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THESIS

.uwn'l’rltflul I 1‘
“I I7. 3.3.4 t '3‘ II

THE PHYSIOLOGY AND VARIATIONS or cmcospgngg BETICOLA
IN PURE CULTURE

Thesis presented in partial fulfillment
for the
Degree of Master of Science,

Michigan State College.

By
’7

Finley G. kgrmer.
1988

THESIS

 

 

 

ACKNOWLEDGMENTS

The writer is indebted to Dr. G. H. Coon.
and Dr. E. A. Beeeey for advice and eugb
geetione given throughout this etudy, and
furthermore to Dr. Occne for criticien.end

correction of the manuscript.

1(fi3_2§3

TABLE OF CONTENTS

IntrOduotion. . C . O O O O O O I O O

Horphology of the organism.
Mycelium . . . . . . .
Conidicphcres. . . . .
Conidia. . . . . . . .

“9 thOd. O O O s s o s s s s s s s 0

Cultural characteristics. . .
Growth on media. . . . .
Light relations. . . . .
Temperature relations. . .
Formation and germination of
Loss of color. . . . . . . .

cases.
0

cusses
..s.

sgssss

zonation.._....o..
Types of zonation. . .
Light relations. . . .
Temperature relations.

The study of variant forms. . . . ,. .
Variants obtained from C. beticola

Physiological acti'ItIes. . .

Pathogenicity . . . . . . . .

Morphological characters. . ;

Variants obtained from C. beticola
Variants obtained from CT SetIcoIa
Variants obtained from UT‘betIcoIa
Aberrant form 0-19“. . . .
Aberrant form 0-80 v/O . .
Variants obtained from C. beticola
Attempts to produce varIEn s . .
Founding. . . . . . . . . .
Illuminating gas. . . . . .
Freezing. . . . . . . . .
Poisoning with chemicals.
Mechanics of sector formation.
Apparent reversions. . . . . .
Discussion . ... . . . . . . .

O
O
s
s
O
O
O
O

swan. O O s O O s o s s s o O O O 0
Bibliography.............

Supplement. . . . . . . . . . . . . . .

Stéain°cl11

strain.c;9:
strain 0-8.

strains 0-19

0 O O O O O
0 O O 0

strain 0-17

0 O O O O O O O O O C 9 O O O O C 0 O

O O O O O 0 O O O O O O O O O

O O O O O O O O O O O

D)

secessssossOsssssss

The physiology and variations of Cercospora beticola

in pure culture
Introduction

Oercogpora beticola is a cosmopolitan fungus causing
leaf spot of sugar beets (Beta vulgarie) and of allied
plants such as the garden beet, mangold-wurzel, swiss chard,
and other members of the family Chenopcdiaceae. It is a
common parasitic disease of the wild beet (Beta maritime)
in Europe (8), and is sometimes found as a leaf spot disease
on (Martynia Louisiana), a species of plant belonging to a
family widely separated from the Chenopcdiaceae. This plant
is a native of the central and western plains states and is‘

a common weed in the best fields of Colorado.

The disease is evidenced as small brown spots, with reddish
purple margins which later become ashsnpgray at the center due
to production of ccnidia. These spots are scattered irregularly
over the sumface of the leaf, but may become so numerous as to
coalesce and cover a large portion of the surface, and may
often cause a large portion of the leaf to die. In time with
severe infections the leaf blade assumes a parched appearance,
begins to discolor gradually from the distal portion toward
the petiole and finally becomes brown, crisp and somewhat
curled or rolled. The outer or older leaves are the first to
show these effects and wither away after the petioles wilt.

To replenish this deficiency of leaf area the plant produces

 

 

 

 

-3—

new leaves at the center of the crown. Consequently the
crown becomes considerably elongated, and the root with early
infections remains abnormally small because the food reserves
of the plant are consumed in replacement of leaf tissue rather
than for growth or storage. As a result of the high crown,
wasteful topping must be practiced to eliminate certain
organic salts that prevent the sugar from crystallising
after it has been extracted (18). This results in a two-
fold loss, namely, reduction in sugar content and tonnage.

Ceroospora leaf spot has long been recognised as being
one of the most destructive foliage diseases of the sugar
best but its importance has in some beet growing areas been
minimised because with certain rainfall distribution its
damage is small. The fungus never causes complete destruction
of the plant as is the case with many of the root-rot fungi,
or the deformities of the foliage that characterize the virus

diseases, especially curly-top. Infection by Cercospora

 

beticola occurs in cycles thus enabling the hhost partially
to recover by producing new foliage so that the severe effects
of the disease on the growing plant are emphasized only in
severe infections such as occur during wet seasons and in the
irrigated regions of the west where rainfall and excessive
use of ditch water.form epidemic outbreaks.

Of the American workers, Pool and McKay (14,15) have
contributed largely to our knowledge of the etiology of the
disease. They have correlated epidemics of leaf spot with

- 3 -
climatic conditions, primarily the influences of temperature,
humidity and other environmental factors on the production
and dissemination of ccnidia. Aside from the reports of
various experiment stations on the results of experiments
for the control of the disease, there is little American
literature dealing with Cercospora beticola.

This paper is a report of the results of studies of the
reactions of the fungus to various cultural conditions,
carried on during 1986-1927 at Michigan State College, and
at the united States Department of Agriculture Field Laborb
atory at Rocky Ford, Colorado. The contents of the paper
fall naturally into two sections, the first dealing with the
morphology, cultural characteristics and physiological reactions
to environment of the normal fungus, and the second dealing
with the abnormal or variant forms that it has produced.
The physiological studies were started in the fall of 1936,
the work on variant forms being taken up the following fall.
Infection experiments have been undertaken only in connection

with the study of certain variant forms.

“orphology of the organig!

Hycelium The mycelium formed in pure culture varies
greatly according to the food constituents of the medium.
The young hyphae are hyaline and tubular with thin walls and
septae only at long intervals, and filled with a homogeneous
finely granulated cytoplasm. As they grow older they become

greenishsbrown in color, build heavier walls and the cytoplasm

- 4 -
becomes coarsely granulated and filled with large highly
refractive globular bodies which are soluble in ether.
Septae are formed at short intervals and the myselium often
becomes gnarly, tortuous and irregular in shape. The aerial
mycelium is umually more regular, somewhat smaller and less
deeply colored, often almost hyaline. Sclerotiumplike bodies
composed of loosely massed hyphae are often observed in the
submerged mycelium at the point of contact with the petri-
dish. These bodies appear to be formed by abnormal division
and growth of a few cells and are more prevalent in newly
isolated cultures. They resemble very closely the sclerotium-
liks bodies formed in the substomatal spaces of the leaf
from which the conidiophores arise. However, several of the
masses have been isolated and kept in distilled water andhoth
cultures bum fructification has never been observed.
Conidiophores Fructification in pure culture results
in abnormal conidiophores. Normal conidiophores are fasciculate,
simple, erect and flavous, arising from a few celled stroma in
the subatomatal cavity of the leaf. Under field conditions,
conidiophores are normally short and non-septate, although
septation and elongation into hypha-like structures may occur
in the older conidiOphores (Plate I, Fig. 1). This latter
condition is especially true on artificially inoculated plants
grown under humid conditions in the greenhouse. The simple

conidiOphores may have one or two indistinct septae near the

-5-

base. They vary from 35 - 80 microns in length, and their
average width is 4 - 5 microns. The older hyphaelike conidio-
phores are sometimes as much as 180 microns long with septae
30 - 40 microns apart. In artificial cultures the younger
conidiophores are short, simple and nonpseptate, arising
directly from a submerged filament (Plate I, Fig. 1). At the
apical end is seen a distinct scar, circular in outline,
which indicates the point of attachment of the spore. Older
conidiophores may have several of these scars along the
surface with a geniculation at each scar. then the conidio-
phore is mature, and having borne a spore is put under favor—
able conditions, it sends out new growth frmm just beneath the
scar. From this new growth a conidium is produced and
abscissed leaving a distinct bend. The older conidiophores
are long, septats and hypha-liks, resembling the older
structures found on plants. As the conidiophore begins to
decline, conidia-like branches arise, these branches and the
conidiophores serving as permanent hyphae (Plate I, Fig. l).

Conidia Abnormal conidia varying from one or two-celled
structures to extremely long multieeptate hypha-like filaments
are sometimes produced (Plate I, Fig. 3). As a rule, conidia
are typically obclavate to needle shaped, hyaline, multi-
septate, and show typical circular attachment scars. Conidia
from normal green cultures have a maximum length of 360 microns
and a minimum length of 55 microns, the average length being

140 microns, and the average number of septae per conidium

- 5 -
being 10.8. The spore contents are finely granular, non-

vacuclated and practically hyaline, conidia produced in pure
culture being slightly more transparent than those produced

on sugar'beets.

Methods

Leaf spot material was obtained from the sugar beet
growing districts of Colorado, California, Minnesota, Iowa,
Nebraska and Michigan, all of which are more or less subject
to severe epidemics of the disease.’ Single spore cultures
were obtained by the dilution plate-method, the individual
conidia being marked as soon as germination could be detected
by microscopic examination. The germinated spore in a minute
block of agar was transferred to corn meal agar in test tubes
and furnished the stock cultures used in the experimentation.
Cultures were identified by microscopic examination, cultural
characteristics, production of conidia and in some cases by
passage through plants, in which case the re-isolation was
compared with the stock culture. Cultural studies have been
made from both test tube and petri dish cultures, plate cultures
being used most extensively since they are more easily observed.

Sugar beets used in artificial inoculation work were
grown in pots in the greenhouse and inoculated by spraying
spore and mycelial suspensions upon the leaves. The plants

were then placed under bell jars and put in a temperature and

‘See 'Cultural strains of Cercospora beticola' in Supplement.

-7-
humidity chamber since moderately high temperature and humidity

have been found to be favorable for infection.

Cultural characteristics

Many kinds of media have been used in the writer's study
of Cercospora beticola since a careful delineation of the
cultural characteristics of this organism on artificial media
is needed to furnish ready means for its identification and
in distinguishing the races or strains from one another.

This work has also given information concerning conidial
production and various physiological phenomena of the organ-
ism, and it will be indicated by the work to be presented
that certain variants depart strongly from the mean behavior
of the species.

9393311. 93 _m_e_d__i_a The fungus was found to grow well on
most media employed. Plantings from corn meal agar cultures
were made as shown in the following summary and the growth
characters observed frequently but the results here given
represent a summary of the observations and may be taken as
the normal behavior of cultures which have grown for a ten day
to two week period. The small mycelial threads are almost
translucent but as they grow older the hyphae manufacture a
pigment which varies in color with the character of the medium
from a dark gray through the different shades of green to
black, but most generally is an olivaceous green in the sub-

merged and gray-green in the aerial mycelium as found on

- 3 -

corn meal agar. A reddish tint is sometimes produced with
certain media, evidently due to some soluble pigment produced
by the fungus. The following records were made after a

ten day period from cultures growing under bell jars at

room temperature under diurnal light conditions.

1. Corn meal agar: Submerged mycelium olive green, soned,
sclerotium-like bodies; aerial mycs ium greenish-
gray, abundant, fluffy, somewhat clumped. Diam.

9 m/m. No typical spores.

8. Cat meal agar: Submerged mycelium pea green, shallow,
zoned; aerial mycelium sparse, white tuft in
center. Diam. 18 m/m. A few typical spores.

3. Potato dextrose agar: Hycelium almost black, shallow
in substratum, soned faintly, exuding amber
colored fluid; aerial mycelium profuse, variously'
colored, gray background, felty and matted.
Diam. 17 m/m. No typical spores.

4. Lima bean agar: Greenish-black submerged mycelium;
aerial mycelium heavy, felty, black at border,
white in center. Diam. 81 m/m. Typical
spores in new growth.

5. Bean pod agar: Black in substratum; aerial growth
abundant, cottony, white in center, dark at
border. Diam. l? m/m. A few spores.

6. Prune juice agar: Deep green in substratum, zoned
faintly, sclerotiumslike bodies; aerial growth
sparse, white and fluffy at center. Diam.
8e m/m. No spores.

7. Malt extract agar: Dark green in substratum, mycelium
distorted, full of globular bodies, dispersed
reddish pigment in medium; aerial myceliflm‘
abundant, fluffy, white to gray. Diam. 85 m/m.

No spores.

8. Plain agar: Submerged growth sparse, pale green; little
or no aerial mycelium. Diam. 88 m/m. No spores.

(I

Q

- 9 -

9. Neutral red agar: Growth shallow and tufted; aerial
mycelium.short, grayish-black. Diam. 7 m/m.
Numerous short irregular spore-like structures,
germinated like conidia. '

lo. Litmus lactose agar: Black in substratum; grayish-
black, low, felty aerial growth. Diam. 18 m/m.
No spores. A

ll. Endo's agar: Hycelium in substratum dark, takes up
red pigment; aerial mycelium short, grayish-
brown. Diam. 15 m/m. No spores.

18. Coon's synthetic agar: Rich green in substratum;
, aerial growth abundant, felty, uniformly
_ grayish-white. Diam. 85 m/m. No spores.

13. Nutrient agar: Submerged mycelium green, amber colored
fluid exuded throu grayish-white, felty
aerial mycelium. iam. 15 m/m. No spores

14. Patel's crystal violet a r: Creenish-brown in
substratum; gray, elty aerial mycelium.
Crystal violet reduced to yellow by growth of
the fungus. Diem. l8 m/m. No spores.

15. Raw vegetable media: Potatoes, carrots, sugar beets,
garden beets, turnips and sweet potato plugs in
sterile test tubes - no growth. Beet leaf
petioles in test tubes - slight grayish aerial
mycelium at point of inoculation, slight penetra-
tion of tissues, epidermis must be broken for
growth.

16. Bouillion broth: Inoculum placed on surface. Surface
growth gray and felty. Submerged mycelium hyaline
and irregular. No spores.

1?. Nutrient broth: Characteristic grayish-green growth
at surface of liquid. No spores.

18. Sugar solutions: Sucrose, dextrose, mannnse, maltose,
levulose, galactose and glycerine in 1.C% solutions
in sterile distilled water. Inoculum placed on
filter paper cones in preparation dishes. No
growth.

18. Nutrient sugar agars: Nade up from nutrient broth to
which 1.07% sugar and 1.0% agar agar had been added.

(I

30.

31.

33.

33.

34.

35.

36.

37.

38.

30.

- 10 ..

Check - growth pellicled, black. Levulose and
glycerin - same as the check. Mannose - greenish-
gray, felty and matted. Diam. 14 m/m. Galactose -
deep greenish-gray, felty and matted. Diam. l5 m/m.
Dextrose - rowth felty, light gray to gray. Diam.

8O m/m. Ma toss - more fluffy, less matted, greenish-
gray. Diam. 88 m/m. Sucrose - similar to maltose,
slightly lighter in color. Diam. 80 m/m. No spores
found on any of these media.

Nutrient sugar agars with dyes: Brom crysol purple -
tolerated by the fungus with a variable degree,
slight indication of reduction of dye on sucrose,
dextrose, maltose, levulose and galactose. Brom
thymol blue - intolerant to the dye, growth very
slight on all sugars.

Beet leaf broth: Growth felty in pellicle at surface,
black. A few atypical spores.

White rice: Mouse gray, olive green at points of contact
with test tube. No spores.

Castor bean seeds: Myselium intensely black, full of
oil globules. No spores.

Corn grains: Mycolium black with short greenish-gray
felty aerial growth. No spores.

Lima bean seeds: Mycelium greenish to black; short
velvety mouse gray aerial growth. No spores.

Peas: Very similar to lima bean seeds, slightly darker
and more velvety. No spores.

Sunflower seeds: Similar to growth on castor bean seeds.
No spores.

Potato plugs: Tough black growth in substrata; cottony
gray aerial mycelium. No spores.

Carrot plugs: Mouse gray to white aerial growth;
greenish-black mycelium in substratum. No spores.

Beet leaf petioles: Olive green in substratum; ashen
gray fluffy aerial mycelium. A few atypical spores.

- 11 -

31. Beet leaf agar:‘ Greenish-black in substratum, distinct
zones; aerial mycelium sparse, low, mealy, deep
green. Poor vegetative growth. Abundant typical
spores.

Corn meal agar has been found to be the best differential
medium used in these studies of Cercospora beticola. The
fungus produces a characteristic olive‘green colony” with
greenish-gray aerial mycelium. Alternaria 32. is sometimes
obtained as a contamination in dilution plate isolations of
Cercospora_beticola from the sugar beet leaves. In color
and appearance of submerged growth it resembles Cercospora
beticola‘but is readily recognised by the conidia which are
very abundant on corn meal agar. Beet leaf agar'has been of
value in studying conidia production in pure culture, but is
not a good nutrient medium for vegetative growth. .

Influence g; depth 2; medium and amount 2; nutrientg
In order to determine the effects of the depth of medium and
quantity of nutrients upon the organism the following tests
were carried out. Petri dishes were supplied with 18 cc.
and 85 cc. and deep culture dishes with 50 cc. of the nutrient
agars listed in Table I and inoculated with strain 0-8.
’Preparaticn of best leaf agar:

1. Young sugar beet leaves dried at 40° C. and finely

powdered.

8. 15 grams of best leaf flour and 15 grams of agar agar

cooked slowly in distilled water for 30 minutes.

3. Strained through cheese cloth and made up to 1000 cc.

4. Autoclaved at 15} pressure for 80 minutes.

"Following general usage in plant pathology, the so—called
'pseudo-colony' of a fungus is termed a 'colony'.

- 18 -'

Parallel cultures placed under similar light and temperature
conditions under bell jars gave such similar results as to
make separate records unnecessary. As will be seen from

the table, differences in linear growth on plates of differb
ent depth of medium were practically negligible at the end
of a ten day period but favored the 85 cc. plates after
twenty days, with the 18 cc. plates showing the least growth.

Table I
Influence of depth of medium on linear growth of g; beticola

 

Agar medium 18cc. 85 cc. 50 cc.
16 85 IE 86 IE 56

da s 'da s _d%%g dags dags dags
Oat meal " 6 7

 

 

 

Litmus lactose 18 87. 18? 48 18 50>
Potato dextrose 80 70* 18 68 88 78
Bean pod 18 64 18 70‘ 81 70
Corn meal 80‘ 68 80 76 80 68
Malt extract 88 67 85 88 18' 80
Beet leaf 18 55 18 48 18 48
Plain 17 61 18 68 18 66
Coon's synthetic 85 88 85 80 83 80
Neutral red 18 84 10 36 ll 36
Prune juice 80 74 81 76 18 68‘
Lima bean 81 71 17 68? 18 76
Endo's .. 14 38 14 45 15 45

Average 18.6 58 18.5 64 18.5 68

 

(Values represent diameter of colony growth in millimetere.)

Since the cultures were grown on solid media there was
no accurate method of weighing the actual amount of mycelial

growth so observations were made on the density and amount of

- 13 -
aerial growth and the depth to which the submerged mycelium
had penetrated the substratum at the end of the experiment.
At this time the growth had attained the greatest depth in
the 50 cc. dishes and the aerial mycelium was profuse and
matted. There was a general tendency for the aerial mycelium
to become lighter in color as further growth occurred. From
the experiment the conclusion can.be drawn that the amount
of nutrient present affects the growth materially after the
first ten days in that an abundance of food permits further
growth of the mycelium formed during the initial period of
growth. The 18 cc. plate cultures had probably consumed
the available nutrients during the initial growth and the
fungus had entered a period of quiescence until more food was
available. Depth of medium probably affects the cultural
characteristics only in so far as the quantity of nutrients
is concerned.

.2133! relations Since many organisms react in a specific
manner to light stimulus, the following tests were carried
on to determine the effects of light upon various strains of
Cercospogg'beticola. Strains 0-11, 0-13 and 0-83 were planted
in petri dishes containing 15 cc. of corn meal and potato
dextrose agars and kept under the different conditions of
light as indicated in Table II.

- 14 -
Table II
Growth of Cereosppra beticola in relation to light

 

"_ Colony diam. m/m
Condition Temperature Medium Strain '_-Dgys of rowth

 

 

 

 

 

 

 

 

of growth 6 0 14
Potato 0‘11 43
dextrose g-gg 45
r -
Darkness 320-34° c. fin 1:11 41—36 -
2;: 3%: ’ 2:
m Potato 6-11 '2 13 34 36
Ordinary dextrose g:%g 8 17 31 48
day ight o_ o r
conditions 31 33 0' 'gfin 5-11 9 17 as
meal 0-13 11 80 87
a 0-33 13 39 36
Potato 5-11 8 16 88 40
Light from dextrose 0-13 8 16 87 38
100 watt 3‘0 0 C 0‘33 3__1i__25_—§§——
nitrogen '35 ° Corn ’11 ll 81 35
filament meal 0-13 10 18 84
bulb m 0-33 11 30 34

 

 

It is evident from the records given in the table that
light relations are not very marked on the growth of Cercospora
beticola. These findings are similar to those of Klotz (11)
in respect to Cercogpoga a ii, a closely related species.

' Linear growth is slightly more rapid in darkness than it is

in either light or diurnal conditions of light but this
difference is so small that it is probably negligible.

Growth on corn meal agar was abundant, and matted in all

forms under the different conditions, differing only in color
of mycelium which was grayish-white under continuous light,

3 light greenish-gray under diurnal light and a dark greenish-

- 15 -
gray in continuous darkness. Growth was more rapid on
potato dextrose agar, being abundant and felty, but the
color contrast was lacking, all cultures having light
greenish-gray aerial mycelium. Observations made at differ-
ent times showed only a difference in color of mycelium be-
tween cultures grown in light and darkness, cultures in light
being somewhat lighter colored.

Sporulation in some fungi has been found to be a direct
response to light stimulus. Coons (7) found that pycnidia
were produced by_§lenodomus fuscomgculans only when cultures
were grown in light. Hedgecock (19 determined that spores
were produced in darkness in artificial cultures of
Cephalotheciumggggggg;and other fungi, while Gallemaerts

according to Bisby (1) found that light inhibits mycelial
growth and thus stimulates sporulation in the same fungus.
Leonian (18) found that light was required for’the production
of pycnidia in species of Sphaeropsidales. According to
Bisby (1), Melts reports that conidial cushions are almost
entirely absent in cultures of Sclerotinia fructiggna when
grown in darkness, and himself concludes that light affects
the tips of the hyphae giving them a brief check or resting
period resulting in conidia formation.

To study the effect of light on conidia production,strains,
0-11, 0-13 and 0-83 were planted on 18 cc. of best leaf agar
in 850 cc. flasks and kept under the conditions described in .
Table II when the data were taken.

- 15 -
Table III
The effect of light on conidia production in.gg_beticola

 

Strain Light 84-85° c. Diurnal 21-23° c. Darkness ss-s.°c.

Growth Spores Growth Spores Growth Spores

 

 

 

0-11 84 ++ typical 88 +++ typical 87 ++ typfimd

?0-ll . 85 do 81 dd 87 do
0-13 i as do as do as do

0-13 86 do 83 do 85 do

0-83 84 do 84 do 85 do

0-83 84 do 84 do 84 do
A70 25e8 83e3 35e3

 

(Numerical values represent diameter of colony growth in m/m.)

Conidia were produced under all conditions of light and
darkness but cultural washings indicated that they were
produced most numerously under diurnal conditions. This will
probably explain the slightly slower linear growth as being
due to checking of the_mycelial filaments resulting in conidia
formation under fluctuating light.

(Temperaturg.5elationg The occurrence of epidemics of
Cercospora leaf spot during the late summer months would
indicate that high temperatures are necessary for its optimum
development. A nine compartment differential thorn-regulator
was employed in studying the relation of temperature to growth
of the organism. The low temperatures were obtained by keeping
ice packed in one end and the high temperatures by the use of
a small electric heater which was placed in the opposite end.
This gave a series of fairly constant temperatures varying by

steps from 10-40° C. Inoculum from corn meal agar cultures of

- 17 -
strains 0-11, 0-13 and 0-83 was transferred in duplicate to
15 cc. of agar in petri dishes and incubated for a period of
ten days, when the data were taken. .As there were only slight
differences between strains in a given medium, the data have
for simplicity and convenience been averaged and recorded
collectively for each temperature range.
Table IV
The effects of temperature on growth of 0. beticola

 

Temp. Médium. DI*’. 0?

Comp. range agar Colony mam Character of growth
X. 7e he ‘

 

 

 

 

 

 

 

I. 8-IC°C Corn m. - 3 -- Too slight to measure
Nutrient - - - do
Pot. dex. - - - do

8.13-18°8 Corn m. “:73 8 5 Poor, deep olive green
Nutrient ll 8 4 , do
Pot. dex. 15 10 4 do

3.17-18°U Corn m. ’16 I? 13 Fair, green, aerial slight
Nutrient lg 15” 13 do , mouse ggay, 'gc
Pot. dex. 7 do , greenis (gray, 0

3-18-8138 Corn m. 86 IE'ClBC ACOod, desp olive green

Nutrient 18 l7 13 do , dark my
Pot.dex. 86 84 88 do ,ggreen sh-gn_y

 

 

 

 

5. 88:83°C Corn m. “IE—'15'CI4 ‘Like (4)'
Nutrient 16 15 14 do
Pot. dex. 88 86 84 do g, sli htl better
8. 84 ~88°8 Corn m. 88 83 88 ‘—Cptimum, like (37*Inv color
Nutrient 18 16 14 do
Pot. dex. 30 88 87 do

 

 

7. 87 -88°C Corn m. 38 85 86 Profuse, pale growth
Nutrient 35 15 14 do , white aerial growth
Pot. dex. 38 30 30 do ,1 white and felty~_

8. 38- 33:5 CCBrn m. 14 8 8 ‘Poor, aerial pale,_heavy
Nutrient 7 g 3 do , white throughout

 

Potgdex. 15 dog, aerial white, heavy;
Corn m. - No growth

Nutrient - do

POtedQXe - do

8. 37-4050

These data indicate that the fungus grows well between

80° and 30° 0., temperatures above or below these limits being

- 13 -
detrimental to the best growth of the organism. The optimum
temperature as determined by the size and color of the

mycelium is around 84-86° 0. Good growth takes place below

the optimum but with a gradual decline in rate of growth.
Temperatures above the optimum seem to stimulate abnormal
growth, the organism becoming pale and producing considerable
aerial mycelium.

Pool and McKay (14) report that conidia production on
the host plant is greatly influenced by temperature and
humidity. Humidity is apparently the determining factor since
sporulation occurs over a wide range of temperature but only
under conditions of high humidity. However, heaviest sporula-
tion occurs at temperatures at the lower limits for optimum
growth of the organism, which was between 84-880 C. on best leaf
agar, slightly higher than for ordinary culture media.

Duplicate transfers of strains 0-11, 0-13 and 0-83 were
made on best leaf agar in petri dishes and inoculated at the
various temperatures of the thermo-regulator for ten days.

Table V i

The effects of temperature on conidia production
in cultures of Cercoeppralbgticolg

 

Comp. Temp. Colony Diam. mlg Character 0

 

 

(°0.) Max. Av. Min. of growth Spgres

. 6-10 - - - ‘Cfione None
8 . 14-16 7 6 5 Poor None
3. 17-18 l4 13 11 Fair + short, robust
4. 19-80 80 18 16 Good ++ typical
5. 88-83 80 18 13 Good +++ typical
6. 84-86 83 18 15 Optimum +++ longer than in (5)
7. 87-89 88 18 15 Abnormal + few and long
8. 38-34 14 10 5 Poor 0 few and hypha-like
8. 37-40 - - - Death None

 

- 19 -

Formation and germination.2£ conidia Conidia production
on artificial media has never been reported in any of the
literature reviewed, although it has been known to occur in
other members of the genus Cercospora (8,11). Typical conidia
have been found on.beet leaf agar and more or less normal spores
have since been found on several media. However, such instances
are rare and the conidia are so abnormal that they could be
readily overlooked. Conidiophores and conidia have been
described and compared with those produced on plants under
'Morphology'.

Spores are produced over the entire surface of the mycelial
growth but they are seen in greatest numbers at the borders of
the colony, due probably to the formation of hypha-like conidio-
phores and the abscissing of the conidia. Apparently light and
temperature play minor r6les in sporulation, conidia being pro-
duced over a temperature range of 17-88° 0. with the heaviest
spore production occurring at about 85° C. which is the lower
limit of temperature for optimum growth. Humidity with proper
nutrients is probably the determining factor, heaviest sporula-
tion taking place at 60% humidity (14) with very few conidia
being produced in a dry atmosphere. High temperature stimulates
linear growth, conidia often exceeding 860 microns under these
conditions. Short robust spores with few septae are character-
istic of low temperatures.

Conidia produced in artificial cultures germinate in the
usual manner (Plate I, Fig. 8). The end cells germinate first,

- 30-
sending out long slender hyaline sometimes branching germ
tubes, and then shortly afterwards the intermediate cells of
the conidium germinate. It is seldom that more than 50% of
the conidial cells germinate, dessioation of the non-viable
cells apparently taking place. Infection on sugar'beets can
be produced as readily with these conidia as it can be with
conidia produced on the plant.

Heavy fructification on best leaf agar is indicated by a
dark olive green to black mycelium in the substratum, with
sparse aerial growth which is dark, short and mealy like. There
is a tendency for cultures to lose their power of conidia pro-
duction when grown in artificial cultures continuously for long
periods. Loss of vegetative vigor and color are generally
indicative of partial sterility. Achromatic cultures resulting
as gradual or sudden variations are invariably sterile although
conidia much smaller than normal have been observed in a few
instances. Heavy aerial mycelium is usually accompanied by low
conidia production.

From the data given the conclusion can be drawn that conidia
production in artificial cultures of Cercospgra beticola is a
response to a food stimulus influenced more or less by other
factors such as humidity, light and temperature.

L31; g_f_ 93195 Loss of the characteristic green color
through reduction in the amount of greenish-brown pigment in
the mycelium has occurred commonly when the fungus has been kept
in culture on artificial media for long periods of time. Factors

which seem to influence this phenomenon are high temperatures,

- 31 -
dessication in old cultures and quality and quantity of
nutrients. None of the factors when tried experimentally

has induced permanent loss of color. Cultures have been re-
peatedly grown at high temperatures resulting in pale colored,
almost achromatic growth which immediately became normal green
when transfers were made from these cultures and grown under
normal conditions.

Observations on the effects of dessication in relation to
loss of color in subsequent transfers were made by taking in-
oculum from normal olive green cultures that had dried out with
age. The following table shows the results of transfers made
from the original stock culture of several forms, over differ-
ent periods of time. Transfers were made on corn meal and
potato dextrose agar slants and incubated at 840 0. for ten

days before observations were made.

Table VII
Viability of g_._ beticola in cultures

Tin sTe r °WW
Strain 58° °1 Corn mail 'Pot. dex. ’00rn meal 0 . rex.

 

 

 

°“1t“r’ a r agar agar a

U-I 15731788 Normil lo growth Normal 805581
0-8 10/31/86 do Normal do do

0-3 10/31/36 do do do do

0-4 11/1/86 do do do do

0-5 11/1/86 do do do No growth
0-10 11/5/86 do do do Normal
C-ll ll/5/86 do do do 'do

0-13 11/5186 do do do do

 

These data show that only normal cultures were obtained

from eighteen month old cultures that were completely dried out.

_ 33 -
Subtransfers from these cultures after they had grown for
thirty days gave only normal forms with no indication of loss
of color.

Growth on certain media produces cultures that are pale
green in color; this being particularly true of a 1.8% wahsed
plain agar medium. Strains 0-6 and 0-17 were grown on plain
agar medium and small quantities of inoculum were taken from
the edge of the colony to obtain as near a homogeneous culture
as possible. This inoculum was transferred to petri dishes
containing 1500. of plain agar’medium and allowed to grow for
fifteen days when the cultures received the treatments indicated
in Table VII.

Table VII

Treatment of pale cultures with amino acids

 

Treatment Strain 0-6 Strain 0-17
Leucins CNo’ohange Same asCC:8-
Tyrosine Greenish-black ' do
Alanine New growth light green do
Glycine No change do
Cystine do do
Asparaginic acid New growth normal green do
Corn meal agar Normal culture do
Check No change do

 

This experiment was run in duplicate and repeated with
similar results. It indicates that aminonitrogen is avail-
able and has a tendency to'gring out the green pigment.
Achromatic cultures of unknown origin and the aberrant form of

strain 0-1 have been treated similarly with negative results.

- 33 _
These data indicate that the abnormally pale forms
studied are not of the same nature as those which are ob-
tained either through continuous culture or the aberrant
forms, in that they immediately return to the normal green

color when grown under favorable conditions.

Zonation

The formation of zones, i.e., production of concentric
rings of light and dark mycelium, is a peculiarity exhibited
by many fungi growing in artificial cultures. Structural
characteristics of the colony growth such as varying density
and amount of spore massing, grouping of pycnidia and sclerotia,
mycelial branching, color, etc., due to differential growth
are largely responsible for the occurrence of this phenomenon.
These effects have been attributed to various casual agencies
such as light relations, temperature relations, resting periods,
staling products, mycelial crowding, alkaline medium and varia-
tions in the.amount of nutrients. Stevens and Hall (17) em-
phasize the fact that zonation may be induced by fluctuation
of the above external factors but that it is largely dependent
upon the fungus.

Zonation was first observed in test tube'culturesbut later
was found to be very pronounced in petri dish cultures. By cor-
relating the number of bands with the age of the colony it
could be seen that the phenomenon was in some way associated

with diurnal fluctuations. Linear growth is normally slow in

- 34 -
Cercospora beticola, consequently the bands are narrow and
often indistinct. This together with the fact that zonation
occurred in cultures in.which there were no conidia formed
and was not due to fructification on best leaf agar, insti-
gated experiments to determine the characters involved and
the causative factors responsible for the expression of this
differential growth.

‘11223‘ g zonation Several types of zonation have been
observed, the most common being due to the differential rate
of linear growth of the submerged mycelium. In this type the
aerial mycelium is often felty, showing a uniform color and
texture throughout the colony. and can be removed without
destroying the concentric rings as they are in the submerged
mycelium only. Microspopic examination shows that the dark
bands are due to a large number of mycelial threads and the
light bands to a small number of mycelial filaments. It seems
that in this fungus the dense crowding of the filaments re-
sulting from their repeated branching inhibits growth, re-
sulting in a period of quiescence, followed by the onward
growth of a few of the more vigorous scattered hyphae. This
process is repeated indefinitely, the rapidity of succession
of zones being dependent upon the relation existing between
the rate of branching and increase in linear growth which is
controlled by fluctuating external conditions. The mycelial
filaments in the dark bands are irregular and somewhat distorted
and twisted, while the filaments in the lighter bands are smooth,

- 35 -
even and regular. Associated with this type we often find
aerial growth appearing in concentric rings which coincide

with the zones in the submerged growth. This type is especially
prominent on lima bean agar but has not been associated with
sporulation but rather with clumping of the aerial mycelium.
Occasionally cultures produce a red pigment in the medium,

which often occurs in distinct regions or zones.

Light relations That zonation in artificial cultures of

 

certain fungi is a direct effect of fluctuating light conditions
has been conclusively proven by several investigators. However,
a general rule for the specific effect on all fungi cannot be
made. Then too, density and grouping of fruiting bodies have
been the characters most commonly associated with the formation
of zones. Since spores are not produced in cultures of 935-
cospora beticola on standard media, no certainty could be placed
on the influence of light without observing its effects under
conditions where other factors~were controlled. In conjunction
with the experimental work on relation of light to zonation,
observations were made on the effects of red and blue wave
lengths since Miss Parr (13) has shown that Pilobulus gave
varying phototropic responses for different portions of the
spectrum. Hedgecock (10) found that blue light was comparable
to daylight and red light to darkness in his studies of
Cephalothecium‘gggggg. According to Bisby (l), Gallemaerts
working with the same genus obtained contradictory results in
that red, orange and.blue lights all gave the same reaction as
diffused daylight.

- 35 -
Egperimental data Data on zonation of Cercggpora

 

beticola in relation to fluctuating light conditions and dif-
ferent wave lengths of light are given in Table VIII, page 87.
Culture dishes of the same diameter and depth were used through-
out the experiment. Twenty cubic centimeters of agar were
poured into each petri dish, thus assuring the same volume

and depth of medium and the same atmospheric conditions for
all cultures. Eight different media and four different strains
of Cercospora beticola were used, inoculum being placed in

the center of each quadrant of the petri dish. The cultures
were incubated in special chambers designed for studying light
relations. The temperature was found unot to vary beyond the
limits of 88-840 0. at any time that readings were taken.

All joints in the chambers were sealed with modeling clay,

thus assuring that light was admitted only through the glass
filters. Diffused daylight was admitted through a north ex-
posure, and the red and blue lights were obtained by passing
natural light from the same source through glass filters which
cut out all except the red and blue lights respectively. Con-
tinuous light was furnished from a 110 v.-100 w. nitrogen fil-
ament lamp, as was the light for the 10 and 30 minute exposure.
Observations on zonation were made after the cultures had grown
for twelve days.

The exposure of cultures of Cercoggora beticolg to diffused

 

daylight alternating with darkness caused zonation in all

cultures on all media except plain agar, and no zonation was

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+ + + o o o o o o o o o o o + + + + o o o o + + + 4 neon mzflg
+ + + + + + I u u t o o o o + + + + t o t o + + 4 + Hams pmo.
+ + + + u u n u u o a o o o + + + + c o t o + + + 4 mama poem
e + + o o o o o o o o o a + + + o o o o + + + .Hmd .aom
+ + + + u n u n a a t s ,b. o o o + + + + o o o 0 + + v + mea Show.

.nHE om .nws 0H, mammxamd 9: Ha mzofipfldmoo pg Ha dog pg,HH mdap graces

Mo momsmomxm pnmfia mdofimflpnoo woodmapnoo qumdfim mmqummd mmmthmw Hmmm

mwfla mmmmmamm

 

Hmsafiflfl .{Hmmhdfinw

mHOOHpmp.um QH :ofipwqou so pamfia Mo poowgm

HHHP manna

    

- 33 -
observed on this medium under any condition. However, due to
density of the aerial mycelium the zones were somewhat marked
on some media. Concentric rings were especially prominent on
corn meal, oat meal and best leaf agars on which the fungus
produces only a moderate amount of aerial mycelium in twelve
days. Allowing two days for the fungus to start active growth,
zonation was correlated with diurnal fluctuations, there being
ten distinct rings formed over a twelve day period. Blue
light alternating with darkness gave comparable results with
daily fluctuations while on the whole zonation was not caused
by red light. Very faint traces of zonation were observed

in some cultures exposed to red light as was the case in
cultures kept in constant darkness. NCultures kept in contin-
uous light gave no evidence of zonation. Distinct growth
bands were induced on oat agar and gaint‘bands on corn meal
and best leaf agars by exposing cultures for 10 minutes but a
30 minute exposure was required to cause zonation in cultures
growing on the other media.

The 10 and 30 minute exposures were made every two days,
the growth of the colony being marked each time. The narrow
dark 'bands were found to be produced beneath the marks indi-
cating the point of growth at the time of exposure to light,
and the wide bands of light colored mycelium were produced
during periods of darkness. This indicates that light has an
inhibiting effect on the hyphae at the growing tips, causing
profuse branching to form the dark growth bands.

- 39 -

_Tgmperature relations Investigators disagree as to the
part played by fluctuating temperatures in causing zonation.
Chaudhuri (4) concludes that there is no correlation between
periodic external conditions and zonation. He found that
cultures of Verticillium albo-atrgg produced Zones in constant
darkness only at 35° C., but that zonation was evident in
cultures grown in constant light at 31-330 0., indicating that
different conditions of light and temperature influenced zon-
'ation but that the phenomenon itself was induced by other
causative factors. Hedgecock (IQ was unable to attach any
significance to fluctuating temperatures in relation to zon-
ation in Cephalothecium roseum and other fungi. Conversely,
Bisby (1) reports that temperature plays an important part in

the zonation phenomenon.‘ He shows that zonation was induced
in Fusarium 32; grown in constant darkness by alternating
temperatures between limits of lO-35° C. He attributes this
to differential growth of mycelium and not entirely to conidia
production- Substantiating this, Christensen (6) produced
distinct bands in cultures of Helminthgsporium gativum by
alternating temperatumes in both constant light and darkness.

Using the same type of petri dishes and media prepared at
the same time as was used in the light experiments, an attempt
was made to determine the relationship of fluctuating temper-
atures and zonation in cultures of Cercospora beticola. The
same four strains were planted as in the previous experiment

and all petri dishes were carefully wrapped to exclude light.

- 39 -
Set (A) was kept in an incubator at 84° C., Set (B) was
alternated between temperatures of 84° C. and 13° C. every
48 hours, and Set (C) was kept at a constant temperature of

13° c. in a chamber of the ice box.

Table II

Temperature in relation to zonation in cultures of g; beticola

 

 

 

 

 

 

34 c e 13-3;v 67‘ ' 13° C e
Agar Constant Alternating_ Constant
Medium ‘_"§train C- fitrain Ce Strain Ca
1 8 30 37 l 8 30 87 l 8 30 37
‘Uirn meaIfi 5 5' 5 0 + + + +. 45* O 0
Potato dextrose O O O O + + + + O O O O
'Beet leaf 0 O O O + + + + O 0 0 0
Cat meal 0 0 O O + + + + 0 O O O
Lima bean O 0 O O + + + + O O O 0
Halt extract 0 C O O +' + + + O O 0 O
Prune juice 0 O C O + +~ + + O O C 0
Plain agar 0 O O O + + + + O O 0 O

 

The experiment was repeated using only corn meal and cat
meal agars'but using more culture forms. Set (A) and (C) were
kept as before‘but Set (B) was given different lengths of ex-
posures to the various temperatures, i.e., five days at 34° C.
and twelve hours at 13° C; five days at 31° C. and twenty-four
hours at 13° 0. and then five days at 34° 0. before the observ-
ations in Table x were made.

Alternating temeratures produce distinct growth bands,
low temperatures inhibiting growth to such an extent that dark
narrow bands of much branched irregular mycelium.are produced,
while wide light colored bands are produced from growth at high

temperatures. Growth was good at 34° C. with no traces of son-

ation but was so slight at 13° C. that it could scarcely be

measured.

- 31 -
Table x
The effects of temperature on zonation in strains

of §;_beticola

9 Corn meag agar a Cat me a r
Strain ZZOU,“"§§:I%$‘CEE"Ig°c'

 

 

 

Cone Alternating Con- Con- Alternating Con-
stant stant stant stant
5-1 :5* +‘distinct 5 5 + distinct 5
c-a O + trace 0 0 do 0
C-3 0 + distinct O 0 do 0
C-4 0 do 0 O ---- 0
C-5 0 do 0 O + distinct 0
C-6 0 do 0 0 do 0
- C—7 0 + very faint O 0 t trace 0
C-8 0 + distinct O 0‘ do 0
C—9 0 do 0 O + distinct O
C-lC 0 do 0 0 do 0
Call 0 do 0 0 do 0
C-lz O --—- O O + trace 0
0-13 0 + distinct O O 9 distinct O
C-la O + trace 0 0 do 0
C-19 0 do 0 O + trace 0
C-85 O --—- s 0 do 0
C-28 O + trace 0 O + distinct 0
0-34 0 + distinct O 0 do 0
C-36 0 do 0 0 do 0
C-37 0 do 0 0 do 0
C-38 0 do 0 0 do 0

 

A set of plates from the 84° C. chamber were kept under a
bell jar in the laboratory for two weeks after’this experiment
was completed. They were exposed to daily fluctuations of light
and temperature, but concentric rings did not form in mycelium
that was formed under constant temperature and darkness. Zon-
ation did occur from the margin outward in all growth formed
under fluctuating conditions. This would seem to eliminate the
possibility that zonation in cultures of Cercospora beticola
is due to staling products or aging of the culture.

According to Bisby (l), Milburm has submitted evidence to

show that medium and not daily fluctuations is the agent involved

- 33 -
in zone formation in artificial cultures of gypocrea rufa.
Cerccgpora beticola growing on different media varies in
color, amount and density of mycelium which materially affects
the expression of zonation, but from observations made during
the light and temperature experiments and on many other cultures,
medium has had no pronounced influence on zonation. _

Observations were made of zonation in cultures growing on
18 cc., 85 cc. and 50 cc. of agar as outlined in Table I, page
18, to determine the effects of amount and depth of nutrients.
At the end of twelve days, zonation was pronounced in all cul-
tures growing on 18 cc. of agar, with only traces appearing in
some cultures growing on 85 cc. of agar. Hedia that produce
a heavy felty mycelium did not show zonation at this time. At
the end of thirty days zonation was very pronounced in most
cultures on 18 cc. of agar, quite distinct on 85 cc. and only
faintly visible on 50 cc. of agar. Variation in width of zones
was quite marked on the different depths, despite the fact that
differences in linear growth were practically negligible. It
was universally true thatzthe widest sense were produced on the
thin substrata and the narrowest zones on the deepest substrate.
Hence, it appears that medium is a contributing factor only in
so far as it affects the expression of growth rings and is not

a causative factor in itself.

- 33 -
The study 2; variant forms

 

The attention of the writer was first called to variant
forms in Cercospora beticola by the occasional occurrence of
sectors of different colored mycelium in colonies of the organ-
ism growing on plates. At first no particular attention was
given to the aberrant sectors until it was observed that they
seemed more or less permanent in subsequent transfers if in-
oculum was carefully selected from the outermost regions of
growth so as to take only the aberrant mycelium. These var-
iants always appeared as fan-shaped or wedge-shaped sectors
and were noted in practically all of the strains under attenp
tion. The aberrants differed from the parental form not only
in the color of the submerged mycelium.but in the rate of
growth of this mycelium. The aerial growth differed also in
type, amount, color and density. In the early part of the in-
vestigation, only these sectors showing marked differences were
selected since they lent themselves more readily to the study.
Later, sectors were selected from the various strains under
observation which differed less in the color variation. The
preliminary transfers of these isolations showed them to be
more or less constant, and it seems that the writer is here
dealing with a similar phenomenon to that observed and studied
by Stevens (16) and Christensen (5,6) in artificial cultures.
of Helminthosporium sativum, and also by Caldis and Coons (3)
in several other species of fungi.

The aberrant forms were isolated from the parental form

by picking a few filaments of the derived fungus from the border

- 3g -
of the colony at the center of the sector. If care is taken,
pure variant material can be picked out in this way in practi-
cally all cases. The variant was then grown on the same plate
with the parental form for a number of times in order to test
the purity of the material. Out of twenty—odd such selections,
in only one case does it seem likely that a portion of the
normal mycelium was transferred along with the variant and .
in this particular instance the first planting gave evidence
of the mixture. In a few instances spores were produced on
the variant forms, in which case single spore isolations were
secured. These cases are indicated in the charts. Earpho-
logical studies were made of the variant of strain C-l. Study
of the other forms was limited largely to the physiological
activities.

The following system was used in keeping the records of
the various cultures used in this work. The various strains
of Cercospora‘beticola used in this work were labeled C-l, c-z,
C-3, etc. Variant forms were labeled v/l, v/8, etc., to in,
dicate the number of times the material had been transferred.
The original parental material was always grown in parallel

cultures on the same plate and labeled as O/l, 0/8, etc.
Thus (C—l v/7) refers to the variant form of strain one of
Cercospora beticola in its seventh transfer and (C-1 0/7) re-
fers to the same strain of Cercospora beticola but is the normal
form which has been grown in parallel culture with the aberrant

mee

- 35 -
Variants obtained from Cercospora beticola strain 9:;

On October 31, 1986, single spored isolations C-1, C-8
and C-3 were made from diseased material collected at Rocky
Ford, Colorado. These cultures apparently alike were carried
through several transfers as stock cultures, until January 11,
1987 when ten day cultures on beet leaf agar in petri dishes
showed achromatic fanpshaped sectors in strains C-1 and 0-3.
Conidia were formed in the normal green mucelium but more were
found on the colorless forms, so on February 15, 1987, myce-
lial transfers were made from the aberrant sectors as (l v/7)
and(3 v/l) to corn meal agar. The variant form C-3 apparently
reverted en masse to the normal form‘but the aberrant form
(1 v/l) remained white with very sparse aerial mycelium. The
variant (3 v/l) was discarded, being considered not permanent,
bum the variant form of strain C-l was transferred to corn
meal (1 v/8) in a parallel culture with the original form
(1 0/8). On March 14, 1987, transfers from the pair (1 0/3)
and (1 v/3) were made to corn meal agar in test tubes. No
further work was done with these forms until October 8, 1987,
when transfers (1 O/4) and (l v/d) were made to corn meal agar
plates. During this period of almost six months the cultures
had become dried and it was necessary to pour nutrient broth
into the test tubes. The growth thus obtained was typically
green and achromatic. .

The most obvious difference between the normal form and

its derivative obtained from strain C-l was the color of the

- 35 _
colony and the amount and density of the aerial growth (Plate
Ix, Fig. 1). These differences were so marked that the var-
iant could easily be mistaken for a contamination rather than
Cercospora beticola. However, physiological and morphological
studies have proved beyond a doubt that the aberrant form is
Cercospora beticola. Moreover, these studies have brought out
certain aberrant characteristics not discernible under ordinary
observation.

Physiological activitigg Parallel cultures of the normal
and the aberrant forms have been grown on many media under_a
wide range of environmental conditions (Plate III, Fig. 1).

On artificial media the physiological characteristics have been
constant throughout twenty transfers over a period of sixteen
months. Table XI shows the rate of linear growth and other
cultural characteristics of the two forms growing on twelve
different media. The data were taken after ten days growth.

The contrast in linear growth was quite marked, varying some-
what with the medium, but generally being more rapid in the var-
iant form. Amount and density of the aerial mycelium also var-
ied. As will be seen in the table, it was abundant but appressed
and felty and on other media almost lacking. With continued
cultivation there has been a gradual decrease in the amount of
aerial mycelium produced.by the variant form on corn meal agar
until at present it is entirely absent except for a tuft in the
center. Passage through the host plant has had a general ten-

dency to restore the color and amount of aerial growth, isolations

 

 

 

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Hmwamm dvmnmanSm E\a Hmwamm dowamapwm E\E
.aman[ .amHnt adwcms
pumfiambx Haemoamm Mama

 

 

H10 snow Hmpmonmm new mom AO\> anov Show panama» mHOOHpmn.dm.Mo mpgoaw

. HM manna

- 33 -
from plants being pale green with considerable aerial myce-
lium.

Pathogenicity Infection of plants by this variant has
been very difficult to secure, evidently due to its low vir-
ulence. Inoculatione from beet leaf agar cultures have been
tried repeatedly under the most favorable conditions for in-

fection. A few scattering spots have been formed'but conidia
were not produced, the spots remaining as small brown flecks.

Morphological characters Camera lucida drawings of
the submerged mycelium from cultures on various media are
shown in Plate 11, Pig. 1,2. The mycelium of the aberrant
form is characterized by being smaller and less tortuous, and
more hyaline with fine granular cyloptasm, with fewer of the
large translucent oil globules indicating a different food
storage capacity than the parental form. The loose sclerotium-
like bodies so often found in the submerged mycelium of the
normal form have never been observed in this aberrant form.

As previously stated, the length of conidia and the
number of septae per conidium in Cercospora beticola are varb
iable factors depending: somewhat upon nutrients, humidity and
temperature. The variant form of strain C-l has been practi-
cally sterile throughout its cultural history but a few very
small conidia were found produced on (C—l v/7) growing on best
leaf agar. leasurements were made of twenty conidia picked
at random from washings of the culture and compared with a
like number from (C-l O/7), the homodegous culture of the

original material.

- 39 -
Table III
A comparison of conidia produced by the variant (C-l v/7)
and its parental form (C-l 0/7)

 

F fio. ofwseptaeu‘ Length in m7m Width In
crm flax. Aver. ‘Hin. HE}. Iigr. Amiga, m[m

- O 7 88 #15.8 II 860 138 55 4-5

C-l v[? 6 4.6 8 58 35 84 3.8-4.3

 

The data given indicate that the differences in average
size of conidia and the number of septae per conidium between
the parent and variant forms are very great. The spores pro-
duced by the variant form are more hyaline with more finely
granulated cytoplasm than those from the normal form. No
difference could be noticed in germination of the conidia in
hanging drops of distilled water and nutrient broth. Bub-
cultures arising from single conddia obtained from the variant
culture gave colonies indistinguishable from their source.

Further occurrenc_e_ 9_f_ variant fprms _i__n_ 93.3.32 9:; On Dec-
ember 8, 1987, an achromatic sector was observed in a petri
dish culture of strain C-l whichtp to this time had been car-
ried in stock culture. A mycelial isolation was made from the
variant sector which was recorded as (C-l A v/O). On January 5,
1988, culture (C-l 0/6) growing on prune juice agar gave rise
to an aberrant sector (C-l B v/O) which was very similar to
(C-1 A v/O). These two variants resembled (C-l v/O) as origin-
ally isolated and so these new variants were not studied in

such detail but were transferred just often enough to maintain

- 4o -
new growth at all times. They are now in the sixth and fifth
transfers respectively and so far have shown no tendency to
revert to the normal form. As in (C—l v/O) there has been a
.steady decrease in the amount of aerial mycelium and from all

appearances these derivatives of strain C-l seem identical.

Variantg obtaingg from Cercospora beticola.gjgaig‘gzg

Diseased material showing heavy infection of Cercospora
leaf spot was received from Grand Island, Nebraska, on Sep-
tember 8, 1987, from which single spored cultural strains C-9,
C-18 and C-14 were obtained by the dilution plate methodl Var-
iant forms have arisen from strain C—9 at two different times,
but were not recognized as like forms until cultures growing
on corn meal agar were compared. After this similarity was ob-
served, comparisons were made on several media and the similar-
ity of behavior has been constantly noted.

Aberrant form 91 Cercospora beticola strain ng_(C—9 v/O)
This aberrant form as outlined in the chart (Plate IV, Fig.8)
originated as a fan-shaped sector in strain 0—9 growing on‘beet
leaf agar. The sector was white with sparse, sterile aerial
mycelium. sub-cultures of the aberrant and parental forms
(9 v/l) and (9 0/1) were made on corn meal agar. Growth in
the substratum was light green with fine mycelium. Zonation
was very pronounced under exposure to ordinary daylight. Aerial
mycelium was almost absent in the younger growth but appeared

In
as a white tuft in the center of theAday old cultures, becoming

- 41 q

grayish-white in older cultures. These aberrant cultural
characteristics are in contrast with the heavy olive green
submerged mycelium and the gray-green aerial growth of the
parental form. Further, the original form also produced a
reddish tinge in the medium, and had abundant aerial mycelium
which in older cultures had a tendency to clump. This variant
form.has been carried on artificial media in parallel transfers
with its parental form (C-9 O/O) through fifteen successive
transfers over a period of six months. A sector of the var-
iant form in transfer (8) reverted to the normal form as is
shown in the chart. The aberrant and its parental form were
grown on best leaf agar in transfer (8) for the first time since
the occurrence of the variant form. The aberrant character-
‘istics were still present, growth being almost colorless, zon-
ation very pronounced and the aerial mycelium sparse and prac-
tically sterile.

Passage through plants has twice been successful without
any marked change in pathogenicity. The slight difference in
virulence of the two forms can probably be explained by the ab-
sence of conidia in the inoculum of the aberrant form (C-9 v/B).
Conidial production in spots produced by the aberrant form took
place as in the parental culture, and the spots when old became
typically ashen—gray from the conidiophores and conidia that
were produced.

Tsclations of single spores were made from these inoculated

'plants to see what effect passage through the host plant had on

- 43 -
the cultural characteristics of the aberrant form. The color
of the submerged mycelium which arose from such isolations was
practically restored to the normal olive green of the parental
form. It was somewhat lighter in color than the parental
form (C-9 O/O) after passage through plants, but had lost the
pale yellowish green of the variant form (C-9 v/l3) which had
been grown continuously on artificial media. Other aberrant
characteristics were apparently unaltered. Zonation was more
pronounced and no sclerotiumvlike bodies were Observed in the
submerged mycelium of the re-isolated aberrant form. Aerial
mycelium was not produced in these cultures until about the
tenth day and then only as a white tuft in the older growth,

a thing which was very characteristic also of the aberrant
form (C-9 v/l3). .

‘Additional aberrant forms pf Cercosppra beticola (C-9 A v/O) -
.(9-9 B v10) These variant forms originated in cultures of
strain C-S growing on.beet leaf agar after the organism had,
been passed through the host plant as is shown in the chart
(Plate V, Fig. 1). As previously stated, these isolations re-
semble the variant form (C-9 v/O) very closely and have been
considered identical with the first variant obtained from this
strain of Cercospora.beticola. As the chart shows, they have
received essentially the same treatment as the first variant
isolated (049 v/O) since their isolation and have behaved very
similarly so a detailed discussion will not be given. Passage

-‘3-
through plants failed to reveal any marked difference in
pathogenicity and produced comparable results with those ob-
tained in the first variant form (C-9 v/O).

The salient features of the work with the variants from
this form seem to be, (1) the same sort of variant has been
thrown three times by this culture, (8) the depth of color
which is a more or less characteristic thing for the variant
varies with the medium and passage,through plants tends to
restore this color, and (3) the mycelial characters of the
variant are more or less constant, apparently being unaltered

by passage through the natural host plant.

Variants obtained from Cercospora beticola strain Gag

Cultures C-7 and C-8 were obtained by single spore isola-
tions from diseased material received from Chaska, Minnesota,
on September 1, 1987. They were typical Cercospora cultures
with olive green mycelium in the substrata and gray-green aerial
mycelium. Culture Cg? has never given rise to aberrant forms
‘but three distinctly different variants have been obtained from
form C-e. On January 1, 1988, thirty-nine transfers of C-8
were made on thirteen different media for the purpose of study-
ing the effect of type and amount of nutrients on the cultural
characteristics of Cercospora beticola. Cultural variations
were pronounced but all transfers except one apparently were
homogeneous. This transfer on 85 cc. of litmus lactose agar

in a petri dish produced an aberrant sector, which at ten days

..44 -
‘was darker colored, with low, compact aerial growth (Plate VII:
Fig. 8). Mycslial transfers from the aberrant sector to corn
mean agar (C-8 v/l) produced a colony deep olive green in color,
with dense deep greenish-gray aerial mycelium which in older
cultures develops a pronounced tendency to clump into loose
masses. The normal form C-8 O/l) on corn meal agar was slightly
lighter colored, producing a wine red tinge in the medium. It
has light colored, less abundant aerial mycelium, which shows
less tendency to form in clumps than the variant form (C-8 v/l).
Cultural washings from (C-8 O/B) and (C-8 v/6) on best leaf
agar indicated that conidia production was heavier in the aberb
rant form. Inoculation on plants produced heavier infection in

the aberrant form, fifteen days after inoculation. Passage
through sugar beets did not affect the aberrant characteristics
in subsequent transfers. There was a slight tendency toward a
deeper color in both the normal and the aberrant forms that had
passed through plants over those carried continuously on arti-
ficial culture media through thirteen transfers.

Aberrant form 19-8 A v19), The transfer of strain C-8 on

 

12 cc. of oat meal agar of January 1, 1938, was abnormally pale
green with very sparse aerial mycelium. A transfer was made to
corn meal agar to determine the effect of nutrients on color.
This culture was normal green with abundant greenish-gray aerial
mycelium. On January 30, 1938, a transfer was again made to

oat meal agar which developed into a pale green culture having

.. 45 .
very little aerial mycelium, with a large achromatic sector
with abundant loose aerial mycelium. A transfer from the
aberrant sector to corn meal agar (6-8 A v/l) produced a pure
achromatic culture with a reddish tinge in the substratum.
A sector of the culture reverted to the normal as is shown in
the chart (Plate Iv, Fig. l) but subsequent cultures from the
achromatic portion have remained constant through nine trans-
fers on artificial media. Inoculatione of the aberrant form
(0-8 A v/7) on sugar beets produced a very light infection.
The diseased areas were typical, producing a few normal conp
idia. Isolations from these plants produced only normal green
cultures comparable with those isolated from plants inoculated
with culture (C-8 A O/7). In this form we have aberrant char-
acteristics that were constant on artificial media'bum were
not constant through plants indicating that the variation was
in some way connected with malnutrition of the fungus.

Aberrant £932 Lc-s s vLO) The plate transfer of the
parental form (C-8 A 0/3) gave rise to an aberrant sector which
had yellow mycelium. Aberrant form (0-8 A v/O) was white, hence
this variant appeared to be distinct from it. This variant has
remained constant through five successive transfers on corn

meal agar.

- 45 -

Variants obtained from Cercospora beticola strains C-19 and C-30

 

Diseased material was received from Grand Island, Nebraska,
on September 4, 1937, from which single spore culture strains
C-18, C-19 and C-30 were obtained by the dilution plate method.
These cultures were carried in stock cultures without produc-
ing aberrant forms until January 1, 1938, when variant sectors
appeared in plate cultures of strains C-19 and C-30 growing on
corn meal agar.

Variant obtained _f_1_-<_>_m Cercospora beticola 35.29.39 9:19
.jC-19 7/21 The variant sector in strain C-18 was yellow,
producing a red pigment in the substratum, with sparse light
colored aerial mycelium, while the rest of the colony was normal
olive green in the substratum with greenish-gray aerial mycelium.
When sub-cultured from mycelium to corn meal agar the aberrant
form became a pale yellowish green producing a pronounced wine
red tinge in the substratum, with grayishwwhite aerial mycelium
in a tuft at the center of the colony. The parallel transfers
of the normal form (C-l9 O/l) was normal in all respects.

These characters were constant throughout the eleven transfers
on corn.meal agar. On prune Juice agar the aerial mycelium in
both the parental and the aberrant forms was lighter in color
and more felty. when grown on beet leaf agar the aberrant form
becomes almost achromatic in the substratum with pure white
aerial mycelium. No appreciable difference in pathogenicity

of the two forms for sugar beet could be noticed through

- 47 -
artificial inoculations. Conidia were produced in the
typical small brown diseased areas on the plants inoculated
with both the normal and the variant forms. Cultures isolated
from the plants inoculated with the variant form were some-
what darker in color than the line carried continuously on
artificial media. The color of the submerged mycelium was
somewhat lighter green than the color of the normal form and
the wine red pigment of the aberrant form was much more pro-
nounced in those isolated cultures than it was in the form
kept constantly under artificial culture. The aerial mycelium
was changed from a grayish-white to a dark gray but no in-
crease in density was noted in the organism passed through
plants.

Aberrant £253 $9-19 A v/O) On February 1, 1938, a
transfer of C-19 was made from the stock culture of December
30, 1937 for comparison with ((2-19 O/3). An aberrant sector
identical with (C-19 v/O) was produced in this transfergso
on February 10, 1938, a mycelial sub-culture was made from
this sector and recorded as (C-19 A v/l). Since the time of
isolation this aberrant form has received the same treatment as
the variant (C-19 v/O) and has been found to be identical with
it in every respect, so much so in fact that they cannot be
distinguished from each other.

In these two variant forms we have sudden variations

occurring whose stability at least in part depends somewhat

upon the medium. The normal color is at least partially

- 43 -
restored when the variants are cultured on the natural host
plant. It is interesting to note that the color of the parb
ental form was somewhat intensified by passage through sugar
beets. The other aberrant characters were apparently unaltered
by passage of the variant forms through the natural medium.
Aberrant fggmgfic-ao v/O) This form originated as an
aberrant sector in strain C-3O as stated above. The growth
in the substratum was a pale yellowish green in contrast with
the olive green of the normal portion of the culture. The
aerial mycelium was abundant, loose and uniformly yellow, as
contrasted with the greenish-gray, rather sparse aerial growth
of the parenta1.form. There has been a gradual decrease in
color of the submerged mycelium.until in the eleventh trans-
fer of the variant form (C-3O v/ll) it is almost the color
of the aerial mycelium. Ihen grown on best leaf agar the
aberrant form becomes almost achromatic with abundant felty
almost sterile aerial mycelium. The aberrant form seemed to
produce a lighter infection on sugar'beets than did the parb
ental form. The symptoms were typical, the diseased areas
becoming ashen gray with conidia. Isolation of the variant
form from inoculated sugar beets showed that passage through
plants had restored the normal olive green color but did not
affect the type of aerial mycelium, which was more abundant
and felty that it was in parallel cultures of the normal form
(C-SO O/O). The color had been changed from yellow to a

grayish yellow. Transfer (3) of the aberrant form produced
a sector which was normal in every respect, through two suc-

cessive transfers on artificial media.

Variant obtained from Cercospora‘beticola strain C-l?

 

Culture strains c-15, C-16 and C-17 were isolated by
the dilution plate method from diseased material received‘
from Hason City, Iowa, on September 4, 1937. Approximately
one-half of a transfer of C-17 growing on a best leaf agar
plate of January 3, 1937 was achromatic with sterile white
aerial mycelium, and the rest of the colony was normal dark
green with dark colored aerial growth. Ihen grown on corn
meal agar the variant became pals yellowish green in the sub-
stratum with sparse light colored aerial mycelium. Zonation
was faint in the aberrant form and very pronounced in the
parental form. Sub-cultures to best leaf agar as indicated
in the chart (Plate V, Fig. 3) were somewhat lighter in color
than the originallform (C-l? v/O) but were not pure achromatic
as was the aberrant sector. There was no appreciable differ-
ence in pathogenicity between the two forms when inoculated
on sugar‘beets. Passage through plants had a tendency to in,
tensify the colors as described in the discussion of variant
form (C-9 v/O) but did not eliminate the aberrant character-
istics. These variant characters are somewhat variable on
media but have been constant throughout the culture on arti-
ficial media.

- 50 -
Attempts tg_produce variants
As detailed by Brierley (3), various workers have pro-

duced 'mutations' in Aspergillus niger and Penicillium glaucum

 

by treating the cultures with various chemicals. Brierley (3)
has tried to repeat this work, using strains of the same
fungi, without success. Stevens (16) reports unsuccessful

attempts to produce 'saltations' in Helminthosporium sativum

 

by wounding and various other methods. The writer has carb
ried out the following outlined attempts to produce aberrant
forms in normal cultures of Cercospora beticola.

wounding The tips of actively growing hyphae in cultures
C-1 and C-37 growing on corn meal agar were killed by means
of a hot platinum wire in several places around the border of
the colony. The inpured areas soon outgrew the injury, the
margins of the colonies presenting a uniform appearance. These
results are strengthened by the fact that variations have never
‘been found to arise in cultures at the point from which inoculum
has been procured with a hot needle.

Illuminating g2; Since gas poisoning of cultures is
always a possibility under laboratory conditions, the writer
sought to determine if this factor was responsible for sector
formation in normal cultures. Two week old cultures C-1 and
C-37 were placed in a series of air-tight chambers and treated
with illuminating gas for 4, 8, 8, lO and 34 hour periods.

The gas was allowed to pass through the chamber for thirty

- 51 - .
minutes, thus forcing out all of the air. Cultures exposed
to 4 and 6 hour treatments were only slightly inhibited in
growth as compared to the checks. Eight and ten.hour treat-
ments inhibited growth completely but did not kill the organ-
ism as inoculum put on corn meal agar produced normal growth,
while twenty-four hour treatment killed the fungus. The
cultures were kept in the laboratory for three weeks after
treatment without any sign of variation, nor have any of the
subsequent transfers given rise to aberrant forms.

Freezing- Since many of the aberrant forms under observ-
ation have appeared during the winter months, cultures of the
organism were frozen with carbon dioxide gas in an attempt to
induce formation of variant sectors by exposure to extremely
low temperatures. The methods of freezing were varied, one to
thirty minutes continuous freezing being applied to some cul-
tures while others were alternately frozen and thawed for
different lengths of time. The treated cultures were allowed
to grow for ten days and then sub-cultures were made from the
frozen areas, but none of the treated cultures or the sub-
sequent transfers gave any indications of throwing variants.

Poisoning with chemicals Vigorously growing cultures
of the fungus were treated with chemicals chosen because they
were strong oxidizing or reducing agents in an attempt to in,
duce the throwing of variants. Small crystals of 1302207
and drops of solutions of KNOS, H303 and CHOH were placed at

- 53 -
different points around the margins of the colonies. In

this manner different concentrations were obtained as diffus-
ion of the chemicals through the medium took place outward
from the points of application. The fungus was killed at

the points of application, and growth was inhibited to a var-
iable degree depending upon the concentration of the chemicals,
but subsequent growth was normal, with no indication of var-
iants. Sub-cultures made from different portions of the
treated colonies gave only normal forms.

These attempts and many others of a similar nature have
been tried at various times by the writer but always with
negative results. They indicate that the coarse methods used
were not so directed as to upset the equilibrium within the
fungus cells, rather than exhausting the possibilities of
success through methods of this sort.

Hechanics g; sector formation

 

Artificial sectors were produced both by mixed planting
and implanting of two forms in an attempt to show the mechan-
ism of sector formation. Stevens (16) used these methods in
an attempt to demonstrate that ordinary transfers might be
mixtures of two forms or races, thus explaining the appear-
ance of 'saltations'. He obtained no segregation of forms in
mixed plantings and only partial success by implantings.

Implantings of mycelium from an achromatic form at the

border of a normal green culture gave rise to typical sectors

- 53 -
only when the implanted material had a chance to start active
growth before the advancing mycelium of the normal form en-
gulfed it, in which case the planting was obscured. If the
implanted material starts growth a typical wedge-shaped sector
is formed. There seems to be very little repulsive influence
between forms of Cercospora beticola as two forms will grow
parallel with each other but with very little intermingling of
hyphae. The forms can be isolated in pure culture from the
borders of the colony.

. Mixedplantiggg Acting upon the knowledge that the var-
iants were frequently more rapid growers than the parental
form, mixed plantings were made to determine whether or not
the two forms would segregate and remain distinct. Equal
amounts (one square centimeter) of agar and its contained my-
celium from each form were thoroughly mixed under sterile con-
ditions, and the mixture used as inoculum on corn meal agar
plates. Several forms have been treated in this manner but
strain C-1 and its variant, and strain C-8 and its variant
have shown the best contrast. Segregation takes place in the
form of several wedge-shaped sectors resembling the normal
and the aberrant sector from which the forms were obtained
(Plate VIII, Fig. 1,3).

The results of this experiment seem very important in
explaining what happens in the case of naturally developing
sectors. If in a culture two elements of different potential-

ities develop, then if the growth rates are approximately the

- 54 -
same or if one is slightly greater than the other, there will
be produced sectors of the one form in the other. The results
from mixing the mycelium together would indicate that a small
amount is sufficient to inaugurate a sector; perhaps only a
single cell would be sufficient if not repressed by the sur-
rounding growth.

Apparent reversions

Several instances of apparent reversion of the aberrant
characteristics have been observed. Such reversions always
occurred as normal sectors in the aberrant form, when the
variant characters were colony color, amount and density of
the aerial mycelium and partial sterility. Achromatic sectors

have appeared on best leaf agar that immediately reverted
en masse to the normal form bud these variants have not been
considered as being stable and only reversions of the stable
forms through sectors are here considered.

Reversion has been found to be most common in forms Just
isolated from the aberrant sector. Aberrant form (C-la v/O)
has shown a great tendency to split up into normal and variant
forms. Continued selection for the contrasting types has fin-
ally resulted in an achromatic variant (0-13 v/7) that has been
stable through the last three transfers. Variant form (C-3O v/S)
developed a normal sector in a five day culture that was ident-
ical with the parental form in every respect through two sUb-

sequent transfers and the variant form has shown no further

- 55 -

tendency to split into the two forms. Variant forms (C-S v/l),
(C—l4 v/3) and C-37 v/l) all showed tendencies toward revert-
ing to the parental form but selections for the aberrant char-
acteristics have resulted in phenotypic cultures.

The production in these variant forms of the duplicate
of the parental forms, under conditions where it seems that
mixture of the parental material was excluded, seems very sig-

nificant.

Discussion
The development of variations in the mycelium of Cercospora

beticola has been noticed throughout the two years of study of

 

the rarious strains.‘ These variants have appeared as wedge-
shaped sectors in normal cultures, being most noticeable for
their lack of color, sparse aerial mycelium and sterility on
beet leaf agar. Some of these have reverted more or less to
the normal form in color but the mycelial characters have re-
mained constant, intensity of color only being influenced by
the medium on which the organism is growing. Some of the aber-
rant forms (C-l v/O) in particular have been remarkably perm-
anent on artificial media. Passage through plants has a tend-
ency to intensify the color of the mycelium. Variant forms
(C-l v/O) and (C-8 v/O) were pure white when growing on arti-
ficial media but became almost normal green after passage
[through sugar‘beets. The density and amount of aerial myce-

lium and type of growth in the substratum were influenced very

- 53 -
little by passage of the aberrant forms through the host
plant.

There has been an extensive literature developed on varb
iations occurring in pure cultures of asexual fungi. Brierley
(3) and Stevens (6) have compiled complete bibliographies on
the subject from which material has been extensively used.
Contradictory views as to the nature of these variant forms
have been expressed by various investigators. However, the
‘bulk of the literature merely reports the occurrence of such
variations along with certain morphological characteristics
and physiological reactions without dealing extensively with
the genetic nature of the variations. One group of invest—
igators believe these variant forms to be true “mutations“ or
'saltations' comparable to the so—called bud mutations which
occur in apparently pure line cultures of plants, of which
view Stevens (16) and Christensen (5,6) are the strongest
exponents. Opposed to this view are those who believe, with
Caldis and Coons (3) that they are modified forms of a more
or less permanent nature. They have based their belief upon
the assumption that if a change in the genetic constitution
. of the organism (as Brierley (3) has defined a mutation) had
actually taken.place, the very common occurrence of reversion
to the normal form would not then be likely.

whether or not the variations studied and recorded in

this paper are "mutations" or 'saltations', or are merely

- 57.-
modified forms, it has been found that they are of only a
semi-permanent nature. The actual cause of their development
remains as yet unknown, but the changes are of such a type as
to suggest nutritional disturbances as playing an important
role. The sparse aerial mycelium can also possibly be ex-
plained as a result of attenuation of the organism through
malnutrition.

In the absence of exact knowledge of the life history of
the organism other than the asexual stage, conclusions as to
the actual nature of the variant forms cannot be drawn, but
at least it can be said that the claim that variants represent

"mutations” is at least far from being proven.

Summary
Cercospora beticola is the fungus causing Cercospora leaf

. spot of sugar beets.

The organism was found to grow well on most standard cul-
ture media. Light apparently plays a minor role in its devel-
opment while temperature affects materially the vegetative
growth.

Conidia production in artificial cultures was found to
be a response to a nutrient stimulus, light and temperature
influences being flexible.

Zonation in artificial cultures is due to the differential
growth of mycelium and not to conidia production. It has been
induced by fluctuating light and temperature conditions. Other

- 53 -
factors influence but do not initiate zone formation.

Variant forms appearing as wedge-shaped sectors were
isolated from known single spore cultures of Cercospora
beticola. These aberrant forms have been constant on arti-
ficial media. Passage of the aberrant forms through sugar
beets has had a tendency to restore the normal color but has
not materially affected the morphological characteristics.

Attempts to produce variants by wounding, exposure to
illuminating gas and low temperatures and by treating with
chemicals have been unsuccessful.

The behavior of these variant forms indicates that they
may be looked upon as modified forms with nutritional distur-

bances playing a role in their development.

1.

3.

3.

4.

5.

6.

7.

9.

10.

11.

is.

- 59 -
Bibliography

Bisby, G. R.
1935. Zonation in cultures of Fusarium discolor
sulphureum. In lycologia 17:89:95.

Brierley, I. B.
1930. On a form of Botrytis cinera with colorless
sclerotia. In Phil .‘Trans. Roy. Soc. London, Ser. B.
210:83-114.

Caldis, P. D. and Coons, G. B.
1936. Achromatic variations in pathogenic fungi.
In Mich. Acad. Sc. Vol. VI.

Chaudhuri , He
1933. A study of growth in cultures of Verticillium
albo-atrum. B. et. Br. In Ann. Bot. 3 : .

 

 

 

Christensen, J. J.
1936. Physiological specialization and parasitism of

He§gdnthosporium sativum. In linn. Exp. Sta. Tech.

Christensen, J. J.
1935. Physiologic specialization and mutation in
Helminthosporium sativum. In Phytopath. 15: 785-795.

Coons, George H.
1916. Factors involved in the growth and pycnidium
formation of Plenodcmus fuscomaculans. In Rept. Journal
Agri. Des. V. 5, No. IE.

Coons, George H.
1936. Beets of primitive type in disease control.
In Yearbook of Agri. 1936.

D r .M.
uggé :1 geaf spot of the best. In Cornell Agr. Exp. Sta.
. 6 .

Hedgecock, G. G.
1 OS. Zonation in artificial cultures of Ce halothecium
and other fungi. In Rept. Mo. Bot. Gard. 17:IIB=IIV.

Klotz L. J.
1935. A study of the early blight fungus Cercospora
521 0 FIGS. In “1011. Agri. Exp. Stae Techs “1e 0e 63s

Leonian, L. H.
1934. A study of factors promoting pycnidium formation
in some lphaeropsidales. In Am. Jour. Bot. 11:19-50.

13.

14.

15.

16.

17.

18.

- go -

Parr, Rosalie
1918. The response of Pilobolus to light. In Ann. Bot.
33:177-305e

Pool, Venus I. and McKay, M. B.
1918. Climatic conditions as related to Cercospora
beticola. In Journ. Agri. Res. Vol. VI, we. 1.

Pool, Venus I. and HcKay, M. B.
1916. Relation of stomatal movement to infection by
Cercospora beticola. In Journ. Agri. Res. Vol. V, we. 33.

Stevens, F. L.
1933. The Helminthosporium foot rot of wheat with observ-
ations on the morphology of Helminthosporium and on the
occurrence of saltations in the genus. In Bul. I11.
Nat. Hist. Surv. 14:78-184.

Stevens, 1“. L. and Hall, J. A.
1909. Variations of fungi due to environment. In Bot.
GEZe v01e ‘8, NOe 1.

Townsend, C. O.
1915. Field studies of the crown gall of sugar‘beets.
In Us 8. Dept. of Agri. Bule 303.

- 51 -
Supplement

Cultural strains of Cercospora beticola

 

The following is a list of the single spore isolations
used in the study of Cercospora‘beticola. The cultures have
been termed strains because of the variability in culture
characteristics existing between the different isolations.

The occurrence of variant sectors is indicated in each strain.
Some of these variants have not been studied in detail but all
have been isolated in pure culture.

C-l: Isolated October 31, 1936, from diseased material
collected at Rocky Ford, Colorado. Aberrant forms (C-l v/O),
(C-l A v/O) and (C-1 B v/O).

6-3: Duplicate culture of strain C-l. Aberrant form
(0-3 V/O)e

C-S: Duplicate culture of strain C-l. Aberrant form
(C-S V/O)e

C-4: Isolated October 31, 1936, from material collected
at Rocky Ford, Colorado.

C-5: Duplicate culture of strain C-4.

C-6: Isolated July 6, 1937 from diseased material re-
ceived from Oxnard, California. Aberrant form (C-S v/O).

C-7: Isolated September 1, 1937, from material received
from Chaska, Minnesota.

C-S: Duplicate culture of strain C-7. Aberrant forms
(C-S v/O), (0-8 A v1!) and (C-8 B v/O).

C-9: Isolated September 3, l937rfrom material received
at Grand Island, Nebraska. Aberrant forms (C-9 v/O), (C-9 A v/O)
and C-9 B v/O).

C-lO: Isolated November 5, l936vfrom material collected
at East Lansing, Michigan. Aberrant form (C-lO v/O).

_ 53 -

6-11:
form (6-11

6-13:

6-13: D licate culture of strain
form (6-13 v/gg.

C-14:
(0-14 v/O).

6-15: Isolated September 4, 1937,
trom Mason City, Iowa.

6-16:

C-17: Duplicate culture of strain
(6-17 v/O) and (6-17 A v/O).

6'18: Isolated September 4, 1937,
received from Grand Island, Nebraska.

6-19: Duplicate culture of strain
(6-19 v/O) and (6-19 A v/O).

C-SO:
(C-SO‘v/o).

Du licate culture of strain
v/O)

Duplicate culture of strain

Duplicate culture of strain

Duplicate culture of strain

Duplicate culture of strain

6310. Aberrant
C-9.

6-10. Aberrant
6-9. Aberrant form

from material received

C-15.

6-15. Aberrant forms
from diseased material
6-18. Aberrant forms

C-18. Aberrant form

6-31 - 6-35 inclusive were Michigan State College Experiment
Station stock cultures and were not used very extensively so

individual strains will not be listed.

6-36: Isolated September 8, 1937,
at Rocky Ford, Colorado. Aberrant form

0-37:
6-38:

from material collected
(6-36 v/O).

Duplicate transfer of strain 6936.

Duplicate transfer of strain 6-36.

 

- 53 -

Explanation.g£;plates

 

Plate 1. Camera lucida drawin s of conidiophores and
conidia. Fig. l, Conidiophores, (a? copy from Duggar, ‘
(b) from plants, (c) young conidiophores produced in cul-v
ture, (d) older conidiophores in culture. Fig. 3, Conidia
from different sources, (a) germinating conidia.

Plate II. Camera lucida drawings of the mycelium of
strain C-1 and its variant form growing on various media.

Plate III. Fi . 1, Chart showing cultural history of
variant form (C-l v50). Fig. 3, Chart showing cultural
history of variant form (6-8 v/O).

Plate IV. Fig. 1, Chart showing cultural history of
variant form (6-8 A v/O). Fig. 3, Chart showing cultural
history of variant form (6-9 v 0).

Plate v. Fig. 1, Chart showing cultural history of
variant forms (C-9 A v/O) and (6-9 B v/O). Fig. 3 Chart
showing cultural history of variant form (C-17 v/O .

Plate VI. Fig. l, Variant forms (C-19 v/O) and
(6-19 A v/O) showing cultural history in chart form.
Fig. 3,/Chart showing cultural history of variant forms

0-80 V 0 e

Plate VII. Fig. 1, (a) differential growth rings in
" the submgrged mycelium induced by alternating temperatures

of seats= 6., (b) Cultures grown at continuous temperature
of as C. Fig. 3, (a) variant sector in culture of strain
6-8 growing on litmus lactose agar, (b) the parental form,
(c) the variant form in pure culture.

Plate VIII. Mechanics of sector formation. Fig. 1,
Mixed plantings of strain 6-1 and its derivative, Fig. 3,
Mixed plantings of strain C-8 and its derivative.

Plate IX. Fi . l, (a) strain C-l, (b) variant from 6-1,
(0) strain 6-8, (d? variant form (C-S v/O), twenty day cultures.
Fig. 3 (a) strain C-l, (b) variant form (C-l A v/O), (0) strain
0-13, (a) variant form (0-13 v/O).

Plate x.’ Fig. l, Reversion through sectors in variant
form (6-13 v/O). Fig. 3, (a) strain 6-8, (b) variant form
(c-s B v/O), (0) strain 0-20, (a) variant form (c-zo v/lO),
all cultures growing on corn meal agar.

 

 

 

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