions deficient in nitrogen, phosphorus,
potassium, iron, and boron. Minus boron leaves contained more total amino
acids than the check leaves, but low potassium leaves accumulated the highest
total free amino acid level. Arginine accumulated only in the leaves deficient
in phosphorus.

The most promising effect of nutritional environment on the susceptibi-
lity of pear trees to E. amylovora was found on trees grown in a high level of
calcium. Neither the plus calcium nor the minus boron trees became infected
with the bacteria. Those trees deficient in phosphorus or iron as well as the
check trees appeared to be quite resistant. Trees grown in high levels of
molybdenum, copper, potassium and nitrogen appeared to be the most suscept-

ible to an E. amylovora infection and spread.

LOWELL N. LEWIS A$TRACT - 3

Studies on the bacteria, itself, showed that E. amylovora readily util-
ized aspartic acid, glutamic acid, asparagine, glutamine, baa-alanine, and
gamma-amino butyric acid as a sole source of nitrogen in a synthetic culture
medium. However, there appeared to be no correlation between the foliar
concentration of the individual amino acids or the total amino acid level and
the susceptibility of the tree to a fire blight infection.

Tests on the bactericidal effect of certain synthetic amides indicated
that bromoacetyl valinamide may be a good bactericide for E. amylovora.
This compound gave better control than agrimycin under the conditions of this

experiment and showed no phytotoxic effect on the tree.

STUDIES ON THE NUTRITION OF BARTLETT PEAR TREES

(PYRUS COMMUNIS L.) AND FIRE BLIGHT (ERWINIA

 

AMYLOVORA. BURR . )

 

By

—. i‘

\‘7'
LOWELL N.“ LEWIS

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

DOCTOR OF PHILOSOPHY
Department of Horticulture

1960

cw /"

7/02/91

\/

ACKNOWLEDGEMENTS

The author wishes to express his sincere gratitude to Dr. A. L.
Kenworthy for his encouragement and guidance throughout the course of
this program.

The author also desires to express his thanks and appreciation
to Dr. N. E. Tolbert, Dr. E. J. Klos, Dr. G. P. Steinbauer, and Dr. A.
E. Mitchell for serving on the guidance committee and for their help and
advice throughout the investigation. Special thanks also goes to Dr. D. R.
Dilley and Dr. C. L. Bedford for their generous assistance and the use
of their laboratory facilities.

Special acknowledgement is also made to the author's wife, Alice,
for her help in typing and editing the manuscript, her assistance in the
laboratory work, and her constant inspiration and encouragement through-

out the entire period of college training.

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . .....

REVIEW OF LITERATURE . .

GENERAL PROCEDURE - EXPERIMENTS I. 11, AND III: METHOD
OF ESTABLISHING PEAR TREES IN VARIOUS NUTRITIONAL
ENVIRONMENTS ................. . . . .

EXPERIMENT I - A Study of the Effect of Nutritional En-
vironment on the Mineral Composition of Leaves . . .

Procedure ...........
Results....................
Nutrient Element Composition of Leaves . .

Deficiency and Toxicity Symptoms of Stems and
Leaves ........

Deficiency and Toxicity Symptoms on Roots.
EXPERIMENT II - A Study of the Free Amino Acid Levels
in Pear Leaves and Shoots as Related to Nutritional En-
vironment . . . ...........
Procedure . . .
Results .
June Samples of Growing Shoots . . . .
August Samples of Mature Leaves (Table IX) .
EXPERIMENT III - A Study of the Natural Occurring and
Synthetic Amino Acids in Relation to Growth of the

Bacteria in Synthetic Culture . . .

Procedure. . . . . . . .....

Page

12

15
15
16

16

26

28

29
29
32
32

37

40

40

TABLE OF CONTENTS CONT'D

Results .

EXPERIMENT IV - A Study of the Natural Occurring and
Synthetic Amino Acids in Relation to Growth of the
Bacteria in Synthetic Culture . . .

Procedure.
Results .

EXPERIMENT V- Testing Selected Compounds for their
Bactericidal Effect on E. Amylovora in Synthetic Culture
andonTrees. . . . . . ..... .. .. .

1. Culture Studies . .
Procedure . . .
Results . .
2. Field Studies . .
Procedure.
Results . .
May Field Test . . .
June Field Test .
DISCUSSION .

Nutrient Absorption and Response .

Amino Acid Accumulation as Related to Nutrient Environ-
ment .

Susceptibility of Trees to _E_. amylovora .

Dependence of E: amylovora Bacteria on Amino Acids .

Possible Bactericides for E. amyIOVOra.

Page
40

45
45

46

51
51
51
52
56
56
58
58
59
60

60

66
69

71

74

TABLE OF CONTENTS CONT'D

SUMMARY. . . .

LITERATURE CITED .

INTRODUCTION

The production of Bartlett pears (Pyrus communis, L.) in Michigan

 

could be greatly increased if it were not for the constant threat of the fire

blight organism (Erwinia amylovora, Burr.). This disease limits production

 

because of the damage it does to the trees, and because of management prac-
tices used by many growers to prevent its occurrence. In many instances
the pear orchards are grown in sod with little or no fertilizer. Fire blight,
therefore, has cut heavily into the potential of the pear industry in Michigan
and many other states.

Certain notations in the literature (34, 40, 58) suggest that the restric-
tions in growth of pear trees to reduce fire blight infection may result in a
reduced level of certain naturally occurring nitrogen compounds and thus re-
duce growth of the fire blight organism after inoculation.

Therefore this investigation was planned to study (I) the nutrition of
pears as related to fire blight susceptibility, and (2) the requirements of the

organism in culture as a possible lead to a means of controlling Erwinia

amylovora.

REVIEW OF LITE RATURE

Pear Nutrition

 

Nutrient element balance is a most essential factor in plant growth;
and any violation of this balance may result in a deficiency or toxicity of the
elements involved. Many studies have been conducted on the problem of
elemental balance in pears especially with nitrogen, phosphorus, potassium,
magnesium, iron, manganese, boron, zinc, and copper.

Early work on the nitrogen, phosphorus, and potassium requirements
of pears was done by L. Proebsting (49). He concluded that nitrogen was the
only fertilizer required for pear trees grown under the conditions of his ex-
periment. The trees which received high nitrogen were lower in phosphorus
but not low enough to indicate phosphorus deficiency. This nitrogen-phosphorus
relationship was also observed by E. Proebsting (50). Neither potassium nor
phosphorus fertilizers increased the level of their respective elements in the
leaves. Baxter (2) was not able to increase the nitrogen in pear leaves even
though large amounts of nitrogen were applied to the tree. According to
Boynton (10), pears can usually tolerate a low phosphorus level. Bryant and
Gardner (15) reported one instance of pears responding to phosphorus ferti-
lizer. These trees were growing in a soil which was believed to contain little
available phosphorus.

Wallace (67) described potassium deficiency in pears as a marginal

leaf scorch appearing first on older leaves and noted the similarity of the
symptoms on pear with those on apple. Reuther (51) found that the leaves of
Bartlett pears showed deficiency symptoms when the potassium level was less
than 0. 5 percentl. Baxter (2) showed that the potassium content of pear leaves
was lowered by soil applications of nitrogen, magnesium or calcium. Con-
versely he noted that calcium and potassium applications lowered the percent
magnesium in the leaves. However, the sum of magnesium, calcium and
potassium remained about the same over a wide variation in the nutrient status
of the tree. Vidal and Herrera (65) noticed the potassium /magnesium (K/Mg)
interaction in their work too. They found that a K/Mg ratio of 3 to 4 indicated a
potassium deficiency, while a potassium excess might cause a K/Mg ratio as
high as 23.

Harley (23) also commented on the K/Mg relationship. He found that
leaves showing symptoms of magnesium deficiency contained about 0. 05 per-
cent magnesium. Leaf potassium was about 50 percent higher in the magnesium
deficient trees than in the normal appearing trees. Harley described magnesium
deficiency symptoms on pear leaves as oblong islands of almost black tissue
arranged in a rather orderly fashion between the parallel veins on both sides
of the midrib.

From a study of Japanese pears grown in sand culture, Sato (55) ob-

served the following concentrations of major elements in leaves showing

1All percent and ppm values in this thesis are based on dry weight unless
otherwise indicated.

deficiency sumptoms: nitrogen - 0.83%, phosphorus - 0. 07%, potassium - 0. 27
to 0.41%, calcium - 0.66%, and magnesium - 0. 09 to 0. 11%. Kenworthy's
data (33) indicated that the optimum level for major elements in healthy orch-
ard grown Bartlett pear leaves was: nitrogen - 2. 50%, phosphorus - 0. 135%,
potassium - 1.45%, calcium - 1. 90%, and magnesium - 0.397%. Sato (56)
found about the same levels of leaf nitrogen, phosphorus and potassium in
healthy pear trees grown in sand culture.

The problem of lime induced chlorosis has made iron the focal point
of many studies. Boynton (10) has described iron deficiency in pears as con-
trasting green leaf veinlets and main veins followed by yellow leaves and finally
white leaves with brown necrotic lesions; the tree may also exhibit thin shoot growth
and considerable dieback. Several workers (37, 63, 66) have noticed the high level
of potassium and low level of calcium in iron deficient plants. Linder and Harley
(37) pointed out, however, that the potassium-calcium interaction was not asso-
ciated with iron deficiency due to low iron but rather with iron deficiency induced
by lime, iron-manganese balance upset or phosphorus-iron balance upset. Vidal
and Herrera (65) also studied the importance of the iron-manganese ratio. They
found that iron deficiency resulted in a diminuation of the ratio compared to a
healthy tree. The data of Kenworthy (33) showed this ratio to be about one in
healthy pear. leaves. Vidal and Herrera also noticed that iron or manganese defi-
ciencies resulted in high levels of nitrogen, phosphorus and potassium. Bennett
(3) also reported that iron chlorosis was accompanied by a high level of nitrogen

in the leaf.

Manganese toxicity symptoms have been described by Grasmanis (20)
from observations on the Josephine pear. There was an internal bark necrosis,
cracking of the outer bark, and rolling of bark cuticle. Tissue showing these
symptoms contained 23 ppm of manganese on a fresh weight basis compared with
5 ppm in the healthy leaves.

Boron deficiency symptoms have been described by many researchers.
Keinholz (32) described them as shortened shoot growth, fewer basal leaves,
dwarfed terminal leaves, defoliation except for the terminal leaves, twig die-
back, and bark cankers on the younger branches. Kobernuss (35) similarly
described the symptoms and added possible swelling of the root tips. The actual
level at which boron becomes deficient may be debatable. According to Compton
(16), the foliar level of boron in deficient trees was 14 ppm. Woodbridge, Carney
and McLarty (70) found that leaves from healthy trees contained 10. 3. ppm of
boron while leaves from deficient trees had 0. 5 to 1 ppm. They pointed out that
low soil moisture may accentuate boron deficiency.

Bruno (14) found that inadequate moisture may also cause signs of a zinc
deficiency to appear when the zinc supply seems adequate. An abundance of
water may retard development of the zinc deficiency symptoms. Mulder (42)
showed that high calcium or phosphorus levels in the soil tend to bring on zinc
deficiency. According to Bollard (6), Bouldgt 3L. (8), and Trocme (64), the
threshold value for zinc deficiency is about 10 to 14 ppm. Healthy trees had

16 to 30 ppm of zinc in their leaves. Woodbridge (69) described zinc deficiency

as a cause of delayed bud opening, modified rosette, small, elongated and uni-
formly yellow leaves with the symptoms often distributed irregularly on the
tree.

Leaves from pear trees deficient in copper may contain between 3 and
7 ppm according to Oserkowsky and Thomas (45). Bould _et _a_l_. (9) found that the
value for copper deficiency was about 5 ppm. They found yellow to necrotic leaf
tips, and dieback of shoots with retention of dead terminal leaves typical of copper
deficiency. Dieback of the terminal shoots with copper deficiency was also des-
cribed by Jones (31) and Harris (24).

Kenworthy (33) also established levels of essential trace elements in
Bartlett leaves: manganese - 133 ppm, iron - I40 ppm,_copper - 54 ppm, and

boron 23 ppm .

Amino Acid Levels as Related to Nutrition

 

Most of the previous work on the relationship between mineral nutrition
and amino acid levels has been with crops other than pears.

Bollard's work (6) on amino acids found in the xylem sap of angiosperms
and gymnosperms showed that the following amino acids were almost always
present: aspartic acid, asparagine, glutamic acid, glutamine, serine, threonine,
methionine, valine, leucine, and gamma-aminobutyric acid. Arginine, alanine,
tyrosine and phenylalanine were sometimes present in trace amounts. In a study
of the amino acids found in the xylem sap of apples, he found that aspartic acid,

asparagine, and glutamine predominated.

Boynton Still. (11) studied the effects of potassium and magnesium defi—
ciencies on amino acid levels in Sparkle strawberry leaflets. Control plants
contained a total of 502 ug of amino acids per gram of dry tissue, the magnesium
deficient plants contained 504 ug per gram and the potassium deficient plants, 1392
ug per gram. In the control treatment about 90 percent of the amino acids were
gamma-aminobutyric acid (27. 7%), alanine (15. 7%), arginine (11. 8%), glutamic
acid (11. 6%), serine (7. 4%), and threonine (5. 0%). Glycine, asparagine, glutamine,
histidine, lysine, proline, valine, tyrosine and pipicolic acid made up the other 10
percent.

Plants deficient in magnesium contained about one-half as much gamma-
aminobutyric acid, about 50 percent more serine and glutamic acid, and slightly
more asparagine and pipecolic acid. All others were present in small amounts as
they were in the control. The leucines were present in magnesium deficient plants
but not in the controls. In potassium deficient plants only 6. 2 percent of the total
amino acids was gamma-aminobutyric acid, 36. 1 percent was arginine, 7. 0 percent
glutamic acid, and 16. 1 percent aspartic acid. The concentrations of the other
amino acids were similar to the checks except for the presence of the leucines in
the deficient plants.

Deficiencies of the elements, calcium, magnesium, potash and phosphorus,
caused a sharp increase in the total free amino acids in tobacco according to Steinberg
e_t al_. (59). Several other workers (18, 21, 52, 54) have observed an increase in total

free amino acids in potassium deficient barley and alfalfa plants. Richards and

Berner (52) and Richards and Coleman (53) found an accompanying decrease in
aspartic and glutamic acids. Richards and Coleman also noted the accumulation
of putrescine in potassium deficient barley leaves.

Richards and Templeman (54) reported that the most pronounced char-
acteristic of phosphorus deficiency in barley leaves is an accumulation of amides
accompanied by an increase in the total free amino nitrogen. They also found a
general drop in all amino acids with nitrogen deficiency. Similar results were
obtained by Gregory and Sen (21).

Possingham (47) reported that tomato plants deficient in zinc, copper,
manganese and iron contained more total free amino acids than control plants,
but the molybdenum deficient plants were about the same as the controls in this
regard. The following table summarizes Possingham's results on the increase
in specific amino acids which accompany various nutrient deficiencies:

Deficient Element Amino Acids Increased

 

Iron Asparagine, glutamine and pipe-
colinic acid

Zinc Asparagine, glutamine and beta-
alanine
Manganese Pipecolinic and other amino acids

except the amides
Molybdenum Beta-alanine
Certain variations from these results have been reported by other workers.
Steward and Pollard (60) found an accumulation of arginine in iron deficient blue-
berry and apple. Brown (13) suggested that arginine accumulation was typical of

iron deficient plants.

Margolis (39) found that tomato plants deficient in manganese accumulate
the amide, asparagine, and Hewitt _e_t El: (28) found an accumulation of both amides
in manganese deficient cauliflower plants. Hewitt (27) later. reported that glutamic
and aspartic acids were generally the principal amino acids accumulated by man-
ganese deficient plants.

Most workers (28, 39, 47) agreed that a molybdenum deficiency may be
characterized by a reduced concentration of amino acids.

The relation between amino acids and nutrition has been summarized by
Possingham (47): Except for amall anomalies, the array of amino acids remains
the same regardless of the nutrient status, but quantitative relations do change

considerably.

Fire Blight Susceptibilitl as Related to Nutrition

 

According to Heald (25) the fire blight disease was first reported in the
New York Hudson River Highlands about 1780 and has spread from that area
throughout the United States and southern Canada. In 1878, Burrill, in Illinois,
established the bacterial nature of the disease. The bacteria is presently known

as Erwinia amylovora (Burr.) and will be referred to as E_. amylovoga in this

 

report.

The close relationship between the succulent growth of the tree and its
susceptibility to fire blight quickly became apparent. Hildebrand and Heinicke
(30) reported an increase in fire blight on shoots of Delicious, Cortland and

Rhode Island Greening apple trees following application of nitrogen fertilizer.

They also noted that orchard cultivation increased growth and fire blight.
Fisher (17) reported that mulching increased leaf nitrogen and the development
of fire blight in the trees. He observed no correlation between application of
phosphorus and/or potassium and fire blight development; however, the leaf
potassium level in his study did not exceed 1. 45 percent. Other workers (12,
44, 57, 61) have also commented on the close relation between susceptibility and
the vigor of the trees.

Nightingale (44) felt the balance between carbohydrates and nitrogen was
more important than nitrogen alone in determining the susceptibility of the trees
to fire blight. E. amylovora grew poorly on agar slants of the juices from trees
high in carbohydrate and low in nitrogen, but slants from trees low in carbohy—
drates and high in nitrogen favored bacterial growth. The addition of asparagine
to the medium from the low nitrogen high carbohydrate tree also favored bacterial
growth. The amino acids tyrosine, cystine, and glutamic acid did not improve
bacterial development. Link and Wilcox (38) also noted the importance of the
carbohydrate-nitrogen balance in fire blight resistance.

The use of asparagine to increase tree susceptibility to fire blight was also
studied by Ark (1). When a dormant Bartlett pear tree was inoculated with aspara—
gine before a bacterial inoculation, the shoots became infected with E. amylovora,
while those inoculated without an injection of asparagine showed no infection. All
_E_. amylovora isolates studied by Ark utilized asparagine as a sole source of

nitrogen in media studies. A few were able to use alanine, leucine, and proline,

10.

II.

but none of the isolates utilized glycine, valine, iso—leucine, cystine, cysteine,

tyrosine, tryptophane, or glutamic acid.

Spraying to Control Fire Blight

 

The use of spray materials has grown from bordeaux sprays in pre-war

days to the present day use of various antibiotics and organic compounds, as

well as the copper sprays. A few of these compounds and their effectiveness

are listed below:
Compound
Bioquin 1
Dithane D-14

2-Pyridinethione-
l -oxide

Naban
Tersan
Dithane Z-78
Streptomycin
Terramycin

Agrimycin

Control

Fair

Fair

Best

Fair

Good

Fair

Good

Good

Fair

 

Reference

 

(48)

(62)

(22)
(58)
(58)
(26,62,68)
(41, 68)
(68)

(17,19)

In spite of all this work, Goodman (19) summarizes the results as nega-

tive, " It is suggested that antibiotic sprays, although capable of providing blossom

type control are ineffective against the late or mid-season form of twig infec-

tion. "

12.

GENERAL PROCEDURE - EXPERIMENTS I. II and III

Bartlett pear trees were grown in various nutrient solutions to supply
material for studies on the effect of nutritional environmentvon the concentration
of certain elements in the leaves, the concentration of amino acids in shoots and
leaves, and the susceptibility of the tree to E. amylovora. Twenty-three nutrient
solutions of high, low, and intermediate levels of nitrogen, phosphorus, potas-
sium, calcium, magnesium, iron, zinc, boron, manganese, copper and molyb-
denum were used, and each treatment was replicated three times.

Nutrient solutions for the various treatments were obtained by using the
Hoagland (29) solution as a check and varying its composition to supply the other
nutrient levels (Table I). Minus solutions were void of the element being studied
except for low nitrogen. This treatment was composed of only 20 percent of the
nitrogen found in the check solutions. In 1958, high level solutions of nitrogen,
phosphorus, potassium, calcium, and magnesium contained five times as much
of their respective element as the check solution. The trace elements, iron,
zinc, boron, manganese, copper, and molybdenum, were 25 times as concen-
trated in their respective high level solutions as the check solution. In 1959 the
level of some of the plus solutions was dropped to keep the trees alive. High
nitrogen was dropped to a three-fold concentration, and high iron, zinc, and
boron were dropped to a fifteen-fold concentration.

Stock solutions were prepared by dissolving C.P. grade chemicals in
deionized water, and the nutrient solutions were made from these.

One-year-old Bartlett pear trees on a French seedling rootstock were

Composition of Nutrient Solution used in 1959 and 1960

TABLE I

 

 

 

Ppm
Treatment ,
N P K Ca Mg Fe Mn B Cu Zn Mo

Check 210 31 235 200 49 5 . 5 . 5 . 02 . 05 . 01
-N 42 31 235 200 49 5 .5 . 5 .02 .05 .01
3N* 630 31 235 200 49 5 . 5 . 5 . 02 . 05 . 01
-P 210 - 235 200 49 5 . 5 . 5 . 02 . 05 . 01
SP 210 155 235 200 49 5 . 5 . 5 . 02 . 05 . 01
-I( 210 31 - 200 49 5 .5 .5 .02 .05 .01
SK 210 31 1175 200 49 5 .5 .5 .02 .05 .01
-Ca 210 31 235 - 49 5 .5 .5 .02 .05 .01
5Ca 210 31 235 1000 49 5 . 5 . 5 .02 .05 .01
-Mg 210 31 235 200 - 5 . 5 . 5 .02 .05 .01
5Mg 210 31 235 200 245 5 . 5 . 5 . 02 . 05 . 01
-Fe 210 31 235 200 49 - .5 .5 .02 .05 .01
15Fe* 210 31 235 200 49 75 . 5 . 5 . 02 . 05 . 01
-Mn 210 31 235 200 49 5 - . 5 .02 .05 .01
25Mn 210 31 235 200 49 5 12.5 -5 -02 .05 .01
-B 210 31 235 200 49 5 .5 - .02 .05 .01
158* 210 31 235 200 49 5 .5 .5 .02 .05 .01
-Cu 210 31 235 200 49 5 . 5 . 5 - . 05 . 01
25Cu 210 31 235 200 49 5 . 5 . 5 . 50 . 05 . 01
-Zn 210 31 235 200 49 5 .5 .5 .02 - .01
ISZn" 210 31 235 200 49 5 . 5 . 5 . 02 . 75 . 01
-M0 210 31. 235 200 49 5 . 5 . 5 . 02 . O5 -
25Mo 210 31 235 200 49 5 . 5 . 5 . 02 . 05 . 25

 

 

*These treatments were 5N, 25Fe, 258. and 252m in 1958.

13.

14.

selected for this study. The trees were pruned to reduce weight variation to
105115 grams and to a standard tree height of 24 inches.

In May 1958, the trees were planted in IZ-inch clay pots using coarse
quartz sand according to the procedure described by Bergman (5).

One quart of the proper solution was applied to each tree twice a week.
The trees were watered with deionizing water as often as needed to prevent
wilting, or about three times a week. The deionized water prevented the toxic
accumulation of minerals in the quartz media. During the winter the trees
were stored in a 38 to 42‘ F storage. They received about one pint of distilled
water per month to prevent dessication. The trees were grown under these

conditions for three years - - 1958, 1959 and 1960.

15.

Experiment I
Procedure

The influence of nutritional environment on the concentration of elements
in the leaf was studied using the Bartlett pear trees described on page 14
Most of the leaves were removed from each tree in early September in 1958
and 1959 for nutrient element analysis.

The samples from 1958 were analyzed for potassium with flame photo-
metry in the Department of Horticulture. All further analyses on the 1958 samples
were made in laboratories of the Department of Agricultural Chemistry. Nitro-
gen was analyzed by the Kjeldahl method. Phosphorus, calcium, magnesium,
iron, boron, manganese, and copper were determined spectrographically.

Samples from 1959 were analyzed by the Department of Horticulture.
Nitrogen was determined by the Kjeldahl method and potassium by flame photo-
metry. Phosphorus, calcium, magnesium, iron, zinc, boron, manganese,
copper, molybdenum, and aluminum were analyzed with a "quantograph"l (34).

The leaf composition data was treated statistically as a completely ran-
domized block. Difference between treatment means was based on a minimum
required difference (MRD). The MRD is obtained by multiplying the standard
deviation of the treatment mean by the value in the studentized range table

corresponding to the degree of freedom for the error term (f) and the number

1Photoelectric spectrometer as manufactured by Applied Research Laboratories,
Glendale, California.

16.

of averages (a) being compared. For this study 'f' was 44 and 'a' was 23.

All values were compared with those of the check treatment for significance.

Results
1. Nutrient Element Composition of Leaves

Data pertaining to the mineral composition of the leaves harvested in
1958 and 1959 are given in Table II for nitrogen, phosphorus and potassium;
Table III for calcium, magnesium and iron; Table IV for boron, manganese and
copper; Table V for zinc, molybdenum and aluminum.

In the following elaboration of the data, all comparisons were based on
a composition difference between the particular treatment and the check.

NitroEn: The concentration of leaf nitrogen was higher in 1958 than
in 1959 regardless of the treatment. The greatest decrease in leaf nitrogen
occurred fer the minus nitrogen treatment which did not significantly affect
the concentration of nitrogen in the leaf in 1958, but did have a significant
influence in 1959. The concentration of nitrogen in the leaf was not significantly
altered by any of the other treatments in 1958 or 1959.

In 1959, trees grown with a minimum of nitrogen had the lowest concen-
tration of leaf nitrogen (I. 34%); while the check trees had 2. 06 percent. The
high nitrogen trees contained 3. 28 percent, which was the highest concentration
of nitrogen observed.

Trees grown in minus calcium solutions were also higher in nitrogen

TABLE 11

Influence of Nutritional Environment on Leaf Composition”
(Percent Dry Weight)

 

 

 

 

Nitrogen Phosphorus , Potassium
Treatment 1958 1959 1958 1959 1958 1959
Check 2. 81 2. 06 .18 .13 2. 24 1. 65
-N 2.52 1.34 .26 .16 2.47 1.89
3N” 3. 66 3. 28 .29 .16 1. 52 0. 96
-P 2. 60 2. 09 .12 .07 2. 25 2.08
51> 2.79 2.33 .32 .55 1.41 0.75
-K 2.39 2.07 .32 .22 0.37 0.49
5K 2. 47 1. 80 .21 .14 3. 64 4. 68
-Ca 3. 27 2.50 . 29 .15 2. 33 1. 99
5Ca 2.67 2.06 .17 .11 1.96 1.76
-Mg 2. 71 2. 22 .23 . 16 2. 46 1. 98
5Mg 2. 80 2. 07 .21 . 13 1. 43 1. 39
-Fe 2. 47 2. 08 .22 . 14 1. 79 1. 65
15Fe*"‘ 2. 49 1. 85 .27 . 14 1. 28 1. 24
-Mn 2. 72 2. 06 .24 . 12 2.10 1. 85
25Mn 2. 43 2. 09 . 27 . 15 .83 1. 53
-B 2. 58 1. 90 .22 . 13 2.01 1. 52
153'” 2.80 2.21 .23 .14 1.53 1.53
-Cu 2.60 1.94 .25 .12 1.71 1.21
25Cu 2. 75 2.10 .20 . 12 2. 08 1. 83
-Zn 2. 84 2.18 .21 . 12 2. 38 1. 59
lSZn*"‘ 2. 60 2.12 . 23 . 13 1. 87 1. 57
-Mo 2. 50 2.17 .23 . 14 2. 30 1. 79
25Mo 2. 82 2.10 . 23 . 14 2. 30 1. 79
MRD 5% .68 .36 N. s. .03 .69 .35
MRD 1% N. s. .48 N. s. .04 .91 .46

 

 

 

 

 

 

 

 

*All treatmentE-are an average of three figures except -Ca and 15 Fe for
1959 which are an average of two figures.
”These treatments were 5N, 25Fe, 258, and 25 Zn in 1958.

Influence of Nutritional Environment on Leaf Composition”

TABLE 111‘

(Percent Dry Weight)

 

 

 

 

 

 

Calcium Magnesium Iron

Treatment 1958 1959 1958 1959 1958 1959

Check 1. 40 0. 83 .33 . 24 .0364 . 0164
-N 0. 86 0. 75 .34 . 17 .0268 .0153
3N” 0. 68 1.14 . 35 . 36 .0348 .0183
-P 1.27 0.67 .33 .21 .0259 .0170
51> 0.78 1.00 .45 .32 .0291 .0149
-1< 1.01 1.05 .54 .34 .0303 .0139
5K 0. 53 0. 41 . 22 .08 .0227 . 0152
-Ca 0. 63 0. 44 .31 . 32 .0886 .0202
5Ca 1.31 1.03 .35 .24 .0234 .0173
-Mg 1. 00 0. 81 . 14 .04 .0201 .0154
5Mg 1. 06 0. 84 .79 .37 .0218 .0167
-Fe 1.31 1.31 .36 .36 .0211 .0137
151:6M 1. 07 0. 98 .64 .30 . 0886 . 0544
-Mn 0. 99 0. 81 . 32 . 24 .0242 .0157
25Mn 1.10 0. 81 .32 .25 .0241 . 0156
-B 1.22 .82 .34 .25 .0222 .0153
15B“ 1. 30 1.12 .48 .26 .0307 .0145
-Cu 1.02 0.94 .50 .25 .0213 .0146
ZSCu 1. 55 0. 86 .44 . 32 .0238 .0148
-Zn 1. 31 0. 96 . 36 . 22 . 0268 .0149
ISZn’" 1. 06 0. 73 . 35 . 22 .0230 . 0148
-Mo 1. 04 0. 89 . 44 . 25 .0271 . 0170
25Mo 1. 28 0. 73 .38 . 25 .0250 .0165
MRD 5% .47 .36 . 18 . 16 .0163 .0061
MRD 1% .63 .47 . 25 . 21 . 0247 . 0081

 

*All treatments are an average of three figures except -Ca and 15Fe for

1959 which are an average of two figures.
“These treatments were 5N, 25Fe, 25B, and 252m in 1958.

18.

TABLE IV

Influence of Nutritional Environment on Leaf Composition*
(Ppm Dry Weight)

 

 

 

 

 

 

Manganese Copper

Treatment 1958 1959 1958 1959 1958 1959
Check 32 29 32 29 13 7
-N 39 50 40 34 15 9
3N" 40 62 28 21 15 9
-P 66 39 35 32 14 13
SP 49 58 32 38 15 7
- K 95 54 34 47 14 8
5K 25 26 29 33 16 7
-Ca 52 78 34 33 15 10
5Ca 45 23 29 23 16 10
-Mg 60 49 35 38 12 8
5Mg 41 30 25 29 17 7
-Fe 53 58 35 31 13 9
15Fe“ 91 64 31 31 15 10
-Mn 21 14 26 31 15 9
25Mn 225 311 36 29 16 9
-B 38 30 17 13 13 8
153M 52 39 112 155 17 7
-Cu 37 34 25 30 12

25Cu 49 31 31 29 22 11
~26 49 29 31 27 13 11
1528” 40 28 32 25 24

-Mo 43 37 27 31 16 8
25m 49 36 32 33 17 7
MRD 5% 49 24 16 11 4 4
MRD 1% 65 31 21 15 7 N. s.

 

 

 

 

_L—

”All values are an average of three figures except -Ca and 15Fe for 1959
which are an average of two figures.

MThese treatments were 5N, 25Fe, 253. and 25211 in 1958-

19.

TABLE V

Influence of Nutritional Environment on Leaf Composition”
(Ppm Dry Weight - 1959)

I

 

 

 

Treatment Zinc Molybdenum . Aluminum
Check 26 . 3. 5 136
-N 27 3. 9 146
3N 29 4. 9 146
-P 31 4. 0 193
SP 26 5. 1 99
-I( 32 5. 6 105
5K 26 2. 4 143
-Ca 30 3. 7 170
5Ca 26 4. 6 174
-Mg 25 4. 0 128
5Mg 27 4. 5 I81
-Fe 27 6. 3 162
15Fe 27 4. 6 177
-Mn 23 3. 9 204
25Mn 28 3. 2 153
-B 23 4. 2 183
153 24 4. 6 126
-Cu 25 4. 7 139
25Cu 27 3. 4 107
-Zn 24 4. 8 213
152m 35 3. 0 177
-Mo 27 4. 5 151
25Mo 22 2. 6 209
MRD 5% N. S. 1. 4 73
MRD 1% N. S. l. 9 96
W - m

 

 

*All values are an average of three figures except -Ca and 15Fe which are an
average of two figures.

21.

than the check trees. This increase was not significant in 1958, but it was

by 1959.

Phosphorus: The concentration of phosphorus in the leaf was slow to

 

respond to the various treatments. In 1958 there was no significant difference
between the concentrations of leaf phosphorus over the entire experiment.
Some trends were evident and these became significant in 1959. The concen-
tration of phosphorus was generally higher in 1958 than it was in 1959.

Treatments void of phosphorus resulted in the lowest concentration of
leaf phosphorus (0. 07%) while the check trees contained 0. 13 percent. When
an abundance of phosphorus was supplied to the tree, the phosphorus in the leaves
reached the highest concentration (0. 55%).

The leaf level of phosphorus was higher than the check in the trees grown
in either an‘excess or deficiency of nitrogen. Both of these treatments resulted
in a leaf concentration of 0. 16 percent in 1959.

The data also indicated that trees grown in a potassium deficient environ-
ment accumulated phosphorus in their leaves. These trees contained 70 percent
more phosphorus than the check trees.

Potassium: The concentration of leaf potassium was higher in almost all
trees in 1958 than 1959. In some instances response to treatments were similar
for both years, but in others the data from the two years did not indicate the
same effects.

The greatest effect on the concentration of potassium in the leaf was

22.

observed on trees grown in the absence of potassium or in an excess of potas-
sium. In 1959, trees grown in the absence of potassium contained only 0. 49
percent leaf potassium. Trees grown in check solutions contained 1. 65 percent
leaf potassium, and the high potassium trees contained 4. 68 percent leafpotas-
sium. In 1958 when the check valuewas 2. 24 percent, the high potassium
leaves contained 3. 64 percent and the minus potassium leaves contained 0. 37
percent.

In 1959, the percent potassium in the leaves increased when trees were
grown in solutions lacking nitrogen, phosphorus or calcium. These effects
were not evident in 1958.

The percent potassium in the leaves decreased under the influence of
high levels of nitrogen, phosphorus, and iron in 1958 and 1959. In 1958, high
boron trees'also contained less leaf potassium, but the 1959 data did not indicate
this effect. In 1959 trees grown in a minus copper solution contained less potas-
sium than the check trees while the‘ 1958 data did not show this effect.

Calcium: The check trees contained more calcium in 1958 than they
did the following year, but this trend was vague for other treatments.

Data for both seasons showed that the lowest concentrations of leaf
calcium resulted when trees were grown in solutions deficient in calcium or
with an excess of potassium.

There appeared to be quite a difference between the effect of the nitrogen

treatments on calcium in 1958 and 1959. In 1958, both the trees grown in high

23.

nitrogen (5N) and those grown in low nitrogen contained less leaf calcium than
the check trees. The 1959 high nitrogen treatment (3N) tended to increase the
concentration of calcium in the leaves. The minus nitrogen treatments for
1958 and 1959 and the checks for 1959 were almost equal.

Minus iron trees contained about the same percent of leaf calcium as
the check trees in 1958 (1. 34% and 1. 40%, respectively). In 1959, however,
the leaves from trees grown in an iron deficient nutrient solution contained
1. 31 percent calcium compared with 0. 83 percent for the checks.

Magnesium: In 1959, the only treatments which appeared to significantly

 

alter the concentration of leaf magnesium were minus magnesium and high
magnesium. Leaves from these two treatments contained 0. 04 percent and

0. 87 percent magnesium respectively, and the check leaves contained 0. 24
percent. The 1958 data indicated a similar response to variations in the mag-
nesium level, although the general concentration was higher for most treat-
ments.

In 1958, the minus potassium treatment increased the magnesium con-
centration in the leaf to 0. 54 percent, compared with the 0. 33 percent in the
check leaves. This trend was evident in 1959, but it was not significant.

High iron treatments (125 ppm) in 1958 resulted in almost twice the
concentration of leaf magnesium as that found in the check leaves. However,
high iron (75 ppm) in 1959 did not seem to effect the leaf magnesium concen-

tration.

24.

£931: During both years the high iron treatment resulted in an accumu-
lation of iron in the leaf; however, the greatest accumulation occurred in 1958.
In 1959, the leaf concentration of iron was less for all treatments. Low levels
of iron decreased the percent of iron in the leaves, but the difference was not
significant either year.

In 1958 the minus calcium trees accumulated almost as high a foliar
concentration of iron as the high iron trees. Minus calcium leaves tended to
contain more iron than the check leaves in 1959, UK the difference was not
significant.

Mangnese: The data for both years showed that the highest concen-

 

tration of manganese resulted from the high manganese treatment. Minus
manganese treatments tended to decrease the leaf manganese concentration,
but the differences were not significant.

The concentration of leaf manganese was also increased when the environ-
ment was deficient in potassium, calcium, or iron, or when an excess of nitro-
gen, phosphorus, or iron was available. Only the effects of minus potassium
and high iron were significant in both 1958 and 1959. The effects of minus
calcium, minus iron, and high nitrogen and phosphorus were significant only
in 1959.

Boron: In 1958, the only treatment which significantly affected the con-
centration of boron in the leaves was high boron. The 1959 data showed an

increase in the concentration of leaf boron due to the application of solutions

25.

high in boron or low in potassium. Trees grown in the absence of boron con-
tained only 13 ppm of boron in their leaves in 1959, as compared to 29 ppm
from the check treatment.

Copper: The concentration of copper in the leaves in 1958 was almost
twice that found for most treatments in 1959. In 1958 the highest concentration of
copper was in the leaves from the high zinc treatment (24 ppm), second was the
high copper treatment (22 ppm) and the check trees contained 13 ppm. In 1959,
the check trees contained only 7 ppm, high copper trees - 11 ppm, and minus
phosphorus trees - 13 ppm.

.2332; The concentration of zinc in the leaves of Bartlett pear trees was
not altered significantly by any of the treatments.

Molybdenum: The highest concentration of this element was found in

 

trees grown in an absence of iron. Other treatments which increased the con-
centration of molybdenum were minus potassium, high phosphorus, and high
nitrogen. None of thetreatments significantly decreased leaf molybdenum
below the check value.

Aluminum: The check trees contained 136 ppm of aluminum. The highest

 

concentration (213 ppm) was found in trees grown in the absence of zinc. High
molybdenum trees were next with 209 ppm. None of the treatments decreased

leaf aluminum below the concentration found in the check treatment.

26.

2. Deficiency and Toxicity Symptoms of Stems and Leaves
During 1958 and 1959 observations were made on the influence of
nutritional environment on the general appearance of the pear trees grown
in various nutritional environments. Some of the trees exhibited symptoms
which could be associated with the excess or deficiency of a certain element.
Nitrogen: Trees grown in a low nitrogen solution were generally re-
tarded in stem and leaf growth. The leaves were smaller than the checks,
and light green to yellow in color. High nitrogen trees had very dark leaves
of good size, growth was about the same as that of the check trees.

Phosphorus: The low phosphorus trees developed foliar symptoms in

 

1959. The leaves were small and the adaxial faces tended to fold together along
the midrib. There was some irregular yellowing of the leaves with necrotic
patches. Defoliation began in late August on lower limbs and moved apically.
There was little twig growth in 1959 or in 1960. Axial buds in 1959 became
visible much later than in the check trees, and in 1960 there was no bloom
although the check trees did flower.

High phosphorus trees had a foliar color that was extremely dark.
Growth was slightly less than on the check trees.

Potassium: Potassium deficient trees showed an inward rolling of the
leaves and a dull gray to brown marginal scorch. The symptoms were most
prevalent at the base of a twig and diminished toward the tip.

High levels of potassium resulted in foliar symptoms resembling those

of magnesium deficient trees.

27.

Magnesium: Leaves on the minus magnesium trees showed patches

 

between the lateral ribs that were either chlorotic or necrotic. These areas
formed a pattern similar to a "Christmas tree” shape. Symptoms seemed to
be most prevalent at the base of the stems.

High magnesium trees had a very desirable appearance.

Calcium: Calcium deficient leaves resembled potassium deficient
leaves except for the color of their necrotic areas. On the low calcium leaves
this area was more of a bright brown.

High calcium trees appeared healthy and made good growth. The leaves
tended to roll slightly and defoliation preceded that of the check trees by 2 to
3 weeks. The petioles and midribs became red in August.

Iron: Low iron trees exhibited less growth and branching than check

 

trees. By mid-July there was also a prevalence of bright yellow leaves. Some
leaves were yellow only along the margins, while other leaves were entirely
yellow. Usually the area next to the midrib remained green.

High iron trees were severely stunted in twig and leaf growth. The
leaves showed patches of necrosis most of which were between the lateral leaf
veins at the leaf's margin. The midrib and the fruit showed an abundance of
red coloring.

Copper: One tree which had a leaf copper value of 5 ppm, showed
symptoms of copper deficiency in 1960. There was from two to three inches of

dieback on each of the shoot terminals. The dead twig and attached leaves

28.

were black and curled, but the leaves did not fall off.

Other Treatments: Low levels of boron, zinc, manganese and molybdenum

 

resulted in growth inhibition, reduced branching and small leaves. There were no
symptoms which were specific for any one element.
High levels of boron, zinc, manganese, copper and molybdenum inhibited

growth slightly but did not result in any specific symptoms.

3. Deficiency and Toxicity Symptoms on Roots

Most of the roots from the trees grown in the various nutritional treat-
ments appeared uniform in their growth and were dark brown in color when
examined after the 1960 growth period.

High calcium trees seemed to have the best root system. The roots
were extensive, well branched, fine, and colored similar to the check trees.

Trees from the high nitrogen treatment had a very poor root system.
The roots had grown very little since planting, they were coarse and the branch-
ing was sparse.

The poorest roots were on the trees grown in a high iron solution. They
had made almost no growth, and branching was limited to short wiry laterals.
The root system was very coarse and black in color.

Minus copper treatments also resulted in poor root growth although the
texture and color were about the same as the check roots.

High phosphorus treatments did not alter root growth appreciably, but

the color was more of a reddish brown.

29.

Experiment 11

Procedure
The amino acid composition of the growing shoots and the mature leaves

from the pear trees described on page 12 was studied to ascertain the effect of
nutritional environment on the amino acids in the tree.

On June 6, 1960, a two- gram sample of the growing shoots were collected
from most of the pear treesl. The tissue was immediately placed in 50 ml of
70 percent alcohol at 80' C for 10 minutes. The alcohol solution was decanted
and 50 ml of water added. After ten minutes at 80° C, this solution was decanted
and added to the alcohol extract, the combined extract filtered, and brought to
volume with 80 percent alcohol in a 200 ml volumetric flask. Four gram samples
0f mature leaves were taken from selected treatments on August 3, 1960, after
all terminal growth had ceased. Alcohol and water extracts were also prepared

from these samples.
To permit the development of better chromatograms a portion of the sample

eXtracts were then purified by use of the following modification of the procedure

described by Plaisted (46):

1. 25 ml of the sample was added to 9 ml of Dowex 50 x 12
resin in a 250 ml wide mouthed flask.

2. Resin and sample were agitated for 10 minutes; the resin
was allowed to settle and the solution decanted.

3. The resin was washed twice with 25 ml of 80% alcohol.
Samples were agitated for two minutes per washing and
solutions were decanted after the resin settled.

‘

l

‘

BeCause of insufficient terminal growth not all trees were sampled.

30.

4. The resin which then contained the amino acids was
washed twice with 25 ml of 4N ammonium hydroxide
in 80% alcohol. For each washing, samples were
agitated two minutes. After the resin settled, the solu-
tions were decanted and saved for analysis. ’1'

5. The resin was again washed with 25 ml of 80% alcohol
with a two minute agitation. The resin was allowed to
settle, and this solution was decanted and saved for
analysis. A similar wash was then done with water,
and this was also saved. *

6. The resin was then regenerated with a hydrochloric acid
washing.

The purified sample was dried under vacuum in a freeze-dry apparatus con-
sisting of a "Cenco High Vac 14" vacuum pump hooked through a glass lyophyli-
zation trap to an approximately 4 liter "Machlett and Sons freezing trap". The
dry sample was dissolved in a 10 percent isopropyl alcohol and brought to a

1 ml volume.

100 111 of the concentrated sample (equivalent to 50 milligrams of fresh
tissue) was chromatographed on sheets of 18 by 22 inch Whatman No. 1 paper
with water saturated phenol in the first direction and n-butanol-propionic acid-
water in the second direction (4).

Identification of the developed amino acid spots was based on three
methods (4): (1) Identification of the amino acid spot with the specific ninhydrin
color; (2) comparison of the relative Rf values as stated in the literature; and
(3) by cochromatography with known amino acids and known Cl4 labeled amino
acids. To develop the ninhydrin color, each chromatogram was dipped in 40 ml

*The additional washes were used to insure complete removal of the amino acids.

31.

of an acetone solution containing 2 ml of glacial acetic acid and 80 mg of
ninhydrin, and dried in an ethanol atmosphere at 60° C for 15 minutes (40).
After identification, a quantitative analysis was performed on each spot (40).
The spot was cut into approximately 1 cm squares, and allowed to stand for

30 minutes in 0. 6 ml of a 5% ninhydrin in butanol solution in a tightly stoppered
test tube. To each tube, 5 ml of 70% alcohol containing 70 mg of CaSO4' SHZO
per liter were added and the tubes were shaken for 15 minutes at a rate suffi-
cient to agitate the paper squares in the tube. The optical density of each solu-
tion was determined at 515 mu1 with a Coleman Spectrophotometer Junior.
Colorimeter tubes were standardized previously to an optical density reading
of .2691005.

A standard curve was established for each amino acid by spotting known
amounts of the amino acid on the chromatographic paper and following the same
method of development and elution. These data were then used to translate
optical density readings into micrograms of amino acids.

The data for treatments for which all replicates were sampled were
statistically analyzed for minimum required differences (MRD). These treat-
ments were check, high nitrogen, high phosphorus, minus potassium, minus
and high calcium, high magnesium, high iron, minus and high zinc, minus and

high boron, minus and high manganese, and high molybdenum.

1The 515 mu maximum absorption peak for the eluted amino acid solutions was
determined with a Beckman Model DK-2 Spectrophotometer.

32.

Results

Data from the samples of growing shoots taken for amino acid analysis
in June, 1960 indicated a wide variation within treatments. This variation made
it difficult to observe amino acid variations that might have resulted from
nutrition. The results of the August, 1960 sampling seemed to be more uniform
within treatments.

In most cases the following amino acids1 were present in pear shoots
and leaves: aspartic acid, asparagine, glutamic acid, glutamine, serine, glycine,
threonine, alanine, and amino acids at the chromatographic location of gamma-
amino butyric acid, leucine, cysteine, and arginine. Because of the large amounts
of glutamic acid and glutamine, the trace amount of ninhydrin color at the Rf of
gamma-amino butyric acid has been designated as this derivative of glutamic
acid (43). g

1. June Samples of Growing Shoots

 

Variations in tree nutrition significantly affected the concentration of
glutamic acid, serine, alanine and the total concentration of the free amino
acids (Table VI). The concentrations of asparagine, glutamine, cysteine, and
arginine (Table VII), and aspartic acid, glycine, threonine, gamma-amino
butyric acid, and leucine (Table VIII) did not vary significantly because of in-

sufficient sample and wide biological variation. However, trends among these

1In all instances, reference to the presence of amino acid means only that a
chromatographic spot, believed to be synonymous with the indicated compound,
was present.

TABLE VI

Effect of Nutrition on Amino Acid Levels” in Pear Shoots — 1960
(Micrograms per 100 Milligrams Fresh Tissue)

 

 

Total Amino Glutam ic

 

 

. l .
Treatment Acids Acid Serine A anine
Check 100 12 8 6
-N*"' 44 14 8 6
3N 188 26 22 8
—P** 98 10 6 4
SP 192 22 18 20
-l( 102 10 8 6
5K“ 224 28 20 20
-Ca 80 14 8 8
5Ca 98 26 16 18
-Mg“‘* 46 10 8 4
5Mg 102 22 10 12
-Fe** 66 10 4 6
15Fe 96 _ 20 8 16
-Zn 104 ' 18 10 ' 10
ISZn 104 12 10 12
-B 76 12 10 8
158 42 8 8 10
-Mn 94 14 8 8
25Mn 72 14 10 12
-Cu*"‘ 90 20 10 10
25CuM 102 20 12 14
-Mo"“" 116 26 12 12
25Mo 80 20 8 14
MRD 5% 72 12 8 8

 

 

 

4

*Data given are an average of three numbers.
”Data were not included in statistical analysis as all replicates could not be
sampled.

TABLE VII

Effect of Nutrition on Amino Acid Levels” in Pear Shoots - 1960
(Micrograms per 100 Milligrams Fresh Tissue)

 

 

 

Treatment Asparagine Glutam ine Cysteine Arginine
Check 58 2 tr tr" ’3‘ *
-N"”" 4 2 tr tr
3N 72 22 tr - -
-P** 60 2 tr tr
SP 94 18 tr - -
-K 64 2 tr tr
5K" 42 32 tr - -
-Ca 32 2 tr tr
5Ca 38 6 tr - -
-Mg""* 6 4 tr tr
5Mg 40 4 tr - -
- F e“ * 30 2 tr - -
15Fe 26 14 tr - —
-Zn _ 44 4 tr . tr
ISZn 54 4 - - - -
- B 26 4 tr tr
15B 6 4 tr - -
-Mn 40 10 tr - -
25Mn 16 6 tr - -
-Cu** 28 6 tr - -
25CuM 32 10 tr - -
-Mo** 38 8 - - - -
25Mo 18 12 tr - -

 

 

*Data given are an average of three numbers. Statistical analysis indicated
no significant differences.
“Data were not included in statistical analysis as all replicates could not be
sampled.
"**The symbol LI: indicates a visible but unmeasurable spot on the chromatogram.

TABLE VIII

Effect of Nutrition on Amino Acid Levels” in Pear Shoots - 1960
(Micrograms per 100 Milligrams Fresh Tissue)

 

 

Gamma -am ino

Treatment Aspartic Acid Glycine Threonine Leucine

 

butyric
Check 10 2 2 tr trio; >3
-N”* 10 tr tr tr tr
3N 26 8 4 tr tr
-P** 12 2 2 tr tr
SP 14 2 2 tr tr
-K 10 2 tr tr tr
5KM 60 8 10 tr tr
-Ca 10 6 tr tr tr
5Ca 10 2 2 tr tr
-Mg"‘* 12 2 tr tr tr
5Mg 10 2 2 tr tr
- Fe” 12 tr 2 tr tr
15Fe 8 2 2 tr tr
- Zn 14 2 2 tr , tr
ISZn 8 2 2 tr tr
-B 10 2 ~ 2 tr tr
153 4 2 - tr tr
-Mn 14 tr tr tr tr
25Mn 10 2 2 tr tr
-Cu*"' 12 2 2 tr tr
25CuM 10 2 2 tr tr
-Mo** 16 2 2 tr tr
25Mo 6 2 tr tr tr
w

 

 

*Data given are an average of three numbers. Statistical analysis indicated
no significant differences.
MData were not included in statistical analysis as all replicates could not be
sampled.
***The symbol tr indicates a visible but unmeasurable spot on the chromatogram.

36.

latter amino acids are interesting and suggestive for future analysis.

The high potassium trees had more amino acids than all others with
a total of 224 ug per 100 mg of fresh tissue; this figure represents only one
sample so its value is questionable. High phosphorus with 192 ug of amino
acids and high nitrogen with 188 ug had the next highest concentrations. The
check contained 100 ug of amino acids per 100 mg of tissue. Low concentra-
tions of total amino acids were found in trees grown with high levels of boron
and with low levels of manganese.

Nutrition of the trees seemed to alter the concentration of glutamic acid.
This was significantly so in high nitrogen and high calcium trees where the
concentration of glutamic acid was almost twice that of the check. High potas-
sium and minus molybdenum trees showed the same trend.

Samples of the shoots from the trees grown in high levels of nitrogen,
phosphorus, and calcium contained 22, 18 and 16 ug of serine per 100 grams of
fresh tissue. These values were significantly larger than the concentration of
serine in the check trees (8 ug). The high potassium treatment also seemed
to increase the concentration of serine in the growing tip.

High levels of phosphorus, calcium, and iron resulted in a significantly
greater accumulation of alanine than was present in the check trees. Check
samples contained 6 ug of alanine while high phosphorus trees contained 20 ug,
high calcium - 18 ug, and high iron - 16 ug. High potassium and copper trees

also seemed to accumulate alanine.

37.

Although the asparagine concentration was not significantly altered by
nutrition, some trends were evident. The minus nitrogen, minus magnesium,
and plus boron trees were lower in asparagine than other‘treatments. They
contained 4, 6, and 6 ug respectively, while the checks contained 29 ug. The
plus phosphorus trees had the highest level of asparagine (47 ug).

Glutamine levels also showed trends although they were not significant.
High nitrogen, phosphorus and potassium trees were all higher in glutamine
concentration than the check trees.

The cysteine spot was not evident in the samples from trees grown in
a solution deficient in molybdenum or containing an excess of zinc, but was
present for all other treatments.

The check and several of the treatments seemed to contain a trace of
arginine. This was true with the minus nitrogen, phosphorus, potassium,

calcium, magnesium, zinc and boron treatments.

2. August Samples of Mature Leaves (Table IX)
Aspartic acid levels were similar in the minus nitrogen, phosphorus,
potassium and iron treatments. In the‘check trees, however, there was only
a trace of aspartic acid and in the minus boron trees 3 ug were measured.
Glutamic acid was similarly affected. Check trees contained about 1 ug
and minus boron trees about 5. 5 ug. Minus nitrogen, phosphorus, and potassium
treatments were intermediate.

Low levels of potassium seemed to account for an accumulation of

TABLE IX

Effect of Nutrition on Amino Acid Levels” in Mature Pear Leaves - 1960
(Micrograms per 100 Milligrams Fresh Tissue)

 

 

 

 

Amino Add Check -N Titemmem -1< -Fe -B
Aspartic acid trM 0. 5 1. 0 0. 7 1. 5 3. 0
Glutamic acid 1. O 2. 3 2. 7 3. 4 3. 3 5. 5
Serine 0. 4 0. 5 0. 4 3. 9 0. 5 1. 7
Glycine 0. 2 tr 0. 2 0. 2 tr 0. 4
Threonine tr tr tr tr tr tr
Alanine 4. 7 3. 0 l. 5 3. 9 1. 8 3. 2
Arginine ' - - - - I. 5 - - tr tr
Asparagine - - - - - - 13. 2 tr - -
Glutamine tr tr tr 7. 9 tr tr
Gamma-amino

butyric acid tr tr tr tr tr tr
Leucine tr tr tr tr tr tr
Cysteine - - - - - - tr tr tr
Total Amino Acids 6. 2 6. 3 7. 0 32. 9 7. 0 13. 2

 

 

‘1—
*Each figure is an average of three values except minus nitrogen and minus

iron which are averages of two figures.
"The symbol 2 indicates a visible but unmeasurable spot on the chromatogram.

 

 

39.

serine. A deficiency of other elements did not seem to affect the serine con-
centration, but the minus potassium trees contained 3. 9 ug compared with 0. 4 ug
in the check trees.

Glycine and threonine did not seem to be affected by variations in the
nutritional program.

The arginine spot was very evident in the samples from minus phosphorus
trees. Samples from the check trees did not show this spot and minus iron and
minus boron had only traces of it. The minus phosphorus trees averaged 1. 5 ug
per 100 mg of fresh tissue.

Alanine seemed to be less abundant in the minus phosphorus trees (1. 5 ug)
and the minus iron trees (1. 8 ug) than in the checks (4. 7 ug). The other amino
acids observed were not greatly affected by nutrition.

Both of the amides, asparagine and glutamine, also accumulated under
the condition of low potassium. Check trees showed only a trace of glutamine
and no asparagine, but the minus potassium samples contained 13. 2 ug of
asparagine and 7. 9 ug of glutamine.

Gamma-amino butyric acid and leucine were present in trace amounts in
all treatments. Cysteine was not present ‘in a noticeable quantity in the check,
minus nitrogen, and minus phosphorus treatments. A trace of cysteine was
present in minus potassium, minus iron, and minus boron trees.

The total amino acid levels were highest in the potassium deficient trees
(32. 9 ug) next highest in the minus boron trees (13. 2 ug) and about the same in

the minus nitrogen, phosphorus, iron, and check samples.

40.

Experiment III
Procedure

This experiment was conducted to study the influence of nutrition on
the susceptibility of pear trees to E. amylovora. On June 7, 1960, three
actively growing shoots on each of the pear trees described on page 12 were
inoculated with E; amylovora. The culture of bacteria used for the inoculation
was 24 hours old and had been established by seeding plates of Emerson's
medium (Table X) with infected plant tissue. The culture was transferred to
the trees by use of the stab type inoculation described by Lamb (36).

Scoring for susceptibility to E. amylovora was based on the percent of
inoculations which became infectious in combination with the percent of the total
length of twig, limb, and trunk infected by the disease. The sum of these two
percentages was designated as a susceptibility value. The percent'of infectious
inoculations was counted 30 days after inoculation, and the percent of the tree

infected was measured 60 days after inoculation.

Results

The susceptibility values used to indicate the ease with which E. amylovora
infected the trees in each nutritional treatment are shown in Table XI as the sum
of the percent of infection based on total inoculations and the percent of infected
wood. A graphic representation of the susceptibility values as they compare

with average susceptibility for this experiment is shown in Figure 1.

41.

TABLE X

Composition of Media for E. Amylovora

 

 

 

Medium Components ' Amounts
Synthetic* KZHPO4 500 mg
M3504 250 mg
KCl 250 mg
Fe-Versenol 7. 5 mg
Thiamine hydroxide . 08 mg
Sucrose 15 g
Water 500 ml
Emerson's (liquid *"‘ NaCl 125 mg
Peptone and beef extract .500 mg
Yeast extract 50 mg
Dextrose 50 mg
Water 100 m1
Emerson's (solid)** . Same components as liquid

plus 1. 5% agar

 

 

*This medium was derived from Czapek's medium, Difco Manual.
** Emerson's medium was recommended by Dr. Dwight Powell, University
of Illinois.

TABLE XI

Influence of Nutritional Environment on the Susceptibility of Pear Trees to
_E_. Amylovora

 

 

 

 

Treatment Percent Take of Percent of Wood ‘ Susceptibility
Inoculat1ons Infected Source
Check 33 25 58
-N 67 26 93
SN 100 69 169
-P 22 9 31
SP 56 46 102
-l( 44 42 86
5K 84 91 175
-Ca 44 83 127
5Ca 0 0 0
-Mg 33 73 106
5Mg 78 78 156
-Fe 33 22 55
15Fe 44 I 71 115
-Zn 67 68 135
lSZn 67 26 93
-B 0 0 0
158 50 36 86
-Mn 89 65 154
25Mn 67 60 127
'C“ 50 74 124

25M0 100 75 175

 

Treatment

Figure I. Effect of Nutritional Environment of Fire Blight Susceptibility

Chec ~
-N

3N

SCI
'MS
5Mg
-Fe

15Fe

 

25116 (a
50 100 150 2

Susceptibility Scores

00

43.

44.

Low susceptibility was found for 12 of the 23 treatments. None of the
inoculations on the high calcium or low boron trees developed into an infection.
The minus phosphorus trees also indicated a high degree'of resistance. The
check trees, which received a balanced nutrient solution, and the minus iron
trees were about equally resistant, and both were well below the rest of the
treatments in the experiment.

The four most susceptible groups of trees resulted from high levels of
nitrogen, potassium, molybdenum and copper. However, trees from treat-
ments deficient in manganese or zinc, and the high magnesium trees were

also highly susceptible.

Experiment IV

Procedure

Various amino acids were used as a sole source of nitrogen in synthetic
liquid medium (Table X) inoculated with _E. amylovora to determine which amino
acids could be utilized by the bacteria as a source of nitrogen. The medium
was devised to insure that no other source of nitrogen was available to the
bacteria except one of the amino acids being studied.

After the amino acid had been added to the medium, the mixture was
adjusted to a pH of 6. 920. 1 with sodium hydroxide or hydrochloric acid and
sterilized. Heat sterilization of the medium appeared to result in deamination
of the amino acids so the medium was sterilized by filtering it through a Sietz
germicidal suction filter equipped with pads of 0. 5 micron porosity. The solu-
tion was transferred to a 125 ml flask and allowed to stand 24 hours. If turbi-
dity did not develop during this period, the medium was assumed to be sterile
and ready for inoculation.

All glassware was autoclaved for 30 minutes at 20 pounds pressure and
120‘ C. Other techniques necessary to ensure asceptic conditions were followed.

A stock of the bacterial was maintained on a slant of Emerson's solid
medium (Table X). Twenty-four hours prior to inoculating the media being
studied, a tube of Emerson's liquid medium was seeded from the stock. A
four millimeter diameter loop of this medium was used to inoculate each

_—‘—.n---——--

1The original culture of _E. amylovora used in these studies was obtained from
Dr. Dwight Powell, University of Illinois.

 

46.

flask of synthetic media containing separate amino acids.

The infected flasks were incubated for 48 hours at room temperature.
After incubation, the growth of the bacteria was measured by recording the
percent transmittance of the medium in a colorimeter at 525 mu using sterile
medium as a standard.

Seventeen naturally occurring L-amino acids, some D- forms of
these acids and five synthetic amides were studied (Table XII). The concen-
tration of each compound was adjusted to provide 210 ppm nitrogen. Tests

were replicated at least three times.

Results

Data showing the ability of _E_ amylovora to utilize various amino acids
as a source of nitrogen are given in Tables XIII and XIV.

The first results obtained from this experiment indicated that only the
amides, asparagine and glutamine, would support the growth of _E_ . amylovora,
and that aspartic and glutamic acid would not. It is believed that these results
were erroneous. The first tests were conducted on a medium that contained
sodium carbonate as a buffer to obtain the desired pH. Autoclaving the medium
resulted in a return to a pH of about 4 (typical of a water solution of aspartic

or glutamic acids) which appeared to be too low for _E_. amylovora to grow.

 

When this experiment was performed as previously described (page 45)

the bacteria grew well (Table XIII) when the source of nitrogen was asparagine,

TABLE XII

Compounds Tested for Bactericidal Effect and/or Support for Bacterial

 

 

 

 

 

 

Growth
:1:
Amides of Amino Acids Moles per Liter x 10-2
Glycyl glycine amide 0. 61
Alanyl asparagine 1. 52
Glycine amide 1. 52
Nicotinamide 1. 52
Chloracetamide 3. 00
Acetamide 1. 32
D-asparagine 0. 76
L-asparagine O. 76
DL-asparagine 0. 76
L-glutamine 0. 76
DL-glutamine 0. 76
Amino Acids Moles per Liter x 10’2
Leucine 1. 52
Arginine 0. 38
Lysine 0. 76
Glycine 1. 52
Alpha-Alanine 1. 52
Serine 1. 52
Phenylalanine 1. 52’”
Tyrosine l. 52
Cysteine 1. 52
Histidine 1. 52
Valine 1. 52““
Tryptophane 0. 76
Beta-Alanine l. 52
D-aspartic acid 1. 45
L-aspartic acid I. 45
DL—aspartic acid 1. 45
Gamma-amino butyric acid 1. 45
Alpha-amino butyric acid 1. 45
L-glutamic acid 1. 43
DL-glutamic acid 1. 43
Ammonium chloride 1. 52

 

*Concentration adjusted to add about 210 ppm of nitrogen to the medium.
MPartially insoluble.

TABLE XIII

Compounds which Supported Growth of _E_. Amylovora

 

 

Compound Percent Transmittance“
D-asparagine 58
L-asparagine 38
DL-asparagine 47
L - glutamine 66
DL- glutam ine 47
L-glutamic acid 58
DL-glutamic acid 35
D-aspartic acid 82
L-aspartic acid 38
DL-aspartic acid 33
Gamma-amino butyric acid 35
Beta-alanine 5
Alanyl asparagine 64
Glycine amide 81
Ammonium chloride 58

 

 

*Values are an average of three readings.

TABLE XIV

Compounds which did not Support Growth of _E_. Amylovora

 

 

Compound Percent Transmittance*
Cysteine 100
Serine 90
Tryptophane 91
Glycine 92
Alpha- alanine 90
Phenylalanine 90
Histidine 90
Leucine 95
Valine ' 88
Alpha-amino butyric acid 100
Nicotinam ide 8 8
Acetam ide 95
Chloracetam ide 98
Glycyl glycine amide 95
Arginine 88

 

*Values are an average of three readings.

50.

glutamine, aspartic acid or glutamic acid. A comparison of the D- and L-
forms of these amino acids showed slight variations in their ability to support
growth. The organism appeared to favor L- asparagine and L- aspartic acid
over their D- forms, and DL- glutamine or glutamic acid over their L- forms.

The two other amino acids, gamma-amino butyric and beta-alanine,
supported the growth ofE. amylovora in culture. To a lesser degree, alanyl
asparagine and glycine amide were satisfactory nitrogen sources.

The rest of the amino acids and amides tested apparently were not

satisfactory sources of nitrogen for the organism (Table XIV).

'D

51.

Experiment V

This experiment was performed to test the bactericidal activity of a
host of natural and synthetic nitrogenous compounds through the use of culture
studies and field studies.

1. Culture Studies

 

Procedure

 

The synthetic liquid medium (Table X) used for these studies was
identical to the one used in Experiment IV except for the addition of one gram
of asparagine per liter of medium. The medium thus prepared, satisfactorily
supported the growth of E. amylovora.

The bactericidal action of each compound was tested by adding it to the

complete medium and inoculating the mixture with E. amylovora. Steriliza-

 

tion, inoCulation, and pH adjustment were all carried out as described in
Experiment IV by the use of germicidal filters. If the medium remained clear
after being incubated at room temperature for 48 hours, the compound was
considered to have bactericidal activity. The development of any turbidity in
the medium was evidence enough to drop the compound from further consider-
ation.

All amino acids which did not support growth of I; amylovora in

 

Experiment IV were tested for bactericidal qualities in the same concentration

used in the studies in Experiment IV. In addition, a group of 48 compounds

52.

obtained from the Union Carbide Chemicals Company, were tested for
bactericidal qualities. The concentration of each compound (Table XV)
used in the test was established so that each compound would add 3. 44 mg

of amide nitrogen to 40 ml of the medium.

Results

Of all the compounds tested, seven appeared to have bactericidal
activity. The rest of the compounds did not inhibit growth of the bacteria
at the concentrations used. The active compounds were checked further to
determine their minimum effective concentration. These compounds and
their minimum effective concentrations are listed in the table below. Those
marked with an asterisk (*) were active at the lowest concentrations, and so

were used in the following field studies.

Compound Concentration
(Moles per liter)

 

CH3-cH- C\H-CONH2
CH3 N\l-l
O :1" C'CHzBr

*Bromoacetyl valinamide 5. 25 x 10'5

CH3-CH-C\H~CONH2

CH3 NH
\
c-cnzm

04

Chloroacetyl valinamide 5. 60 x 10'3

Compound

CCHZ-CH-CONHZ
|
NH

OQC-cnzm

Chloroacetyl phenylalaninamide

ClCHz-CONHZ

Chloroacetam ide
0

II

I
OzN ©CHOH-CH-CH20H

* Chlo ramphe nicol

0
Q g—cnznr

* Bromacetophenone

o
,, CH-(CH3)2

t
l' ‘N-CH-CDNH2

\
‘0

*Alpha-maleimido isovaleramide

53.

Concentration
(Moles per liter)

 

5.20 x10.3

5.15 x 10'3

1.25 x10"5

1.25 x10-6

5.35 x 10‘

TABLE XV

Compounds Tested for Bactericidal Effect on _E_. A_r_ny_13vora

 

 

Moles per Liter

 

. Compound ( x 10-3 )
DL-methioninamide crotate 5. 35
DL-bromoacetyl valinamide 5. 40
N (n-Butyl)-N' -(alpha-( -p-chloro-phenylglycinamido)) urea 5. 45
DL-alpha-Aminobutyramide hydrochloride 5. 45
DL-Stearoyl valinam ide 5. 35
DL-Alaninamide hydrochloride 5. 45
DL-N- Carbamyl valinamide 2. 52
DL-alpha-Chloropropionyl valinamide 5. 35
N-(2-(3-Methyl) butyramido) carboxamidomethyl pyridinium

chloride 5. 35
Sarcosinam ide hydrochloride 5. 45
2-(3-Phenyl-5-isopropyl) thiohydantoin 5. 45
Chloroacetylglycyl valinamide 5. 45

N-(Ethylexalyl) valinamide 5
DL-alpha-Chloropropionyl leucinamide 5
2-(3-Ethyl-5-isopropyl) thiohydantoin 5
N -Phenyl-N' -(alpha-isovaleramide) urea 5. 45
Alpha-(Maleimido) isovaleramide 5
DL-Maleylvalinamide 5
DL-alpha-hydroxybutyramide 5
Orotic acid salt of DL-valinamide 5

N - Benzoyl~DL-valinamide 5. 35
N-Acetyl-DL-valinamide 5. 05
DL-Chloroacetylvalinamide 5. 60
DL-Norvalinamide hydrochloride 5. 40
N-carbethoxy valinamide 5. 30
Chloroacetyl- DL-phenylalaninamide 5. 20
DL-p-chlorophenyl glycinamide hydrochloride 5. 35
2-(3' -Indoleacetam ido) acetamide 5. 50
DL-Serinamide hydrochloride ' 5. 35
DL-Methioninamide hydrochloride 5. 45
I-Methyl-Z, 6-dicarboxamido piperidine hydrochloride 2. 74
DL-Valinamide hydrobromide 5. 45
DL-Stearoylglycinamide 5. 35
DL-Phenylglycinamide hydrochloride 5. 40
DL-Sorbyl phenylalaninamide 5. 25

DL-Glycylvalinamide hydrochloride 5. 30

TABLE XV Cont' (1

Compounds Tested for Bactericidal Effect on Ii). Amylovora

 

 

Compound _ Moles per Liter
( x 10.3 )
DL-Valinamide hydrochloride 5. 40
N-Carboxamidomethylmorpholine hydrochloride 5. 55
DL-O-ethylserinamide hydrochloride 5. 35
DL-Isoleucinamide hydrochloride 5. 40
DL-Leucinamide hydrochloride 5. 40
2-Methylalaninamide hydrochloride 5. 45
2-(3' -Indoleacetamido)-4-methyl valinamide 5. 30
DL- sorbylglycinam ide 5. 35
Lauroylsarcosinamide 5. 35
DL-Phenylalaninamide hydrochloride 5. 40
DLralpha-hydroxyphenylacetamide 5. 40
DL-alpha-hydroxypropionamide 5. 35
*N-Chloroacetyl asparagine 5. 60
*Benzoyl glycine amide 5. 20
*Chloramphenicol 6. 25
*Bromacetophenone 0. 63

 

*All compounds were supplied by the Union Carbide Chemicals Company
except those marked with an asterisk. '

55.

56.

2. Field Studies

 

Procedure

The compounds which appeared to have the best bactericidal qualities
were checked further in a field study in the following manner: A randomized
block experiment was established in a block of newly set one-year-old
Bartlett pear trees. One-half of the treatments were sprayed on the trees
prior to inoculation with E amylovora and one-half after inoculation. Treat-
ments made before and after inoculation consisted of two concentrations of
each compound being tested (Table XVI). Agrimycin was applied at the re-
commended rate before and after inoculation to afford a comparison with a
material being used commercially. All treatments were replicated twice.
Four trees were inoculated but left unsprayed to serve as checks.

The before inoculation sprays were applied on May 25, 1960. Thirty-
six hours later each tree was inoculated in one growing shoot as described in
Experiment III. Sixty hours after inoculation the second half of the treatments
were sprayed on the trees. Each tree was sprayed with about 75 ml of solu-
tion. The results of this study were recorded ten days after inoculation.
Trees were graded as either infected or non-infected.

Cold weather and rain which prevailed made it desirable to repeat the
experiment. Care was taken to asceptically remove all fire blight from in-
fected trees as well as the inoculated shoot on trees which did not become

infected. Each tree received the same compound in both experiments to

 

57.

TABLE XVI

Treatments for Bactericidal Sprays in Field Studies

 

 

lst Sprayl 2nd Spray2 3rd and 4th Sprays3

Compound COHC- ' 1391‘“ Come. - ppm Conc. - ppm
Chloramphenicol 200 800 800
Chloramphenicol 29 80 -
Bromoacetyl

valinamide 200 1250 1250
Bromoacetyl

valinam ide 20 125 -
Alpha-maleimido

isovaleramide 400 1000 1000
Alpha-maleimido

isovaleramide 200 200 -
Bromacetophenone 25 250 250
Bromacetophenone 2. 5 25 -
Agrimycin 300 300 300

W

The concentrations in this column were used only in the pre-inoculation spray
in the first field study.

2The concentrations in this column were used in the post-inoculation in the
first field study.

3The concentrations in this column were used in the pre- and post-inoculation
sprays in the second field study.

58.

prevent an interaction or carry-over effect. Only one concentration (Table
XVI) of each compound was used and each treatment including check was
replicated four times. The agrimycin treatments were replicated three times.

On June 6, 1960, the before inoculation sprays were applied. Thirty-
six hours later three growing shoots on each tree were inoculated with E.

amylovora. Thirty-six hours after inoculation the post inoculation treat-

 

ments were applied. Eleven days after inoculation the results were recorded

on the basis of percent of inoculated shoots infected per tree.

Results

May Field Test: The check trees in this test were infected at every

 

inoculation point. None of the trees sprayed with agrimycin were infected.
The trees sprayed with bromoacetyl valinamide after inoculation showed no
signs of infection. When this compound was sprayed on before inoculation,
there was one point of infection with the high concentration and one point with
the low concentration.

Bromacetophenone sprays after inoculation also resulted in an absence
of infection. When sprayed on before inoculation, only one of the trees receiv-
ing the low concentration was infected.

All of the trees sprayed with Chloramphenicol after inoculation were
infected. None of those treated with this compound before inoculation were

infected regardless of the concentration.

59.

Trees sprayed with alpha-maleimido isovaleramide after inoculation
were infected 100 percent, and 75 percent of those sprayed before inoculation

became infected.

June Field Test: Three of the four check trees were infected at each of

 

the three points on inoculation. The fourth tree was not infected. Spraying
with agrimycin after inoculation prevented development of fire blight in two
of the three trees. The third tree was infected at all points of inoculation.
When agrimycin was sprayed on before inoculation, one twig of a possible six
became infected.

None of the inoculated twigs sprayed with bromoacetyl valinamide
showed signs of becoming infected. This was true for the trees sprayed be-
fore as well as after inoculation.

Chloramphenicol sprayed twigs showed no signs of infection when the
trees were sprayed prior to inoculation. Of those trees sprayed after inocula-
tion, one tree appeared to be infected at all three points of inoculation.

Three twigs on one tree sprayed with alpha-maleimido isovaleramide
became infected. Two trees sprayed after inoculation became infected at all
points of inoculation.

Three of the four trees sprayed with bromacetophenone before inocula-
tion became infected at all points of inoculation. TWO of the four trees sprayed

after inoculation also became infected at all points of inoculation.

60.

DISCUSSION

Nutrient Absorption and Response

 

The complete Hoagland solution appeared to be generally satisfactory
as a nutrient source for pear trees. The following comparison of the leaf com-
position of nutritionally healthy pear trees grown in Michigan orchards (33)
and the pear trees from this experiment suggests that the trees received an
adequate amount of most elements except calcium, magnesium, manganese
and copper from the nutrient solution. The level of calcium, magnesium,
manganese and copper in the leaves suggests that the concentration of these

nutrients should be increased in the basic Hoagland solution.

 

 

Element Kenworthl's Data Check Treatment
Nitrogen - % 2. 50 2. 06
Phosphorus - % 0.13 0.13
Potassium - % l. 45 1. 65
Calcium - % 1. 90 . 83
Magnesium - % 0. 4O . 24
Manganese - ppm 133 29
Iron - ppm 140 164
Copper - ppm 54 7

Boron - ppm 23 29

61.

In general an increase or decrease in the concentration of an element
in the nutrient solution resulted in a similar change in the leaf concentration
of that element. These changes were not always significant, especially in the
trace element category, but the trends usually followed the treatment. There
were also many instances, especially within the major elements, when an
interaction between two or more elements seemed to be present.

Varying nitrogen in the nutrient solution affected the concentrations of
leaf nitrogen and potassium. As the concentration of foliar nitrogen increased
from 2. 06 to 3. 28 percent the concentration of potassium decreased by the
same factor from 1. 65 to 0. 96 percent. A similar trend was noticed by Baxter
(2). Low leaf concentrations of nitrogen did not seem to alter the potassium
concentration in the leaves, and the potassium concentration did not appreciably
alter the percent nitrogen in the leaves.

Proebsting (49) noticed that applications of nitrogen fertilizers on
Bartlett pear trees decreased the phosphorus content of the leaves, but a
nitrogen-phosphorus interaction was not observed in this study.

Nitrogen deficiency symptoms were recognized in this experiment on
trees with leaves containing 1. 34 percent nitrogen. Sato (55) observed nitrogen
deficiency at 0. 83 percent leaf nitrogen in the Josephine pear.

The concentration of phosphorus in the nutrient solution seemed to
influence the concentration of leaf phosphorus and potassium. Minus phosphorus

trees contained an average of 0. 07 percent phosphorus. This level was

62.

accompanied by an appearance of deficiency symptoms which were probably
attributable to the low concentration of phosphorus. This value agrees with
that established by Sato (55) for the phosphorus deficiency level.

The concentration of potassium seemed to be indirectly related to the
concentration of phosphorus. However, only a potassium deficiency increased
the phosphorus concentration; an excess of potassium did not seem to affect
the leaf phosphorus level.

Potassium levels in the leaf were generally dependent on the quantity of
potassium in the nutrient solution. Leaves from the minus potassium treat-
ments exhibited deficiency symptoms as described by Wallace (67) when the
percent potassium was 0. 49. Reuther (51) observed potassium deficiency
symptoms at the 0. 5 percent level, and Sato (55) stated that symptoms may
occur in leaves having between 0. 27 and 0. 41 percent potassium.

Toxicity symptoms observed on the high potassium trees could have
been due either to a potassium excess or to a magnesium deficiency. The
magnesium level (0. 08%) was below the level (. 09 to . 11) set by Sato (55) and
approaches the level observed in the minus magnesium trees (0. 04) of this
experiment.

Calcium was the only major element which was not increased signifi-
cantly in the leaf by an increase in the quantity of calcium in the solution. The
high calcium solution contained 1002 ppm of calcium of which about 40 percent

was in the calcium sulfate form. According to Bergman (5), high levels of

63.

calcium sulfate did not increase the concentration of calcium in grape petioles.
Perhaps this form is not suitable for nutrient solutions. Minus calcium treat-
ments resulted in a calcium level of 0. 44 percent and in the occurrence of
deficiency symptoms. Sato (55) observed deficiency symptoms in pears at

the 0. 66 percent level. Trees grown in the high calcium solution showed
symptoms which may have been due to the accumulation of the relatively in-
soluble calcium sulfate in the root zone.

Low magnesium treatments reduced the concentration of magnesium
in the leaves to 0. 04 percent. These trees exhibited deficiency symptoms
similar to those described by Harley (23) for trees which contained 0. 05 per-
cent magnesium. Sato (55) felt that leaves containing 0. O9 to 0. 11 percent
magnesium or less would show deficiency symptoms.

Baxter (2) reported that soil applications of magnesium and calcium
decreased leaf potassium and that appliCations of potassium and calcium had
a similar effect on magnesium. He also felt that the sum of the potassium,
calcium, and magnesium concentrations in the leaves remained about the
same. Since high calcium concentrations were not attained in the leaves, it
was difficult to evaluate the effect of calcium. However, the influence of
potassium and magnesium seemed to follow the pattern described by Baxter
in that the concentration of potassium in the leaf increased as the level of
m agnesium in the solution decreased. The reverse was true when the mag-

nesium levelof the nutrient solution was increased.

64.

The sum of the potassium, calcium and magnesium concentrations in
the check trees was 2. 72 percent. Most treatments did not vary from this
figure by more than 10 percent of this sum. The most unequal sums of these
three elements were found in the trees deficient in potassium (sum - 1. 88%)
or with an excess of potassium (sum - 5. 17%). The potassium excess was
so great that both magnesium and calcium would have had to be less than zero
to maintain the equality. Trees grown in an iron deficient nutrient solution
seemed to have a high sum (3. 32%) and trees with an excess of phosphorus
had a low sum (2. 07%).

Low iron treatments did not seem to affect the level of iron in the leaf,
but high iron treatments instigated a toxic accumulation of iron (544 ppm).
Root and top development were very poor in the high iron trees, and necrotic
spots were evident on the leaves.

Interestingly, the trees grown in a nutrient solution lacking iron exhibited
an accumulation of calcium. These trees exhibited some chlorotic symptoms
which have been associated with lime induced iron deficiency in other reports.
Here it would seem that the calcium accumulated as a result of iron deficiency
rather than a cause of it.

Either a high or a low level of iron in the solution resulted in an in—
crease in leaf manganese. The manganese level was still less than the value
indicated as essential by Kenworthy (33). However, the manganese level in

the nutrient solution did not seem to affect the concentration of iron in the leaf.

65.

Minus iron treatments also accounted for the largest accumulation of
molybdenum observed in this study (6. 3 ppm).

Nutrient solutions containing 25 times the normal amount of manganese
resulted in the accumulation of 311 ppm of the element in the leaves. Accord-
ing to Grasmanis (20) this should have induced toxicity symptoms, but none
were observed. Withholding manganese from the trees lowered leaf manganese
to 14 ppm, but it did not induce any particular symptom other than reduced top
and leaf growth. This symptom was typical of most of the trees grown on low
or high levels of trace elements.

Trees grown in high and low levels of boron exhibited no particular
symptoms of deficiency or toxicity even though they ranged in concentration
from 13 to 155 ppm.

Oserkowsky and Thomas (45) felt that 7 ppm was the threshold value
for copper deficiency. The check trees and minus copper trees in this experi-
ment averaged 7 ppm in 1959, and may have been dangerously low in copper.
In 1960, one minus copper tree developed symptoms similar to those described
by Jones (31) and Harris (24). This tree contained only 5 ppm of foliar copper
in 1959. Thus, it appears that the critical value for the copper level in pears
grown under these conditions was about 5 ppm. Increasing the copper level in
the nutrient solution 25 times over that of the check solution raised the foliage
concentration to 11 ppm. Trees deficient in phosphorus had the highest con-

centration of copper on a percentage basis. Since these trees made almost no

66.

growth, there was probably less dilution of the copper absorbed and therefore
only appeared that there was an accumulation of copper.

Zinc concentrations in the trees were not affected significantly by the
level of zinc in the nutrient solution, but trees grown in an absence of zinc
accumulated the highest level of aluminum in their leaves. Aluminum was
also more concentrated in the trees grown in a high molybdenum solution. The
amount of molybdenum in the nutrient solution did not significantly alter the
concentration of molybdenum in the leaf.

There is often no explanation for the interactions between elements
that are evident under various nutritional environments. It is important, how-
ever, to recognize their existence in diagnosing nutritional disorders and

making fertilizer recommendations.

Amino Acid _A_ccumulation as Related to Nutrient Environment

 

Growing Shoots: Variations in the nutrient solution content also

 

affected the accumulation of free amino acids present in the growing tips of

pear trees. There seemed to be little qualitative variation of the amino acids
under these conditions, but there were quantitative responses. The amino

acids present in the most generous concentrations were aspartic acid, glutamic
acid, serine, glycine, threonine, alpha-alanine, asparagine and glutamine.
Cysteine, gamma-amino butyric acid, and leucine were present in trace amounts
in most samples. Traces of arginine were present in the check trees and a few

minus treatments.

67.

Trees with high concentrations of nitrogen, phosphorus, and potassium
accumulated the largest total amounts of free amino acids. The high nitrogen
trees achieved this accumulation through an increase in” concentration of each
of the more plentiful amino acids. High phosphorus trees accumulated all of
the plentiful amino acids except glycine and threonine. The high potassium
value represented only one sample and so should not be emphasized.

The reason for the large amounts of total free amino acids in the
shoots of pear trees growing on high levels of phosphorus and potassium is
not known. It is possible that a high phosphorus and potassium nutritional
condition may have stimulated the photosynthetic production of the amino acids,
but that the resultant nutritional inbalance prevented complete utilization of the
amino acids for protein synthesis.

Steinberg (59) found an accumulation of total amino acids. in tobacco
plants deficient in phosphorus, calcium, magnesium or potassium. Other re-
ports (11, 18, 21, 52, 54) also have shown this to be true with potassium deficient
plants. The accumulation of free amino acids in the high phosphorus trees
could be partially due to the low concentration of potassium in its leaves. The
minus magnesium and plus potassium trees did not follow this pattern.

The lower concentrations of free amino acids occurred in trees high in
boron, low in magnesium or low in nitrogen. The low level of free amino acids
in these trees was due mainly to the low level of asparagine. There was about

90 percent less asparagine in these trees than in the check trees.

68.

Since asparagine usually serves as an ammonia detoxification mechanism
in plants, the trees low in nitrogen and magnesium or high in boron may not
have had any excess of ammonia. The low nitrogen trees probably did not
because nitrogen was not available to be absorbed in large quantities. The
minus magnesium and high boron trees contained just as much total nitrogen
as the check trees or high phosphorus trees. Thus, the condition of these
trees must have favored a rapid assimilation of the nitrogen into amino acids
and thence into protein.

Mature Leaves: The nutritional environment also induced variations

 

in the amino acid levels in mature leaves from pear shoots which had ceased
growth. These variations were not always consistent with those in the actively
growing shoot.

Those trees grown without boron or without potassium had the highest
total free amino acid levels in their leaves. An increase in the acidic amino
acids, aspartic acid and glutamic acid, accounted for most of the increase due
to low boron. Potassium deficient trees accumulated asparagine, glutamine
and serine. These amino acids, and especially the two amides probably accumu-
lated as an ammonia detoxification mechanism. Thus, it would app ear that the
potassium deficient pears were either not utilizing nitrogen for protein syn-
thesis or that the low potassium level was equivalent to a starvation effect and
protein breakdown resulted in an excess of ammoniacal nitrogen.

Phosphorus deficient trees were also very interesting in that they showed

69.

a significant accumulation of arginine. The alanine concentration was one-
third of that in the check trees, and there were 1. 5 ug of arginine compared to
none in the checks. None of the other treatments showed an accumulation of
arginine.

Many of the changes in amino acid composition of pear leaves or
shoots as a consequence of an abnormally high or low level of an essential
element cannot be explained. Detailed investigations on certain of these
changes might contribute to an understanding of the biochemical function 1111119
of the respective element. It is also possible that variations in amino acid

levels could be an indication of an approaching element imbalance.

Susceptibility of Trees to E. amylovora

 

 

Trees grown in a nutrient solution containing an excess of calcium and
those grown without boron did not become infected with E. amylovora even
though each tree was inoculated three times. Minus phosphorus, minus iron
and the check trees were also quite resistant to the disease.

The high calcium and the check treatments resulted in similar trees
with a healthy appearance. The other treatments which resulted in resistant
trees would be impractical as the trees grew poorly. The high calcium trees
did not accumulate an excess of calcium in their leaves; in fact, the high
calcium trees were not significantly different than the check trees for any
nutrient element. Neither was there any difference in the concentration of the

total free amino acids between these two groups. It should not be assumed

70.

that there was no physiological difference between the check and high calcium
trees. It is possible that the high level of calcium altered the metabolism of

the tree sufficiently to increase resistance to _E_. amylovora.

 

The striking effect of the minus boron and high calcium treatments
point out the importance of further study with these treatments to learn more

about their effect on resistance of pear trees to _E_ amylovora.

 

The trees most susceptible to _E_. amylovora were those grown with
high levels of molybdenum, copper, potassium and nitrogen. The high nitro-
gen trees contained the largest concentration of leaf nitrogen and so follow
the observations of several workers (17, 30, 59, 61) that high nitrogen levels
accentuate a fire blight infection. However, the level of nitrogen in the other
most susceptible trees was no different from the level found in the least sus-
ceptible trees. In fact the minus nitrogen trees which contained the lowest
concentration of leaf nitrogen were about average in susceptibility. Thus,
the axiom that excess nitrogen will increase susceptibility to fire blight does
not include the prevalent feeling that a minimum of nitrogen will lessen sus-
ceptibility. The same can be said for the other elements as a deficiency of any
element except phosphorus, iron, boron, and potassium resulted in trees of
average or above average susceptibility.

No obvious difference in elemental composition of the leaves was ob-
served which would explain the extremes of susceptibility which seemed to

result when the nutrition of the tree was altered. The only two treatments

71.

which exhibited similar levels of resistance with no significant differences in
the elemental composition of the leaves were high calcium and check. Perhaps
the balance of elements found in the check and high calcium trees is the best

pattern for a resistant tree.

Dependence of E. amylovora Bacteria on Amino Acids
The experiments to determine which nitrogen sources would support

the growth of E. amylovora were initiated because of reports in the literature

 

(1, 44) that this organism had a nearly obligated requirement for asparagine.
For the present research, it was postulated that the growth of an organism
whose nitrogen requirement was so specificshould be controlled through the
inhibitory action of an analogous compound. Furthermore, it was postulated

that the spread of _E. amylovora in the plant might be correlated with the avail-

 

ability of asparagine in the growing shoots and leaves. Asparagine was found
to be present in large amounts in the pear shoots (Experiment III) and it was
felt that the organism utilized this reservoir of asparagine directly for its
growth.

However, _E_. amylovora was able to grow not only on asparagine but
also on glutamine, aspartic acid, glutamic acid, gamma-amino butyric acid,
beta-alanine, alanyl asparagine, glycine amide, and ammonia (Table XIII).
This disagreement with Ark's data (1) is believed to be due to a more careful

control of pH.

72.

There is, however, a remarkable correlation in structure of the
amino acids which the organism can utilize. Aspartic acid and glutamic acid
are two very similar dicarboxylic amino acids, and asparagine and glutamine
are their corresponding amide derivatives. These four compounds were found
in Emeriment III to be major amino acids in the shoots which would be avail-
able to the organism. Further, the utilization of gamma-amino butyric acid
and beta-alanine by the organism may be directly related. Gamma-amino
butyric acid is formed in plants and micro-organisms from glutamic acid by
enzymatic decarboxylation (4) and beta—alanine would be the decarboxylation
product of aspartic acid; however, this pathway for the formation of beta-
alanine in plants has not been found (43).

The growth of the organism on alanyl asparagine and glycine amide was
poor and these compounds are not naturally occurring substrates. Growth on
ammonia was good, but little or no nitrogen exists in plants in the ammonia
form. Therefore, the growth ofE; amylovora on six amino acids seems re-
lated strikingly in that aspartic acid, asparagine and beta-alanine are chemi-
cal analogs and glutamic acid, glutamine, and gamma-amino butyric acid are
similarly related to each other, and all are among the constituents of the
dicarboxylic amino acids. The lack of growth on all the other amino acids
accentuates the uniqueness of the growth on the dicaroxylic amino acids.

Future research should be directed toward a study of the metabolism

of _E_. amylovora to determine the reason for its obligated requirement for the

73.

dicarboxylic amino acids. These amino acids are common constituents pro-
duced from the citric acid cycle in most organisms, and it is unusual to find
an organism that requires an independent source of them for growth.

Since four of the amino acids utilized by the bacteria made up the
majority of the total free amino acids in the pear shoots, it was suspected
that only the total concentration of the free amino acids in the shoots and
leaves might be an important factor in regulating the susceptibility of the
trees to E. amylovora. However, the data indicated that neither the quanti-
tative nor qualitative aspects of the free amino acid level were factors in fire
blight susceptibility.

Three of the treatments containing the highest total level of amino
acids were high nitrogen, phosphorus and potassium. High nitrogen and
potassium trees were of about average susceptibility. The lowest levels of
total free amino acids were found in minus nitrogen, minus magnesium, and
high boron, and these trees were of average susceptibility or a little lower,
but were not lowest in susceptibility.

Thus it seems that there was usually enough of one of the utilizable
amino acids present to support growth of the bacteria regardless of the nutri-

tional program .

74.

Possible Bactericides for E. amylovora

The working hypothesis that a chemical analog of asparagine might in-
hibit the growth of _E_. amylovora was not possible after the discovery that
other related amino acids would also support growth. However, the structural
relationships between these amino acids (page 72) were so similar that the in-
hibitory effect of unnatural amide structures was explored. These compounds
were selected because they contained an amide, and, in general, a substituted
constituent on or near the amino group. It was hoped that a structure some-
what similar to asparagine would be accepted by a specific and limiting enzyme
in the organism and blo'ck its action and thus block the growth of the organism.
This hypothesis was tested first in synthetic culture studies and then in field
studies.

J Of all the potential bactericides checked, the only one which seemed to
be as good as the material presently being used by orchardists (agrimycin)
was bromoacetyl valinamide.

When agrimycin sprays were applied after bacterial inoculation, three
twigs of the eleven inoculated became infected. Sprays prior to inoculation
resulted in one infected twig of a possible eight. Altogether, 21 percent of
the inoculated twigs became infected. Similar bromoacetyl valinamide treat-
ments reulted in one infected twig of a possible sixteen when the sprays were
applied before inoculation; the same ratio resulted when the treatment was
applied after inoculation. In this case about 12 percent of the inoculated twigs

were infected.

75.

Check trees, which were not sprayed with a bactericide, had 13 infected
twigs out of the 16 inoculated or about 81 percent infection.

Chloramphenicol acted as a good preventive bactericide; none of the twigs
inoculated became infected. However, when it was applied after inoculation,
about 31 percent of the inoculated twigs became infected.

None of the compounds tested exhibited any phyto-toxicity in the quanti-
ties used.

All of the compounds which showed bactericidal effects had several in-
teresting structural similarities. (1) In general, the compound contained a
chloro or bromo substituent on a carbon adjacent to a carbonyl group. This halo-
gen could carry a partial positive charge and would be reactive with sulfhydryl
amino acids or -SH sites on a protein molecule. (2) This active halogen is ad-
jacent to the amide group with which it conceivably might react to form a ring
structure which in turn might be responsible for the biological activity. (3) Chloram-
phenicol is an inhibitor of growth because it blocks the incorporation of the amino
acid-RNA complex into protein. Thus bromacetyl valinamide may likewise be blocking
growth at some step in amino utilization.

Future work on these compounds should include investigations on the site
of action of bromacetyl valinamide not only for _E_. amylovora inhibitors but also
as an inhibitor in studies on the biochemistry of protein synthesis. Similar com-
pounds are available, and they should be checked for possible bactericidal acti-

vity.

76.

When a compound is perfected which will penetrate pear leaf and stem
tissue and kill E: amylovora without damaging the tree or the fruit, growers
will be able to grow pears without the fear of fire blight. Until that time, the
present spray program, coupled with a well balanced and substantial nutri-

tional program should result in a minimum of fire blight problems.

77.

SUMMARY

Bartlett pear trees were grown in a nutrient solution sand culture at
various levels of nitrogen, phosphorus, potassium, calcium, magnesium, iron,
zinc, boron, manganese, copper, and molybdenum during the 1958, 1959 and
1960 seasons. These trees were used to study the effect of nutrition on the
mineral composition of the leaves, on the free amino acid levels in the grow-
ing tips and older leaves, and on the susceptibility of pear trees to fire
blight.

As an auxiliary study, the causal organism of fire blight, _E_. amylovora,
was grown in synthetic media to ascertain its ability to utilize natural and syn-
thetic amino acids and amides as a source of nitrogen. Various other synthetic
amides were tested for their bactericidal effect.

The following paragraphs contain a summary of these studies.

1. Decreasing the amount of an element in the nutrient solu—
tion resulting in a corresponding decrease in the leaf concentration of that ele-
ment in all cases except iron, zinc, copper, and molybdenum. Increasing the
amount of an element in the nutrient solution increased leaf concentration ex-
cept for calcium, zinc, and molybdenum. In a few instances the concentration
of one element seemed to be dependent on the concentration of another. Mag-
nesium, calcium, and potassium concentrations seemed to be closely inter-

related. Potassium and phosphorus showed an indirectly proportional relation-

 

78.

ship. There were other instances of low magnitude elemental interactions.

2. The free amino acid composition of the growing tip of a
pear tree also seemed to be related to the nutrient element composition of
the leaves. Increasing or decreasing total leaf nitrogen resulted in a similar
change in the free amino acid concentration. High levels of phosphorus or
potassium in the leaf were associated with an increase in total free amino acids
similar to that found for trees high in nitrogen. Either increasing leaf boron
or decreasing leaf magnesium resulted in amino acid levels equally as low
as those in the minus nitrogen trees.

3. The free amino acid levels were also studied in the mature
leaves from non- growing shoots of trees grown in solutions deficient in nitrogen,
phosphorus, potassium, iron, and boron. Minus boron leaves contained more
total amino acids due mainly to an increase in the concentration of glutamic acid
and aspartic acid. Low potassium leaves accumulated large quantities of as-
paragine and glutamine and thus had the highest total free amino acid level.
Arginine accumulated in the leaves deficient in phosphorus but not in any of
the other treatments.

4. There was some variation in susceptibility to _E_): amylovora
based on the nutrient element environment of the trees. Trees grown in a high
level of calcium did not become infected with E amylovora at all, but these
trees did not indicate any accumulation of calcium in their leaves. Trees defi-

cient in boron did not become infected either. Those trees deficient in

79.

phosphorus or iron as well as the check trees appeared to be quite resistant.
Trees grown in high levels of molybdenum, copper, potassium and nitrogen
appeared to be the most susceptible to an _Ei. amylovora infection and spread.

5. _E_. amylovora readily utilized aspartic acid, glutamic
acid, asparagine, glutamine, beta-alanine, and gamma-amino butyric acid
as a sole source of nitrogen in a synthetic culture medium. Consistent with
these findings, there appeared to be no correlation between the foliar con-
centration of individual amino acids or the total amino acid level and the sus-
ceptibility of the tree to a fire blight infection.

6. Culture studies and field tests indicated that bromoacetyl

valinamide may be a good bactericide for E2. amylovora. This compound gave

 

better control than agrimycin under the conditions of this experiment and

showed no phytotoxic effect on the tree.

10.

ll.

80.

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