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. This is to certify that the

..; {3? _‘ thesis entitled
Studies with Cycloheximide: Toxicity

_. to Wood Rotting Fungi and Utilization
'3} by Tissues and Organic Compuunds

: ; presented by

.; William Klomparens

a.
’11

-' J 4

7" ‘ '
. ”l.

-k‘ I

f “i

I. 7

r. has been accepted towards fulfillment

of the requirements for
7‘ p _M..S._degree in Wathology
a 5-

.&

 

 

STUDIES WITH CYCLOHEXIrIDE: TOXICITY TO woco-

f<

ROTTIRG EJLCI, AID ULILIZA‘LQI E” TISSUES APB

ORGANIC COmPOUhDS.

bv

I!

William chmoarens

Submitted to the School of Graduate Studies of
Michigan State College of Agriculture and Ap-‘
plied Science in partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE

Department of Botany

and Plant Pathology

1950

ACKNOWLEDCELEIT

The writer wishes to express his gratitude to
Dr. John R. Vaughn for timely help and suggest-
ions, and to the Upjohn Company, Kalamazoo,
Michigan for its financial aid, without which

this work could not have been done.
6

244562

lETFODUCTICK ...............
LITERATURE REVIEW ...............
MATE lIALS Alli) L'JI'HC_)S ...............
EXPERIKENQAL RESULTS ...............
DISCUSSIoi ...............
CQICLYSICNS ...............
SUMMARY ---—-—'--7 .....

LITERATURE CITED ———————————————

Page

(1)
INTRODUCTION

Since l9h8 when-cycloheximide was found to be
of potential value as a fungicide, practical exper-
iments have shown it to be effective in many cases,
both with and without phytotoxic effects, Rose mil-

dew, Sphaerotheca pannosa Ltallroth) Leveille var.

 

rosae Weronchine, was found to be controlled by a

l

‘% ppm. concentration, but with some injury to new

leaves; bean mildew, Erysiphe polygoni, was con-

 

trolled by a 10.0 ppm. with no phytotoxic effects.
As a turf Spray, the antibiotic apparently was ver y
effective, and no phytotoxic effects were observed,
from the use of applications as high as 1000 ppm.
Cycloheximide at 10.0 ppm. indicated superior con-

trol of cucumber scab, Cladosporium cucumerinum, in

 

one field trial. The antibiotic demonstrated fur-
ther promise for control of cherry leaf spot, Hig-

ginsia hiemalis, under field conditions.

 

Inasmuch as the early experimental work demon-
strated practical uses and possible applications,
an attempt was made to determine the action of the
antibiotic when in contact with organic tissues and
compounds. The aims of the experimental work were
partially to investigate the constancy of the fung-

icidal activity of the antibiotic under varied en-

vironmental conditions, and also a study of retent-

(2)

ion, absorption, recovery, or possible translo-
cation in living and non-living tissues. It was
also hoped that a correlation could be establish-
ed between cytological aberrations produced in on-
ion roots with the absorbed quantity of the anti-
biotic.

Eundamental knowledge of this type should be
of vital interest to scientists dealing with anti-

biotics, and to those interested in fungicides.
LITERATURE REVIEW

Cycloheximide, an antibiotic produced by Strep-

tomyces griseus, as a by-product in Streptomycin

 

production (h,7,15,16), was initially suggested as
a possible plant fungicide in l9h8. In this first
fungicidal experiment (5), powdery mildew,Erysiphe
polygoni, on greenhouse beans, was found to be con-
trolled effectively by cycloheximide concentrations
between 5.0 and 10.0 ppm. Mention was also made of
possible phytotoxic effects by higher concentrations.
In l9h9, in Michigan, the effect of the anti-
biotic on seed germination in petri plates, and
action and control on rose plants with mildew under
greenhouse cenditions was investigated (13). Radish
seed inhibition ranged from 100% in those soaked in

100 ppm. cycloheximide to only a delay in germin-

ation at 1.0 and 5.0 ppm. The other seeds tested
(pea, wheat, and bear) were inhibited less by the
100 ppm. concentration but displayed the same pro-
portional change as the concertration was lowered.
lhe rose mildew was controlled by 2% ppm., but the
increasing of the concentration to 5, 75, and 10.0
ppm. caused injury to young and new leaves. Eur-
ther investigations showed excellent inhibition in

agar plates against Honilinia fructicola, Clado-

 

sporium cucumerinum and Bones so. Also, a recheck

 

of the bean mildew trial (l9h8) resulted in con-
trol at 10.0 ppm., and no phytotoxic effects.

Another possible application for the anti-
biotic was described in igha by vgughn and Eamner
(12). As a Spray for such turf diseases as " Dol-
lar Spotss", "Brown Patch%," and "melting Cut,"
the cycloheximide appeared to be at least the e-
qual of the fungicides commonly used, and possi-
bly superior - particularly for the "Melting

A ll .‘ “
out, paused by Belminthosporium 3p.. However,

 

the spray trials were limited by changin; weath-
er, and should be repeated for more definite re-

Slllts 0

1"Dollar Spot" - caused by Sclerotinia homoeo-
gfr and the brown patchV'caused by Pel-
calaria filamentosa 3 Corticium solani.

 

 

 

F“!

de geeuw and Vaughn, in 1950, reported the cy-
cloheximide as the best apparent Spray in a single

field experiment with cucumber scab, Cladosporium

 

cucumerinum (2). The antibiotic was used at 10.0

 

ppm. and compared with Crag 658, Tribasic copper
sulfate, Dithane, and Zerlate.

As a postharvest Spray for brown rot,Honilinia

 

fructicola, in concentrations of 2.0, 5.0, and 10.0

 

ppm., four sprays of cycloheximide were compared
with liquid lime sulfur and sulfur dusts by Peterson
and Cation (8). After cold storage for two days,
and common storage for four, the peaches sprayed
with 2.0 ppm. cycloheximide showed 2% rotted, the
5.0 ppm. Sprayed peaches 0% rotted, the 10.0 ppm.
1.0 % rotted, the lime sulfur sprayed and dusted
peaches 57 rotted, and the unsprayed peaches 7%
rotted. In another test, under conditions of
heavy inoculum, a 20.0 ppm. cycloheximide spray
was compared with wettable sulfur and to grower
sprayed liquid lime sulfur. The cycloheximide
sprayed peaches showed 8h% rotted, the sulfur
sprayed 67S rotted, and the grower sprayed 89%,
rotted.

In other trials with peaches, a 20.0 ppm.
spray of cycloheximide was found to cause crack—

ing in firm-ripe Halehavens, and possibly to

dissolve the Skin coloring matter. The severity
of these actions was less with lower concentrat-
ions. The same report indicated that cyclohexi-
mide may be a superior spray for cherry leaf spot,

Higginsia hiemalis, in that 1.0 ppm. almost com-

 

pletely controlled heavily spotted Montmorency
leaves, and these same trees held a high percent-
age of noticeably greener leaves for a longer
time than the trees sprayed with "Tennessee 26",
"Ferbam," "Crag Cherry Fungicide EhlB," and "Nab-
am". ‘
Gottlieb et al, in 1950 (5) in a report based

on visual observation of growth in liquid media of

six pathogenic fungi, all but one - Monilinia fruc-

 

ticola, were inhibited by a 100.0 ppm. cyclohexi-
mide concentration. The report also stated that
in phytotoxicity tests with tomato, bean, peach,
geranium, and strawberry; the strawberry alone was
not injured by 100.0 and 1000.0 ppm. concentrat-
ions.

In Canada, eight fungi were tested for Spore
and mycelium inhibition, and for the effect of the

antibiotic on pea seed germination by Wallen et a1

(1h). Using the agar streak method of Waksman and

Reilly, spore germination varied from complete in-

(6)

hibition in the case of Ascochyta pisi Lib. at 1.0

 

ppm. to slight inhibition at 50.0 ppm. with £3213
cillium sp.. However, using the filter paper disk
method of Vincent and Vincent, which consisted of
placing disks saturated with cycloheximide near
the growing colony, no inhibition was found in con-
centrations up to 255 ppm. It was also shown that
soaking infected pea seeds in varying concentra-
tions of cycloheximide did not control the inter-
nal parasite A. pisi; but the pea seeds were in-
jured as shown by reduced germination as the con-
centration increased.

The same workers alSO investigated the ac-
tivity of the cycloheximide on germinating pea
seeds. In the lower concentrations, an abnormal,
discolored root, and a long shoot resulted, while
in concentrations from 10.0 ppm. and up, there
was little or no germination.

Recently Whiffen tested thirty-three plant
pathogenic fungi in cultire for effect from var-
ious concentrations of cycloheximide (17). If
spores were produced, suspensions were streaked
on agar plates; in the case of fungi not spor-
ulating, a mycelial disk was used as inoculum.

The concentration needed for complete inhib-

ition after hd and 72'hours was noted. The

antibiotic was found to cause complete inhibition
for 72 hours in concentrations of less than 1 ppm.

with Ustilago tritici, and ranged to 100.0 ppm.

 

for 72 hour inhibition when used against Ramularia

 

pastinaceae and Fusarium lycopersici.

 

 

MATERIALS AED METHODS

Since broad objectives were chosen with the
hope that facts concerning absorption, retention,
possible translocation or utilization, and/or re-
covery might be obtained, the experimental work
was divided into six separate parts dealing with

different phases or dissimilar organic compounds.

PART ONE

Biological Assay Technique

Inhibition of linear fungal growth on agar
plates containing varying amounts of cyclohexi-
mide*, forms the basis for assaying unknown a-

mounts of the antibiotic (see TAELE I).

* Crystalline, pure chemical used, unless other-
wise stated.

Disks, five millimeters in diameter, of Poria

 

microspora* taken from a six day old culture - were
seeded in 2% malt extract agar (Difco, dessicated),
and incubated at 300 C until the necessary measure-
ments were made. The medium was made up in single
lots, either 500ml. or 1000 m1., and the desired
dilutions made by pipetting from a standard 1000.0
ppm. cycloheximide solution into smaller flasks.
The smaller flasks, containing enouwh medium for
four plates, were autoclaved, and the medium pour-
ed into previously labelled Petri dishes. Pour
replications were used in every case. The inocu-
lum pieces cut with a curved, sharpened, brass tube
were seeded - fungus side up - as near the center
of the plate as possible. After incubation at 500
C for forty-eight hours, the first linear measure-
ments were taken with the aid of a Specially con-
structed box, illuminating the culture from beneath
so as to define the periphery of the colony.

The growing colony was measured and the growth
recorded (see TABLE I) at twenty-four hour inter-

vals for six consecutive days.

* Poria microspora : Poria monticola, recently has
become welléknown as destructive to wood in service,

(1).

 

 

(9)

PART TWO
Retention by Wood

The retention of cycloheximide by non-living
tissues was investigated by using Basswood, Tilia
glabra Vent. chips, and the bio-assay technique.

Forty Basswood chips (5/16 x 13/8 x 2 inches),
were submerged in one liter of 200.0 ppm. cyclo-
heximide for a period of three minutes. The sol-
ution remaining was retained for subsequent bio-
assay (see TABLE III) and the chips dried to ap-
proximately the original moisture content. In re-
.peated leaching trials, the chips were placed in
one liter of distilled water and shaken vigorously
for fifteen minutes. The chips were re-dried.and
the solution saved for bio-assay for cycloheximide
content (see TABLE III). The control followed the
procedure with chips not previously soaked in cy-
cloheximide, but was determined only once as there
was no evidence of either stimulation or inhibition
in the assay plates.

Chromated zinc chloride (DuPont) leaching was
also investigated. A biological assay table was
set up (see TABLE II) and leaching trials with sub-
sequent assays were attempted. The procedure was
identical to that followed with cycloheximide ex-

cept for the autoclaving. .The chromated zinc

(10)

chloride precipitates under conditions of high heat
so the chips were sterilized with propylene oxide
(6), submerged in sterile distilled water, shaken
vigorously as before, and under sterile conditions,
the leaching water was added to warm, sterile med-
ium for pouring into the assay plates.
The leaching trials with the chromated zinc

chloride were discontinued, due to the resistance

of the compound to leaching under these conditions.

PART THREE

Leutritz Technique

The Leutritz method of evaluating wood pre-
servatives (10) was employed, using varied 100.0
ppm. treatments of cycloheximide and Ponderosa
pine test blocks.

In this method, two feeder* chips,Pinus pal-

 

ustrig, were placed i inch apart on approximately

two inches of soil in flat-topped jars. The jars
were sterilized, and the feeder chips seeded with

Ioria micrgspora as an inoculum disk placed be-

 

tween the chips. After five weeks, when the chips

% Feeder chips of Pinus palustris served to "feed"
inoculum to the test blocks.

(11)

were covered with mycelium which had also pen-
etrated the soil, the previously weighed test
blocks were placed ; inch apart upon the chips
with a treated and an untreated test block in
the same jar.

The treatment of the test blocks (approx-
imately l/fl x l x 2 inches Einug BEEQEEBEE ) was
as follows:

1. Cold soak in 100.0 ppm.* crude cyclohex-
imide solution for twenty-four hours.

2. Cold soak in 100.0 ppm crystalline cyclo-
heximide water solution for twenty-four hours.
5. Hot and cold bath*% for one and one half
hours in crude cycloheximide water solution.

A. Hot and cold bath in crystalline cyclo-
heximide water solution for one and one-half
hours.

5. Hot dip (three minutes) in 100.0 ppm.

crude cycloheximide plus crankcase oil (at approx-

imately 9hOC.).

The jars were held for one hundred and twenty

* All treatments with 100.0 ppm.
as Hot and cold treatments averaging 950 C. and
cooled in the same solution.

days in a humidity chamber which averaged 280 C
and 87; relative humidity? Loss in weight by the
treated and untreated blocks is presented in TABLE

IV.

BART POUR

Synthetic vs Organic Medium

Two types of liquid medium were employed in
this part of the experimental work.-

Coon's synthetic broth (9) consisting of suc-
crose, 7.2 grams; dextrose, 5.6 grams; KNOB, 2.02
grams; MgSOu, 1.25 grams; KHQPOA, 2.72 grams; per
1000 cc. of distilled water, was used in comparison
to 2% malt extract liquid medium. Four concentra-
tions (0, l, 10, and 20.0 ppm. cycloheximide) were
used with each type of medium and four replicates
containing 50 ml. of medium per flask.

Eggggigg gp. was grown on a 2% malt extract
agar plate and cut with the 5 mm. rod for seeding
purposes. The seeded flasks were shaken contin-
uously for four days, after which the mycelium

was strained, dried and weighed (see TABLE V).

*Leutritz jars containing quercus ruhra blocks,
and tomes igniarius var. laevitagus as the wood-

 

 

 

rotter were discarded. There was nearly a 1002
failure of the fungus to spread to either the
treated or the untreated blocks from the feeder
chios.

The liquid media, and the mycelium, were retained
for future antibiotic assay, as in TABLES VI and

VII respectively.

PART PIUE
Onion Root Reaction

Onions which had been germinated for thirty-
six hours in tap water, were transferred to flat
dishes holding 100 ml. of distilled water or 100
ml. of 100.0 ppm. cycloheximide in distilled water.

Pour onions in each dish were left for periods
of 1,2,h and 6 hours in the cycloheximide solution
and in the distilled water checks. At each inter-
val the onions were removed, and the solutions re-
tained for biological assay. After a one minute
wash in running water the onions were returned to
100 ml. of fresh distilled water for a twenty-
four hour leaching period.

Growth patterns for comparison to TABLE I are
found in TABLE VIII.

Although the roots were retained for assay,
the absorption results were too indefinite to make

this step of any value.

PART SIX
Cultural Inhibition

The inhibition in culture was determined with
the method described in Part I (Biological assay
technique).

The graphs represent a single repetition with
the exception of Graph I constructed from the re-
sults of TABLE I, from which all unknown cyclo-
heximide concentrations were estimated.

is based upon drv weight of Poria

It

Graph III

microspora growth in 50 m1. of 2; malt extract

 

liquid media. Twelve flasks ior each 01 five cy-
cloheximide concentrations - 0.0, 0.01, 0.1, 1.0
and 10.0 ppm. were held for seven days at room
temperature, after which three of each concentra-
tion were removed and the mycelial dry weights
noted. weight determinations were made for four
successive days. Thus the graph is based upon
three replications, while Graphs IV thru VII of

Lentinus lepideus, Tomes igniariis var. laevig-

 

 

atus, Lenzites trabea, and Schizophyllum commune

 

 

as the reSpective fungi, are with four replicates,

and based upon linear measurements.

EXPERIMETTAL PESULTS
ihe master table (IAELL I) was established
from the constant linear growth rates of Poria

microspora on malt extract agar plates containing

 

known cycloheximide concentrations. The numerous
replicated trials showed the method to be adequate-
ly accurate to check the experimental work that was
undertaken.

The choice of the funéus was fortunate as its
hith susceptibility to the antibiotic allowed ac-
curate determinations of a small amount of cyclo-
heximioe. ihe susceptible fungus further allowed
an almost unlimited range for detection merely by
using dilutions for the higher concentrations.

The fungus served admirably also in that its my-
celial growth on agar plates is comparatively

flat and quite consistently forms an almost per-
fect circle. The malt extract (Difco, dessicated)
as a nutrient source was also found to yield re-
sults consistently within the ranges of the twenty-
four hour periods, even when made up from two dif-
ferent lots. A burette technique for dilution in-
to single plates under sterile conditions was found
to be less accurate than making dilutions 01 a vol-
ume sufficient for the replication of four.

In periodical assays of the 1000 ppm stock

(15a)

solution of cycloheximide, it was found that the
antibiotic did not lose determinable amounts of
fungicidal activity when stored in the refriger—
ator for periods up to three months.

TABLE I, and Graph I, indicate that the deter-
mination of a 0.01 ppm. conbentration is below the
scope of accuracy of the method due to overlapping
of growth ranges. However, the 0.5, 1.0 and 2.0
ppm. cycloheximioe growth ranges show clear cut, in-
dependent ranges at the twenty-four hour periods.
This gave quite accurate determinations when used
as the standard for comparison to Crowth rates in
plates cintaining unknown cycloheximide concentra-
tions.

flhe plates with 5.0 ppm. concentration showed
no growth for the six day period, but did show
slight growth aiter the sixth day and-would recover
if the inoculum was transferred to a plate contain-
ing no cycloheximide. The plates containing a 10.0
ppm. concentration, however, inhibited all growth
and the inoculum, when transferred to a pure nutri-
ent agar plate after the seventh day, would not re-
cover.

It was noted that, except in 10.0 ppm. and up,
growth on plates containing cycloheximide was pre-

ceded by a marked lighter colored diffusion zone.

(16)

TAFLE I

Growth rates of Poria microsppra in 2% malt ex-
tract agar plates containing cycloheximide.
Master table used for bio-assays

 

Rate of growth in mm.*

 

 

 

 

 

 

 

Day 2 5 h 5 6
0.0 ppm. 12.5 22.5 58.0 h7.6 61.5
Renae 9.5 17.2 25.2 57.7 5u.5
to to to to to
1h-5 25~5 39-3 h9-5 63-2
0.01 ppm. 10.7 20.1 51.6 A2.2 52.7
Range 8.2 18.5 2.14.02 56.? LL90?
to to to to to
12.2 22.7 35.2 h5.0 56.7
0.5 ppm. 0 8.7 15.7 20.7 25.0
Range 8.2 11.2 17.7 25.5
to to to to
12.0 17.5 28.5 51.7
1.0 ppm. 0 6.5 9.5 15.5 18.0
Range 5.2 9.2 10.2 15.7
to to to to
7.2 12.5 17.0 22.0
2.0 ppm. 0 0 6.0 9.0 11.0
Range 5.1 8.5 9.7
to to to
7-2 9-5' 12.7
5.0 ppm. 0 O O 0 0

 

10.0 ppm. and up - no growth.

 

* The daily measurements include the 5.0 mm.

inoculum disk, and are based upon six trials

with replicates of four.

(17)

The diameter of the zone varied inversely as the
concentration of the antibiotic.
Table II, and Graph II demonstrate the growth

rates made by Poria micrOSppra in plates containing

 

chromated zinc chloride. As previously mentioned,
the chromated zinc chloride was to be used as one
commonly employed water soluble wood preservative
in contrast to the antibiotic, cycloheximide, as a
wood preservative. Since attempts to analyze the
original dipging solution of chromated zinc chlor—
ide after the three minute dip indiCQted there was
still the original 5% solution present, and a trial
calculated 10 yield leaching iigures showed no
leaching — even when concentrated ten times, this
part of the experiment was discontinued. The table
and graph do show a possible stimulation ty very
low concentrations of chromated zinc chloride, and
a sharp, delimiting toxicity line as opposed to the
gradual suppression with cycloheximide when slight
dilutions are made.

flable II and Graph II are, however, based upon
much larger concentration variations. In ppm. the
C.2fl chromated zinc chloride would be 2000 ppm. and
the ,3% would be equivalent to 5000 ppm. The crit-

ical toxicity point for Poria microSpora in culture

 

is between 0.2% and 0.53 or 2000 and 5000 ppm. of

(18)

TABLE II

Growth rates of Poria microspora in 2% malt extract«
agar plates containing chromated zinc chloride.

 

Rate of growth in mm.*

 

 

 

 

 

Day 2 5 h 5 6
0.0% 12.5 22.5 58.0 A7.6 61.5
Range 9.5 17-2 25-2 57.7 58.5
to to to to to
lh-B 25-5 59-3 h9-3 65-2
0.1% 15.5 26.0 58.9 u8.2 60.2
Range 11.2 22.2 57.5 R5.0 59.7
to to to to to
1k.7 29.5 59.2 k8.5 66.2
0.2% 6.5 1k.6 21.7 50.5 57.7
Range 6.0 12.2 1h-2 28.2 52.2
to to to to to
7-7 16-7 26-0 55-7 39-7
0.5%
w 0 0 0 0 0
Range

 

0.h% and up - no growth

 

* Each figure represents linear measurements of
Poria microspora taken at twenty—four hour in-
tervals, and are an average of twelve plates;
e.g. they represent three trials with four
replicates.

(l9)

chromated zinc chloride, as opposed to 10.0 ppm.
or less in the plates containing cycloheximide.

TABLE III, showing results of the assay for
cycloheximide content in the solution having had
the chips submerged in it for a three minute dip,
indicateS‘that the active antibiotic content was
, materially reduced - by mechanical adsorption by
the wood, chemical alteration, or a combination of
these factors. Two dilutions, made for 0.5 ppm
and 1.0 ppm. growth rate comparisons to TABLE I,
show that very little over 100.0 ppm. cyclohexi-
mide remains from the original 200.0 ppm concen-
tration. The dilution made for a 1.0 ppm. read-
ing fits the 0.5 ppm. dilution as found in TABLE I,
and the 0.5 ppm. dilution of the dip solution falls
just below the 0.01 ppm. range. The two, cross-
checking each other, lead to the conclusion that
100.0 of the 200.0 ppm. cycloheximide was retained
by the wooden chips.

Leaching trials I through V, in which the un-
diluted leaching water was used in the medium, in-
dicate a leaching of between 1.0 and 2.0 ppm. for
the first leach (l), slightly over 0.5 in (2),
slightly less than 0.5 ppm. in (5), and progres-
sively less in trials (h) and (5). The original

solution, diluted_for 1.0 ppm. reading, falls

(20)

TABLE III

Linear growth rates of Poria microsporg on media
containing cycloheximide leached from Tilia glabra

 

 

chips.

Rate of growth in mm.

 

 

 

 

Day 2 3 u 5 6
s 11ti
bifdreogip 0 6.2 9.0 15.0 19.2
Solution ‘ h
after dipel) 8.2 12.5 21.0 25.5 52.5
2) 0.0 8.2 15.2 19.2 22.7
Leaching
trials
1 0 5.7 7.5 10.2 15.2
2 O 8.2 12.2 1707 2007
5 6.0 12.0 20.2 25.5 50.2
h 6.5 15.0 21.0 27.2 5u.7
5 '8.0 16.0 22.2 51.2 59.5

 

* Solution after dip: l)- diluted for 0.5 ppm.
reading. 2)- diluted for 1.0 ppm.
The solution before the dip was diluted for
a comparison with 1.0 ppm. in Table I, all
others were assayed undiluted.

(21)

perfectly in that category in TABLE I.

The results of a modified Leutrita technique
(10) experiment are shown in TAELE IV. Treat-
ment (5), the 1% hour hot and cold bath in water
solution of the crude antibiotic, gave evidence
of cycloheximide being an excellent water soluble
wood preservative. The twenty-four hour cold soak
solution prepared from the crystalline product,
(Treatment 2), yielded results which indicated that
the antibiotic was valueless as a preservative, as
the treated blocks were more severely rotted than
the untreated in both replicates. The twenty-four
cold soak in crystalline prepared solution (Treat-
ment A) was also found to be.an ineffective wood
preservative. Although the treated blocks lost
less weight than the untreated, the resulting fig-
ures were inconclusive. Treatment (5), an oil
base cycloheximide solution, was entirely incon-
clusive, varying between the replicates.

All results may have been affected by the high
humidity at which the jars were held, particularly
as the antibiotic was used as a water soluble wood
preservative. High humidity may lead to a higher
loss in weight percentage when using the Leutritz
technique (10, page 11).

Northern white cedar, Thuja occidentalis é.

 

(22)

TABLE IV

Percentage loss in weight of Quercus rubra blocks

 

after being subjected to rotting conditions used

in the Leutritz technique.

Treatment*

(1)

% loss in weight

 

 

 

 

Cold soak in crude cycloheximide Treated 11.5
solution for 2h hours. Check 67,8
Treated h.l
Check 61.6
(2)
Cold soak in crystalline cyclo- Treated 15u,7
heximide solution for 2h hours. Check 99.7
Treated 70.5
Check h8.1
25) _
Hot and cold bath in crude cyclo- Treated 1.6
heximide solution for 15 hours. Check 109.9
Treated 1.9
Check 72.9
(A)
Hot and cold bath in crystalline Treated 8Z.0
cycloheximide for 1% hours. Check in .9
Treated 2.1
Check .9
(5)
Hot dip (5 min.) in crude cyclo- Treated 5 .3
heximioe plus crankcase oil. Check .8
Treated , 109.2
Check 118.

 

* Test blocks removed after 120 days in a humidity

chamber averaging 28°C. and 87% relative humidity.
All treatments with 100 ppm. cycloheximide - Poria
microspora as the test fungus.

 

fence posts were given treatments identical to those
listed for the Leutritz trial, and were placed be-
neath greenhouse benches in four replications. After.
seven months, visual observations yield no indica-
tions of the superiority of one treatment over the
others.

In TABLE V, in which increase in mycelial
weight of Fusarium pp. in synthetic and organic
liquid media is compared, the Coon's synthetic me-
dia is shown to be superior to the 2% malt extract
when used with no cycloheximioe concentration. Al-
though there is a greater weight increase in the
synthetic medium flasks containing 0.0 ppm. cyclo-
heximide, the flasks of this same medium containing
10.0 ppm and 20.0 ppm. cycloheximide show consider-
ably less mycelial growth than in the 2% malt ex-
tract with a 10 and 20 ppm. cortent.

The synthetic medium is evidently superior to
the malt extract liquid medium when the antibiotic
is not present, but inferior for producing mycelium
when the antibiotic is added, 1.6. the fungus is
less inhibited in 2% malt extract medium containing
cycloheximide than it is in the Coon's synthetic
medium containing the same amount 01 cycloheximide.

The difiicult analysis 0' TABLE VI, in which

the antibiotic assays of the two media is presented,

(2h)

TABLE V

Comparison of Fusarium 13. dry mycelial weight
increase grown in two different media contain-
ing cycloheximide.

 

 

 

 

 

ppm. Coon's Synthetic 2% Malt Extract
% . Grams % Grams
Increase Increase Increase Increase
0.0 h200.0 0.071h 3hll.7 0.0580
1.0 3929-h 0.0668 5652.9 0.0621
10.0 2u05.8 0.0u09 269h.1 0.0u58
20.0 2017.6 0.05u5 2576.u 0.0u58

 

yields evidence of antibiotic presence, but also the
presence of at least one possible metabolic product.
The synthetic medium originally containing 1.0 ppm.
cycloheximide, and the synthetic medium check, which
were diluted one part of substrate with one part
fresh 2% malt extract medium, showed no evidence of
either cycloheximide or a metabolic product. Both
fit well into the check range of the assay table
(TABLE I). If the antibiotic were present in 100%
active forn,‘the original l.0 ppm substrate assay
should fit the 0.5 ppm. range in TAELE I. The malt
extract check medium however, gives evidence of the
presence of a toxic metabolic product when assayed'
in the same manner. Both the check and the orig-
inal 1.0 ppm. medium gave considerable inhibition.
The check media for the 10.0 and 20.0 ppm. cy-
cloheximide flasks, were diluted one part of sub-
strate to nine parts of fresh and one part to nine-
teen parts of fresh medium respectively. The growth
on assay plates shows definite stimulation of the
linear growth rates when compared to the check
growth in TABLE I. Stipulating that the stimula-
tion is not only acting upon growth in the check
media flasks but also upon the noticeably inhibited
growth on the plates containing media from the 10.0

and 20.0 ppm cycloheximide flasks, TABLE VII is

(26)

TABLE VI

Growth rates of Poria microspora on 2% malt extract
agar containing fifibstrate Tram two oiflerent media
which had supported iunpal growth in cycloheximide
concentrations.*

 

Rate of growth in mm.

Coon's Synthetic

 

 

 

 

 

 

Day 2 5 u 5 6
from flasks
containing
1.0 ppm. 11.0 22.7 55.5 05.2 u8.5
0.0 ppm. 15.0 20.5 57.0 07.5 59.0
10.0 ppm. 7.5 16.5 25.5 29.5 55.0
0.0 ppm. 16.0 28.0 uu.0 55.5 6u.2
20.0 ppm. 5.5 11.5 19.0 2u.7 29.5
0.0 ppm. 18.2 50.0 u5.6 5.7 6h.5
Malt Extract
1.0 ppm. 7.0 11.5 20.0 28.2 55.5
0.0 ppm. 7.2 12.0 21.2 52.0 no.0
10.0 ppm. 10.7 20.7 29.7 57.2 A5.2
0.0 ppm. 17.0 28.2 ui.7 5u.2 67.0
20.0 ppm. 10.5 19.7 28.7 56.5 85.5
0.0 ppm. 17.0 50.2 hl.7 5b.? 65.?

 

* Media in which cultures had been grown was di-
luted, hoping to avoid action by possible met-
abolic waste products, i.e.; original 1 ppm.
media diluted 1 to l to yield 0.5 ppm. cyclo-
heximide if the original amount is available,
and check media also diluted 1 to 1.
ppm. diluted (1 to 9 and l to 19) to yield 1
ppm. and comparable dilutions with the 0.0 media.
Dilutions made with fresh 2% malt extract. Re-

plicated four times in all cases.

10 and 20

(27)

TABLE VII

Revised liquid media assay showing growth rates
after removal of the stimulation factor.

Coon's Synthetic

 

 

 

 

 

10 ppm. (day) 2 5 ll 5 6

% stimulation 28 25 16 12 u
Revised rate 5.h l2.h 19.7 26.0 55.6
20 ppm.

% stimulation u5 5k 15 1h 5
Revised rate 5.0 1.6 16.1 21.2 27.0
2% Malt Extract

10 ppm.

% stimulation 56 26 10 1h 9

Revised rate 6.6 15.5 26.] 452.0 hl.5

20 ppm.

% stimulation 56 55 10 15 7
'Revised rate 6.6 12.8 25.8 51.0 h2.§

 

presented with figures showing the percentage stim—
ulation in the check plates and that percentage
subtracted from the stimulated growth as found in
TABL: VI. After the elimination of the stimulation
factor, the assaLS, all of which were diluted for
comparison at 1.0 pom. concentration in TAELE Vii
are as follows: the 10.0 ppm. cycloheximide in
Coon's synthetic medium assays at slightly less
than 0.5 ppm. indicating that 50% of the cyclohexi-
mide was unavailable or altered; the 20.0 ppm. syn-
thetic medium assays directly in the 0.5 ppm. range
in T ELE 1, indicating that 75% of the activity of
the antibiotic was missing. The 10.0 ppm. cyclo-
heximide in malt extract assay shows a concentra-
tion of the antibiotic slightly more than 0.01 ppm.
cycloheximide, indicating that 0.1 ppm was present
or approximately 99% was inactive. The 20.0 ppm.
in malt extract assays at nearly the same level as
the 10.0 ppm. flask. The difference between the
theoretical 0.1 ppm. present in the supposed 10.0
ppm. malt extract medium and the 0.2 ppm. in the
20.0 ppm. cannot be determined by the method used.
It is obvious that there is much higher retention
of the antibiotic by the malt extract medium. The
possibility that the antibiotic was previously

utilized by the fungus is unlikely, since the

synthetic medium was the more suitable medium when
no antibiotic was added.

Since the media retained or altered the anti-
biotic, or it was taken up by the fungus, an assay
on the dried mycelium was made. The results in
TABLE ViII indicate that the cycloheximide was not
present, in detectable amourts, or was altered so
as to eliminate any chance of anti—fungal action in

’the assay. Had the 0.1 grams of mycelium added to
the 100 ml. of media been one thousandth part pure'
cycloheximide, it could have been assayed as an
approximate 0.1 ppm. concentration. The Lrowth
rates all fall within the check range as found in

Ta

m
m

The results in TAELE X indicate that no anti—
biotic was taken up by the onion roots when they
were immersed in the 100 ppm. cycloheximide for
the 1,2,h and 6 hour periods. However, the water
in which the roots were leached for twenty-four
hours and assayed undiluted, gave indications as
follows: the one hour dip assayed very close to
0.5 ppm., two hour dip at 0.5 ppm., four hour dip
at slightly over 1.0 ppm. concentration, and the
six hour dip at 1.0 ppm. cycloheximide concentra-
tion. .

V

The results indicate that very little, if any,

(50)
TABLE VIII
Assay of mycelium grown in two different media

containing cycloheximide.*

Cgpndgngynthetic

 

Rate of growth in mm.

 

Day 2 5 Ll i 6
From flask
containing
0.0 ppm. 15.5 2b.? 55.5 u8.5 62.5
1.0 ppm. lh.5 26.0 55.0 i7.7 60.0
10.0 ppm. 11.0 21.0 28.7 58.7 52.7
20.0 ppm. 10.3 12.Z_i 29-Tn_ L2.Z 55.5

 

Malt Extract

 

0.0 ppm. 10.7 21.5 52.0 u5.0 56.2
1.0 ppm. 10.0 19.2 29.7 A2.0 56.0
10.0 ppm. 11.2 20.5 51.2 u5.5 57.5
20.0 ppm. 15.2 26.0 56.2 A6.7 59.7

 

* The washed mycelium from the Coon's synthetic

broth and from the malt extract liquid media

were collected after weighing, pulverized, and

0.1 gm. added to 100 m1. of 2% malt extract
agar for the assay with Poria microspora.

 

of the antibiotic was taken up, but positive evi-
dence of inhibitory material, possibly cyclohexi-
mide in the leaching water, is shown with the tech-
nique used.

when diluting one milliliter from the 100 ppm.
original soaking solution to 99 of pure water, the
possibility arises that ten, twenty, or more ppm.
could have been taken up and that the 0.8 or 0.9
ppm. could not be differentiated from 1.0 ppm. by
the bio-assay technique. in the assay of the leach-
ing water, however, the undiluted use would allow
assaying of the small amounts.

The indications are therefore, that the amount
of antibiotic taken up in the original dip could
not be detected by dilution from a concentration as
high as 100 ppm., but that 0.5 ppm. was leached
from the one hour and two hour dips, and 1.0 ppm.
from the four and six hour dips. Unfortunately the
lack of data as to the quantity absorbed allows no
conclusions as to whether all or only a percentage
of the antibiotic was removed in the subsequent
leaching process.

The assay of the check plates gave no evidence
of stimulation or inhibition from the presence of
the onion roots for the 1,2,; and 6 hour periods.

Graph 111 based upon the dry weight of mycelium

.UopsHHnds finance momma oucH oopmaomaoodm *$
.H mqm4e dH somHAmQEoo .Edd o.H you oopSHHm *

 

 

 

o.mm m.ae m.Hm m.am o.mH s m m.am m.na s.ma m.m o s m
0.:m o.H: m.om e.om pros s : o.ma e.ma e.m o o s :
m.mm o.mm m.mm m.mm ~.ma s m o.mm m.Hm o.ma ~.m o s m
n.mm m.4: o.mm o.mm m.aa .mg a s.mm m.am o.ma o.ma m.m .sm a
sammmm 5.3mm
noon :m axon :m
0.3m 0.4: s.am m.am o.aa s o m.mm m.sa o.ma m.s o s p
o.em m.m: s.am 0.0m m.aa s : m.am m.ea m.ma o.o o s o
o.mm m.s: o.mm m.:m >.ma s m m.mu s.mw 0.3H e.o o s m
».mm s.»: 0.0m 0.4m m.ma .mg a m.nm o.ma 0.:a ~.m . 0 .mm H
case same
e m n m m an o m in m m to

 

*amXconc momma noHHHpmwm *mfip .Edm 0.00H

.QOHpSHom wsmsewoa m no no man pooh Cease Cm mm pond seen on: mean; doapoanm
ecfiEHxQSOHoho m2wcfimpdee mopwam amom pomauxc mama Rm do whoomoaOHE mfiaom no mopma apnoea

 

xH mqm<8

”-1 AV

sour-x“- ' '9‘

A
\ H
\N
V

grown in liquid 2% malt extract media, shows a grad-
ual suppression of total growth — grading through

the suppression of a growth peak - to total inhib-
ition in 1.0 and 10.0 ppm. cycloheximide. The graph
also demonstrates that the antibiotic is more effect-
ive in the liquid media which contains no agar.
Growth is not totally inhibited in agar plates con-
taining the same, 1.0 ppm. concentration of the

cycloheximide (see TABLE

H

and Graph I).
Graphs IV through VII show the inhibition by
cycloheximide of four common wood-rotting fungi.

All except Schizophyllum commune were totally in-

 

hibited by the 2 ppm. concentration.

DISCUSSION

The bio-assay technique developed for use in sub-
sequent trials probing the action of the antibiotic,
was found to be an accurate standard for determining
very dilute, unknown cycloheximide concentrations by
comparison of growth rates in agar plates.

The Leutritz technique showed that the crude cy-
cloheximide was an effective wood preservative. How-
ever, despite the high retention by wood, and extremely
high toxicity of the antibiotic to wood rotting fungi,
crystalline cycloheximide treatments were not as con-
clusive as the treatments with the crude product. In
a leaching comparison, chromated zinc chloride gave
evidence of resistance to leaching, but needed two
thousand times the concentration for the same cultural
inhibition as cycloheximide.

Possible explanations for the inconclusive re-
sults are found in the very high humidity at which the
jars were held, and in the fact that the extremely
toxic substance leaches out in amounts sufficient to
permeate the soil water with a lethal dosage of the
antibiotic.

An explanation for the superiority of the crude
product as a preservative should be sought. Also,
since the leaching trial was undertaken using the

crystalline antibiotic, there is a possibility that

(35)

the crude cycloheximide preparation would not yield
the same leaching results.

The results also indicate that cycloheximide may
be of value as a killing agent to be added to inorganic
preservatives and water repellents.

The experimental results also showed that the cy-
cloheximide was-not as active as an inhibiting agent
when in a highly organic media. Greater fungal growth
was found in a synthetic broth containing only sucrose
and dextrose as carbohydrate source, than in a 2% malt
extract media. It was definite that one or more sub-
stances found in the malt extract - proteins, various
carbohydrate and nitrogen sources, and enzymes or
prowth regulators - served to alter or retain the anti—
biotic. Agar also seemed to retain the cycloheximide,
as a.lD ppm. concentration would inhibit all growth in
a liquid medium, but would not totally inhibit in the
same medium in agar plates. There was however no
apparent recovery of the cycloheximide from the dried
mycelium which had obviously absorbed and altered or
'closely retained the antibiotic.

The above facts indicate that further work could
narrow the number of organic compounds showing high re-
tention, and might yield more valid recovery data if a
larger mycelial mass was assayed. A leaching study on

growing mycelium should also be undertaken.

The lack of positive absorptive results when
onion roots were dipped in 100.0 ppm. cycloheximide
solutions for one, two, fcur and six hours suggests
that more valid results might be gained by lowering
the concentration of the original dipping solution.
The lower initial concentration should allow an assay
if only 10.0 or 20.0 ppm. cycloheximide are taken up
by the growing tips. This could be aided further by
the use of more onion roots per 100 m1. of solution
to increase the proportional surface area. Valid re-
sults as to the amount of removed antibiotic activity
compared with that leached from the roots would yield
the quantity actually held by the roots.

The onion root absorption was undertaken, not
only for purposes of determining action of the anti-
biotic when in contact with the growing tips of higher
plants but with the hope that its absorptive power
could be correlated with cytological aberrations found
to be caused by the antibiotic. Wilson (18 1950) found
that cytological recovery of the antibiotic was impos-
‘sible from initial dippings in concentrations up to 200
ppm. cycloheximide. Also, the cytological results in-
crease after the onion roots are removed from the cyclo-
heximide solution and placed in the pure water.

As previously mentioned, a low concentration in the

original onion root dip solution should yield a valid

(37)

assay as to the quantity of the antibiotic absorbed;
this coupled with the assay on the leaching water
could lead to correlating graphs constructed from
cytological data and from the retention data.

Another line for possible continued study would
be the investigation of the noticeably lighter colored
zone produced by the fungi growing in the malt extract
agar plates containing cycloheximide. There is a
possibility that the zone represents removal or alter-
ation of the antibiotic and is a diffusion gradient
established by the fungus prior to its linear growth.
Whatever the cause, it should be investigated for cy-

cloheximide content.

CONCLUSIONS

It was found that with a biological assay using

Poria microSpora and 2% malt extract agar, it was

 

possible to detect cycloheximide concentrations in
amounts as low as 0.5 ppm.

The cycloheximide was shown to be forcibly ab-
sorbed by the wood used and could be leached out in
quantities very near 1.0 ppm.. Despite the high re-
tention and toxicity, the crystalline antibiotic was
not an effective wood preservative in all trials at-
tempted, however the crude preparation showed excell-

ent preservative properties.

Cycloheximide was also shown to be somewhat less
effective as an inhibiting agent when used in highly
organic media. The assay of the medium in which the
funLus was grown indicated that from 50% to approximate-
ly 99% of the antibiotic was removed or inactivated by
the growing fungus. The synthetic medium containing
cycloheximide suppressed fungal growth more than the 2%
malt extract medium with the same antibiotic content.
The negative results in assaying the dried mycelium is
an indication that antibiotic activity of the cyclohex-
imide was altered or destroyed.

The antibiotic also gave indications of being ab-
sorbed by growing onion roots. The amount absorbed was
impossible to determine due to the high initial concen-
tration, but the leaching data showed that 0.5 ppm. cy-
cloheximide was leached from the one and two hour dipped
roots, and 1.0 ppm. from the four and six hour dips.

An assay of onion roots treated in this manner indicated
that the amount of cycloheximide present was too small
to be assayed, or that the antibiotic was in an altered,
non-toxic form.

Substantiation of the data indicating that the anti-
biotic is considerably less active when in contact with
organic matter, was found in the replicated liquid

culture growth of Poria microspora. The funtus was

 

totally inhibited by a 1.0 ppm. concentration in the

(59)

liquid culture, but not by this same concentration in
agar plates.

Cycloheximide was shown to be highly toxic against
four representative wood-rotting fungi, using linear
measurements on agar plates as a basis for comparison.
Three of the four were totally inhibited by a 2.0 ppm.

concentration.
ESIJiiiiiifilf

Pertinent facts brought out as a result of this

work are as follows:

1. The antibiotic can be assayed biologically for
concentrations as low as 0.5 ppm..

2. Cycloheximide was extremely toxic to wood-
rotting fungi in very low concentrations, and
was an effective wood preservative in two
trials, but of doubtful value in others.

3. Cycloheximide is forcibly absorbed by wood
tissue, and released in minute quantities on
subsequent leaching in water.

A. The antibiotic is less effective as a fungal
inhibitory agent when in highly organic media

as compared to a synthetic medium.

5. Cycloheximide is absorbed by growing onion roots,

and can be recoVered by leaching these growing
0

roots.

(1+0)

6. Recovery of the antibiotic from dried my-

celium and dried onion roots was not

possible under the concitions of the ex-

periment.

Suggestions for further research are as

follows:

a.

Ca

d.

Study of possible absorption,
translocation, and recovery in
higher plants.

Repetition of wood-rotting tests

to prove or disprove that crude
cycloheximide was superior to the
crystalline product for wood pre-
servation. A leaching study with
the crude product would be valuable.
Investigation of possible selective
retention by organic compounds in
nutrient media.

Study of the mass effect upon re-
tention by mycelium and higher plant
tissue.

Attempted recovery from larger masses

of organic tissue.

Diameter in millimeters

(1+1)
Graph I

Linear growth of Poria microspora in 2% malt extract
agar plates containing cycloheximide.

 

 

 

0.0 ppm. cycloheximide
0.01 " " '
O. II I!

1.8 .. ..

2.0 II II

Diameter in milliliters

(42)

Graph II

Linear growth of Poria microspcra in 2% malt
chromated zinc

 

 

extract agar plates containing
chloride.

4'

 

 

 

and up

Weight in milligrams *

(1+5)
Graph III

Cultural inhibition of Poria microspora in 2%
malt extract liquid media containing cyclohex—
imide.

 

 

 

“‘-‘ 0.0 ppm. cycloheximide

n...- 0.01 n u

..._...o 0.1 N I!

~---- 1.0 and 10 ppm. cycloheximide

* Increased to mg. for graphing.

Diameter in millimeters

 

(1:14)

Graph IV

Linear growth of Lentinus lepideus in 2% malt

 

extract agar plates containing cycloheximide.

 

 

ppm. cycloheximide
II
n

II
II II

NHOO
o O O o
OOKDO

Diameter in millimeters

('45)
Graph V

Linear growth of Fomes igiarius var. laevigatus
in 2% malt extract plates containing cycloheximide.

 

 

 

 

“—-' 0.0 ppm. cycloheximide

—.___- II II
.._...._0 3:3 II II
A II II

_" 2.0

Diameter in millimeters

(A6)
Graph VI

Cultural inhibition of Lerzites trabea in 2% malt
extract agar plates containing cycloheximide.

 

 

 

ppm. cycloheximide
II II

II II
II II

.o
.5
.0
.o

O
NI—‘OO

Diameter in millimeters

(1+7)
Graph VII

Cultural inhibition of Schizophyllum commune in
2% malt extract agar plates containing cycloheximide.

 

 

 

ppm. cycloheximide
II II

BIBLIOGRAPHY

1. Davidson, Ross W., Frances F. Lombard and Ray R.
Hirt. Fungi causing decay in wooden boats. Myco-
logia, WIX, No. 5, 315-527, iay- June 19K7.

 

 

2. de Zeeuw, Donald J., and John R. Jaué hn. An anti-
biotic of potential value in field control of“ cuc-
cumber scab. Plant Dis. Reptr. Vol. 5E, No. 17 Jan.
15. 1950-

 

 

 

5. Pelber, Irma M., and C. L. Hamner. Control of mil-

dew on bean Elants b means of an antibiotic. Bot.
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h. Ford., J._I., and E.E. lea ch. flcti d1 one, an anti-
biotic Irom btrentomlces gri seus. Jour. Am. Chem.
SOC. (0.1245-1cc6, 194;'.

 

 

 

5. Gottlieb, David, H.H. Hassen, and M.E. Linn.
Acti-dione as a plant protectart. Phytopath. hO:
21o - 21;, Le

 

 

C1.

6. Hansen, H.i., an Wm. C. Snyder. Ca
ilizati :n oi biological naterial for use as culture
1 _____.-

 

.. '-_....._ L _ .-. _‘
mec a. antooath. 27: :5) - ‘{U, may

\

". Le ach, E.L. J.H. ford and Alma J. Whifien.

’ ’
Acti dione, an antihiotic from Strepiomgces griseus.
Jour. Am. Chem. ooc. by: E7h - 475, 1947.

 

 

 

3. Petersen, D., anc D. Cation. Ix ploratorl ex-
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oI peach brown rot and cherrv leaf spot. Plant
DLseg se Reoorter, Vol. 3!, no. 1, Ta n. 15, 1950.

 

 

 

 

 

 

9. Rawins, Thomas E. thtotatholopical Methods.
John Wiley & Sons, Inc., London, 173:, page 92.

 

lC. Richards, C. Audrey, and Ruth m. Addoms. Lab-
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Preliminarl compari.son 23 agar and soil culture
technigues using imp*eanated wood blocks. re—

print - Amerlc:n IOOO‘ -Preservers' Association. 19h7.

 

 

 

ll. Unpublished.

EIELIOCRAPEY

12 . Vaughn, J. R., and C. L. hamner. Che present
status LI cycloheximlce (Acti-dione) as a fungicide.
PPDC. JL' 0 UJCQ 110171.. bCLSDCG, V'Qlo ELL, 1914.90

 

 

 

15. Vaughn, J.R., Dockwood, J. L., G.S. Randwa, and

 

 

 

Chis. firmner. ”he actl on oI Acti- ~di one Ln plant
tissue and upon certain IurLi. Euich. Agr. xper.
Eta. ¢;art.1u1., V131 :1, no. _ ,hSé-né)i, ay 19h9.

1a. mallen, v. R., 3? Sutton, and A. J. Skolko. Ihe

ggiect LI Acti- -dione on the prowth of certain patho-
EPJC Iungi and Ln the germination LI ea seed.

bhytopath. ‘hO: no. 2, 156-160, reb.195

 

 

 

 

 

15. Whiffen, Alma J., N. Bohonos, and R.L. Emerson.
Uhe product on LI an anti- Iungal antibiotic bv’Strep-
tcumyces griseus. Jour. Bact. 52: 010-611, 19 o.

 

 

 

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18. \Vilson, G. B. Erom conversation at Michigan State
COllegeo

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