AN ECONQECQL EYALEIAHON 05F “FEE
ARLEEGFMEEC ENFWENCE 0*?
JUGLANS NIGRA GR

gvcemsmw gsmmm

 

This“ for ”M Dogma of M. S.
MICHIGAN STATE UNEVERSETY
Robe-fl Roger Sherman. in
3.97!

 

ABSTRACT
AN ECOLOGICAL EVALUATION OF THE ALLELOPATHIC

INFLUENCE OF JUGLANS NIGRA ON
LYCOPERSICON ESCULENTUM

BY

Robert Roger-Sherman, Jr.

The allelopathic influence of one plant on another is
important not only from an ecological standpoint, but also
from the standpoint of plant development and landscape
plantings. The black walnut, Juglans nigra, is one of many
plant species which exerts an allelopathic influence on
other plants. This has been noted for many years, but the
primary dispersal mechanism has not been determined.

The primary objective was to determine the primary
method of toxin entry of Juglans nigra and to isolate the
toxic material in wilted plant tissue. Toxins mayenter
the environment by crown or leaf leachates, fog drip,
volatilization, plant residues, or root exudates. Root exu-
dates seemed to be the primary mode of entry. The presumed
toxic substance, juglone, was not confirmed as being present
in wilted tomato plants grown beneath Juglans nigra trees.
However, certain extraction paper chromatograms did produce
the characteristic color test for juglone, but the substance
could not be positively found through elution and rechroma-

tography.

AN ECOLOGICAL EVALUATION OF THE ALLELOPATHIC
INFLUENCE OF JUGLANS NIGRA ON

LYCOPERSICON ESCULENTUM

BY

Robert Roger Sherman, Jr.

A THESIS

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

MASTER OF SCIENCE

Department of Horticulture

1971

_G¢?ofiq

ACKNOWLEDGEMENTS

The author wishes to extend thanks to Drs.
Harold Davidson and Roy Mecklenburg for their
guidance and advice throughout the course of the
Masters program, and to Professor Clarence E.
Lewis for his assistance in plant nomenclature.

The author is very grateful to the Ann Arbor

Garden Club, Ann Arbor, Michigan, for partial
financial support of this study.

iii

TABLE OF CONTENTS

LIST OF TABLESOQOOO'OOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOC

LIST OF FIGURESOOOO0.0.0....O...OOOOOOOOOOOOOOOOOOOOO

 

PURPOSECO......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

INTRODUCTION.........................................
LITERATURE REVIEW....................................
EXPERIMENTAL PROCEDURE...............................
Fruit Residues..................................
Leaf Leachates..................................

Root ExudateSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO
Chromatographic Studies.........................

RESULTSQOOOOOOOOOOOOOCOOOOO0.0.0.0.....00............

FrUit Regiduesooooooooccooooooooooooooooococo-o.
Leaf Leachates..................................
ROOt EXUdateSQOocoo.ooooeooocoo-00.000.000.00...
Chromatographic StUdiesoooooooooooooooooocoo-coo
DISCUSSIONC......................0...................
SWARY.’............................................

LITERATURE CITEDOOOOOOOOOOOCOOOOOO0.0.000000000000000

APPENDIX.....O0.0......00.0.0000...0......00.0.0.0...

iv

Page

LIST OF TABLES

TABLE Page

I. Plants Reported to Produce Growth—Inhibitors of
Known coulPOSitionOOOOOOOOO0.00.00.00.00....00.. 19

II. Plants Reported to Produce Growth-Inhibitors of
Unknown Composition............................ 21

III. Plants Reported Not to be Tolerant of Juglans
nigraOOOooooooOOOOOOOOOOOOQOOOOOOOOO00.00000... 26

LIST OF FIGURES

FIGURE Page

1. Average number fruits per plant with increasing
distance from the walnut tree trunks........... 48

2. Average total plant weight, in grams, with
increasing distance from the walnut tree

trunks...00......00000000OOOOOOOOOOOCOOOCOCOCOO 49

3. Data relative to average total number of fruits
and average total plant weight................. 51

vi

PURPOSE

The purpose of this study was to determine the influ-
ence of the various plant parts of one genus on the
growth and develOpment of another plant genera. The influ—
ence will, of course, vary between plant genera. The
inhibitory action of one plant upon another probably occurs
quite frequently in natural ecosystems. This action may
also occur in areas in which unrelated plant genera are
brought together for their aesthetic value or crop produc-
tion.

Juglans nigra was chosen for this study since it has

 

an extensive history of inhibiting the growth of some other
plants, specifically tomatoes. Much work has been done in
relation to the toxic influence of Juglans nigra, and the
question as to which plant part is responsible for the toxic
action is still unanswered. Those interested in growing
plants for their aesthetic value are still in doubt as to
whether or not to plant beneath black walnut trees.

This work, relative to the mode of toxin entry by
Juglans nigra, was concentrated in three areas; 1) plant '
residues, specifically fruit, 2) leaf leachates, and 3) root

exudates.

The following plants were selected for study:

1. Lycopersicon esculentum (tomato)

2. Pyracantha coccinga (Pyrapantha)

The studies were conducted at the Michigan State Univer—
sity horticulture farm, swine farm field, Rose Lake Wildlife
Research Area, and the Graham Experiment Station in Grand

Rapids, Michigan.

INTRODUCTION

Throughout time it has been observed that certain
plants, including Juglans nigra, inhibited the growth of
other plants when grown in close association. The nature
of the factors involved in this action have been misunder-
stood by many interested in growing plants for food and
their aesthetic value.

Much of this inhibitory action, exerted by one plant
upon another, is thought to be due to the natural release
of biochemicalby—products. The release of such compounds,
which may or may not be toxic, by higher plants is charac-
terized as allelopathy. Ecologically, allelopathy is
important relative to its influence on succession, dominance,
species diversity, community structure, and productivity.
It is the purpose here to evaluate the allelopathic nature
of various plant parts of Juglans nigra relative to

Lycopersicon esculentum.

LITERATURE REVIEW

For the past several hundred years, individuals in
agriculture and related fields interested in the science
of growing plants have noted that certain plant species
inhibit the growth of the same or other genera and/or
species of plants. This growth inhibition, usually
characterized by a stunting of the plant coupled with a
chlorotic appearance of the leaves, seemed to occur widely
throughout the plant kingdom with one of the many pre-
sumed causative species being the black walnut, Juglans
nigra L. Juglans nigra tends to exhibit both a homologous
(inhibiting its own species) and a heterologous (inhibit-
ing other plant genera and/or species) type of inhibitory
action (24).

One of the first individuals to refer to the problem
of growth inhibition relative to the black walnut was the
Roman philosopher Pliny. He stated in his "Natural History."
that "the shadow of the walnut tree is poison to all plants
within its compass," and "that it may kill any plants with
which it comes in contact" (28). In the 17th century,
Evelyn rejected the idea of a toxic substance being pro—

duced by the walnut, stating that the roots were below the

plow layer affecting neither the cultivation nor the growth
0f plants beneath the walnut (28). Another of the early
advocates of naturally occurring plant inhibitors or
phytotoxic substances was DeCandolle (17), who in 1832
observed this toxic action. DeCandolle found that spurge
inhibited or reduced the growth of flax, flax reduced the
growth of wheat, and oat yields were reduced by thistles
(68). He believed that excretions of organic material from
the roots were the cause of the growth inhibition.

The hypothesis of naturally occurring growth inhibitors
was closed by Liebig in 1852, with his studies of soils’
relative to micronutrients (17,24). Liebig believed that
nutrient balances were essential for plant growth. He found
that by addition of certain elements, poor growth could be
alleviated and that poor growth could be induced by removal
of certain elements. His theory, the "Law of Minimum,"
meaning that plant growth is limited by the element present
in the least amount relative to its need by the plant, was
postulated in the early part of this century. During the
early portion of this century, Schreiner(72) found that
soils may be unproductive due to the presence of organic
compounds which were toxic to growing plants. Due to this
fact, he was largely responsible for reOpening the question
of naturally occurring growth inhibitors. Excretions of
plant metabolism products from roots and other plant

tissues may, if allowed to remain in the soil, exert a toxic

effect on succeeding crops. Schreiner also found that
meristematic portions of root tips in some soils became
discolored, swollen, or slimy (72).

Currently, this toxic plant-to—plant interaction is
referred to as allelopathy, which was first coined by
Molisch in 1937 (8). Previously, most plant interactions
were thought to be of a competitive nature for water,
light, and nutrients. Allelopathy meant originally the
interaction of all plant organisms and microorganisms as
well as the interactions of plants relative to the produc-
tion of products derived from plant metabolism processes
(8). This increased the amount of study involving the
relationships between higher plants.

All plants may, at some stage of development, produce
substances which are toxic to other plants or which may
induce a growth response, either stimulatory or inhibitory
(2). The degree of toxicity may be relative to the concen—
tration of the toxic substances produced. The concentration
of the material may, in turn, be increased or decreased
relative to various environmental factors, microorganisMs,
Soil types and structures, soil pH, nutrient and moisture
availability, and the plants' ability to metaboliZe the'
physiOIOgically active substance (57). Physiologically
active substances are also called growth substances and
therefore can affect plant growth when applied in small

amounts. The production of such substances is regulated in

plants, as with cytokinins, I.A.A. (indol—acetic acid),
and gibberellins.

Phytotoxic substances must also be released into the
environment as well as being of a high enough concentration
to cause injury. Many organic and inorganic substances
which may be physiologically active are released as by-
products of metabolic processes. An example of metabolic
by-products are phenolic compounds (57). Entrance into
the environment can be through exudates from roots and root
parts, leachates from leaves and possibly other above ground
parts, and decomposition of plant residues, all relative to
breakdown or decomposition by various environmental condi-
tions, soil conditions, and microorganisms. Upon entrance,
the physiologically active materials may cause reactions
between plants and other plants, plants and other living
organisms, as well as reactions of plants or living organ-
isms to themselves and their environment (59). This well-
known definition of ecology seems to suggest that substances
Which are physiologically active and which are released into
the environment can and do play a role in the distribution
of plants within their various ecosystems through their
allelopathic nature. However, for toxic substances to exert
an allelopathic effect. the rate at which they enter the
environment must exceed the rate that they are converted to

a less toxic substance.

Since plants growing together use a mutual medium, it
is presumed that there is some root competition. Because
of this, the roots were one of the first areas to be con—
sidered.

Bedford and Pickering observed that grass had an
inhibitory effect on the growth of fruit tree seedlings,
and in some cases it caused death. The foliage of apple
seedlings was chlorotic, and plant growth was poor. They
disregarded such factors as soil temperature, poor or
changeable soil aeration, increases in carbon dioxide, and
increases in alkalinity or acidity. It was concluded from
this that the grass produced a toxin which was leached into
the soil from the leaves, excreted from the roots of the
grass, leached into the soil from the decay of grass debris,
or by an alteration of the bacteria content of the soil
relative to grass decay (2).

Cubbon (68), like Pickering, believed that the grass
produced a toxin since his work with grapes showed a sig-
nificant inhibition of the plants relative to size. The
hypothesis of Bedford and Pickering was not accepted by
Russel, who found that injury characterized by slow root
growth, poor nutrient absorption, chlorotic and premature
abscission of leaves, and slow fruit maturity could be over-
come with applications of ammonium sulfate (40). He
therefore concluded that the grass had the ability to tie

up excess nitrogen present in the soil.

Ahlgren and Aamodt (1) also working with grass, found

 

that Kentucky bluegrass (Poa pratensig) is extremely

sensitive to clovers and Agrostig alba (Redtop). When grown

 

in close association, the bluegrass was almost eliminated
relative to growth in a pure stand indicating that perhaps
a substance is excreted by the roots of Agrosti§_a1ba.

Fletcher determined that Sesamum indicum (Sesame)
failed to reach maturity when sown within two feet of
Sorghum vulgare (Sorghum) (22). Sesamum died after reach-
ing a few centimeters in height and failed to reach maturity.
This was similar to Pickering's results with the apple, and
Fletcher, like Pickering, believed a substance was given
off by the roots. Fletcher was criticized since he believed
that bacteria may have been responsible for the toxic
action observed in water cultures and that an environmental
stress or lack of nutrients may have been responsible for
the action in the field.

Schreiner and Shorey (72), working with soils, found
that dehydroxysteric acid could be isolated from poor soils.
It was believed to be removed by decomposition and the pre-
cipitate was found to be insoluble in water. They were
also able to isolate carboxylic, oxalic, salicylic acid,
and vanillin from nonfertile soils (72).

Substances produced by quackgrass (Agropyron repens)
rhizomes may be harmful to other plants based on research

by Kommendahl in which the rhizomes, when mixed with soil,

10

caused stunted and chlorotic alfalfa seedlings. When
compared to oat soil leachings, the quackgrass—rhizome soil
leachings reduced wheat growth. The rhizomes were fre-
quently found to harbor root rotting fungi, which are
responsible for some seedling blights and root rots.

A combination of Helminthosporium sativum and quackgrass
rhizomes was more inhibitory than the fungus alone, a fungus
which resulted in an 80 per cent loss in stands of wheat,
oat, or barley seedlings (37).

Through root leachings and related experiments, Bonner and
Galston (5,6) found that guayule is inhibited by its own
species but not by tomato extracts. These experiments
carried out indicate that the substance is organic in nature
and that it is derived from the roots. Bonner found that
nutrient solutions in which guayule was grown contained a
substance inhibitory to the growth of other guayule plants
when applied as a soil drench. Upon isolation, the substance
was found to be transcinnamic acid. In similar experiments
dealing with the soil in place of nutrient solutions, no
substances containing cinnamic acid were found, indicating
that perhaps it was destroyed by microbial activity.
Infertile soils then might be responsible for the failure
of a system to destroy, change, or reduce the concentration
of the toxic material present.

Proebsting and Gilmore (66), concerned with the diffi—

culty of replanting peaches (Prunus persica) in old field

11

sites, grew seedlings in two different soils and found
that when the roots were added to virgin soil, there was
a decrease in growth. They also discovered that roots of
cherry and apricot trees promoted a reduction of lateral
growth but not height growth. It was concluded that the
bark of the root and not the wood was responsible for the
toxin produced.

Since root systems of plants are in close association,
it is conceivable that products of metabolic processes cap-
able of causing inhibition or death could be excreted from
living roots. It is also possible that substances once in
the pedosphere, or soil, can be acted upon by various
environmental factors, soils and soil types, and microorgan—
isms in such a way that a more toxic or less toxic substance
is present than that Which was first excreted. This could
also account for a build-up of phytotoxins capable of severe
damage to other or the same species of plants and micro—
organisms in the ecosystem.

Aside from root contact and excretion of phytotoxins
by the roots, another and more visual method of entrance
into the environment is available. This is a method known
as crown leaching or release of toxins from above ground
parts. This includes leachates of leaves, stems, flowers,
and fruits. Tukey (76) has shown that leachates are
extremely important as a mechanism for the release of

phytotoxins. Tukey and Mecklenburg found that inorganic

12

and organic materials can be leached from foliage by rain,
dew, or mist (75). Once they are leached, it has been
determined that nutrients lost are replenished by root up-
take (48). Roger del Moral (18,19), has determined that
fog drip also leaches metabolites from the crown of
Eucalyptus globulus (tasmanian blue gum) and hence serves
as another crown release mechanism. The fog drip alone
seemed to be toxic to many herbs when naturally collected
beneath Eucalyptus globulus, indicating that absorption

of terpenes to soil colloids or leaching of phenolic acids
from litter may not need to be combined with the fog drip
to develop injury. It has also been observed that nutrients
can be returned to the part from which it was removed and
that calcium is leached by a process of ion exchange and
diffusion which involves not only the outside but also the
inside of the leaf (49).

The leaves of Artemisia absinthium (wormwood), when
crushed and mixed with soil, retarded the germination of
.giggm sativum (peas) and Phaseolus species (bean) (23).
Funke observed that thyme bordering Artemisia was reduced
in size. Muller (52) found the leaves and twigs of Artemisia
to be toxic in that they exerted an inhibitory effect on
the germination of Avena fatua (oat) seeds. Salvia leu-
cophylla, as well as Artemisia californiga contain terpenes
which, upon volatilization, inhibit the growth of seedlings

in the California Chaparral. McPherson (47) found that

13

shrubs of Adenostoma fasciculatum produce toxins through
normal metabolic processes which are in turn dissolved by
rains and leached to the soil. Volatile materials produced
by leaves of Salvia leucophylla (salvia) inhibit root
growth, hypocotyl growth, and production of lateral roots

in germinating herb seeds (54,56). The leaves reduced
oxygen uptake by mitochondria and the number of lateral root
initials in Cucumis sativus (cucumber) (55).

The leaves of Encelia faringsa (encelia) have been
found to contain a substance that will inhibit the growth
of tomatoes. The leaves were found to be less toxic when
mixed with fertile soil than when mixed with infertile soil.
Leaves which had been chOpped prior to mixing with the soil
exerted more of a growth inhibition than those leaves which
were mixed in the unchOpped state (25).

Bennett (3) determined that Thamnosma montana
(thamnosma) contained toxic substances, three of which were
toxic or inhibited the growth of tomato plants. Upon isola-
tion, two of the compounds were found to be identical to
byakangelicin, having an emperical formula of C15H1505(OCH3)
and isopimpinellin, with the emperical formula of
C11H403(OCH3)2. The most lethal of the compounds was de—
termined to be a derivative of an isobergaptene nucleus, which
is an isomer of the other two compounds. The rate for
lethality was determined to be 12-15 mg./l. Tomato growth

was also found to be inhibited by extracts of Franseria dumosa,

14

and the plants were characterized by wilted leaves and a
destroyed root system. Muller (53) also found that
Franseria dumosa, Thamnosma montana, and Encelia farinosa
inhibit the growth of tomatoes grown in water solutions.
Muller concluded that the toxic action is due to a lack of
microbial activity, build-up due to absorption to soil
colloids, or inactivation of microorganisms relative to
substances released by the leaves. Went (78,79) observed
that certain annuals depend on certain shrub species in
desert situations and were found to grow Where the largest
amount of organic material had accumulated.

Mergen and others (51), have found that certain woody
species also produce substances in the leaves which, when
extracted, exert an inhibitory effect. Brown (11) found
that of water extracts from 56 plants commonly found in a
Jack Pine forest, some inhibited germination, while others
stimulated germination. Cornus canadensis (dngood). Eiggg
resinosa (red pine), and Pteridium aquilina (pteridium)
stimulated gigu§_banksiana (jack pine) germination, whereas
Prunus pumila (sand cherry), Gaultheria procumbus (winter-
green), and Solidago jungga (golden rod) had an inhibiting
effect on germination. Peterson (64) found that dry leaves
of 531mi§.§ngnfitifiolia.(sheep—laurel) contained a substance
inhibiting the formation of primary root development in
black spruce. The substance was soluble in ethanol as well

as water (50). Mergen determined that the toxicity of

15

Ailanthus altissima (Tree of Heaven) decreased in stem

and rachis extracts as compared to leaflet extracts. This
might indicate production of the toxin in the leaves or
translocation to the leaves from another part of the
plant. The substance also moved upward when applied to a
cut stem, and the substance was located in the xylem of
the stem and veins of the leaf. If the substance were to
move by translocation, this might account for wilting dur-
ing periods of rapid growth (51).

Aside from plant interactions occurring under natural
conditions as with root systems and those that can be
observed, leaves and associated leachates, there is a third
phase or means of environmental entrance by toxins. This
third phase of toxin entry is plant residues. This phase.
which we can and cannot see depending on the season and
stage of decomposition of the material, is related to en-
vironmental factors and microorganisms.

Guenzi et al. (29,32,45) found that wheat, oat, sorghum.
and corn residues exert an inhibitory effect on wheat seed-
ling growth, which is important in stubble-mulch farming.
In the residues, the phenolic acids (ferulic, p-coumaric,
syringic, vanillic, and p-hydroxybenzoic) were present (30,
31). It was also noted that Wheat, oat straw, soybean,
sweetclover hay, corn and sorghum stalks, and bromegrass,
and sweetclover stems all contain substances capable of

inhibiting the germination and growth of sorghum, corn, and

16

wheat. Autoclaving of the soil decreased seed germination
and shoot growth, indicating that soil microorganisms may
have an influence on the toxic action of allelopathic sub-
stances. It may be that acids are released in localized
areas of the soil during ideal decomposition conditions.

Patrick (61) determined that inhibitory substances are
released when peach root residues are combined with micro-
organisms. He found that the substances inhibited the
respiration of excised root tips and that they influenced a
necrotic condition of root meristematic cells. Toxic sub-
stances were not produced in soil which had undergone
autoclaving, nor were they found in sour cherry (Prunus
cerasus), tobacco (Nicotiana sp.) or pepper (Solanaceae)
root residues.. Patrick and Koch (62) theorized that some
of the poor growth could be related to an increased sus—
ceptibility of the tissue to normally nonpathogenic organ—
isms due to a weakening of the plant relative to the
phytotoxin.

Dried apple root bark was observed by Borner to reduce
apple (Mglgg sp.) seedling growth in nutrient cultures.
Five phenolic compounds were isolated, of which only
phlorizin could be considered a bark constituent. The other
four compounds were breakdown products. Two of the four
breakdown products displayed an inhibitory action, indicat-
ing that products of decomposition may also influence plant

growth in an inhibitory manner (7).

1?

Hook and Stubbs (13) examining 588 seed trees found
a definite reduction in the growth of the understory vege-
tation beneath cherrybark oak (guercus falcata var.,
'pagidaefikua'), swamp chestnut oak (Quercus michauxii),
Shumard oak (Quercus shumardii). This reduction in growth
of the understory vegetation was not as pronounced under
yellow peplar (Liriodendron tulipifera), sweetgum (Liquidamber
stygaciflun), white ash (Fraxinu§_americana), green ash
(Fraxiums lanceolata) or loblolly pine (Pinus taeda). The
retardation was more prevalent in low, wet areas than in dry
areas. Root exudates, or crown leachates, were presumed to
be responsible for the phytotoxic action, even though the
understory growth was not repressed under all of the oaks.

Jameson (34) has determined that Blue grama, Boutelaria
gracilis, radicles are inhibited by extracts of the foliage
of juniper, ggniperus monosperma.

Rice et al. (60,81) have shown through experimentation
that sunflower, Helianthus annuus, inhibits its own seedlings
and other early weeds in old—field succession situations in
central Oklahoma. Applications of extracts from decaying
leaves, root exudates, leaf leachates, and soil extracts
have all displayed an inhibitory action to seedling growth
and seed germination. Digitaria sanguinalig (crabgrass) was
found to be inhibitory to itself and to Hglianthus annuus
seedlings (l). The decaying crabgrass seemed to have no

effect on germination, indicating that perhaps the toxic

18

substance was exuded by living roots. This type of situa-
tion emphasizes the fact that more than one plant part
could be responsible for allelopathic expression. Micro-
organisms, species of plant releasing the material, stage
of maturity of the material, water content of the soil, pH
of the soil, and length of time subject to various orders
of decomposers may all influence the degree of toxicity
exerted by plant litter or residues (62). Phytotoxic sub-
stances need not only be available, they must also be
released into the environment or in the case of the soil,
the pedosphere, (where they can be reacted with actively grow-
ing plants. It is important to know from which plant part
or parts phytotoxins are released and the influence it
exerts on other plant species aside from the normal aspect
of light, water, and nutrient competition. AllelOpathic
substances are important not only ecologically relative to
plant community diversity but also important in commercial
and noncommercial plant production. Since there are many
plants either producing toxins or presumed to be producing
toxins, the diversity of plants relative to phytotoxins is
important. Tables I and II, taken from Garb et a1. (6,22,
24,32,51,60,61,66,8l) list some of the plant species
reported to exhibit an allelopathic nature.

As-early as the time of Pliny (28), the Roman philos—
opher, it was observed that the black walnut was toxic in

some way to plants growing beneath its crown. Since that

l9

mo>moa

0:» cw Houwnwgcw

ucmumwmmu

H03oamcom 0cm
.mumo .moaumn

ucmumwmmu

Amuoouv

madness .saaa
.coflco .uouumo

CHOU
.Hmmmmm .oumEou

Amuoouv
mmammam .AHHH

CHHmESOU

spasmoamucmn
ImxoguoelmlamumUMIm

AH0>OHU ummev
.mm msuoawamz

 

Amwamocov
smocwnmm maamocm

 

 

Anson mxcouv

 

 

 

 

 

 

 

mmmnnmo .mnouo .cowco .uouumo cHHmEooo mwaomavwmoomo xmuoomwo
Anuoouv

ucmumammu MMHMMHM .mawa Arson mxcouv

mmmflflmo .mmwnu .cowco .uouumo saunasoo mumuooo x>amumwo
m0>mma «so Amuoouv. Amwcmmuomv

ca Houflnfinsw mmadavmom omen? cannons maaomwmmmuo mflcmmumm
Awumov

nsomoaoaog memo cfiumHomoom newcomm mcm>¢
Amuwawmcmv

oumeou cwowaomcmxmmn mannam muwammcd
Anm3on
Iosvmmm mcwcmoummv

choc .mmmno cwcoemcmououm maaaumnasonmcoemod

mucmaaoo wouwnwncH manna HouwnwncH mo mouoom ucmam

Hm\ocm mucmam

cowuflmomeoo Hmowamnu

 

 

 

.ZQHBHmomZOU Z3OZM m0 mmOEHmHMZHImBZOMG WUDQOMm OB Gmfimommm mBz¢Am

H mqmdfi

 

20

 

no>coH
may a“ nouananca

msomoaoaon
ucmumflmmu ouuaou

Anuoouv
oumeou .cofico

Amuoouv
mmcflavomm uc0£3

oumeou

Amuoonv
oumeou .cowco

CHOU . GQQHO

Anuoouv
mmcflaoowm anon?

mom .oasmmom

Amuoouv
oumEou .COHco

meow ownuomcumm
sflaawcm>

msmaoss osmummmuwn
cues Ammoocmomsmodo

cfiaamchEHQOmfl
deadammcmxmhn

Ufiom oaQuommumm
cacOEmcmouonm
raucous

ofiow oaemcswomcmuu

oflom oanuommumm

manganese puma:

coon maaflcm>

Anamosemnuv
monocoe mamocamaa

 

Ammwuuon
Sum camucsozv
msflummooom monuom

Amsououusnv
.mm usasosssmm

Aamom
nacsaaoo mswwm

Aoaommowv

 

 

Esumucwmum Eswcwguuwm

AUGHH mmsmnov
.mm monuflo

21

TABLE II

PLANTS REPORTED TO PRODUCE GROWTH-INHIBITORS
OF UNKNOWN COMPOSITION

 

Plants and/or

 

 

Plant Source Parts Inhibited Comments
Acer spp. (Maple) Triticum spp. (Wheat)
A rostis alba Phleum pratense
(Redtop) (Timothy)
Poa pratensis
(Kentucky bluegrass)
Chewing's fescue
Ailanthus altissima Pinus spp. (Pine)
(Treeéof-Heaven) Betula spp. (Birch)
Antennaria fallax Antennaria fallax homologous

(Cat's-ear)

Artemisia absinthium
(Wormwood)

Aster macro h llus(Aster)
Avena spp. (Oats
Backhousia angustifolia
(Backhousia)

Bromus inermis
(Brome Grass)

Castanea dentata
(Chestnut)

Castanogsis sempervirens
(Chinquopin)

Citrus spp.
Cornus spp. (Dogwood)

Encelia farinosa
(Encelia)

(Citrus Fruits) Citrus spp.

 

Senecio (Groundsel) Datura and
gall—am

resistant

Aster macrophvllgg
tomato crown gall
Araucaria cunninghamii
(Hoop Pine)

homologous

brome grass seedlings

Triticum spp. (Wheat)

Ribes spp.

homologous
Triticum spp.(Wheat)
Lycopersicon esculentum

(Tomato)
Zea mays (Corn)

continued

 

TABLE II-—continued

22

 

 

Plants and/or

 

 

 

 

 

 

 

Plant Source Parts Inhibited Comments
Encelia frutescens Lyconeggicon
(Encelia) W
(Tomato)
Erigeron pulchellus Erigeron pulchellus homologous
(Poor Robin's Plantain)
Eucalyptug_spp. herbaceous spp.
Euphorbia flax
Franseria dumosa Lycopergicon
(White Bur-sage) esculentum
(Tomato)
Fraxinus americana Pinus strobug
(White Ash) (White Pine)
Grevillea robusta Grevillea robusta homologous

(Silk oak)

Helianthus scaberrimus
(Sunflower)

Juglans cinerea
(Butternut)

Juniperus osteospgrma
(Juniper)

Kalmia angustifolia_
(Sheep-laurel)

Larrea tridentata
(Creosote Bush)

Leersia hexandra
(Zacate)

Liriodengron tulipifera

(Yellow Poplar)

 

Triticum spp.

Helianthu§,scaberrrmu§

Potentilla fruticosa
(Shrubby Cinquefoil)
Pinus mugo 'mughus'

(Swiss Mountain Pine)

Bouteloua spp.
(Blue Gramma)

Picea mariana

(Black Spruce)

Lchpersicon esculentum
(Tomato)

rice

(Wheat)

continued

 

TABLE II——continued‘

23

 

Plants and/or

 

Plant Source Parts Inhibited Comments
Lolium multiflorum Kentucky bluegrass

(Domestic Ryegrass) Chewing's fescue

Malus spp. (Apple) Malus spp. (Apple) homologous

Parthenium argentatum

(Guayule rubber)

Pinus spp. (Pine)

Poa pratensis
(Kentucky Bluegrass)

Prosopis juliflora
(Mesquite)

Prunus persica
(Peach)

Prunus pumila
(Cherry)

Prunus serotina
(Cherry)

Prunus spp.
(Cherry)

Quercus spp. (Oak)

Robinia pseudoacacia
(Black Locust)

Salvia spp.
(Salvia)

Salix pellita
(Willow)

gyrus spp. (Pear)

Parthenium argentatum homologous

Triticum spp. (Wheat)

Poa compressa
(Canada Bluegrass)

foliage and roots

Lycopersicon esculentum

(Tomato)

Prunus persica homologous
(Peach)

Pinu§_banksiana
(Jack Pine)

Pinus banksiana
(Jack Pine)

Prunus spp.
(Cherry)
Triticum spp. (Wheat)

Quercus spp. (Oak)
Triticum spp. (Wheat)

homologous

Brassica spp. (Mustard)

Pinus strobus (White Pine)

Hordeum vulgare (Barley)

Many species

Pinus banksiana

(Jack Pine)

continued

TABLE II--continued

24

 

Plant Source

V7

Plants and/or

Parts Inhibited Comments

 

Sarcobatus vermiculatus

(Greasewood)

Sorghum vulgare
(Sorghum)

Tridax procumbens
(Tridax)

Trifolium spp. (Clover)

Viquiera reticulata
(Viguiera

Lycopersicon esculen—
tum (Tomato)

other grasses

other weeds

Poa pratensis (Kentucky
Bluegrass)

Lycopersicon esculentum
(Tomato)

 

25

time, many observations relative to the toxic action of

the walnut have been made and probably will continue to

be made as long as plants are grown beneath Juglans nigra.
L. The American Indians and the early pioneers were prob-
ably the first groups to experiment or work with the black
walnut (82). They found that the black walnut husks would
make a permanent brown dye for clothing when they were
macerated in water (20). Salves and other various oint-
ments were also made from the husks by these early settlers.
In 1749, Kolm, perhaps one of the first people to observe
the growth of the walnut, noticed that as soon as the
fruits ripened in the fall, the black walnut leaves fell
(36). It was also noted that cherry, apple, flax, and some
vegetables planted in close proximity of the walnut, wilted
and eventually died. One belief, which may not have been
completely incorrect, was that dew reacted with vapors
which were blown by the wind to nearby plants, which in
turn were poisoned. After many considerations, it was
finally theorized that the roots of the black walnut ab—
sorbed all the water and nutrients in the immediate area,
thus starving other plants beneath its crown. The many and
varied reports of the toxic action relative to Juglans
nigra indicate that there may indeed be a phytotoxic plant
interaction. Based on these observations, Table III has
been prepared depicting some plants that have been reported

not to grow beneath the crown of Juglans nigra. The table

 

TABLE III

PLANTS REPORTED NOT TO BE TOLERANT OF JUGLANS NIGRA

 

Scientific Name

Common Name

 

Trees:

Betula alba

Elaeagnug angustifolius
Ilex verticillata
Magnolia soulangiana
Malus 'Hopa'

Malus app 0

Pinus resinosa

Sambucus canadensis
Viburnum sieboldii

Shrubs, Vines and Flowers:

Azalea spp.
Chrysanthemum morifolium
Hydrangea spp.
Potentilla fruticosa
Prunus tomentggg.
Pvracantha spp.
Rhododendron spp.

Rosa chinensi§"Minima'
Syringa x persica
Syringa vulgarig
Vaccinium corymbosum
Viburnum opulus

 

Vegetables and Others:

Alfalfa spp.

Beta vulgaris
Lycopersicon esculentum
Solanum_tuberosum

Vigna sesguipedalis

 

White Birch

Russian Olive

Black Alder,Winterberry
Saucer Magnolia

'HOpa' crab

Apples

Red Pine

Common Elder

Siebold Viburnum

Azalea

Chrysanthemum
Hydrangea

Shrubby Cinquefoil
Nanking Cherry-
Pyracantha (Firethorn)
Rhododendron

Fairy Rose

Persian Lilac

Common Lilac

“Highbush Blueberry

European Cranberry Bush

Alfalfa
Sugar Beets
Tomatoes
Potatoes
Asparagus

 

27

was prepared from the following references: 9,10,12,13,
21,24,26,27,35,36,41,42,44,46,58,67,69,70,7l,73. Table
III does not necessarily represent all plant genera or
species that may be involved.

Davis determined through experimentation that the
toxic substance present in Juglans nigra is juglone (16).
It was found to be toxic to tomatoes, alfalfa, and apples
when injected into the stems. The substance was found in
all parts of ggglgns nigra. A variance in the location
of juglone within the plant was determined by Lee and
Campbell (39). They noted that the amount of juglone
varied in the leaves and in the fruit husks of different
black walnut varieties. It is hypothesized that the sub-
stance was translocated from the leaves to the hulls at
different times during the year.

Juglone is a napthoquinone, and only one has been
isolated, having a formula of 5-hydroxy—l,4-napthoquinone,
C10H503 (14,43). Juglone has a molecular weight of 174.00
and is slightly soluble in water, being more soluble in
chloroform, benzene, alcohol, and ether (74). Diethyl
oxide and ether have been found to be the best extractants
from walnut hulls (16).

It is conceivable that a toxic substance present in
the soil could kill living cells by modifying the soil
solution and perhaps effecting absorption of water and

nutrients. MacDaniels and Muescher concluded that the soil

28

pH may in turn effect the absorption of each soil site.
Massey, as cited by MacDaniels, worked with root bark of
Juglans nigra relative to tomatoes grown in nutrient cul-
tures and found after 48 hours that the roots became dis-
colored and then slimy. A similar experiment was carried
out using bushel baskets filled with soil, and it was ob-
served that the soil containing root bark inhibited the
growth of tomatoes, but the soil from the field where former
symptoms occurred did not inhibit growth (41).

A representative of the California Walnut Growers
Association stated that toxic symptoms had been observed
relative to cover crops. It was also noted that applica-
tions of excess nitrogen seemed to remove.all symptoms of
the toxic interaction (41). Davidson (15) of Ohio noticed
that raspberry plants died beneath apple trees but grew
beneath mulberry and persimmon trees. Having heard of the
toxic action of the walnut, he pointed out that he has
apple and walnut trees growing together with no toxic
symptoms present.

Potentilla fruticosa, or shrubby Cinquefoil, has.been
observed in many locales of Vermont, but has nOt been
observed beneath either mature or seedling trees of Juglans
nig£a_(35). The area beneath the walnut trees was clean of
most weeds and very grassy relative to maple or apple trees
in the same area. The area beneath the walnut seemed to

get larger each year. Observations by A. H. Gilbert

 

29

indicated that this action of inhibition is not present
beneath birch, beech,‘maple, cherry, apple, and pine
trees.

Apple trees have, for many years, been observed to
suffer when planted or grown near walnut trees. The
effect is often more pronounced on the side of the apple
tree next to the walnut (9,21,44,70,7l). Schneiderhan,
observing four apple trees surrounding a black walnut,
noticed that one was dead, one was dead on the side nearest
the walnut, one was extremely stunted, and one exhibited
very weak growth. Staymen varieties, however, displayed
much more tolerance toward Juglans nigra, perhaps indica-
tive of some genetic or varietal difference. It was also
noted that corn seemed stunted when grown next to Juglans

Recently, many species of pines have been found to
suffer when grown in close proximity of Juglans nigra.

Red pine, Pinu§_resinosa, began to suffer and eventually
died when planted next to Juglans nigra (7). ‘It grew well
when planted between red oak and did not show any discolora-
tion or needle loss as was displayed next to Juglans nigra.
Bruner (12) observed a loblolly pine forest on acreage
formerly consisting of old homesites and noticed scattered
spot-dying. Upon investigation, it was revealed that each
occurrence was in close proximity to Juglans nigra. The

suffering pines were first characterized by a white flow of

30

pitch which then turned yellowish. The bark soon slipped,
exposing the cambium. This would occur on the side of
the pine adjacent to the walnut. Subsequently, the
majority of deaths were attributed to bark beetle attack
of the weakened trees. Deaths of those not attacked by
bark beetles was attributed to walnut interaction. The
characteristic symptoms were found in the shortleaf pine,
Pinus echinata, as well, but did not seem as severe.

Ericaceous type plants, such as azaleas and rhododen-
drons, which are important in any landscape or nursery
operation, have been observed to die when planted beneath
black walnut trees (9,65,67). In the few isolated cases of
good growth, a rock ledge has been found separating the two
root systems. Pirone (9) observed that many varieties of
Rhododendron catgwhien§§_died but that those without root
system contact did not die. In some instances, soil pH was
used for an explanation. Nitrogenous applications did not
create a growth response, although the symptoms seemed to be
lessened.

Kentucky bluegrass, which is widely planted, has been ob—
served to be one of the plant species enhanced by Juglans nigra
(9,35,38,42,46). More often than not, weed or poverty grasses
are found less beneath walnut crowns relative to maples
(Ag§£_sp.), beech (E333; sp.), oaks (Quercus sp.), and

hickories (Carya sp.)(9,35). This can often be observed on

31

rich as well as poor soil sites, and good varieties of
grasses, such as Kentucky bluegrass, are often found beneath
Juglans nigra.

Peaches, like apples, have been noticed to be sus—
ceptible to the toxic action of walnuts but in some situa-
tions have grown well (35,46). In situations characterized
by poor growth, competition for water and nutrients was
theorized as the cause. McKay noticed that the growth of
plums, was restricted to a lesser degree than peaches.

Two plants, more than any others, have long been
associated with the toxic or inhibitory action exerted by
Juglans nigra. These plants are the tomato, Lycopergicon
sp. and alfalfa, Medicago sp. (4,9,10,13,16,26,27,42,58,67,
73). Tomatoes, often grown by home gardeners, were probably
noticed to be inhibited When planted next to black walnuts,
which may account for the many early observations. Alfalfa
was probably first observed by farmers planting it along
fence rows which are often habitats of black walnuts.

Like many other plant species producing growth inhibi-
tors, the roots of Juglans nig£a_were the first toxin
producing area to be considered as the cause. Brown (10)
found roots inhibited the growth of alfalfa seedlings. The
alfalfa roots were shriveled and brown, and the plant was
chlorotic and stunted. Reinking (67) reported that apple,
quince, Mazzard cherry seedlings, Mahaleb cherry seedlings,

and Western sand cherry seedlings were inhibited in growth.

32

The phloem was found to be discolored, and.he concluded
that the substance exuded by the walnut could remain in
the soil almost indefinitely, perhaps until all roots were
dead, decomposed, and leached from the soil.

Walnut leaves, too, have been considered as the toxic
agent. Perry (63) determined an inhibition of oxygen up-
take in the bean and tomato plant species. He found respira-
tion to be inhibited about 50 percent in each. Bode (4)
hypothesized that the toxin is contained in the leaves of
Juqlang nigra and is leached by the rain to the environment
of other plants. The leaves of Lycopergicon sp., tomato,
were characterized by curling, and the yellowing which
usually occurred, was not present. No inhibition of tomatoes
was displayed when the plants were covered with plastic.

He also determined that nutrient solutions which had con-
tained walnut seedlings promoted tomato growth, and that
catkins first inhibited, then promoted growth after

twelve days.

 

EXPERIMENTAL PROCEDURE

Fruit Residues

The major emphasis concerning plant residues relative
to Juglans nigra was directed toward the fruit it produces,
considering it a means of toxin entry. It was thought
that the characteristic wilt symptoms would occur, provid-
ing a visual interpretation, as well as being an initiation
point for further work with this aspect of black walnut
toxin entry. This was desired since Lee (39) had shown
that certain varieties of walnut fruit do contain various

quantities of juglone.

Study No. 1:

Unhusked walnut fruits were gathered, separated, and
stored in the dark at -20°F on October 15, 1969.

The study was divided into two parts.' Part No. I con-
sisted of the following treatments applied to 6" plants of
LycoEersicon esculentum, variety Spartan Red, and Pyracantha
coccinea ,grown in 4“ styrofoam pots containing a 1:1:1
media mix of soil, sand, and peat:

Part I.

1. Control A--tomato plants in 4" pots.

2. Control B-—pyracantha plants in 4" pots.

33

34

3. Mulch A--application of 20 grams of walnut husks
as a mulch to tomato plants.

4. Mulch A1—-same as 4 above using 30 grams.

5. Mulch B-—application of 20 grams of walnut husks
as a mulch to pyracantha plants.

6. Mulch B1--same as 5 above using 30 grams.

Part No. II consisted of plants planted in a pot with
the bottom removed and subsequently placed upon a second
4" pot of soil containing one of the following treatments:

Part II.

1. Control A--tomato plants potted in the uppermost
pot with two number 8 rubber stoppers
wrapped in tinfoil in the lower pot
for simulation of walnut fruits.

2. Control B--same as 1 using pyracantha.

3. A1--same as l with one 2-inch diameter walnut
fruit in the lower pot.

4. A2—-same as above plus 20 grams of broken husks
applied as a mulch to the uppermost pot.

5. A3--same as 3, however using two walnut fruits.

6. A4-—same as above plus 20 grams of broken husks
applied as a mulch as in treatment 4.

7. B1--same as 3 using pyracantha.

8. Bz-—same as above plus 20 grams of broken husk
mulch.

9. B3--same as 7 using two 2" walnut fruits.

10. B4-—same as 9 plus 20 grams of mulch.

The plants were potted on November 25, 1969, and grown
to maturity. The experiment consisted of two replicates
per treatment with four plants per replicate. Observations

were made twice weekly throughout the experiment, and

35

plants were watered daily as needed. Plants were fertilized

once weekly with a 20:20:20 water soluble fertilizer.

Leaf'Leachates

 

Study NO. 1:

The leachate study at the Rose Lake Research area was
begun on May 26, 1970, with the clearing of grassy areas
and the placement of black polyethylene plastic over areas
approximately 3 feet by 8 feet, which served as growing
plots for tomato plants grown in containers. Four plots
were established beneath mature Juglans nigra trees, 2
beneath Qggrcus velutina trees, 1 beneath Carya cordiformis,
1 beneath 3 seedling trees of Juglans nigra 6 feet in
height, 1 in the open between a mature tree of Juglans nigra
and Carya cordiformis, and 2 plots in the open field, making
a total of 11 plots. On the same day, soil was removed
from beneath mature Juglans nigra trees, of Which half was
sterilized and half was unsterilized. Each soil fraction
was mixed with sterilized peat and sand, forming a mixture
with a 1:1:1 ratio. Tomato seedlings, varieties Roma,
Spartan red, and Campbell, which had been sown on May 20,
1970, were transplanted into steam sterilized No. 10 cans
on June 10, 1970. Drainage holes were provided for by
the use of a can—opener. At planting, each container
fraction of unsterile and sterile soil mix was mixed with

2 tablespoons of osmocote fertilizer, analysis 18-9-9.

36

On June 15, 1970, the containers were set in the field.

Each plot, except those beneath Quercus velutina and Carya

 

cordiformis, contained two plants of each variety; one in
sterile soil and one in nonsterile soil. The Quercus

velutina and Carya cordiformis plots contained 4 plants of

 

each variety, 2 in sterile soil and 2 in nonsterile soil.
The combined total was 42 plants in nonsterile soil and
42 plants in sterile soil.

The containers were watered every day, excluding
periods of precipitation, after field placement with 1 gallon
of water per container. Once every two weeks, each con—
tainer was fertilized with 1 tablespoon of 5-10-10 fertilizer,
and the plants were sprayed weekly with malathion and captan
at the rate of 1 tablespoon per gallon of water for the dura-
tion of the experiment. Observations were made at each

watering.

Root Exudates

StudLNo. 1:

This first study, initiated to determine the influence
of root exudates, was done in conjunction with seedlings of
Juglans nigra. This study took into account both root
exudates and leaf leachates.

Forty seedlings of Juglans nigra were planted, two to a
container in May, 1970. The containers, made of plastic, were

approximately 8 inches in diameter by 10 inches in depth.

37

The seedlings were planted approximately 1 inch from the
sides of the container with approximately 5 inches between
seedlings. The black walnut seedlings were pruned to a
height of 14 inches.

Part A consisted of tomato seedlings, variety Roma,
planted on June 10, 1970, between the black walnut seedlings,
which were allowed to grow upright in the container. In
this situation, the tomato plants would receive root exudate
as well as leaf leachates. The plants were watered from
above every other day as necessary, depending on the pre-
cipitation.

Part B consisted of tomato seedlings, variety Roma,
planted on June 10, 1970, between black walnut seedlings,
which were tied to one side of the container. This allowed
for root interaction but no leachate interaction between
the two plant genera. Part B as well as Part A consisted
of two replicates of five plants per replicate. Two control
plants were grown in similar containers with 3/4 the amount

of soil.

Study No. 2:

Tomato plants of the variety Roma were grown in an Open
cold frame beneath seedlings of Juglans nigra, which were
suspended above the tomatoes on a lath frame., The experi-
mental setup was designed to determine whether or not the
leachates from seedling root systems had any detrimental

influence on growth of the underlying tomatoes. Each tomato

38

plant was grown in a container and placed directly beneath
a walnut seedling in order to catch any leachable root
exudates.

The tomato plants, as well as the seedlings of Juglans
nigra, were watered daily except during periods of rainfall.
Daily watering of the black walnut seedlings was necessary
due to rapidity of container drying caused possibly by wind
exposure.

The experiment consisted of 2 replications of 4 plants
per replication and 2 tomato control plants placed beneath
lath without Juglans nigra seedlings. The plants were
fertilized every other week with 2 tablespoons of 5-10-10
fertilizer per container and were sprayed every week with
a mixture of captan and malathion at the rate of 1 tablespoon

per gallon of water.

Study NO. 3:

 

This experiment, established at the corner of Forest
and College Roads on the campus at Michigan State University,
was undertaken to determine the influence of mature tree
root exudates. This was considered the primary interaction
at this location due to the fact that tomato plants were
planted beneath the mature trees of Juglans nigra. The
walnut trees were planted in a straight east-west row. The
tomato plants, varieties Roma and Campbell, were planted
randomly in rows perpendicular to the walnut trees. Prior

to planting, the plot was tilled twice with a roto-tiller.

39

At planting and throughout the remainder of the experiment,
all plants were fertilized with a 5-10-10 fertilizer at the
rate of 1 tablespoon per plant per week. All plants were
sprayed once weekly with a captan and malathion mixture at
the rate of 1 tablespoon per gallon and were tilled once
weekly. The plants were watered daily excluding days with
.5 inches of rainfall or more. The plants were not watered
on days following 1 inch or more of rainfall. The plants
were observed specifically for wilting twice weekly, at
Which time observational data was recorded. Yield data were

taken at termination of the experiment.

Study No. 4:

The design of this experiment was identical to that of
Study No. 3. However, the location was at the Graham Experi-
ment Station in Grand Rapids, Michigan. The plants were
grown exactly as those in Study No. 3, and data were collected
the day following collection at the College Road location.

The control plants in Studies No. 3 and 4 were grown in pots
and arranged with plastic, having only the plant exposed,
the plant and pot exposed, and neither the plant nor the pot
exposed to leachates of Juglans nigga. None of the control

plants exhibited wilt symptoms.

Chromatographic Studies

Since juglone is considered to be the toxic substance

responsible for the inhibitory action exerted by Juglans

40

nigra, the following studies were undertaken to determine
whether or not juglone could be found in plant tissue of
the wilted tomato plants. The tissue was Obtained from
tomato plants that exhibited wilt conditions in studies
three and four of root exudates. The tissue was gathered
in the field, frozen in dry ice, and moved to a freezing
room for storage at -20°F. The material was then 1y0phi1-
ized and ground with a 40 mesh screen in a Wiley mill.

The extraction consisted of extracting ground,
lyophilized tissue twice with petroleum ether. The first
extraction was for 10 hours and the second extraction was
for 24 hours. The extracts were combined, evaporated to
near dryness and redissolved in chloroform in preparation
for chromatographic application. The previous extraction
procedure applies to all materials tested. However, the
quantity of material extracted and solvent system may vary
with the individual study. All studies utilized systems of
butanol and water 6:1 and isopropanol and water 4:1 with
Part D of Study No. 3, utilizing the above mentioned plus
butanol, acetic acid and water 5:1:2.2, and methanol and

water 7:3.

Study NO. 1:

This study was undertaken to develop the procedure for
comparisons of synthetically pure juglone with extracts of
wilted plant parts, and to determine whether or not juglone

could be identified in tissue of wilted tomato plants.

41

Part A: For a standard, .01 grams of synthetically
pure juglone from K & K Laboratories, Inc., Plainview,
New York, was dissolved in 100 ml. of petroleum ether,
evaporated to dryness, and redissolved in 100 ml. of chloro-
form for ascending paper chromatographic application.
Whatman. No. 1 )grade paper was used throughout the experi—
ment. The solvent used in Study No. 1, consisted of
n-butanol and water in a ratio of 6:1 as described by Lee
(39), and isOprOpanol and water in a ratio of 4:1. A 20
lambda portion of the solvent was applied, and the solvent
was allowed to ascend a distance of 22.5 cm., at which time
the chromatograms were removed, dried, and subjected to
identification techniques consisting of ultraviolet light
and NaOH spray.

Part B: This consisted of chromatographing an extract
of leaves selected from the upper one—half portion of a
nonwilted and a wilted tomato plant in conjunction with the
synthetically pure juglone. One gram of material was ex-
tracted twice with 25 m1. portions of petroleum ether. A
20 lambda portion of each extract was spotted on a separate
paper strip, and each was run in a separate developing
cylinder.

Part C: This consisted of chromatographing an extract
of leaves selected from the lower one—half portion of a
nonwilted and a wilted plant in conjunction with the pure

juglone. The chromatograms were run identical to the

42

standard in Part A, and the material was extracted as in
Part B.

Part D: This part was carried out as the previous
part. However, stems from the upper portion of tomato
plants were used. The extraction method was identical to
Part B.

Part E: The test was carried out as the previous
test, Part D, but stems from the lower one-half portion
of the tomato plants were extracted with petroleum ether.

Part F: This test consisted of chromatographing
redissolved extracts from the ripe and nonripe fruit of
wilted and nonwilted tomato plants, which were grown beneath
trees of Juglans nigra. One gram of the material was
extracted twice with 25 ml. portions of petroleum ether,
evaporated, and redissolved in 3 ml. of chloroform.

All of the extracts chromatographed in Study 1 of the
chromatogram studies were compared to the synthetically
purified juglone. The tests included all of the plant parts
except for the root systems of wilted and nonwilted tomato

plants.

Study NO. 2:

The same extraction procedure was used for tomato tis—
sue as in Study No. 1, and a 20 lambda portion of each
redissolved extract was chromatographed in both previously
mentioned systems of isOprOpanol and water and butanol and

water. This study utilized Juglans nigra leaves as the

43

standard for juglone comparison in wilted tomato plant
tissue. Twenty grams of fresh walnut leaf tissue was
extracted twice with 20 ml. of petroleum ether, evaporated
to near dryness, redissolved in 3 ml. of chloroform, and
chromatographed.

Part B: This test utilized the walnut leaves as a
standard in conjunction with leaves of wilted and nonwilted
tomato plants. The leaves were taken from the upper portion
of the plants and extracted as Part B of Study No. 1.

Part C: This test was carried out identical to Part B,
Study No. 2. However, the leaves were taken from the lower
half of the tomato plants.

Part D: This part of Study No. 2 consisted of testing
the extracted stem material of wilted tomato plants and
comparing the results with the leaf juglone standard. The
procedure was the same as that used in Parts B and C of

Study NO. 2.

Study NO. 3:

Part A: In this study, the walnut husks were utilized
for the juglone identification standard. The material was
extracted and chromatographed as Part A of Study No. 2.

A 20 lambda portion of each extract was chromatographed in
both n-butanol and iSOpropanol solvents in the ratios stated
previously.

Part B: The leaf tissue extracts of wilted and non-

wilted tomato plants were chromatographed in both solvents

44

with walnut-husk extracts used as the identification
standard. The extracts were prepared as in Part B of Study
No. 1.

Part C: This part consisted of ripe fruit tissue
extracted as in Part F of Study No. l. The extracts were
chromatographed using both solvent systems.

Part D: This part consisted of chromatographing ex-
tracts from root tissue of wilted and nonwilted plants and
the walnut husk standard. The root extracts consisted of
extracting 5 grams of lyophilized material twice with 45
ml. of petroleum ether. The extracts were evaporated and
redissolved in 3tmL.of chloroform in preparation for paper
chromatographic application. Four solvent systems,
n-butanol and water, butanol acetic acid and water, iso-
propanol and water, and methanol and water, were used in

this part.

RESULTS

Fruit Residues

The study utilizing the whole walnut fruit as a source
of toxin, for entry into tomato plants, did not present any
visible wilt symptoms aside from occasional water stress.
This slightly wilted condition could be alleviated with
water applications, and therefore was not characteristic of
reported walnut wilt. There were no significant differences
between tomato plants relative to the various treatments,
and all tomato plants grew normally to the extent of produc—
ing fruit, which matured. The pyracantha plants grew well
and showed no significant wilting other than occasional
water stress.

Upon examination of the root systems, it was found that
the soil area surrounding the walnut fruit was darkened in
some cases, due probably to the decomposition of the husk.
Tomato and pyracantha roots were found growing in these
areas, and there was no apparent injury to the roots of

either plant genus.

Leaf Leachates

Throughout the months of July and August, there was

evidence of some wilting. The wilting was more prevalent

45

46

on the plots in the Open field rather than on those beneath
Juglans nigra. Walnut wilt has been characteristically
expressed as a condition which cannot be alleviated even
after watering. In the case of these plots, the plants

did not remain wilted after watering.

On August 30, 1970, all plants were removed from the
research area, and comparisons were made between plants
under the same tree and plants under other tree species
relative to Juglans nigra. All of the plants flowered and
produced fruit which matured on the plants. (There were no
apparent differences between plant height, fruit size, or
color of tomato plants of the same variety, when compared
to plants beneath the same or one of the other trees used
in the study. Throughout the study, the plants in the plot
in the open field exhibited the most wilting, but this was

due to rapidity of drying of the containers.

Root Exudates

Study NO. 1:

The tomato plants grown in conjunction with and without
walnut seedling leachates grew well considering the compe—
tition for water and nutrients relative to the container
size. All of the plants produced fruit which matured on
the plants, and there were no apparent differences in plant
height, fruit amount, size, and plant color relative to
the control plants. The tomato root systems did not exhibit

any browning or stuntedness.

47

Study NO. 2:

All of the tomato plants grew well, flowered, and pro-
duced fruit which matured on the plant. There were no
apparent differences between plant height, fruit size or
plant color of the plants grown beneath the seedlings rela-

tive to the control plants.

Study NO. 3:

The plants grew well and the first sign of wilting,
which occurred on July 25, 1970, was scattered throughout
the planting. The plants were very slow to revive upon
watering. However, they did look fairly turgid on August
1, 1970, following 3 days of very light precipitation.

The plants looked progressively worse during the early part
of August despite daily waterings. In fact, some were
damaged to the extent of browning foliage. The condition
was, as reported by others, spotty with little or no dis-
tinction between tomato variety, row location, or tree drip
line. It was determined that there was an increase in the
average total number of fruit and average total plant weight
with increasing distance from the walnut tree trunks, as
indicated in Figures 1 and 2. This increase dr0pped sharply
at approximately the seventh plant in each row, which
corresponded with the drip line of the trees.

The previous discussion of Study No. 3 is relative to
distance from the tree, whereas the following results are

based on a wilt-rating made at the termination of the

48
3004-

200*

1( “I Study 3

P"#4V////
1 -L

j. a. d. J).
g 1 I l l
15 18 21 24 27 30 33 36
Distance from Tree (ft.)

 

 

 

 

Average number fruits
per plant

 

 

 

 

 

 

 

m 100*' _

.p F

fl

9

H

\H

’44-)

'83 50_ Study4
521 -

a” (-

0”

mo.

3 J-

8 ._____ly. i — L I
fl 5 8 11 l4 17 20

Distance from Tree (ft.)‘

Figure 1. Average number fruits per plant with
increasing distance from the walnut
tree trunks.

20,000‘

15,000

10,000‘

5,000

Average Total Plant Weight
(in grams)

 

5,0004

2,500

Average Total Plant Weight
(in grams)

Figure 2.

 

49

 

 

 

 

 

 

 

 

 

 

 

 

1'
1' Study 3
e r
s- ,14”)
_ -L ‘5
/ -L
4L
J_ l L l‘ 1 l l J,
15 18 21 24 27 30 33 36
Distance from Tree (ft.)
1-
r— u—
w-
Study 4
\\\\\///// -u
-L
l I L J _I ~ J
5 8 ll 14 17 20 23
Distance from Tree (ft.)
Average total plant weight, in grams,

with increasing distance from the
walnut tree trunks.

50

experiment and collection of data. The rating consisted of
the following scale:

1. Very good growth and yield with no walnut roots
less than 24" deep.

2. Good growth and yield with no walnut roots less
than 18" deep.

3. Slight wilt but good growth and yield. No walnut
roots less than 12" deep.

4. Wilted with some leaf browning and poor yields.
No walnut roots less than 6" deep.

5. Extremely wilted with less than 40% green tissue
and extremely poor yields. Walnut roots in top
. 6" of soil.

Figure 3 depicts the data relative to average total
number of fruits and average total plant weight.

Upon examination of the wilted plants, it was found
that in each case the roots of the tomato plant were in
direct contact with roots of Juglang nigra. In this study
a total of 55 tomato plants, representing wilt conditions
3, 4, and 5, were found to have roots in direct contact
with black walnut roots. The remaining 45 plants did not
exhibit wilt symptoms, and of these plants, 23 did not come
in contact with walnut roots. The remaining 22 plants did
not wilt. However, there were walnut roots beneath these

plants, but in each case the roots were at least 18" in

depth.

Study NO. 4:
The data was collected the day following collection in

Study No. 3. Results relative to fruit number and weight

Average Total Weight (in grams)

Study 3
20,000

I

10,000

 

 

S 4 3

51

 

 

l

b

2 1

Wilt Condition

 

 

 

 

 

m
p
o; d- -.
H Study 3
En
H 200-
0)
.Q
E
:3 4J _-
2::
(U
H H
3 Ch
(3H lOOh—
B Q)
Q:
(1)
g) _-
u l _L
0)
> —_
T . , I I 1
5 4 3 2 l

Wilt Condition

Figure 3.

5,000

2,500

100

50

 

 

 

 

 

 

 

 

 

 

Study 4
_ -~ 1'
“F
/b
r/7
5 4 3 '2 1
Wilt Condition
L
Study 4
T
b
‘F‘/
P ‘L
5 4 3 2 1

Data relative to average total number of fruits
and average total plant weight.

Wilt Condition

52

were similar to the results of Study No. 3. These results
were plotted in Figures 1, 2, and 3.

Results of root interactions relative to wilt rating
were similar to those described in Study No. 3. A total of
59 tomato plants representing wilt conditions were found to
have roots in direct contact with black walnut roots. The
remaining 29 plants did not exhibit wilt symptoms and of
these 29 plants, 18 did not come in contact with walnut
roots. The remaining 11 plants did not wilt. However, in
these 11 plants walnut roots were present at a depth of at
least 18 inches.

The wilting in Studies No. 3 and 4 was spotted through-
out the rows in plots. There were no apparent significant
differences between the wilting of any one variety or loca—
tion of the wilting in relation to the walnut trees in
Studies No. 3 and 4. The most significant factor was that
in each case of wilting, the walnut roots were in direct con—

tact, or touching roots of Lyc0persicon esculentum (tomato).

Chromatographic Studies

Study NO. 1:

Fluorescence was found under shortwave ultraviolet
light on all paper chromatograms and absorbance only in the
zones containing synthetic juglone. Juglone was found to
be a brownish spot, whereas the other materials were not

characterized by any absorbance except in the case of

53

overloading. In this instance, an orange area was found.
The Rf value for juglone was determined to be .94 t .02,
while the Rf value of the material was .96 t .02. Each
area was sprayed with 5%1sodium hydroxide, and juglone
appeared reddish-purple, while thete was no change in the
unknowns. The method was considered to be valid since
synthetic juglone was identified. However, there was no
evidence for juglone in the tomato tissue extracts of

Parts A, B, C, D, E, or F when compared chromatographically

to the synthetic material.

Study NO. 2:

Fluorescence was observed under shortwave ultraviolet
light, but the walnut leaf extract was similar to synthetic
juglone, and the tomato tissue extract was not since no
brown area developed. Subsequent spraying with 5% sodium
hydroxide exhibited a pinkish-purple color on the chroma—
togram of the walnut leaf extract. There was no indication
of juglone in any chromatograms utilizing the extracts of
the tomato tissue in Parts A, B, C, or D When compared to

the walnut leaf chromatogram.

Study NO. 3:

Neither Part A nor Part B gave any indication of
juglone in tomato tissue. The fruit tissue of Part C again
gave negative results. Part D deviated from all others

with an increase in the amount of tomato material extracted.

54

Five grams instead of 1 gram were used in this part. Upon
examination under ultraviolet light, a slight band was
observed near that of juglone. Upon spraying with 5%,sodium
hydroxide, a pinkish-purple area appeared at a similar Rf
to juglone. Upon further chromatography using methanol
and water which lowered the Rf values, the tomato tissue
extract area exhibited a drag streak. This was attributed
to an overloading of the chromatogram in an attempt to get
the material to show up. The characteristic color was
evident upon spraying. However, it was a vertical band
due possibly to the drag of the material.

An attempt to elute the material from the paper was
made using petroleum ether and chloroform extracts, evaporat-
ing to near dryness, and rechromatographing the resultant
material. No evidence of juglone in this paper extraction
was seen under ultraviolet light, nor was there a color

exhibited with the 5% sodium hydroxide spray.

DISCUSSION

The characteristic wilt, that has been exhibited by
many plants growing beneath Juglans nigra, was found with
tomato in only one experiment. This consisted of a field
interaction between mature black walnut trees and tomato
plants. The primary purpose of this part of the study was
to determine the influence of root exudates.

The interaction of the other major modes of toxin
entry, leaf leachates, and plant residues, did not inhibit
tomato growth.

The chromatographic studies indicated no juglone, the
presumed toxic substance, in extracts of 1 gram of tomato
plant tissue. However, there was a characteristic color
reaction in the 5 gram extraction chromatogram. Upon elu-
tion and rechromatography, in order to obtain a more pure
substance, no color reaction occurred. This may have been
due to the oxidation of the material, the low concentration
level, or the inability to remove juglone from the paper.

The results of all the studies indicate that perhaps
root exudates are the primary mode of toxin entry into the

environment by Juglans nigra.

55

S UMMARY

Since all plants are made up primarily of chemical
compounds, it would seem that plants could not exist with-
out emission of by-products of metabolic processes. Many
trees shed their leaves in the fall, exemplifying one
possible method of emission. Metabolic by-products may be
4 phytotoxic and may be released by root excretions or
exudates, crown leachates, fog drip, volatilization, and
plant residues.

Upon entry into the environment or immediate ecosystem,
the phytotoxic substances may be acted upon by microorgan—
isms, environmental factors, or absorbed to soil colloids.
These factors may aid in a build-up or subsequent breakdown
of the toxins, and if they are not broken down, it is con-
ceivable that they might be absorbed by living plants.

The resultant action displayed, either stimulatory or
inhibitory, may be relative to the ability of the plant to
metabolize the physiologically active substance.

The allelopathic nature of Juglans nig£a_has been
observed for many years since the Roman empire. The method
by which the toxin is released to the environment has been
considered to be by leaf or crown leachates, plant residues,

or root exudates.

56

57

It was the purpose of this study to determine the
primary mode of the black walnut toxin entry into the
environment and to isolate the toxin, juglone, in tissue
of plants exhibiting this allelOpathic action. Root inter-
actions and subsequent exudates seemed to be the primary
area responsible for the toxicity of the black walnut rela-
tive to tomato.

The toxic material was not isolated from wilted plant
tissue. Further studies will need to be undertaken in
order to isolate the toxin from wilted plant tissue and
to determine anatomically what plant part or parts are influ—

enced by the toxin.

10.

11.

LITERATURE CITED

Ahlgren, H. L. and O. S. Aamodt. 1939. Harmful root
interactions as a possible explanation for effects
noted between various species of grasses and legumes.
Jour. Amer. Soc. Agron. 31:982—985.

Bedford, Duke of and S. U. Pickering. 1914. The effect
of one crop on another. Jour. Agri. Sci. 6:136—151.

Bennett, E. L. and J. Bonner. 1953. Isolation of
plant growth inhibitors from Thamnosma montana.
Amer. Jour. Bot. 40:29433.

Bode, H. R. 1958.‘ AllelOpathy in some juglandaceae.
Planta 51(4):440-480.

Bonner, J. and A. W. Galston. 1944. Toxic substance
from the culture media of guayule which may inhibit
growth. Bot. Gaz. 106:185—198.

Bonner, J. 1946. Relation of toxic substances to
growth of guayule in soil. Bot. Gaz. 107:343-351.

Borner, H. 1959. The apple replant problem. I. The
excretion of phlorizin from apple root residues.
Contr. Boyce Thomp. Inst. 20:39-56.

Borner, H. 1960. Liberation of organic substances
from higher plants and their role in the soil sick-
ness problem. Bot. Rev. 26:393-424.

Brooks, M. G. 1951. Effect of black walnut trees and
their products on other vegetation. West Vir. Univ..
Agri. Exp. Sta., Bull. 347:1—31.

Brown, Babette L. 1942. Injurious influence of bark
of black walnut roots on seedlings of tomato and
alfalfa. N. Nut Growers Assn. Rep. 33:97-102.

Brown, R. T. 1967. Influence of naturally occurring

compounds on germination and growth of jack pine.
Ecol. 48(4):542-546.

58

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Bruner, Marlin.

Cook, M. T. 1921.

59

1969. Walnut and pine don't mix.
For. Farmer 28(4):15.

Phytopath. 11:346.

Daglish, C. 1950.

Wilting caused by walnut trees.

The isolation and identification

of a hydrojuglone glycoside occurring in the walnut.

Bioch. Jour.

Davidson, J. 1939.

47:452-457.

N. Nut Growers Assn. Rep. 30:58.

Davis, E. F. 1928.

Some unanswered questions.

The toxic principle of Juglans

nigra as identified with synthetic juglone and its
toxic effects on tomato and alfalfa plants.
Amer. Jour. Bot. 15:620.

De Candolle, M.

Del Moral, Roger and C. H. Muller.

A. 1832. Physiologie Vegetale T. 111.:

1969.

Fog

drip:

a mechanism of toxin transport from Eucalyptus
globulus. Bull. Torrey Bot. Club.

Del Moral, Roger and C. H. Muller.
pathic effect of Eucalyptus camaldulenais.

Midland Naturalist. 83(1):254-282.

Dengler, H. W.

96(4):467-475.

1970.

The

1958. The folklore of walnuts.
N. Nut Growers Assn. Rep. 49:96-100.

allelo—

Amer.

Fed. Garden Clubs of Mich. 1968. Through the garden

gate. 27(4):

Fletcher, F. 1912.

16—18.

Agri. Sci. 4:245-247.

Funke, G. L. 1943.

absenthium on neighboring plants.

Garb, S. 1961.

Gray, R. and J.

Blumea

Toxic excreta of plants.

The influence of Artemipia

5:

Jour.

281-293.

Differential growth-inhibitors pro-
duced by plants. Bot. Rev. 27:422-443.

Bonner. 1948. An inhibitor of plant

growth from the leaves of Encelia farinosa.
Jour. Bot. 35:52—57.

Greene, K. W.
black walnut.
155.

Atner e

1929. The toxic (2) effect of the

N. Nut Growers Assn.

Rep.

20:

152-

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

60

Greene, K. W. 1931. Toxic effect of the black
walnut. N. Nut Growers Assn. Rep. 22:103—105.

Gries, George A. 1943. Juglone, the active agent in
walnut toxicity. N. Nut Growers Assn. Rep.
32:52—55.

Guenzi, W. D. and T. M. McCalla. 1962. Inhibition
of germination and seedling development by crop
residues. Soil Science Soc. Amer. Proc. 26:456-
458.

Guenzi, W. D. and T. M. McCalla. 1966. Phytotoxic
substances extracted from soil. Soil Sci. Soc. Amer.
Proc. 30(2):214-216.

Guenzi, W. D. and T. M. McCalla. 1966. Phenolic
acids in oats, wheat, sorghum and corn residues
and their phytotoxicity. Agron. Jour. 58:303-304.

Guenzi, W. D., T. M. McCalla and Fred A. Norstadt.
1967. Presence and persistence of phytotoxic sub-
stances in wheat, oat, corn, and sorghum residues.
Agron. Jour. 59:163-165.

Hook, Donald D., and Jack Stubbs. 1967. An observa-
tion of understory growth retardation under three
species of oaks. U. S. Forest Service Res. Note
SoEo-70o

Jameson, D. A. 1966. Pinyon-Juniper litter inhibits
growth of blue grama. Jour. Range Mng. 19:214-217.

Jones, L. R. and Morse, W. J. 1902-1903. The shrubby
cinquefoil as a weed. Ver. Agri. Exp. Sta., 16th
Ann. Rep. 173-190.

Kolm, Pehr. 1942. The characteristics and uses of
the North Amer. Black Walnut Tree (Trans. E. Larsen).
Agri. History l6(l):l49-154.

Kommendahl, T. 1957. Quackgrass can be toxic to cr0p
seedlings. Down to Earth 13(2):4-5.

Kline, L. V. 1949. Tree crops for the Tennessee
Valley. Amer. For. 47:470-472.

Lee, K. C. and R. W. Campbell. 1969. Nature and
occurrence of juglone in Juglans nigra. L. Hort.
Sci. 4(4):297-298.

Loehwing, W. F. 1937. Root interactions in plants.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

61

MacDaniels, L. H. and W. C. Muenscher. 1941. Black
walnut toxicity. N. Nut Growers. Assn. Rep. 31:
172—179.

Massey, A. B. 1925. Antagonism of the walnut
(Juglans nigra L. and Juglans cinerea) in certain
plant associations. PhytOpath. 15:773—784.

 

 

Mathis, C. 1966. Comparative biochemistry of hydro—
quinones, pp. 245—269. In T. Swain, Comparative
phytochemistry, Academic Press, New York.

Mattoon, H. G. 1942. A commercial black walnut
venture. Amer. For. 48:172-174.

McCalla. T. M., W. D. Guenzi and Fred A. Norstadt.
1964. Phytotoxic substances in stubble mulching.
8th Intern. Congress of Soil Sci., Bucharest,
Romania.

McKay, John W. and Harley L. Crane. 1950. Bunch
disease of black walnut. N. Nut Growers Assn.
Ann. Rep. 41:56-62.

McPherson, J. K. and C. H. Muller. 1969. Allelopathic
effects of Adenostoma fasciculatum "chamise" in
the California Chaparral. Ecol. Mon. 39:177-198.

Mecklenburg, R. A. and H. B. Tukey, Jr. 1964.
Influence of foliar leaching on root uptake and
translocation of calcium-45 to the stems and
foliage of Phaseolus vulgaris. Plant Phys. 39(4):
533—536.

 

Mecklenburg, R. A., H. B. Tukey, Jr., and J. V. Morgan.
1966. A mechanism for the leaching of calcium from
foliage. Plant Phys. 41(4):610—613.

Mellanby, K. 1968. The effects of some mammals and
birds on regeneration of oak. Jour. App. Ecol.
5(2):359-366.

Mergen, F. 1959. A toxic principle in the leaves of
Ailanthus. Bot. Gaz. 121:32—36.

Muller, C. H., W. H. Muller and B. L. Haines. 1964.
Volatile growth-inhibitors. Sci. 143:471-473.

Muller, H. and C. H. Muller. 1956. Association
patterns involving desert plants that contain toxic
products. Amer. Jour. Bot. 43:354-361.

62

54. Muller, W. H. and R. Hauge. 1967. Volatile growth
inhibitors produced by Salvia leucophylla: effect
on seedling anatomy. Bull. Torrey Bot. Club.

94(3):182-191.

55. Muller, W. H., P. Lorber, and B. Haley. 1968.
Volatile growth inhibitors produced by Salvia
effect on seedling growth and
95 (5) :415-

leucophylla:
Bull. Torrey Bot. Club.

respiration.
422.
B. Haley and K. Johnson.

56. Muller, W. H., P. Lorber,
1969. Volatile growth inhibitors produced by
effect on oxygen uptake by

Salvia leucophylla:
mitochondrial suspensions.

96(1):89-96.

Bull. Torrey Bot. Club.

 

57. Neish, A. C. 1965. Coumarins, phenylpropanes, and
In Plant Biochemistry edit. by Bonner, J.

lignin.
and J. E. Varner. Academic Press. New York.

1054 p.
and For. Ser. 1951.

58. North Central Forest Exp. Sta.
Walnut and butternut. Bull. F. S. 186.

Fundamentals of ecology.
W. B. Saunders Co., Phila. pp. 3—8.

60. Parenti, Robert L. and E. L. Rice. 1969. Inhibitional
effects of Digitaria sanguinalls and possible role
in old-field succession. Bull. Torrey Bot. Club.

96(1):70-78.

59. Odum, E. P. 1959.

61. Patrick, 2. A. 1955. The peach replant problem in
Ontario. II. Toxic substances from microbial
decomposition products of peach root residues.

Can. Jour. Bot. 33:461-486.

and L. W. Koch. 1958. Inhibition of

62. Patrick, Z. A.
germination and growth by substances

respiration,
arising during the decomposition of certain plant
36:621-647.

residues in the soil. Can. Jour. Bot.

63. Perry, S. F. 1967. Inhibition of respirations by
juglone in phaseolus and lycopersicon. Bull. Torrey

E. B. 1965. Inhibition of black spruce

64. Peterson,
primary roots by water soluble substances in
Kalmia angustifolia. For. Sci. 11(4):473-479.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

63

Pirone, P. P. 1939. The detrimental effects of walnut
on rhododendrons and other ornamentals. N. Nut
Growers Assn. Rep. 30:73—74.

Proebsting, E. L. and Gilmore, A. E. 1940. The rela-
tion of peach root toxicity to the reestablishing of
peach orchards. Proc. Amer. Soc. Hort. Sci. 38:
21‘260

Reinking, O. A. 1946. Tomato wilt caused by black
walnut. Farm Res. 12:2-3.

Rice, E. L. 1967. Chemical Warfare Between Plants.
Bios. 38(2):67-74.

Rudolph, V. J. and P. W. Robbins. 1956. Growth of
planted black walnut on the Michigan State University
campus. Mich. Agri. Exp. Sta. Quart. Bull. 38(4):
520—523.

Schneiderhan, F. J. 1921. (Apple disease studies in
Northern Virginia.) The incompatibility of black
walnuts and apple trees. Vir. Agr. Sci. Bull. 245:
29-30.

Schneiderhan, F. J. 1927. The black walnut (Juglans
nigra L.) as a cause of the death of apple trees.
Phytopath. 17:529-540.

Schreiner, O. and H. S. Reed. 1908. The toxic
action of certain organic plant constituents. Bot.
Gaz. 45:73-102.

Strong, M. C. 1944. Walnut wilt of tomato. Mich.
Quart. Bull. 26:194—195.

Taylor, Glenn G. and R. E. Odum. 1969. Some bio-
chemical compounds associated with rooting of
Carya illinoensis stem cuttings. Amer. Soc. Hort.
Sci. 95(2):146-151.

 

Tukey, H. B. and R. A. Mecklenburg. 1964. Leaching
of metabolites from foliage and subsequent reabsorp-
tion and redistribution of leachate in plants.

Amer. Jour. Bot. 51:737-742.

Tukey, H. B., Jr. 1966. Leaching of metabolites from
above ground plant parts and its implications.
Bull. Torrey Bot. Club. 93:385—401.

77.

78.

79.

80.

81.

82.

64

Waks, C. 1936. The influence of extracts of Robinia
pseudoacacia on the growth of barley. Pub. Fac.
Sci. Univ. Charles [Praha] 150:84-85.

 

Went, F. W. 1942. The dependence of certain annual
plants on shrubs in southern California deserts.
Bull. Torrey Bot. Club 69:100-114.

Went, F. W. 1955. The ecology of desert plants.
Sci. Amer. 192:68-75.

Westfall, B. A., Robert L. Russell and Theodore K.
Auying. 1961. Depressant agent from walnut hulls.
Sci. 134:1617.

Wilson, Roger E. and E. L. Rice. 1968. Allelopathy
as expressed by Helianthus annuus and its role in
old—field succession. Bull. Torrey Bot. Club.
95(5):432-448.

Zim, Herbert S. and Alexander C. Martin. 1952.
Black walnut, p. 136. In Trees, a guide to familiar
American trees, Golden Press, New York.

AP PEND IX

 

L L m 1.... w l f L . is...
.M _.. m 3 3 . a...
t. ed m . we . v at 33 .. .. bah.
mic. Mt... I W .. W. . ITHKNQ,
(3 _ H H _ m . _
V108. 3.2.2.. flying.“ no.“ E._2.h...lon.,mneo.m _ a... .21 I. . -LOJEUbPom
one i. m .2. 3 u - f- - ..,.-!.-. -tsré
9.6 so. “2:. 3.._8.. soars .4 2...... nus . I >1”
.5..." a a. .9. 8. L. 8 £8 r3». .3.”
- . t _ ino—H . . i (I
“3.4....33 _ I _ _ _ 3 3.... .35. 44.3.... . 1......
- .mklofl ,7: mafia w. .. . -

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

      

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. n .42 , >995

HICHIGQN STATE UNIV. LIBRQRIES

II rll'llllllllllllll IllillllllllllII|||||H|HI1|I|1|
l 1

31293101909921