PHYSIOLOGICAL STUDIES AND TRIAL APPLICATION
OP ANTI-FUNGAL ANTIBIOTICS FOR THE CONTROL
OF PLANT DISEASES

In vitro studies by
S.M* RingeX and E*S. Beneke
In vivo studies by
C.B. Kenaga and R.L. Kiesling

Department of Botany and Plant Pathology
Michigan State College

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TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS.............

1

INTRODUCTION
A.

Introduction of in vitro gectfon.....................

2

B.

Intoduction of in vivo section

......... .............

4

In vitro section.....................................

6

Is. vivo section

8

LITERATURE REVIEW
A.

.....................................

IN VITRO METHODS AND RESULTS
A.

Assay testing of antifungal materials ................

11

B.

Stability tests on pyridinethione, rimocidin
and diaphine .......................................

15

C.

Physiological studies with Colletotrlchumphomoides C80A

IN VIVO METHODS AND RESULTS..................................

17
2?

DISCUSSION AND CONCLUSION
IS. vitro studies........................................

39

In vivo studies

4-5

................

♦.........

SUMMARY

A-

In. vitro section......................................

A?

In vivo section.................

49

SELECTED REFERENCES.........................................
APPENDIX

(Plates)

.342.9S8

50

1

ACKNOWLEDGMENTS
The authors wish to express their sincere appreciation to the
Horace H. Rackham Research Endowment for the grant that has provided
the financial assistance for research in antibiotics active against
nlant pathogens.
Sodium pyridinethione, nystatin and M^575» were furnished by The
Squibb Institute for Medical Research, Hew Brunswick, Hew Jersey;
rimocidin, thiolutin, fungistatin X G ,and diaphine were obtained from Chas.
Pfizer and Company, Inc., Brooklyn, New York; and candicidin A was supplied
by the Department of Microbiology, Rutgers University, New Brunswick, New
Jersey.

A large number of the vitamins were supplied by Hoffman-LaRoche,

Inc., Nut ley, New Jersey.

Panogen was supplied by Panogen, Inc., New York.

The writers are indebted to the Department of Entomology for
generously offering the use of a constant temperature room to house
the shakers.

2
INTHODUCTIOH

I n t r o d u c t i o n of in vitro

section.

D u r i n g the nast decade e xt ensive efforts have

been d i r e c t e d toward

the d e v e l o p m e n t of a n t i b a c t e r i a l compounds to control h u m a n and other
a n i m a l diseases.

U nti l v e r y r e c e n t l y little a t t e n t i o n has b e e n dir e c t e d

t ow ard the d e vel op men t of a g ood a n t ifu nga l substance from a n antibioticr
source to co n t r o l plant d iseases due to fungi.

T he

same s it u a t i o n is

true for h u m a n dise as es caused b y fungi.
R e c e n t l y A n d e r s o n a n d G-ottlieb ( 2 ) p u b l i s h e d a survey of the litera­
ture

c o n c e r n i n g antibiot ic s

diseases.

last year,

that m a y be of v alu e

Klomparens a n d Vaug hn* r e p o r t e d the results of

the ir studies on the effect s of three antibiotics,
and

streptomycin,

in c o n t r o l l i n g plant

actidione,

endornycin,

on B o n n i e B e s t Tomato v a r i e t y in ocu lat ed w i t h A g r o b a c ­

terium tumefaciens.

Actidione

in the soil d i d reduce the amount of

i n f e c t i v i t y of the b a c t e r i a a l t h o u g h in all cases gall f o r m a t i o n con­
t i n u e d to develop.

The effect

c a r b o n sources, dextrose,

of fu ngicidal a c t i v i t y w i t h different

lactose a n d maltose,

P u s a r i u m l y c o p e r s i c i was g r o w n in

was f oun d to v a r y w hen

liquid medium.

A c t idi one was f o und

to be more fun gic i d a l w h e n the m e d i u m co nt a i n e d lactose as

the c a rb on

source.
D u r i n g the current 7rear1s studies of an ti f u n g a l substances active
a g a i n s t plant pathogens,
in vivo studies.

there are two distinct ph a s e s — in v i tro a n d

In the first phase,

in a g a r and l i qui d m e d i a to d e te rmi ne

a n umb er of an tib i o t i c s were tested
the effectiv ene ss of the c om pou nd

*
U n p u b l i s h e d report on "Studies of the p r op ert ies of a n t i b i o t i c s —
tr a n s l o c a t i o n a n d p h y s i o l o g i c a l studies w i t h cyclo hex im ide (Acti— d i o n e ),
endornycin, and str ep to m y c i n s u l f a t e . ” sub m i t t e d for the grant from
the Horace H # R a c k h a m R e s e a r c h E ndo w m e n t in 1953» D ep art m e n t of B o t a n y
and Plant Pathology, M i c h i g a n State College.

3
against

the test organisms.

The s t a b i l i t y of the compounds were also

teste d u n d e r v a r i e d e n v i r o nm ent al conditions..

Af t e r these p r e l i m i n a r y

p r o c e d u r e s were done, p h y s i o l o g i c a l studies were u n d e r t a k e n to d e t e r ­
m i n e what effect
condi t i o n s .

the a nti f u n g a l

substance has u n d e r vario us

c o ntr oll ed

4
Introduction of in vivo section
The testing of antibiotic materials of possible antifungal activity
increases in importance as new and perhaps better materials are dicovered.
To date actidione is the only antibiotic material applied to plants for
the control of fungal plant diseases on a commercial basis.

There are

many other antibiotics which are currently being investigated for their
commercial applicability against fungal plant diseases.

Sometimes these

compounds are specific against the causal agent of a plant disease which
is poorly controlled.

However, in vivo tests must be made to determine

the interaction of pathogen, host, environment, and antibiotic.

Surface

and systemic applications are run against the different types of plant
parasites.

Both protective and eradicant foliar applications are carried

out.
The use of antibiotic materials in plant protection programs raises
important questions.

In addition to tests on the activity, stability,

availability, and sticking qualities, tests must be made to determine
host reaction to one or more applications of the antibiotic material.
Another important question is whether these materials are fungistatic
or fungicidal.

Any fungal pathogen used in connection with these anti­

fungal antibiotics might produce lines resistant to these materials.

In

one hundred years of spraying and dusting with the wide spectrum organic
and inorganic fungicides, no resistant fungal lines have arisen.
Therefore, checks must be made to determine if and how fast such lines
might appear if antibiotics are used.

Such antibiotic resistant organisms

are already known in bacterial pathogens of animals.
Because of the nature of in vivo tests in the greenhouse, certain
limitations of space, suitable hosts, pathogens, and time exist.

5
Therefore, hosts such as common garden vegetables and small grain plants
are used; and the test organisms are those which cause foliar disorders.
However, as new tests are made, more organisms are tested since some anti­
biotics are specific in their action against certain fungi.
Not all antibiotics which appeared desirable could be used.

Some of

the compounds were not available in quantities large enough to allow
spray tests to be made.
available.
alcohol.

Others were discontinued and were no longer

Certain antibiotics were not soluble in water or dilute ethyl
Therefore, unless they could be suspended or supplied as a

soluble salt, they were not used.
The in vivo section of this report shows the progress which has been
made in one year.

The data presented is not conclusive, but rather in­

dicates some very interesting lines of work to be investigated.

Among

these problems are the tocicity tests on some of the dicots and the
investigation of lines of causal agents after they were sprayed with anti­
biotics.

With more information on the in vitro stability and temperature

effects, further refinements and interpretations may be made of some of
the spraying results.
need additional study.

Eradicant properties and run off of spray materials

6
In V i t r o L i t e r a t u r e R e v i e w
An exte nsi ve

l ite rature r e v i e w was c o n d u c t e d up to September,

N o p u b l i s h e d i n f o r m a t i o n was a v a i l a b l e c o n c e r n i n g the pos si ble
nutritive
com pou nds .

1953 •

role that

s u b sta nce s might have in a f f e c t i n g the e f f i c i e n c y of a n t i f u n g a l
K l o m p a r e n s a n d Vau g h n * foun d that a cti -d i o n e h a d inc re a s e d

a n t i f u n g a l a c t i v i t y w h e n ffusarinm lvcopersici was g ro wn in l i qui d m e d i u m
c o n t a i n i n g la ctose as the car boh y d r a t e source.

In addition,

they also

o b s e r v e d the same to be the case for endornycin v/hen maltose was
ca r b o h y d r a t e source.
influence

the

E n v i r o n m e n t a l factors other than n u t r i t i o n m a y

the a c t i v i t y rate of a n antibiotic.

L ebe n (26)

in 1 95^ found

that p H a lon e plays a part in r e t a r d i n g p h y t o p a t h o g e n i c fungi w h e n acidic
b u f f e r s are e m p l o y e d I n in, vitro tests.
Hil l b o r n (2h)
pl a n t s
et al

f o u n d R h i z o c t o n i a and Ver tic i l l i u m infections

in tom ato

to be c o n s i d e r a b l y r e d u c e d b y u si ng r i m o c i d i n or thiolutin.

Davisson

(12) r e p o r t e d that r i m o c i d i n inhibits m a n y of the hu m a n pa t h o g e n i c fungi

in v i t r o at c o n c e n t ra tio ns
Onndicidin,

of 1 to 5 ppm.

a new a n t i b i o t i c d i s c o v e r e d b y Le che v a l i e r and his co-workers

(3 1 ), is r e p or ted by them to h ave an a ctivity spectrum a gainst yeasts but
it is only p o o r l y a cti ve against yeas ts but
the filamen tou s

it is only p o o r l y active agai nst

fungi a n d no a c t i v i t y against

report this compo und to be v er y soluble

the bacteria..

in water but it is thermolabile and

shows a m a r k e d loss of p o t e n c y a f t e r b e i n g kept
for

Zh h o u r s

at room temperature.

for the a n t i b i o t i c M^575,

The authors

in a n a qu eou s

so lution

‘Phe minimal inh ibi tio n concent rat io ns

n y s t a t i n and pyr id i n e t h i o n e are 6-12 ppm,

a n d 0 .3 - 1 . 0 p p m r e s p e c t i v e l y for m a n y fungi

.

6-12 ppm,

D i ap hin e hy dr o c h l o r i d e is

ef fec t i v e a g ain st m a n y of the h u m a n p at hog e n i c fungi in vitro at c o n c e n t ra tio ns
o f 100 - 1000 ppm .* **
♦ U n p u b l i s h e d report

on "Studies

of the properties of a n t i b i o t i c s - t r a n s l o c a t i o n

a n d p h y s i o l o g i c a l studies w i t h c ycl oheximide

(Acti-dione),

endornycin,

a nd

7

str e u t o m v e i n sulfate", snbrai bted for the -r^nt from the Horace F. Rac k h n m
R e s e a r c h ^ndowraent In 1 9 r>3 » Departm ent of botany a n d P l ° n t Pn.fc> olo^y, !Ti o hi .ran
State College.
* * I nforav-i tion k i n d l y f u r n i s h e d b ” H. it. Squ ibb and Sons.
***T information k i n d l y s u p pli ed b y Chn.s. P f i z e r and Co.

8
Literature review of in vivo section
The vast majority of antibiotics are derived from "bacterial and
fungal activity (4, 8, 9* 27*

43).

Other antibiotics are d e r i v e d f r o m

various sources* "but these as a group are not important in plant disease
control programs (4* 5, 6 ,15, 35* 37* 40).

In the control of plant diseases

"by antibiotics, four general methods of application are used.
are:

These

soil* systemic, seed treatment* and foliar applications.
In soil applications, antibiotic materials are either applied to the

soil as a drench or mixed in as a dust.

However, for most soils* the

diverse microflora and colloidal complexes such as clays and soil organic
matter absorb or effectively inactivate most antibiotics that have been
tested (16,18*36*42).

Numerous strains of antibiotic producing fungi

and bacteria were added to soils* but these have proven to be Ineffective
in plant disease control (1 6 *3 8 )*
Antibiotics are also tested as systemic fungicides or chemotherapeutants (8 ,10,13*20,25*44).

A chemotherapeutant Is defined as a sub­

stance which is translocated throughout a plant to combat the disease
organism from within.

Davis and Dimond, (11), state that there is no

consistant relation between fungi toxicity and chemotherapeutic activity.
Thus, an antibiotic may have excellent antifungal properties but little
or no chemotherapeutic activity.

This makes it necessary to screen anti­

biotics for both fungistatic and chemotherapeutic activity.

Although

chemotherapeutants hold promise in future disease control programs, they
are not widely used to date.
Antibiotics are tested as seed treatment compounds.

Besults demon­

strate that field control of plant diseases caused by seed-borne fungi
is possible through the use of antibiotics (22,29).

Helixin was included

9
in the United States Department of Agriculture iniform seed treatment
nursery on oats and spring wheat in 1 9 5 3 (32,45).
Many antibiotics are screened for use as foliar sprays.

Several

fungal diseases, which formerly were poorly controlled or not controlled
at all, are now controlled by antibiotics.

Actidione at 2 ppm applied

as a foliage spray controls cherry leaf spot (21,39)*

This antibiotic

is very widely used against a number of turf diseases such as dollar spot,
pink patch, fading

out* copper spot, and brown patch (45).

Powdery

mildew on brambles is also controlled by spraying with actidione at a
concentration of 2-5 ppm (47).

Actidione gives both a protectant and

an erradicant action against many plant pathogens (1 7 ,2 1 ,3 9 )* but may
cause severe plant injury on susceptible hosts and tissues.

Helixin

controls early blight of tomato* and also proved rather effective as a
small grain seed treatment compound (28*29*32).

Although this compound

shows high activity against certain fungi, it is not being produced
commercially.
fractions.

Helixin has been found to contain several different active

Griseofulvin shows promise of controlling early blight in

England (4l).
Some plant diseases caused by bacteria, such as fire blight of pome
fruits, are now controlled by the applications of terramycin and strepto­
mycin (1,23,46).

Streptomycin is used at several different concentrations,

and critical work needs to be done to check this point.
The reaction of the antibiotic sprays is important, and antifungal
activity of a spray material is increased by lowering the pH.

However,

Leben (2 6 ) in work with early blight of tomato showed that this was not
increased antifungal activity on the part of the antibiotic, but rather
the result of the lowered pH alone.

He reports that from 53 to 79 percent

control of early blight was obtained by using acid buffers alone.
Most of the tests with the various antibiotics and organisms
date are in vitro and relatively few are in vivo tests.

11

MET HOD S A N D R E S U L T 3 IN V I T R O

A,

A s s a y test ing of a n t i f u n g a l m ate r i a l s
Methods
Several c o mpo und s were s c r e e n e d to det ermine

t i o n co nce ntr ati on s.

their m ini ma l

inhibi­

T h e f o l l o w i n g is a list of the compounds ass aye d

a n d the m e th ods fo r p r e p a r i n g the stock solutions?
*So diu m u v r i d i n e t h i o n e
*Nystatins

575*

(MC2113)?

v e r y soluble in water.

soluble in a ci d methanol.

raise pH of suspension to pH 11 with NaOH, solution then
occurs.

-Back titrate i m m e d i a t e l y w i t h acid.
Rimocidin sulfate?

'soluble in water.
S t o c k solutions
will be s lightly cloudy.

of 1 mg /ml

mfrHolntins sparingly soluble in water, in magnitude of 50ppm.
S o l u b i l i t y can be incr ea sed to 150ppm b y u s i n g 7 0 ^
methanol*
Diaphine H C L z

v e r y soluble

in water.

C a n dic idi n A?

v e r y soluble

in wat er

(3l)»

VTith the e x c e p t i o n of Diap hin e a n d p y r i d i n e t h i o n e , the compounds l i s t e d
ab o v e are all antibiot ic s.
trations were employed,
d e e m e d necessary.
p o u n d s be

F o r the initial screenings,

s u c h h i g h c onc en ­

that s t e r i l i z a t i o n of the test fu ngi c i d e s weren't

It is r e c o m m e n d e d however,

that the experime nta l

com­

s t e ri liz ed via Seitz- f i l t r a t i o n to p r ev ent b a c t e r i a l i n fec tio n

of the cultures.

This p r o c e d u r e

are c o m p l e t e d in order to d ete r m i n e
il i z e d by a u t ocl av ing .

is f ol l o w e d u n t i l t h e r m o s t a b i l i t y tests
if some

of the c ompounds

can be

In all cases unless otherwise indicated,

s o l u t i o n s were f r e s h l y p r e p a r e d p r i o r to use.
c u s s e d in a n o t h e r p o r t i o n of this report.

s ter ­

the test

Sta bi l i t y tests are dis ­

12
The following fungi were used as test organisms in the physiological
studies»
Collectotrlchum phomoides C8Q&:

isolated from tomato fruits.

Cplletotrichum phomoides 101» isolated from tomato fruits.
Belminthosporium sativum 925 » isolated from "barley in the thumb
area, Michigan.
Lilly—Barnett synthetic glucose asparagine medium (33) w&3 employed
as a basis for physiological studies.

The following constituents were

of C.P. grade:
D-glucose

10 gm

L -asparagine

2 gm

MgS04.7H20

0.5 gm

KH2PCUJ-

1.0 gm
0.02 mg

W /

0.02 mg

MaU

0.01 mg

thiamine
biotin

100 ug
5 ug

pH to 6.0 add distilled H20 to make one liter.
In some instances agar was added to the medium at a concentration of 2$
in order to have both solid and liquid media for the assays..

The medium

was autoclaved at 15 lbs. pressure and at 2^0°F. for 20 minutes.
In the beginning, mycelial inoculum was used.

This was accomplished

by placing a small portion of mycelium from the desired organism in the
center of a petri plate to which the medium and the test compound had
previously been added.

Currently however, spore suspensions prepared in

13
sterile water are used as inocula.

The spore suspensions are prepared

from heavily sparulating cultures.

The spore suspensions; are adjusted

so that a concentration of 400—500 spores per ml of medium will result
when 1 ml is used as inoculum for 20 ml of medium.

Spore counts were

determined with the aid of the Levy Hemacytometer.
Aseptic techniques are maintained throughout the procedure.

One ml

of inoculum is pipetted into a petri plate followed by the medium and
then by the test compound.

The latter is serially diluted so as the

amount used per 20 ml total volume in the petri plate gives the desired
concentration fer the test.
addition of each constituent.

Each plate is gently swirled after the
All assay plates and controls were run

in duplicate and incubated at room temperature.
Results
The results as presented in Table I indicate that pyridinethione
and Rimocidin are most effective against the organisms tested.

The

minimal inhibition concentration for C. -phomoides C80A, C_. nhomoides
101, and H. sativum 925 using pyridinethione was less than 1 ppm
and with Rimocidin, less than 1 0 ppm..

It was on this basis that the

aforementioned compounds were selected f®r pysiological studies.
some cases, duplicate runs were also made on agar.
the results were the same as in liquid.

In

E©r the most part,

The data in Table II indicate

that agar may tie up the compound in some cases s'o that a higher con­
centration than the liquid assay is required to inhibit the organism.
When nystatin is used, the minimal inhibition concentration against

14
TABLE 2
Activities of antifungal agents
Minimal inactivation concentration in ppm in liquid
Compound

C phomoides

C^. phomoides
101

C80A

H. sativum
925

<1,0

< 1.0

< 0.1

>10.0

5 0 .0

>5 0 . 0

>200.0

> 50.0

>200.0

Rimocidin

< 1 0 .0

<1 0 .0

< 10.0

Thiolutin

-7 1 5 . 0

> 1 5 .0

>15.0

Pyridine thi one
Nystatin
m575

Diaphine

-

Candicidin A

-

100 .0-200.0

200.0

5 0 .0

200.0

TABLE II
G-rowth comparison on agar and liauid media.
Fungicide

Nystatin

Organism

Cone, of fungicide in agar Cone, of fungicide in liquid
______
in ppm
e__________ inp p m ________ _
100
100
200
10
50
200
10
50

C_ phomoides
C80A

//

C_ phomoides

//

101

H. sativum

m

925

C.

nhomoides

/

C80 A
Rimocidin

C. phomoides

/

101

H. sativum
925

-

15
C,. phomoides C80A on agar is 100 ppm whereas in liquid, it is 10—50
ppm.

Plates (i, II, IV) show concentration effects on _C. phomoides and

H. sativurn when fungicidal compounds were put in "both liquid and agar
media.
B.

Stability tests on pyridinethione. rimocidin and diaphine
Methods
Stability tests were made on the above listed compounds with

regard to thermostability and "shelf life" of the material in solution
under varying conditions.

In order t® determine whether or not the

fungicides could be autoclave! together with the medium, a series was
set up for comparison of the minimal inhibition concentration of fungicide
sterilized by Seitz filtration with those of the fungicide autoclave!
together with the media.
compounds, pyridinethione

In another series, the "shelf life" of two
and rimocidin, were investigated by placing

10 ppm of the Seitz sterilized materials in either a water solution or

combined with the liquid medium at

* room temperature, and at 3 0 °^.

The 3 0 °C tests were made in shake flasks on the Kershaw Rotary Shaker
(see plate III) in ®rder t© duplicate conditions for the subsequent
physiological studies and to find out if there might be a loss of
potency during an incubation run.
Results

The data in Table III show a definite potency loss when pyridinethione,
rimocidin or diaphine are autoclave! with the medium.
due to a breakdown of the compounds.

This might be

Plate IV is a typical example.

16
depicting potency loss when diaphine was autoclave! together with the
medium.
clnving.

In the case ©f pyridinethione, the media darkened upon autoThe other compounds screened were not tested for stability as

the minimal inhibition concentrations were too high and did not merit
further consideration at this time.

^

The synthetic compound pyridinethione shows complete “shelf life"
stability under the conditions tested, whereas the antibiotic rimocidin
holds up only under refrigeration.

In addition, it is of interest to

report here that an aqueous solution of pyridinethione showed no potency
loss after being refrigerated for 10 weeks.

The "shelf life" results

are for 15 days and are being carried further but the information will
not be ready in time for this report.
TABLE

III

Thermostability of 2 Fungicides
Minimal Inhibition Concentration in ppm
Compound
Sterilized by
_______________________ Seitz Filtration
C. n h o m o i d e s
C80A

Pyridinethione
Rimocidin
Diaphine

H.

Sterilized by
Autoclaving With The Media *

sativum
925

C. p ho moides
C8QA

1 .0

0 .1

1 0 .0

1 0 .0

1 0 .0

15.0

1 0 0 -2 0 0

50

200

H.

sativum
925

5—10
1 5 .0
200

* 15 lbs. pressure and 240
degrees F. for 20 minutes.

_

17
C. P h y s i o l o g i c a l

stu d ies with co lle t o t r i c h u m h h o rrioides C8QA

1. D e t e r m i n a t i o n of harvest

time optimum
M e thods

The

organisms C_. p h o m o i des C 8 0 A . C. p ho mo i d e s

101 an d H. s at ivu m 925

were g r own in liquid m e d i u m to a s c e r t a i n the o p t i m u m harvest
s tan d a r d m e d i u m u s e d is the L i l l y —Barnett
medium.

time.

The

synthetic g lu co se— as p a r a g i n e

F i f t y ml of m e di um were d isp e n s e d into 250 ml erl en m e y e r flasks

w h i c h was

then p l u g g e d w i t h cot t o n a n d a u t o c l a v e d 20 minutes.

lum was p r e p a r e d f r o m po t a t o dextrose
old organism.

A

T he inocu­

slants c o nt ain ing a 3 to 4 weeks

spore s u s p e n s i o n was p r e p a r e d in sterile d ist i l l e d

w a t e r a n d d i l u t e d so that 1 ml of inoc ulu m p e r f l a s k y i e l d e d a concen­
t r ati on of 6 — 7 t0 0 0

spores /ml medium.

a i d of a Levy Hemacytometer.

Spore counts were made w i t h the

Cultures p r e p a r e d in triplicate were incu­

b a t e d in a s t a t i o n a r y f lask series as well as a shake f l a s k series.
l a t t e r series was

on a K e r s h a w R o t a r y Shaker at 1A0 rpm.

w e r e m a i n t a i n e d at 28— 3 0 °C.

in the dark.

out of three flasks were processed.

A l l cultures

At harvest time,

the best two

The m o l d was f i lte red v ia s uction

on a B u c h n e r fu n n e l throu gh a n y l o n filter.
three

The

The my celium was then w a s h e d

times w i th d i s t i l l e d w ater to remove a n y a d h e r i n g nut rie nt

substances

a f t e r w hi ch it was p l a c e d in a n u m b e r e d t ar ed alu min um w e i g h i n g cup an d
d r i e d overnight at 6 0 °C.

The cups x\rith the dr i e d m uce i i u m were p l a c e d

in a d e ssi cat or u n til weighed.
Resu lt s:
As
for

ind ic a t e d in Table

IV, it appears

that the optim um harvest

shake cultures is A days a f te r w h i c h time aut olysis

seems

The s t a t i o n a r y cultures were h a r v e s t e d at a 9 d a y optimum.

time

to set in.

Although

13
m a x i m u m grov/th was not ach i e v e d at the end of 9 days,
was c h o s e n for the sake of expediency.
stat i o na ry cultures
consistency.

The

this

time p e r i o d

typical g r o w t h p a t t e r n in

is a mat— .!ike mass w i t h a sub— surface gela tin ous

On the other hand,

shake cultures pro du ce a pellet

and

g l o b — like growth.
TA B L E

IV

G r o w t h P at ter n
On L i l ly -Ba rne tt S yn thetic Gluco se— A s p a r a g i n e Media
28— 30°C. in the dark
r e c o r d e d as mg dry m old/50 ml m e d i u m
O r g a n i s m ___________ 4 D a y s

2.

6 Days

9 D ays

_0. -phomoides C8QA
Shake
Stationary

198
154

173
155

154
1 76

C. p h o m o i d e s 10T
Shake
Stationary

200
76

189
171

146
177

H. sativum 925
Shake
S t a tio nar y

134
98

120
148

200
204

Ca r b o h y d r a t e v a r i a t i o n effect

on fungicidal activity.

Me t h o d s
P o u r different

c a rb ohy dra te sources were

invest iga ted separately in

c o n j u n c t i o n w i t h v a r y i n g con cen tra ti ons of fungicides

to determine how

n u t r i t i v e e n v i r o n m e n t a l conditions might influence f u ngi cid al acti vit y
towards a p a t h o g e n i c fungus.

The b a sa l m e d i u m u s e d was Lil ly-Barnett

s ynthetic g l u c o s e —asp ar agi ne m e d i u m complete with the e x c e p t i o n of the
c a r b o n sou r c e . *

fhe s t and ar d m e d i u m contains glucose equi val en t

to

*
It should be n o t e d that the sugars e m pl oye d did not s up ply the
o nl y carbon in the m e d i u m but that the L-aspar agi ne might also enter the
ca rbo hy d r a t e m e t a b o l i c cycle.
It was o bse r v e d that the s ta nda rd m e di um
m i n u s the c arb oh y d r a t e (glucose) su ppo rte d l im ite d g r o w t h w h e n tested on
a gar slants.
It is therefore a d v isa ble that an inorganic nit r o g e n source
be utilized.
*t has b e e n found that
p ho moides and H. s ati vum g r o w we 11
on the s t and ard m e d i a m o d i f i e d to contain sodium nitrate in place of the
as par a g i n e .

19

gm of car b o n p er liter; hence
qu a n t i t i e s
10 gm,
The

the f o l l o w i n g sugars were u s e d in the

s p e c i f i e d so as to give

sucrose 9.5 gm. D —xylose

k g m of c a rbo n per liters D -g lucose

10 gm a n d a l p h a - l a c t o s e .H20 1C gm.

or ga n i s m e m p l o y e d f or this study was C> phom oid es

were made un d e r
fications
cides

C80A.

Huns

shake a n d s tat i o n a r y conditions a cc ord ing to the speci­

set up u n d e r the m ethods

of the previ ous

section.

The f u n g i ­

s ele cte d for these studies o n the b as is of greatest a c t i v i t y in

the initial s c ree ni ngs were p y r i d i n e t h i o n e an d rimocidin.

Re s u i t s
The

c ompound py ri d i n e t h i o n e c a u s e d i n h i b i t i o n on shake and station­

a r y cultures at less than 1 p p m w h e n D— glucose,
la cto se were used.
1 a n d 3 ppm.

In the case of D — xylose,

(See p l a t e V.)

sucrose a n d a l p h a -

inh ib i t i o n was b e t w e e n

No m y c e l i u m or at best v e r y sca nt y amounts

were obt ain abl e u n d e r the range of fingici da l concentrations
these

studies

(l, 3.

6 and 10 ppm).

set u p for

C o n s e q u e n t l y , tabulated d ata on

dry m o l d weights a re l a c k i n g for this report.
The r e s u l t s w i t h r i m o c i d i n a re

tabulated in Tables V a n d VI whe r e i n

v a r y i n g a mo unt s of the a n t i b i o t i c were u s e d in c o nj unc tio n w i t h four
d i f fer ent

sugars;

sacchari des

the pen t o s e D — xylose,

sucrose a n d a l p a — lactose.

hexrose D — glucose and the diIn addition,

the data a l s o

i n clu de results u n d e r b o t h st ati on ary and shake conditions.

The p H

readings of the f iltrates made at the time of harvest are i n cl ude d in
the above m e n t i o n e d tables.
Sucrose a p p e a r s to r e n d e r the fungus more
at l owe r c o n c e n t r a t i o n s whereas

susceptible

to the rimo cid in

the anti bio ti c is effective o nly at h i ghe r

20
concentrations
carbohydrate

(10 ppm)

source.

when D — xylo se

or a l p h a — Inctose

is u s e d as

the

The data of Tables V a n d VI have b e e n p l o t t e d on

the f o l l o w i n g g r a p h s t
Graph

I

G r a p h II

V a r y i n g amounts of rim oci din in c o m bi nat ion with di fferent
c a r b o h y d r a t e sources — shake cultures
V a r y i n g amoun ts of r i m o c i d i n in com bin a t i o n w i t h d ifferent
ca r b o h y d r a t e sources — station ary cultures

G r a p h III D i s p a r i t y for X y l o s e a n d Glucose u n d e r s t a t i o n a r y conditions
wh en r i m o c i d i n reaches critical point.
R e f e r r i n g to the p l o t t e d curves the f o llo win g observations

can be mad.e *

1.

In g r a p h I, the a n t i b i o t i c act i v i t y rate is m ar k e d l y incr eas ed
w h e n u s i n g D-glucose or sucrose in co mp ari son to D — xylose or
a l p ha -la cto se .

2.

The curves do not d r o p as sharply in the s t a t i o n a r y cultures as
c o m p a r e d to the shake cultures*

3*

Xy l o s e in s ta ti o n a r y culture seems to sho w a s tim ul a t o r y effect
when r i m o c i d i n is p r e s e n t u p to 3 ppm.
In addition, the xyl ose
control in the s t at ion ary culture has a d r y mol d weigh t of 173
m g i n 11 days as c o mpa red to 85 m g in 6 days for the shake cul­
ture.
C_. -phomoides C80A i s not inhibited at 10 p p m ri mo c i d i n
w h e n g r o w n in xylose -under stationary conditions.

4.

G r a p h III indicates that there is a disp ari ty for x y los e a nd
glucose w h e n the a nti b i o t i c approaches the critical range of
activation.
This is i n dic at ed b y the d o t t e d lines, the fact thaii
no re pli c a t e s agree.
In the case of glucose, there seems to be
a- ten d e n c y for the divergent lines to r e j o i n ag ain at higher
a n t i b i o t i c concentrations.
This is not evident for xylose as
a p p a r e n t l y the concen tra ti ons u s e d were not h i g h enough.

It is i n t e r e s t i n g to note that r i m oci di n is more effective
iting

in inhib ­

-phomoides C 30A w he n it is i nco rp o r a t e d into sucrose med ium

tha n w h e n in glu cos e medium.

Shake flas k cultures

on these

two sugars

at 6 a n d 10 p pm of the a n t i b i o t i c were m a i n t a i n e d over a 5 w e e k period.
F r o m the data p r e s e n t e d in T ab le VII,

it is apparent that w h e n the o r ga nis m

was grown

in a glucose medium,

t r a t i o n or r i m o c i d i n was
sucrose media,
a n d total

g r owt h started at 9 days w h e n the concen­

6 ppm a n d at 15 days w h e n at 10 ppm.

h i g h l y r etarded g r o w t h was a p p are nt

In the

in 27 days at 6 ppm

in hi bit ion was still m a i n t a i n e d at 10 ppm at the end of 38

days at w h i c h time the e xperiment was terminated.

22
TABLE V
Dry mycelial weight of Colietotrichum ohomo ides C80A grown in
liquid medium containing varying amounts of rimocidin in conjunction
with different C sources expressed as mg from the average of the best
2 of 3 replicates.
Incubated at 20-30°C? D-glucose, sucrose.
Ppm of
Fungicide

Stationary 9 days

Shake 4 days
D-■glucose
*
mg
pH

Sucrose
mg

D-glueos e

pH

mg

Sucrose
pH

mg.

pH

0

211.0

7.5

163.0

7.6

176.0

8.1

184.0

7.6

1

180.0

7.5

160.0

7.0

171.0

8.1

180.0

7.9

3

1.5

6.0

0.0

5.2
6.7

5.0

5.0

6.2

0.0

—

6

0.0

0.0

10

0.0

—

xS : o } 126**
12 .0 -j t o **
7.0->

0.0
3.0 J

6.1
6.1
6.1

0.0

TABLE VI
Same conditions as TABLE V except incubated at 30-33°Ci D-xylose, and
lactose.
Ppm of
Fungicide

Stationary 11 days

Shake 6 days
D-acvlose
ms.

eH

D-xylose

Alpha-lac 1 0 se
mg

.

m.

Alpha-lactose
22

pH

0

85.0

5.9

20.0

8.3

172.0

7.5

37.0

8.3

1

51.0

5.5

6.0

7.3

182.0

8.2

37.0

8.4

3

4-6.0

5.8

7.0

7.7

181.0

8.2

25.0

8.2

6

20.0

5.6

2.0

6.7

^ > 3 2 * *

7.2
7.4

11.0

8.0

5.0

5.6

1.0

7.8

7.0
6.4

0.0

10

8 6 ;0 >6;„ .

*pH of filtrate at time of mycelial recovery.
♦♦Creater deviation occurred in these cases when rimocidin was used.

23

T A B L E VII

Protective effect of 2 different sugars incorporated into
the liquid media with riraocidin. Shake cultures at 3°°^.
Recorded as the incubation time required for the appearance of growth.
Rimocldin
inform

Sugar
9

Incubation in days
15
22
27

6

D-glucose

10

D-glucose

-

growth

6

Sucrose

-

—

10

Sucrose

growth

-

growth

38

GRAPH 1

24

Shake, Culture* Growth of C. phomoldes C80A
In Liquid Medium Containing Varying Amounts of Rimocidin Sulfate
With Different Carbohydrate Sources.

180
D-glucose 4 days

160
sucrose 4 days

zag dry mycelium

120

100

80

D-rylose 6 days

20
alpha**lactose 6 days

10
ppm rimocidin

25

GRAPH II

Stationary Culture* Growth of <3. phomoides G80A
In Liquid Medium Containing Varying Amounts of Rimocidin Sulfate
With Different Carbohydrate Sources.

200

180 -

160

m
g dry mycelium

sucrose
9 days

120

D-xylo se
11 days

-

100

alpha- lac t o se
11 days

20

0

1

2

5

6

ppm rimocidin

7

8

9

10

GRAPH III

20

Stationary Culture. Growth of C_. -phomoides CSOA
Showing Divergencies Between Replicate Flasks When
The Concentration of Rimocidin Sulfate Reaches The Critical Point.

200
I>-rylose 11 days

180

160
cose

mg dry mycelium

mo

120

100

80

10
ppm Rimocidin

2?
In vivo methods and results
Four antibiotic materials, thiolutin,

fungi statin XG, nystatin,

and rimocidin sulfate, were tested along with two synthetics, di&phine
hydochloride, and sodium pyridinethione.

The solubility and stability

of thiolutin, rimocidin, diaphine hydrochloride, and sodium pyridine­
thione are discussed in the in vitro section of this report*

Fungis-

tatin XG-, supplied by Charles Pfizer and Co., Inc., was tested for
antifungal properties in vivo.

Further communications with the Pfizer

Company revealed that this antibiotic was not in production nor were
any plans made for future production.

Therefore, nystatin, an antibiotic

produced and supplied by E. R. Squibb and Sons, was substituted in the
testing program for fungi statin XG-.
company as soluble in methanol,
pH2 and pH9*

The compound was listed by the

stable at neiutral pH, and unstable at

A partial spectrum of antifungal activity was furnished

which indicated a fair degree of activity at high concentrations in
the in vivo tests.

Actidione produced by Upjohn Co., and panogen, an

organic mercury compound, produced by Panogen Inc., New York, were added
later to the testing program as treated checks.

All of the antibiotics were prepared in water solutions or suspen­
sions for application to the test plants.

An emulsifying agent, tergitol,

was used to keep thiolutin in suspension at concentrations above 50 PP®
Nystatin and rimocidin were first disolved in two milliliters of 95
percent methanol and then diluted with distilled water to the desired
concentration.

Stock solutions of all the materials were prepared at

a dilution of l/lOOO. Such stock solutions were then stored under
refrigeration at 5° F.

Tive fungal test organisms were used.

These are listed below with

the common name of the disease which they cause.
Scientific name of organism
Alteraaria golani (ELI. and G. Martin)
L. R. Jones & Grout
Colletotrichum phomoides (Sacc.) C80A
Chester
Phoma lipgam (Tode es. Er. ) Desm.

Common name of the disease
Early blight of tomato
Anthracnose of tomato
Blackleg of crucifers

Helminthosporium sativum (Pam. )
King and Bakke

Spot blotch of barley

Puccinia graminis tritici
Eriks , and 3G. Henn.

Stem rust of wheat.

A. solani, P. 1ingam, and H. sativum are included in several in
vitro testing programs, (26,27,28).

They were used in these tests because

of their ease of application to foliage spray programs as well as their
foliar type of infections.

C. phomoides on tomato is included in numerous

spray testing programs and is used in these tests because of its difficult
control, importance as a ripe fruit rot, and as a representative of an
important group of fungi attacking plants.
Two mono cot yledonous hosts, wheat and barley, and two dicotyledonous
hosts, tomato and cabbage, were used in the testing program.

In addition

to these, corn, beans, cucumber, and tobacco were used in phytotoxicity
tests.

These species were used to provide a range of host material in

testing host reaction to antibiotic applications.
were run for all substances tested at 200 ppm.

Phyto toxicity tests

Diaphine hydrochloride

was tested further at 1 0 0 , 5 ^* and 25 ppm.
The materials were sprayed directly on the foliage by means of air
pressure and atomizers or by use of hand spray guns. In the case of
I
cabbage, the wax, or bloom, on the leaves was rubbed off by hand as the
sprays were applied.

This was done to insure contact between host,

29
sprays, a.nd inoculum and to reduce run-off.

Although some run-off was

present on the other plants, by care in application it was not a serious
problem.
A single isolate of each of the test organisms, except stem rust,
was maintained on potato dextrose agar, and used throughout the experi­
ments.

The fungi growing on agar were then ground in a blender, and the

suspension was used as inoculum.

The rust spores were collected from

inoculated hosts which were used to build up the amount of inoculum.
The suspension of ground mycelium or spores was sprayed on the plant with
atomizers or hand spray guns.
The test plants were first sprayed
allowed to dry for several minutes.

with the test organism and

The antibiotic materials were then

applied, and shields of cardboard were used to prevent drift from one
test row to another.

After the antibiotic materials were applied, the

plants were placed in a humidity chamber which was kept moist by a fine
spray of water and air under pressure.

The plants were removed from the

moist chamber after three or four days, and the disease was allowed to
develop.

Headings were taken when the lesions on the checks appeared to

be well developed.

In most cases the readings were based on the number

of lesions per plant.

Where this could not be used, readings were based

on the numerical gradlations of 0— 5 » where 0 indicated no disease
symptoms on all plants and 5 represented each plant having heavy infection*

30
The data presented in this section deals with only two phases o f the
problem.

The first of these is the phytotoxic reactions caused by spray®

ing antibiotic materials on the foliage of the test plants.

Th© second

is the actual control of the pathogens by spraying shortly after the host
plants were inoculated.

In one preliminary run against Sclerotlnia

solerotiorum (Lib. )Bdy. on bean the antibiotic materials were applied to
the soil as drenches.
The initial phytotoxicity tests were made over a wide range of test
plants (Table I) to determine as quickly as possible the host reactions
to antibiotic sprays.

A concentration of 200 ppm was used as a starting

point since it approached the highest concentraion at which control work
is generally feasible from a cost standpoint.

The monocotyledonous

plants as a group were seemingly more tolerant of antibiotic sprays than
were the dicotyledonous plants.

In

no case was any damage noticed on

the graminaceous plants under test.
Several types of injury were found on the dicotyledonous plants
depending on the plant and the antibiotic used.

Thiolutin produced a

downward bending of the petioles and bronzing of the leaves of tomato
plants but on bean it cuased only a slight mottling.

Fungistatin XG

caused a downward bending of tomato leaf petioles, leaf crinkle on
cabbage, slight leaf puckering on beans, and chlorotic spots on cucumber
leaves (Plate V I ).

D iaphine gave a toxic reaction on all dicotyledonous

plants tested (Table I, Plate VI).

In mostcases this consisted of chlorotic

spots or chlorotic leaf margins on the
time of application.

leaves which were Immature at the

On cabbage diaphine produced chlorotic rings which

were similar in appearance to certain virus symptoms.

In most cases,

however, the host plant grew out of the damage, and any newg-owth that

31
appeared was either free of injury or only slightly injured,

^imocldin

caused some slight puckering on bean and cucumber leaves, but the damage
was not severe.

Sodium pyridinothione caused leaf puckering on bean and

marginal necrosis on cucumber leaves (Plate VI).

Actidione caused necrotic

spots on the leaves of most test plants, but such damage is known to
occur at higher concentrations of this material.

Banogen which Is a very

volatile organic mercury gave no pnytotoxic reactions on the plants which
were sprayed with it.

In the only soil application made diaphine was

found to cause injury.

Again this Injury was associated with the leaf

which was immature at the time of the application of the material to the
soil.
Dilution tests were run using diaphine at 200, 100, 50, and 25 ppm
(Table II).

^obacco, cucumber, and beans exhibited chlorosis at all

dilutions tested although the chlorosis became less pronounced at the
lower concentrations.

Cabbage and tomato showed a marked reduction In

phytotoxicity at the lower concentrations although some chlorosis was
still present.
The results of the spray tests against pathogens varies with host
and pathogen.

None of the antibiotics tested to date have exhibited

high levels of antifungal activity in these in vivo tests.

H. sativum.

Erysiphe gramlnls, A. solanl, and C. phomoldes were all poorly controlled
(Tables III, V,VI, VII).

In a number o# cases slight to rather strong

stimulation of infection was found to occur with the use of thiolutin
(Table IV).

'I’he results for P. 1 ingam are as yet inconclusive with good

control occurring In at least one run (Table IV).

In the trials with

P. graminls trltlcl (Table VII) tiolutin, diaphine, and sodium pyridinothione
gave perfect control while rimocidin and nystatin allowed some lesionlng.

32
In the two trials In which panogen and actidione were tried they were
better than any of the other materials.

TpiB indicates that the other

compounds are not as antifungal as might be desired for a spray compounds.

33

Table I. Nummary of the phytotoxicty teats of several antibiotic materials
applied to several different test plants as sprays in concentrations of
200 ppau 0 no damage-— 5 severe damage.
Test
Spray Materials
Plant_____
Thiolutin *unglstfttin Diaphine ^imocldln Na Pyridlnethlone
Old tomato

1

If

2

0

1

Old cabbage

0

If

0

0

0

Old tobacco

0

0

1

0

0

Bean

1

1

3f

1

-1

Cucumber

0

If

2f

-1

1

Com

0

0

0

0

0

Wheat

0

0

0

0

0

Barley

0

0

0

0

0

-1

-1

3

-1

1

0
Nystatin
0

3

1

0

0

0

0

Young tomato
Young Cabbage

0

Barley

0

Young tomato

0

0

3

0

0

Wheat

0

0

0

0

0

3^

^able II. Phytotoxicty teste using diaphine hydrochloride at four
____________concentrations._____________________ ______ _______________
Test plant

ftumbor of plants out of 8 showing chlorosis at$
200ppm.
lOOppm.
50PP*u
25ppm.

Cabbage

8

8

6

2

Tobacco

8

8

8

8

Tomato

8

5

5

**-

Beans

8

8

8

8

Cucumber

8

8

8

8

35

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o a b
t
—1ct(jl)

p a a

d p
.
d * d»

" *P
*

^
t
e
i

o

3

a

ro

a

ro

V
>
l

o
d

P
vjj

ro

3*
3
d

a

PJ
3
o
od
d

|T04

v>i

P
d*
3

v_n
a
a

S
3
a
od
d
O
3
O
d

3*

a
ro

V>l

P
*
d

3a

a

d

H

d
O
aa

P

3

d
d

a*

M
*

o

'3
d
P
3
d
•g
d
3*
O
<8

Resialts of several antibiotic materials sprayed on plants for the control of

<0
o

a

bd co
S3

III.

o
c
*

3 I®

o
3
P
d

Table

O

3

36
Table IV. Be suits of several antibiotic materials applied to Cabbage
for the control of Phoma 1 ingam (blackleg)

Table A
Treatment
_______________
1. Control

!Itetal number of lesions on
eight plants.
6

2. Thiolutin

IS**

3. Fungistatin

IS**

*+* Diaphine

3

5« Rimocidin

l

6. Ra. Fyridinethione
l
*L.S.D. at the .05 level-— 5.01
**L. S.D. at the .01 level— 6.72

Table B
Treatment

Total number of lesions on
eight plants.

1. Control

630

2. Tholutin

118**

3. Diaphine

23^*

U. Systatin

93**

5. Rimicidin

82**

6. Na Pyradinethione

3U**

7. Itenogen
1**
S. Actidione
level—
-67.50
.S.D.
at
the
.05
*L
** L.S.fl.at the .01 level— -"-86.20

37
Table
- Besults of several antibiotic materials applied to tomato leaves
for the control of Collctotrlchum phomoideg (anthracnose)t C30A#
Treatment
-------------

Total number of lesions on
e-ightslants.
_______ ______________

1. Control

7

2. Thiolutin

8

3. Diaphine

6

K

5

Rystatin

5. Rimocidin

U

6. Na ^jrridinethione

3

7. Panogen

l

8. Actidione

1

Table VI # Results of several antibiotic materials applied to tomato leaves
for the control ©f A l t e m a r i a solani (early blight)
Treatment
Total number of lesions on
_______________twelve plants,
__________

1. Control

122

2, Thiolutin

131

3. Fungistatin
U. Diaphine
5. Rimodidin

83
105
76

6. Ka Fyridinethione 171

38

Table VII* Results of several antibiotic materials applied to wheat
leaves for the control of Pucclnla graminls tritlcl (wheat stem ru^t).
Treatment
Average number of lesions per plant
________________ per t r e a t m e n t ________________

1. Control

5.9

2* Thiolutin

0

3. Nystatin

.3

U. Diaphine

0

5. Rimocidin
6. Na pyridinethione

A
0

39
D i s c u s s i o n a n d .co n c l u s i o n s o f the in vitro

sec t i o n .

P r e l i m i n a r y s c r e e n i n g s were c o n d u c t e d on compounds r e p o r t e d to hove
anti-fungal

properties,.

two strains

of the

tomato fruit an thracnose organism,

p h o n o i d e s a n d the leaf
sativum.

Of these,

0.1-1*0 ppm.

were

tested against

C ol letotrichum

spot a n d root rot or ganism of wheat,

fielminthosoorium

three c omp o u n d s were f o u n d to possess f a ir ly good

in h i b i t o r y p r ope rti es .
was 15-30 ppm,

Seve n a n t i — fungal compounds

The mi n i m a l inhibi tor y concent rat ion for thiolutin

for rimocidin,

less

than 10 ppm,

and for pyridinethione,

W h e n t e st ed in b o t h a g a r a n d l iq uid media,

of n y s t a t i n r e q u i r e d f o r the i nhi b i t i o n of
be 100 p p m on a g a r a n d 10 p p m in liquid.
the a n t i b i o t i c m a y be

the c oncentration

p hom o l d e s C80A

was found to

This data seems to indicate that

tied up w i t h the a g a r in such a w a y that its act i v i t y

rate is reduced.
T h e r m o s t a b i l i t y tests showed the two most p r o m i s i n g compounds, pyr id ine thi one
a n d rimocidin,

are i n a c t i v a t e d in the m e diu m w h e n subjected to h i g h temperatures

such as a ut ocl a v i n g .
that

This of course has some d ep r e c i a t i o n on the value

can be p l a c e d on the compounds.

for the active compound s,
This feature

So far as solubility is concerned

p y r i d i n e t h i o n e alone is readily soluble in water.

is p a r t i c u l a r l y desi ra ble when un d e r t a k i n g physio log ic al studies.

A l t h o u g h r i m o c i d i n is not as soluble as pyridinethione,

stock concentrations

of 1 m g/ ml can be p r e p a r e d bv a d j u s t i n g the p H of the su sp ension so that
almost all of the m a t e r i a l will go into solution.
atu re review,

As ment ion ed in the l i t e r ­

no p u b l i s h e d i n f o r m a t i o n is available on pyridi net hi one and

v e r y little c o n c e r n i n g rimocidin.
The r esults
e thione as

on the "shelf life" studies again point towards py ridin—

the c o m p o u n d of choice.

showed c omp let e

B y way of comparison,

p yridinethione

s t a b i l i t y w i t h no pote ncy loss when m a i nta ine d at temperatures

of 5 ° C., r o o m t e m p e r a t u r e a n d at 3 0 °C.

in aqueous

solution as well as when

c o m b i n e d w i t h the s t a n d a r d n u tr ie nt medium for a p er iod of 15 days.

i
R i m o c i d i n on the

ot h e r hand,

r e t a i n e d its full p o t e n c y only when m a i n t a i n e d

at 5°C.
It was d e e m e d ad vis a b l e at this time to c a rry on the p r e l i m i n a r y
w o r k r e g a r d i n g the ways

in w h i c h e n v i r o nm ent al m o d ifi cat io ns may affect

the a c t i v i t y of a n a n t i - f u n g a l agent towards a fungus.
p r e vio us R a c k h a m report,

the car boh y d r a t e studies were continued using

the f o l l o w i n g s ug ars : D —xylose, D-glucose,
The a n t i - f u n g a l m a t e r i a l s

sucrose a n d alpha-lactose.

to be u s e d in conjun cti on w i t h these sugars

from the i n i t i a l s c r e e n i n g program,
It is c o n c e i v a b l e

On the basis of the

selected

were pyr idi ne thi one a n d rimocidin.

that a n o r g a n i s m might be rendered more susceptible

to a n i n h i b i t o r y c o m p o u n d if its g r o w t h (hence its me ta bolic rate) can
be accel era te d.

I n o r der to i n ves tig ate this possibility,, the organisms

inv ol ved w ere g r o w n s i m u l t a n e o u s l y in shake a n d statio nar y cultures,
u s i n g C[. o h o m o i d e s
to det e r m i n e

C 8 Q A a n d 101

, as well as on Helminthos-porium sativum 925

the o p t i m u m i n c u b a t i o n p e r i o d pr i o r to the ph ys iol o g i c a l studies.

Shake cul tu res g r o w n in the st a n d a r d glu cos e-asparagine media for 4 days at
28 to 38°C,

was c o n s i d e r e d to be

the optimum incuba tio n p e r i o d for harvesting.

If a l l o w e d to c o n t i n u e longer, aut ol y s i s
st a t i o n a r y cultures,

sets in (see Table VI),

In

the growt h curves for the above listed organisms continued

to increase over a longer p e r i o d of time, but the rates are m u c h slower than
those

of the shake cultures.

T h e harvest

in the p h y s i o l o g i c a l studies was

time for the stationary cultures

taken as nine days al th o u g h the organisms

i n v olv ed had not a t t a i n e d their maxiumu growth.
p e r i o d was c h o s e n from
from the

s t a t i o n a r y cultures agree

b y L il ly and B a r n e t t
fungi

the e x p e d i e n c y viewpoint.

(3^0

in w h i c h the same

source was used.

The nine d ay incubation
The mycelial dry weights

quite well with the results

obtained

in their w o r k on the ut i l i z a t i o n of sugars by

s t an da rd mediu m w i t h glucose as the carbohydrate

C o l l e t o t r i c h u m nho moi des has a dry weight

of 176-177 mg

41
at the e n d of 9 days as

c o m p a r e d to Lilly's results

and 225 m g for 12 days.

of 155 mg f o r

5 days

F o r H e l m i n t h o s p o r i u m sativum the dry weight at the

end of 9 days was f o u n d to h e

20h mg as c o m par ed to Lily's

results of 217 mg

for 12 days.
Whi le it w o u l d have h e e n a d v i s a b l e to conduct the phy si olo gic al
w it h 2 organisms,

it was felt

studies

that a greater range of variations could be

a c c o m p l i s h e d if o n l y one or ga n i s m was chosen,
a r b i t r a r i l y c h o o s i n g the fungus

hence

the studies were made by

C o l l e t o tri ch um phomoides

C80A as

the test

organism.
It was f o u n d that the w o r k i n g range for pyridinethione,
was too high.

1.0 - 10.0 ppm,

I n h i b i t i o n o c c u r r e d at 1.0 p p m for all the sugars tested w i t h

the e x c e p t i o n of D — xy l o s e u n d e r shake conditions,
was b e t w e e n 1 and 3 ppm.

in whic h case inhibition

It is sugge st ed therefore, that the work ing range

for p y r i d i n e t h i o n e be m o d i f i e d to 0.0 - 1.0 ppm.
The d a t a c o n c e r n i n g the r i m o c i d i n studies
features for discussion..

contain some interesting

The o rga n i s m grows best on D— glucose or sucrose

and ra t h e r p o o r l y on D— xylose or alpha-lactose.
cor re lat ion s m a y be q u e s t i o n e d b eca us e
an d temperature.

of the difference in i ncubation time

The glucose a n d sucrose phase was con ducted at the same

time a n d the p r e s e t

con dit io ns f o r the experiment were followed.

case of the xy l o s e a n d the lactose
temperature

The val i d i t y of true

run, also conducted simultaneously,

in the i n c u b a t i n g room caused some difficulty.

m a i n t a i n i n g a 28 to 30°C.

range,

somewhat r e t a r d the g r o w t h of
i n c u b a t i o n period.

it rose to 33°°.

The harv est

it t o o k 11 days..

Instead of

This was high enough to

time in this la.st instance was based upon

to that of prev iou s runs,

For the sh ake cultures,

the

the organism a n d hence requi red a longer

the g l uc ose con t r o l w h i c h was r u n at the same time.
a growth comparable

In the

When the latter showed

all the flasks were harvested.

this was 6 da^^s whereas for the stationary cultures,

42
Talcing the a b o v e
ihe a c t i v i t y rate
g lucose
x yl os e

into consid er at io n,

some

trends can be

evaluated,

for the r i m o c i d i n is suite r ap i d w hen c o m bi n ed with

or sucr os e

wn il e a n a tt e n u a t i o n is e v i d en c ed when com bi ned with

or lactose*

This can be r e a d i l y a s c e r t a i n e d bv the co rr ss n on di n p

slopes d e p i c t i n g a c t i v i t y rate as p re s e n t e d

in Graphs I a n d II.

The

d if f er e nc e b e t w e e n s'mke and s t a t i o n a r y cultures does not a ope or to m a r k e d l y
influence

the o r g a n i s m ' s s us ce pt ib il i ty to the rimocidin.

The fact that the

controls g r e w m u c h b e t t e r w hen c u lt ur ed w ith glucose or sucrose

than when

c u l t ur e d w i t h xylose

or lactose i ndicates a h i g h me ta bo li c rate for the

former sugars.

seems

act ive
more

tissue

This

to corr el at e well with fungal toxicity.

such as raeristematic grow ing points

Highly

on plants are u su a l l y

sus ce p t i b l e to p h y t o t o x i c i t y effects w h e n sprayed with these test

compounds.

T h i s is d i s c u s s e d in the section on in vivo studies.

a n a l o g y 3®ems to h o l d for jC. o h omo id es C 8 Q A .

Conversely,

Thus the

when this fungus

shows a l o w or m o d e r a t e m e t a b o l i c rate such as w h en grown in xylose or
lactose,

c o r r e s p o n d i n g l y , the s usc e p t i b i l i t y to the compound is not as

m a r k e d a n d r e q u i r e d a g r e ate r co nc e n t r a t i o n f o r inhibition.
po in t

is m r t i c u l a r l y indicated

can u t i l i z e

this

enzyme system.

sugar

A d a p t a t i o n is g ra d u a l but af ter a lag p eri od of about 6 days,

rs v'oll os glucose

agree with those

The or ganism a p pa r e n t l y

only slowly at, first, p er ha ps r eq u ir i ng an adaptive

e sti m a t e d on the b a s i 3 ©f xylose
utilized

in the case w i t h xylose.

This last

controls,

or sucrose.

of L i ll y a nd T arn et t

this sugar can u l t i m a t e l y oe

Our results wit!

(34).

weight of C. -phomoi d e s in s tat io n a r y culture

the xylose controls

T h e y found the dry mycelial
to be 3b ing

5 days and 210 m g

at the e nd of 1 2 days w h ere as our weight in shake culture was 85 mg in

6 days a n d 172 m g at the end of 11 days in stati on ary culture*
s t i m u l a t o r y effect was also n o t i c e d w h e n
t o g e t h e r w i t h r i m o c i d i n at 1 to 3 ppm.

A slight

o h o m o i d e s was g r o w n in xylose
This m a y not be c o ns i d e r e d too

u n u s u a l for smal l a m o u n t s of a n t i b i o t i c s have b e e n kn own to have a sti mul at ory

43
effect

on organisms.

8-rapn III
concentrations
However,

shows a d i s p a r i t y b e t w e e n replicate flasks
of r i m o c i d i n h a v i n g re ta r d i n g effects

c o n tai nin g those

on the

organism.

r e p l i c a t e flasks agree w h e n either no a n t ibi oti c is present

in those a m o u n t s w h i c h have no no tic e a b l e effect ©n the fungus.
and the curve

seem to indicate

at those c o n c e n t r a t i o n s

that

that w h e n the a n t i — fungal

start to affect

this range

is called

from f l a s k to f l a s k mi gh t account
In the case of glucose,

however,

At t h e s e

to come t o g e t h e r as does

That

one

the purpose of

B i o l o g i c a l variations

for this d i s p a r i t y in this critical range.
my ce l i a l weights of replicate flasks con taining

two concentrations,

point of t ot al inhibition.

substance is present

For

the "critical range".

6 p pm of r i m o c i d i n are not too far apart a n d are
at 10 ppm.

The data

the organism adversely,

f l a s k will not a g r e e w i t h a n o t h e r in the same series.
this report,

or is present

still closer together w h e n

the fungus

is reta rd ed almost

to the

the d i spa ri ty curve for xylose does not seem

the curve for glucose

indicates

that the critical

range w i t h this su gar has not b e e n surpassed and higher concentrations of
r imo cid in a p p r o a c h i n g the inh ib i t i o n range are needed to show the joining
t en den cy of the curve.
When fla sk s c o n t a i n i n g glucose a nd rimo cid in at a c o n c e n tra tio n of
10 ppm are i n o c u l a t e d w i t h a spore suspens ion of .C. ohomoides
f ung is t a t i c effect

is o bserved for a p e r i o d of 15 days a f ter w hich time

g row th is apparent.
equal,

If sucrose is substituted, all

the f u n g i s t a t i c

peri od of 38 days,
terminated.

effect is p r o l o n g e d considerably,

in this case for a

e x p l a n a t i o n for this might be a t t r i b u t e d to the

of glucose w h i c h slowly b re aks down the anti bio tic rim oci din

over a p e r i o d of time.

After

its p o te ncy is so altered,

h e l d in ch e c k are a l l o w e d to germinate.
does

other conditions b e i n g

with no visibl e g r o w t h at the time the experiment was

A possible

reducing property

C80A. a

the spores

Sucrose on the other hand,

not c o n t r i b u t e to the r i m o c i d i n breakdown.

that were
a p pa re ntl y

44
The disparity occurring between replicate flasks when concentrations
of rimocidin show a retarding effect needs further investigation to
determine, if possible, the cause of this spread in the curves before
inhibition occurs*

This may also be found to be the case for other organisms

when glucose and xylose are used as a carbon source and rimocidin as the
anti fungal subs tance.
Continued in vitro studies of the test compounds and any new ones in
connection with effects on metabolism of the pathogens will give a better
picture of what takes place.

This includes all of the various combinations

and Variations in the nitrogen sources, growth factors such as vitamins,
studies, and determination of whether the compounds are fungicidal or
fungistatic
It is

proposed that arrangements could be made with the Abbott

laboratories for a tagged compound with radio isotops so that it will be
possible to trace the fate of the compound in the organism, if it is absorbed.
/

It would also be possible to trace what takes place when this material is
put on the surface or in the internal parts of a plant.

45
Discussion and conclusions of the in vivo section.
Because of the relatively short time that these experiments have been
carried, out, emphasis can not be made on these conclusions.

Ho^/g-yer,

there are certain interesting phenomena which have been observed and recorded*
There was a general tendency for the test plants to grow out of the
injury caused by the applications of these antibiotic materials.

The new

growth on the pHants which had exhibited injury was frequently less damaged
than the growth that had been immature at the time of antibiotic application.
The amount of injury on the new growth in the case of diaphine was pro­
portional to the concentrations of the material in the spray.

This

phytotoxic effect of diaphine was translocated through the bean stem
to the leaves that were Immature at the time of application when the
material was applied to the soil as a drench.

However, the leaves which

developed after this initial injury were nearly free of injury.

These

observations seem to indicate that the action of this antibiotic was
against some part of the maturation process and that the mature cells
were much more resistant to this type of injury.

This idea Is further

developed when it is learned that bean plants which were at the primary
leaf stage had to be sprayed with heavy application of diaohine at 200
ppm to cause necrosis of these mature leaves.

These results would

seem to indicate that the activity of this material decreases in con­
tact with living tissue and that it is not translocated to the meristematic
tissues in any large amounts.

The rather interesting chlorotic rings

which were found on cabbage need further investigation as to their
association with the types of injury caused by diaphine.
The rather Inconsistent results in the spray trials may be caused by
several factors.

The temperatures of the greenhouse could be controlled

46
only at night*

No daytime controls

existed.

This resulted In tenroerature

changes on sunny days of from 25°^*. to 30°N*. On cloudy days the temperature
stayed within

of the night temperature.

This temperature effect be­

came more important after the JLn vitro tests showed rimocidin to be very
unstable at high room temperatures.

Another important factor affecting

the results of the control trials Is run-off.

Hun-off is the amount of

active material which either runs off at the time of application or is
washed off by the subsiquent mist anpli cat ions.

Care in applying the

materials was used to avoid as much run-off as possible.

If the mist

particles in the humidity chamber are not fine enough, then the amount of
run-off is greatly increased.

Aig© the more soluble the compounds;the

poorer the sticking qualities;and therefore, the greater the run-off.
The type of lesion produced on the leaves of the host were also
important in the final analysis of the results.

C. phomoldes will attack

the leaves of tomato, but the lesions are small and difficult t© read.
Therefore, in future test programs and studies anthracnoee of bean may be
substituted.

Probably the most significant factor was the low in vivo

fungicidal or fungistatic activity of these materials.

Although some of

these materials exhibited high activity in vitro, such activity may not be
exhibitied in association *ith living plants (9?.

Thi« is the primary

reason for in vivo testing programs.
The trials against the stem rust organism were the only ones that
gave total control in some cases.
sprayed plants.

However, lesions appeared on some of the

These lesions may represent areas that ©scaped coverage.

However, It would be interesting to reisolate from such lesions to determine
if any of them contained fungi which were more resistant to the materials
under test.

Such Isolates have been collected In the case of rust on corn.

47
SUMMARY

l

Summary of in v i s e c t ion
1,

ihe antibiotics , M^+5 75» ri mocidin, thiolutin, diaphine , cendicidin

an* sodium pyridinethione were te-te- b- the nco of a ^ r end liouid media wit
varied concentrations as a

reliminary in vitro test against strains of

Colletotrichum phomoidoo and Helminthosnoriurn sativum. All tcftt organisms
were inhibited at less than 10 worn by sodium pyridinethione.

The other

antifungal substances were less effective.
2.

Hystatin inhibited C_. nhomoides C 80A ,at 100 pum on agar and 10 ppm

in liquid.

This data seens to ind.icate that the antibiotic may be tied

ur with the agar.
3-

In the thermostability tests all the compounds showed a definite

loss of notenc}*- e„fter autoclaving.

However, sodium pyridinethione was the

only compound that showed no ootency loss in an aqueous solution in various
temperature ranges through 3C°C. which is a desirable characteristic.

This.

compound was the only one of those tested that is readily soluble in water.
\

U.

The growth oat tern of C_, phomoides CBOA, _C. phomoides 101. and.

Helminthosporium sativum 925 was determined for both stationary and shake
cultures.
occurred
5.

The maximum growth for shake cultures was 4 days before autolysis
and 9 days for stationary cultures.
C. r>homo ides C80A was found to grow best on D-glucose or sucrose

and. rather poorly on D— xylose or alpha-lactose.
6.

In both shake m h

stationary cultures the antibiotic activity rate

against C. phomoides CBOA for rimocidin was markedly increased when using
D—glucose or sucrose ^n cornuarison to D—xylose or alpha—lactose*
7.

The rate of grov/th of the test organism does not decrease as

rapidly in the stationary cultures as it does in the shake;cultures as the
amount of rimocidin is increased.
8.

A s t i m u l a t o r y effect occurred in stationary culture when rimocidin

is present up to 3 PPm *

4-S

9.

There was a marked disparity for xylose and gliicose when rimocidin

approached the critical range of retardation of C. r>homo ides C80A for
different replicates.

As the concentration of the antibiotic increased,

the rate of growth for the replicates became about equal near the point of
complete inhibition.
10.

The fungistatic activity of rimocidin was maintained for 15 days

when spores of C. nhomoides C80A were put in a glucose medium with 10 ppm
of the antibiotic.

If sucrose is substituted under the same conditions, the

fungistatic effect was prolonged for at least 38 days.

Summary of the in vivo section
Eight antifungal materials obtained, from various sources were
tested on eight plants to determine their phytotoxicity.

Certain of

these materials tested were antibiotics in the strict sense of the word,
but others were synthetic organic compounds.
panogen, was an organic mercury.

One of these materials,

Of all the materials tested, diaphine

exhibited the most consistent phytotoxic reaction at dilutions as low
as 25 ppm.

Some of these reactions appeared similar to the chlorotic

ring spot reactions found associated with certain viruses in plants.
Phytotoxic reactions were associated with the parts of the plant which
were immature at the time of applications.

The gramineous plants tested

exhibited no phytotoxicity when sprayed with antibiotics at 200 ppm.
The materials tested except for actidione and panogen, which were
introduced as checks in the later experiments, were not of high general
antifungal activity.

The in vivo level of activity was lower than that

found in some of the in vitro tests.

Some of the compounds reduced the

number of lesions, but did not totally prevent infection.

The pathogen

should be reisolated from such lesions and its pathogenicity studied
for possible antibiotic resistance.

Good controls with certain materials

were found in blackleg of cabbage and stem rust of wheat.

No signifi­

cant amount of control was found for early blight of tomato, spot blotch
of barley, anthracnose of tomato, and powdery mildew of barley.

Fluct­

uating greenhouse temperatures and run—off of the spray materials were
factors affecting the results.
These findings represent a statement of progress and are subject
to results of further investigations.

Selected References

1# Ark, P.A.

Use of Streptomycin dust to control fire 'blight*
SI. Bis. Rep. 37: 4 0 W + 0 5 . 1953*

2.

Anderson, H.W. and D. Gottlieb.
antibiotics.
Econ. Bot. 6(3 ):

Slant disease control with

29^- 308. 1952 •

3. Blanchard, F.A*
tomatoes.

if.

Aureomycin chemotheraphy of crown gall in
Phytopath. *fl: 95**— 958. 1951.

Brian, S.W.

Antibiotics produced by fungi.

Bot. Rev. 17: 357-*^'30. 1951.

5. Bruckner, B.H. et al.

The partial purification and properties of
antibiotic substances from the sweet potato plant.
J. Clin. Invest.
28 : 89**— 898. 19**9 .

6. Carlson, H.J. et al. Antibiotic substances separated from sumac.
J. Bact. 55: 607* 19**8.
7.

Celino, M.S. and B. Gottlieb.
Control of bacterial wilt of
tomatoes b y Bacillus •pol.vmyxa. Phytopath. if2; if. 1952.

8. Chapman, R.A.

Relation of specific chemotherapeutants to the infection
court. Phytopath, ifl: 6. 1951*

9*

Cooper, W.E. and S.J.P. Chilton.
Studies on antibiotic soil organisms.
I Actinomycetes antibiotic to Pythium arrbenomanes in sugar-cane
soils of Louisiana. Ehytopath. h0: 5^***— 552. 195®.

10.

Bavis, B. Ghemotherapeutic activity may be independent of
fungi tori city. Phytopath. if2: 6. 1952.

11.

_ _ _ _ _ and A.E. Bimond. Altering resistance to diseases with
synthetic organic chemicals. Phytopath. *f2* 563-567* 1952.

12.

Bavisson, J.W. et al. Rimocidin, a new antibiotic*
Antibiotics and Chemo theraphy. 1(5)* 1951*

13*

Bimond, A.E. and B. Bavis.
The chemotherapeutic activity of
bennzothiazole and related compounds for Fusarium wilt of tomato.
Phytopath. b3i *f3-*f*f. 3.953*

l*f»

Evans, E. and D. Gottlieb.
Phytopath. kZx ^ 65. 1952.

15.

Fontaine, T.B. et al. Partial purification and properties of tomatin,
an antibiotic agent from the tomato plant. Arch. Biochem. 12: 395* 19*^7•

16.

Gottlieb, B. The disappearance of antibiotics from soil.
Ehytopath. *f2: 9* 1952.

The role of gliotoxin in the soil.

17.

Gottlieb, D. et al. Actidione as a plant protectant.
Phytopath. 40: 218. 1950.

18

Gottlieb, D. et al.
The production and role of antibiotics in soil.
IV. Actidione and OX&vaciiu Phytopath. 42s 493-496. 1952.

*

En&omycin, a new antibiotic. Phytopath 41:393-^99.

19.

20

.

1951.
Gopalk r ishnan, K. S. and J.A. Jump.
The antibiotic activity of
thiolutin in the chemotherapy of the fusarium wilt of tomato.
Phytopath. 42: 338-339. 1952.

. Hamilton.

21

.

J.M. and M. Szkolnik#
Factors involved in the performance
of Cycloheximide (actidione) against Coccomyces hi email s.
Phytopath. 43s 109- 1953.

22

Henry, A.W. et al.
Control of covered smut of wheat by rapid
seed treatment with an antibiotic. Science 1X5:, 90-91* 1952.

23

Heuberger, J.W. and P.L. Paulos. Control of fire blight and frogeye
leaf spot (blackrot) disease of apples in Delaware in 1952.
PI. Dis. Hep. 37: 81-83. 1953.

24.

Hillborn, M.T. Effects of various chemicals on infection by
Bhi zoctonla solan! and Vertiollliuia albo-atrum.

*

Phytopath. 43(9)1 4?5. 1953*
25.

Hors fall, J.G. and A.E. Dimond.
Microbiol. 5* 209-222. 1951.

Plant chemotherapy. Ann. Hev.

. Leben,

26

C.
Influence of acidic buffer sprays on infection of tomato
leaves by Alternaria solani. Phytopath. 44: 101— 106. 1954.

27.

__________ and G.W. Keitt. Laboratory and greenhouse studies on
antimycin preparations as protectant fungicides. Phytopath. 39: 529-540.
1949.

28.

. Studies on helixin in relation to plant
disease control. Phytopath. 42: 168-170. 1952*

29.

Leben, C. et al.
Small grain seed treatment with the antibiotic
Helixin B. Phytopath 43: 391-394. 1953*

30.

. Helixin, an antibiotic active against certain
fungi and bacteria. Mycologia 44: 159-169* 1952.

31.

Lechevalier, H. et al.
Candicldin, a new antifungal antibiotic.
Mycologia 45(2 ): 155-171* 1953.

32.

Leulcel, R.W.
Comparative tests of seed treatment chemicals on
spring wheat and oats in 1953* ^1* JHs. d©P* 37 * 587-594. 1953*

52
33«

Lilly* V.G. and H.L. Barnett.
McGraw Hill, N.Y. 1951*

34.

..
Bui.

Physiology of the fungi, xii/ 464pp*

The utilization of sugars by fungi.

362T, W. Va. U. Agric. Espt. Sta. 1953.

35. Little, J.B. and K.K. Grobough.

Antibiotic activity of some crude
plant Juices. J. Bact. 52: 587* 1946.

36. Martin, K. et al.

The production and role of antibiotics in the
soil. III. Terramycin and Aureomycin. Phytopath. 42: 294-29 6. 1952.

37. McDonough, E.S. and L. Bell.

Inhibition of growth of Schizonhy^lum
commune by extracts from living trees. Phytopath. 41: 25. 1951.

38. McGahen, J.W.

Soil amendments in relation to the Actinomycete
population and the antibiotic index of sugar-cane soil.
Phytopath. 41: 25* 1951*

39*

Mitchell, A.E. et al.
College. 1954*

Spraying calendar. Ext. Bui 154, Mich. State

40.

Scott, W.E. et al.
The partial purification and properties of
antibiotic substances from banana. J. Clin. Invest. 28: 899-902. 1949*

41.

Sharvelle, B.C.
Systematic fungicides in 1951 British investigations.
PI. Dis. Hep. 36: 35-43. 1952.

42.

Siminoff, P. and D. Gottlieb.
The production of antibiotics in the
soil. I. The fate of Streptomycin. Phytopath. 4l: 420-430. 1951*

43.

Stessel, G.J. et al. Partial purification and properties of the
antifungal antibiotic toximycin. Phytopath. 43: 23— 26. 1953*

44.

Stoddard, E.M. and A.E. Dimond.
Bot. Rev. 15: 345-376. 1949*

45*

D.S.D.A.

46.

Winter, H.P. and H.C. Young.
Control of fire blight of
Ohio in 1953. 1*1* Bi»* Rep. 37: 4 6 > 4 6 4 . 1953*

47*

Young, W.J. and R.H. Pulton.
A field test of several fungicides
for the control of powdery mildew on Lucretia Dewberry in 1951*
PI. Dis. Rep. 35: 540-541. 1951.

The chemotherapy of plant diseases.

The Yearbook of Agriculture. Plant Diseases. 1953*
apples in

A P P E U D
(PLATES)

IX

PlateI
Concentration Series of Rimocidin in p.p.m.,
against Colletotrichum phomoldes C80A, Incubated
for 6 Days at Room Temperature.
Upper left:

Control on liquid.

Upper right:

Control on agar.

Lower, from left to right:
liquid 10 p.p.m.
agar 5 0 p.p.m.

Concentration Series of Uystatin in p.p.m.,
a£ainst Colletotrichum phomoides 101, Incubated
for 6 Days at Room Temperature.
Upper:

On agar media .

Lower:

in liquid media .

Plate II

Concentration Series of* Thiolutin in p.p.m. v
against Colletotrichum phomoldes CgOA, Incubated
for 6 Bays at Room Temperature.
Upper*

on agar media.

Lower®

on liquid media.*

Concentration Series of Compound M^575 in p.p.m.,
in Liquid Media, Incubated for 7 Bays at Room
Temperature.
Upper*

Colle totrichum phoTnoid.es CgOA.

Lowers

Helmintbosporium sativum 925.

Plate III
View of Kershaw Rotary Shaker.

This machine

is standardly used in this project for the
physiological studies involving liquid shake
cultures.

Plate IV
A.

Concentration Series of Diaphine against
Helminthosporlum sativum 925.
Upper*

diaphine sterilized via Seitz
filtration.

Lower?

diaphine sterilized by autoclaving directly with the
media.

Concentration Series of Diaphine against
Colletotrlchum phomoides 1QI.
Uppers

diaphine sterilized via
Seitz filtration.

Lower!

diaphine sterilized hjr autoclaving with the media.

CONTROL

CONTROL

Plate V
Stationary Cultures of Colletotrichum phomoides
CSOA.

General appearance when grown on differ­

ent sugars* without antibiotic.

Concentration Series of Pyridinethione incorp­
orated with B-xylose.

Stationary cultures of

Colletotrichum phomoides CgOA.

GLU C O S £

STATIONARY

Plate VI
A.

Phytotoxicity caused by Diaphine at 200 p.p.m*.
on Cucumber.
Rights

treated plant, showing chlorosis.

Lefts untreated check.

B.

Phytotoxicity caused by Diaphine and Pjrridinethione
at 200 p.p.m.. on Bean.
Right?

diaphine treated plant showing
chlorosis.

Middle?

pyridinethione treated plant
showing puckering.

Left?

untreated check.

Plate VII
Spray Trials for Control of Phoroa lingam on
Cabbage.
Right?

untreated check.

Left?

treated vith thiolutin at
200 p.p.m.

Spray Trials for Control of Phoma lingam on
Cabbage.
Right?

untreated check.

Left?

treated with diaphine at

Plate VIII
Spray Trials for Control of Phoma lingam on
Cabbage.
Right?

untreated check.

Left?

treated with pyridinethione
at 200 p.p.m.

Spray Trials for Control of Phoma lingam on
Cabbage.
Right?

untreated check.

Left?

treated with panogen at

4

Plate IX
A.

Spray Trials for Control of Phoma lingam on
Cabbage.
Right?

untreated check.

Left?

treated with nystatin at
200 p.p.m.

B.

Spray Trials for Control of Phoma lingam on
Cabbage.
Right®
Left?

untreated check.
treated with actidlone
at 200 p.p.m.