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THE EFFECT OF NUTRIENYS AND
ENViRONMENTAL FACTORS ON GROWTH
OF UROCYSTIS COLCHiCl
(SCHLECHT) RABENH

Thesis for the Degree of M. S.
MECHIGAN STATE UNEVERSETY
RUBERTA LQUISE DOW
1968

THESIS
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ABSTRACT

THE EFFECT OF NUTRIENTS AND ENVIRONMENTAL
FACTORS ON GROWTH OF UROCYSTIS COLCHICI
(SCHLECHT.) RABENH.

by Roberta Louise Dow

Malt extract and phytone were the best single nu-

trient sources for growth of Urocystis colchici in liquid

 

culture. Growth increased linearly with all phytone con-
centrations tested. Sucrose, glucose, or mannose were
superior to other simple sugars. A concentration of 30
g/liter was Optimum for sucrose while malt extract was
best at 10 g/liter.

Small quantities of malt extract, phytone, or pep-
tone stimulated growth when used with simple sugars. Ten
grams per liter was the optimum amount of phytone when used
with 30 g sucrose/liter.

More growth was obtained with organic nitrogen
sources than with inorganic nitrogen sources. Of nine
amino acids and four inorganic nitrogen sources added at
a concentration of 0.3 g N/liter to sucrose and mineral
salts, L-asparagine and L-aspartic acid gave the best
growth. Asparagine at 1 g N/liter gave the best growth

of any nitrogen source.

Roberta Louise Dow

Optimum initial pH was studied using two buffer
systems (citrate and phosphate) spanning the pH range 3.0
to 8.0. The Optimum initial pH range for growth was 5.8
to 6.4.

Growth was studied at five temperatures (12, 16,
20, 24, and 28°C) using three different media: sucrose-
asparagine, malt extract-NaNOB, and phytone. The optimum
temperature with sucrose-asparagine was 24°C. Growth was
equally good at 16, 20, and 24°C in malt extract-NaNO3
medium.

Growth in standing culture was equal to that in
shake culture. Three weeks was the Optimum time for

harvest.

THE EFFECT OF NUTRIENTS AND ENVIRONMENTAL
FACTORS ON GROWTH OF UROCYSTIS COLCHICI

(SCHLECHT.) RABENH.

BY

Roberta Louise Dow

A THESIS

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

MASTER OF SCIENCE
Department of Botany and Plant Pathology

1968

TO MY FAMILY

ACKNOWLEDGMENTS

I wish to sincerely thank Dr. M. L. Lacy for his
interest, direction, and encouragement throughout this
study.

Thanks are also due to Drs. E. S. Beneke, D. J.
DeZeeuw, and J. L. Lockwood for their suggestions and
critical evaluation of this manuscript.

Recognition is extended to Dr. N. E. Good for his
helpful advice.

I am also very grateful to my husband, Tom, who

endured and encouraged a student-wife.

iii

TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . iii
LIST OF TABLES . . . . . . . . . . . . . . . . . . v
LIST OF FIGURES . . . . . . . . . . . . . . . . . . vi
INTRODUCTION . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . 4
MATERIALS AND METHODS . . . . . . . . . . . . . . . 13
RESULTS . . . . . . . . . . . . . . . . . . . . . . 16
DISCUSSION . . . . . . . . . . . . . . . . . . . . 37

LITEMTURE CITED 0 O O O O O O O O O O O O O O I O 47

iv

LIST OF TABLES

 

 

 

Table Page
1. Effect of concentration of malt extract on
growth of Urocystis colchici in a basal
mineral salts--NaNO3 medium . . . . . . . 21
2. Effect of simple sugars with malt extract
on growth of Urocystis colchici . . . . . 22
3. Growth of Urocystis colchici on carbohy-
drate sources aIOne and with peptone or
23

phytone O O O O O O O C O O O O O I O O O

Figure

1.

10.

LIST OF FIGURES

Effect of carbon sources on growth of
Urocystis colchici . . . . . . . . . .

Effect of time on growth of Urocystis
COlChiCi O O O O O O O O O O O O O O O

 

Effect of concentration of sucrose on
Urocystis colchici growing on a basal
mineraI saIts--NaNO3 medium . . . . . .

Effect of concentration of phytone on
Urocystis colchici growing in a basal
mineraIsalts--NaNO3 medium . . . . . .

Effect of concentration of phytone on
Uroc stis colchici growing in a sucrose
(30 g7IiterS--NaNO3 (2 g/liter) medium

Effect of amino acids on growth of
Urocystis colchici . . . . . . . . . .

Effect of inorganic nitrogen sources on
growth of Urocystis colchici . . . . .

 

Effect of temperature on growth of
Urocysti§_colchici in three different
media 0 O O O O O O O O O O O O O O O 0

Effect of initial pH on growth of
Urocystis colchici . . . . . . . . . .

 

Effect of concentration of two amino
acids on growth of Urocystis colchici .

 

vi

Page

17

19

24

26

27

29

30

31

33

3S

INTRODUCTION

Smut diseases have played an important role in the
history of plant pathology. Spores of Tilletia caries,
incitant of stinking smut or bunt of wheat, were used by
Tillet (48) to establish the contagious nature of disease
for the first time. It was also with stinking smut that
Prévost (36) discovered that fungal spores were the "seeds"
of microsc0pic plants, which could germinate and give rise
to new individuals. These early empirical studies on smut
diseases served as models for later scientific work in the
nature and causes of plant diseases.

Smut was one of the first diseases to be controlled
by chemicals (26). In the late 1700's EurOpean farmers
used various seed treatments collectively known as 13_
Chaulage, involving chemicals such as lye, ammonia, or
COpper sulphate for the control of stinking smut. The
first control methods for onion smut were develOped in the
early 19th century. Then, as now, control consisted of
either seed protection or in-row soil fumigation. The
earliest control consisted of treating onion seeds with
sulphur and lime (45). The first effective control was
develOped by Selby (42) using a formaldehyde solution
dripped in the row near the seed. The formaldehyde vapors

l

diffused through the soil, inhibited the fungus, and al-
lowed the onion to develop beyond the short susceptible
period. This method with modification is still used today.
More recently, thiram has come into use as an effective
seed protectant (27). Due to the longevity of the fungus
Spores in the soil, this disease cannot be economically
eradicated and therefore remains important in many onion
growing regions despite effective control measures (52).
The taxonomic position of the onion smut fungus is

controversial. Early workers used the binomial Urocystis

 

cepulae Frost because it attacked the common onion Allium
SEES! distinguishing it from g, colchici found on autumn-
crocus (Colchicum autumnale L.) (45, 52). Fischer and Shaw
(14) have chosen to combine Urocystis cepulae, g, allii_and
g. magica with the earlier described species 9, colchici,
based on morphological similarity of their teliospores.

The host range of g. colchici as described by Fischer and
Shaw is limited to members of the lily family. They felt
that smut fungi of similar morphology and symptomatology,
and parasitizing members of the same host family, should

all be considered one Species, whereas a smut fungus similar
in morphology but attacking only members of a different
family should be regarded as a distinct species. The writer

agrees with this concept and uses Urocystis colchici

 

(Schlecht.) Rabenh. for the onion smut fungus in this work.

The onion smut fungus is a soil borne plant pathogen
(l, 3, 45, 52, 55). It overwinters as teliospores, often
incorrectly referred to as chlamydospores, in the soil.

The thick walled teliospore contains a single diploid nu-
cleus and is surrounded by small sterile cells (1, 3, 45,
52). Prior to germination of the teliospore, the nucleus

is believed to undergo meiosis (3). The haploid nuclei
migrate into the branching promycelium, one nucleus passing
into each mycelial branch. Hyphae developing from these
branches directly penetrate the cotyledon of the onion (52).
Systemic infection is apparently associated with penetration
of the mycelium to the meristematic region. The fungus
ramifies through the onion tissue, ultimately forming
teliospores in the cotyledon, bulb scales, or leaves.
Environmental conditions affect the period of susceptibility
and amount of infection (54). Infection is low in warm
climates where mean soil temperatures often exceed 25°C.
Under these conditions the onion seedling develOps rapidly,
shortening the period during which it is susceptible to
infection (54), and the fungus appears to be inhibited
directly by temperatures above 25°C (53).

Little definitive work has been done on the phys-
iology of Q. colchici. The objectives of this investiga-
tion were: 1) to investigate nutritional and environmental
requirements for growth; 2) to develop a defined medium
yielding good growth; and 3) to compare nutritional require-

ments for somatic growth and spore germination.

LITERATURE REVIEW

Early workers believed that smut fungi were obli-
gate parasites. Brefeld (5) was the first to demonstrate

that they could be grown and maintained on man-made media.

Natural Media

Culture media were first made from natural products
that early investigators found readily available. These
extracts or wastes of plant or animal materials served as
carbon, nitrogen, mineral, and vitamin sources with varying
efficacy. Brefeld (5, 6, 7) demonstrated that soil ex-
tracts, dung decoctions, natural fruits and vegetables,
fruit and vegetable juices, meat extracts and malt extract
could be used to culture fungi. Most of these media have

since been used to culture the smut fungi.

Dung and soil decoction.--Sartoris (39) found

 

horse-dung decoction to be an inadequate medium for smut
fungi, supporting little or no growth of Ustilago hordei,

g. heufleri, g, maydis, g, tritici, Tilletia tritici, and

T. foetans. Maire (32) reported that the growth of Ustilago
maydis on horse-dung decoction was made up exclusively of

yeast-like cells. Anderson (1) attempted to grow Urocystis

colchici, the onion smut fungus, on dung decoction agar
but obtained very poor growth. Soil decoction agar sup-

ported even less growth.

Fruits, vegetables, and their_juices.--Urocystis

 

 

colchici has been grown on sterile beans, carrots, potatoes,
and fresh onions (3). Only beans and potatoes were found
to be good substrates. Onion decoction and onion agar have
been used by several workers to culture the onion smut
fungus (l, 3, 13, 45, 53). Onion decoction supported poor
growth according to Anderson (1), whereas the fungus grew
well on onion agar. Potato agar was also used by Anderson

with only fair results. Ling (31) reported that Urocystis

 

occulta grew poorly on sugar beet juice, dahlia tuber juice,
potato or rye plant juice alone or in combination with a
synthetic medium. Carrot agar was used by Potter (35) for

culturing Sphacelotheca reiliana (Sorosporium reilianum).

 

Ranker (37) felt that liquid carrot decoction was such a
good medium for culture of Ustilago maydis that he used it
as his control in experiments to develop a synthetic medium
yielding growth equal to that of a natural medium. Ustilago
maydis was found by Maire (32) to grow better on carrots
than on potatoes. Cider agar was used by Bauch (2) to

germinate and culture Ustilagg bromivora and g. grandis.

 

Meat extracts.--Nutrient media consisting of meat

 

extracts have been very popular in liquid and solid culture

media of the smut fungi. Ustilago avenae, g, bromivora,

 

g. grandis, g, maydis, and g. striiformis develop well on

 

this type of substrate (2, 9, 19, 20, 28, 32). Anderson
(1) found beef-broth agar to be a very unfavorable medium

for Urocystis colchici.

 

Malt extract.--One of the most widely used natural

 

substrates for fungi is malt extract. Potter (35) was one
of the first to use malt extract in the culture of the smut

fungi. He found that Sphacelotheca reiliana developed luxu—

 

riant mycelium on malt extract agar. Hanna in 1929 confirm-
ed this result (17). Thirumalachar (46) used malt extract

agar for culturing Urocystis agropyri. Ling (31) utilized

 

this rich medium for culture of the slow-growing smut fun-

gus, Urocystis occulta. Kniep (21) found 0.5% malt extract

 

agar supported rich growth of Urocystis anemones. Other

 

workers to report optimum growth of smut fungi on this

medium are: Thren (47) working with Ustilago nuda, Leach

 

et_al (28) studying g, striiformis, Lange de la Camp (24),

 

studying Q, tritici, and Zscheile (57) working with Tilletia

caries.

Supplemented natural media.--B1izzard (3) obtained

 

much better growth of Urocystis colchici on onion decoction

 

agar plus sugar, than on onion decoction agar alone. Like-
wise potato decoction enriched with sucrose increased the
growth of this organism (l). Tachibana (44) found potato

dextrose agar (PDA) to be a satisfactory medium for growth

of the onion smut fungus. Urocystis occulta and Urocystis

 

 

agroEyri also produced good mycelial growth on PDA (31, 46).

InVestigators studying Ustilago maydis, g. striiformis, g.

 

nuda (g. tritici), Tilletia caries, T. controversa, and

 

 

Sphacelotheca reiliana routinely used potato sucrose agar
(PSA) or PDA for culture, maintenance, and spore formation
(9, 17, 22, 24, 28, 37, 47, 50, 57).

Halbsguth (16) found that peptone, an amino acid-
rich, carbohydrate-free substance, added to PSA increased
the growth of Tilletia tritici. Sartoris (39) recommended
malt extract enriched with peptone for culturing several

mmtfmgL

Synthetic Media

 

As work continued with culturing fungi and interest
increased in fungal nutrition, more definitive media were
required. Halbsguth (16) stressed the importance of defin—
ing the technique of media preparation. He demonstrated
that decoctions of uncut potatoes, peeled potatoes, and of
potato peelings varied in their ability to support growth
of Tilletia tritici. Decoctions of uncut potatoes were
unable to support any growth whereas the other two supported
good growth. Ranker (37) objected to the wide usage of the
various infusions, decoctions, and extracts for determining
physiologic specialization. He reported that the inability

to standardize media made from complex natural substances

prevented duplication of research. One of the first syn-
thetic media for fungi was develOped by Brefeld (7). It
consisted of glucose,(NH4)§O4 and NH4NO3 in water solution,
poured over cigar ashes.

Polysaccharides.--Polysaccharides must first be

 

broken down into simple sugars before they can be utilized
by an organism. This hydrolysis requires special enzymes
which some organisms do not possess. Soluble starch was
reported by Zscheile (57) to be utilized by Tilletia caries,
while wheat and potato starches gave little growth. The
results of studies by Chung and Trione (10) showed that

only minimum growth of T. controversa occurred on starch.

 

Oatmeal agar, which contains a high quantity of starch,

was reported by Sartoris (39) to be an excellent medium for
culture of several fungi including T. tritici, T. foetans,
and Ustilago maydis. Glycerine, dextrin and soluble starch
were indicated by Volkonsky (51) to support growth of g.
maydis. He reported that the enzymes of this fungus strong-
ly attack a large number of glycosides. Wolf (56), how-
ever, found that g, maydis would not grow on soluble starch.
Glycerine, starch, and dextrin were reported by Blumer (4)

to give better growth of g. violaceae than simple sugars.

 

Mono-and disaccharides.--The simple sugars are the
most thoroughly investigated carbon sources for fungi.
Sucrose, glucose, and maltose appear to be the most effec—

tive for smut fungi (35, 37, 39, 40, 50, 57). Xylose,

glucose, fructose, galactose, maltose, and sucrose were
able to support good growth of Ustilago maydis (51). In
another study, g. maydis grew best on glucose, fructose,
mannose, sucrose, maltose, and trehalose of twenty carbon
sources tested (56).

Halbsguth's data showed sucrose or glucose to be
better sources of carbon than crystallized maltose for
growth of Tilletia tritici haplonts (16). Galactose and
lactose were unable to support any growth of this organism.
Anderson (1) reports favorable growth of Urocystis colchici
with cane sugar (mostly sucrose) in Czapek's agar.

After reviewing the literature on carbon nutrition
one cannot help but-concur with Cochrane (11) who stated:
"the most important single facet of carbon nutrition is the
high degree of specificity; differences between Species and

strains are the rule in fungi."

Organic nitrogen.--Few workers have studied nitrogen
sources as thoroughly as carbon sources. The widely used
natural compounds usually contained enough amino acids to
fulfill the nitrogen requirements of the test fungus. In
most cases where organic and inorganic nitrogen sources
have been tested, organic sources have been superior to the
inorganic forms (4, 10, 25). In all the literature re-
viewed, asparagine ranked among the best nitrogen sources

for growth (4, 10, 16, 41, 56, 57). Halbsguth (16),

10

studying T. tritici, found alanine, allantoin, and aspar-
agine superior to all other nitrogen sources tested.
Blumer (4) tested fourteen nitrogen sources on the

growth of Ustilago violaceae and found fibrin to be super-

 

ior to others tested. Peptone was also found to be an ex—

cellent nitrogen source for U. violaceae. Asparagine and

 

urea gave good growth while nucleic acids were inadequate.
Further work with this fungus demonstrated that DL-serine,
DL-phenylalanine, L-arginine, DL-aspartic acid, L-aspar-
agine, and L-oxyproline were the best of 36 amino acids
tested (41). Amino acid isomers of the D form were poorly

utilized.

Inorganic nitrogen.--Schopfer and Blumer (41) found

 

that (NH4)ZSO4 and ammonium citrate were as good as aspar-

agine for growth of Ustilago violaceae. However, this was

 

only true when vitamins were added to the media. Ranker
(37) found that U, maydis could grow well on NaNO3 and KNO3
but maximum growth was obtained on a medium containing
NH4N03. Lange de la Camp (25) found that T. tritici grew

poorly on all inorganic nitrogen sources tested.

Other Factors

 

The effect of hydrogen ion concentration in media
on mycelial production of the smut fungi has been investi-
gated by some authors. Most of the smuts grew best in

slightly acid media (39). Using media adjusted with KOH

11

or HCL, crude studies were conducted by Sartoris (39) to
determine the Optimum initial reaction for mycelial devel-
Opment of four species of Ustilago. The results of his
narrow range study indicate that pH 6.8 to 7.1 were best.
Ranker's (37) synthetic medium which supported excellent
growth of g, maydis had a pH of 5.6. Wolf (56) reports
that pH 6.0 was good for growth of U, maydis. Preliminary

studies by Blumer (4) indicated that U. violaceae had a

 

strong tolerance to pH. This organism was able to develOp
an average amount of mycelium in media of pH 3 to 8.3. He
conducted most experiments at pH 5.6 which happened to be
the pH after sterilization of his unbuffered media. Trione
(50) used agar media adjusted to pH 6.0 for culturing

Tilletia caries and T, controversa.

 

Temperature.--Different species of smut fungi appear
to favor different temperatures with the majority growing
best around 20°C. This temperature produced well-developed
cultures of T. tritici and T. foetans (39). When cultures
of these fungi were placed at l and 2°C for one week they
would die; 25°C also inhibited the mycelium which would
then disintegrate. An incubation temperature of 19 to 20°C
was used for T. caries by Zscheile (57) who reported that
growth decreased at 15 and 25°C.

While studying four species of Ustilago, Sartoris
(39) found that the optimum temperature varied with species.

2. heufleri grew best at approximately 20°C. The maximum

12

temperature that it could tolerate was around 36 to 37°C.
Haskins (19) also noted that U. maydis grew most rapidly at

30°C. Ustilago violaceae was unable to grow at 28°C.

 

Optimum growth for this species of smut fungi occurred
between 18 and 22.5°C (41). Ling (31) tested the effect of

temperatures ranging from 5 to 25°C on the growth of

 

Urocystis occulta on PDA and malt extract agar. The Op-
timum temperature was near 20°C on both media. Growth
occurred at all temperatures with mycelium produced at
25°C equal to that produced at 10°C. Walker and Wellman
(53) report 18°C to be optimum for mycelial growth of

Urocystis colchici.

 

In summary, little is known about the nutritional

requirements of Urocystis colchici. It grew poorly on

 

sterile carrots, sterile onions, onion decoction, meat
extract, dung decoction and soil decoction (l, 3, 13, 45).
Fair growth was Obtained with sterile beans and potatoes.
Onion agar, onion sucrose agar, and potato dextrose agar
have been most widely used for culturing this slow growing
fungus (l, 3’.13' 23, 45, 53). It has rarely been cultured
on defined media.

Eighteen degrees centigrade was reported optimum

for growth on onion decoction agar (53).

MATERIALS AND METHODS

Urocystis colchici teliospores were aseptically
collected from sori in the leaf lacunae of infected onions
(Allium cepa 'Downings Yellow Globe') using the method
described by Lacy (23). The spores were placed on malt
extract agar plates and allowed to germinate in an incu-
bator at 24°C. Mass transfers were made from these col-
onies to potato dextrose agar (PDA) or potato sucrose agar
(PSA) plates, and were transferred every three weeks so
that mycelia used as inoculum would be in an actively grow-
ing condition.

Unless otherwise indicated, the basal medium con-
tained (grams per liter): KH2P04, 1.0; KZHPO4,

4, 0.01. Carbon and nitrogen sources

0.46; KCl,

0.5; MgSO 0.5; FeSO

4:
were added as described for each experiment in the Results.
All chemicals used were of reagent grade. The medium was
buffered at pH 6.4 with phosphate buffer unless stated
otherwise. Buffer was used initially at 0.1 M concentra-
tion and later lowered to 0.05 M or 0.01 M. Forty ml of
liquid media were placed in 125 ml Erlenmeyer flasks; the

flasks were plugged with cotton and autoclaved for twenty

minutes at 15 psi, 121°C.

13

14

Since the teliospores of U, colchici germinate
poorly or not at all in liquid media (1) it was necessary
to use mycelial fragments as inoculum. Mycelium free of
agar was aseptically scraped from the surface of PDA plates
with a stainless steel spatula. Mycelia were washed 3
times by centrifugation in sterile glass-distilled water
then resuSpended in sterile, glass-distilled water. A
sample of the hyphae was pipetted onto PDA or PSA plates
to assay for possible fungal or bacterial contamination
before using the inoculum. Meanwhile, the rest of the
inoculum was maintained in glass-distilled water at 2°C
(44). Uncontaminated inoculum was fragmented in sterile
distilled water with a Sorvall Omnimixer. After the first
30 second period of fragmentation, the size of the hyphal
fragments was microsc0pically examined. If the length of
the fragments averaged more than 45 u, the inoculum was
fragmented 30 seconds longer. Concentration of the hyphal
fragments was determined with a hemacytometer and adjusted

by dilution to 4 x 106

fragments per m1. One ml of the
inoculum suspension was aseptically transferred to each
flask of the medium under a Microvoid hood, surface-disin-
fested with Chlorox. The lip of each flask was flamed
before entry of the pipet and after delivery of the inoc-

ulum. All flasks were incubated for three weeks at 22°C

unless otherwise indicated. In experiments investigating

15

nitrogen sources, all glassware was acid-washed with con-
centrated HCl and rinsed in tap and then distilled water.

Growth of the fungus was measured by dry weight of
the mycelium. The fungus was collected by filtration on
oven-dried Reeve-Angel, glass-fiber filter papers. The
combined filtrates of all flasks of a treatment were col-
lected and the pH was measured to determine any change
from the original pH.

All experiments were repeated one to three times,

and the data were analyzed statistically.

RESULTS

Effect of carbon sources on growth.--Experiments
were designed to determine which carbon sources were capable
of supporting good growth. Three complex carbon sources,
one polysaccharide, two disaccharides, and four monosac-
charides were added at the rate of 10 g/liter to the basal
medium containing NaNO3 at 2 g/liter as the nitrogen source.

The control consisted of the basal medium plus NaNO Only

3.
D—isomers of the sugars were used. The results (Fig. 1)
show malt extract (rich in carbohydrates, amino acids, and
vitamins) and phytone (papaic digest of soybean meal con-
taining amino acids, vitamins, and carbohydrates) superior
to all others tested. Sucrose, D-mannose, and D-glucose
were not significantly different from each other but were
significantly better than the five other carbon sources.
Dextrin, peptone (carbohydrate-free protein hydrolysate
containing vitamins and amino acids), maltose, and D-fruc-
tose yielded little or no growth. Growth with maltose and

D-fructose was not significantly different from that of the

control.

Effect of oxygen on growth.--To determine if aera-

tion enhanced growth of this fungus, inoculated flasks

l6

 

 

l7

 

CK
FRUCTOSE
MALTOSE
PEPTONE
DEXTRIN l
GALACTOSE I
GLUCOSE
MANNOSE
SUCROSE
PHYTONE
MALT EXT.

 

O 2 4 '6 8 IO I2 IAIO I820222426
MG DRY WEIGHT PER FLASK

Figure l.--Effect of carbon sources on growth of Urocystis
colchici. Carbon sources were added at 10 g/liter
to a basal mineral salts—NaNO medium. Bars joined
by a common line did not diffar significantly at
the 5% level.

 

18

containing the basal medium with malt extract, 10 g/liter,
and NaNO3, 2 g/liter, were placed on a reciprocating shaker
set at 90 strokes/minute. Other flasks were placed on a
shelf in the same room. After three weeks, shaken flasks
yielded an average of 25.3 mg and standing flasks 25.1 mg
mycelial dry weight. Growth in the shaken flasks was in
the form of submerged pellicles while that in the standing
flasks was made up of both surface mycelium and submerged,
loosely-organized clumps. Apparently aeration of the me-

dium is not required for maximum growth of this organism.

Effect of time on amount of growth.--In nutritional

 

studies it is necessary to measure growth at the time when
maximum growth has occurred and prior to autolysis. Malt
extract and sucrose were added at the rate of 10 g/liter to
the basal medium containing NaNO3 at 2 g/liter. Dry weight
measurements were made at weekly intervals. Within one
week good growth was obtained with the malt extract medium
(Fig. 2). It supported maximum growth after three weeks.
Growth began more slowly in the sucrose medium and was at
the highest level after four weeks.

Later studies using phytone and sucrose-phytone
also showed that 3 weeks was Optimum for growth. On the

basis of these results it was felt that a three week period

was most suitable for obtaining Optimum growth.

l9

 

 

 

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Figure 2.--Effect of time on growth of Urocystis colchici.
Ten grams malt extract or sucrose were added per

liter of basal mineral salts medium containing
2 g NaNOB.

20

Effect of concentration of malt extract on growth.—-

 

Malt extract was added at concentrations of 0.1, 0.5, 1.0,
5.0, 10.0, 15.0, and 20.0 g/liter to the basal mineral
salts solution containing NaNO3 (2 g/liter). After auto-
claving, the medium was filtered through Whatman number 1
filter paper to remove any precipitate. The medium was
then dispensed and reautoclaved for 15 minutes.
Concentrations of 0.1, 0.5, and 1.0 g malt extract/
liter showed no significant difference in growth (Table 1).
Five grams/liter yielded nearly five times more growth than
1.0 g/liter and 10.0 g/liter nearly twice as much as 5
g/liter. Ten and 15 g/liter were best but not significantly
different from each other. A further increase in the con-

centration decreased the amount of growth.

Effect of adding a complex nutrient source to a
simple sugar.--To determine whether unknown nutritional
factors in malt extract might have an effect on the uti-
lization of simple sugars, 0.1 g or 1.0 g malt extract
were added to one liter basal mineral salts medium con-
taining 2.0 g NaNO3 and simple sugars, each at 10 g/liter.
Dry weight yields were greater on media containing sucrose,
glucose, or mannose plus 1.0 g malt extract (Table 2) than
with the same sugars alone. Therefore it appeared that
nutritional factors in relatively small amounts of malt

extract could have considerable effect on growth in the

21

presence of some simple sugars. However, 0.1 g malt extract
per liter added to 9 carbohydrate sources showed no effect
on the utilization of simple sugars by g. colchici.

Table l.--Effect of concentration of malt extract on growth

of Urocystis colchici in a basal mineral salts--
NaNO3 medium.

 

 

 

 

Malt extract Dry weighta

g/liter mg
0.1 1.5 a

0.5 3.5 a

1.0 2.7 a

5.0 13.9 b

10.0 26.1 cd
15.0 27.3 d
20.0 22.7 c

 

aBased on Duncan's multiple range test for signif-
icance at the 5% level. Means which have letters in common
are not significantly different.

In other experiments, phytone and peptone (0.1 or
1.0 g/liter) were added to 7 carbohydrate sources, each
used at a concentration of 10.0 g/liter (Table 3). The
basal medium contained 2.0 g NaNO3/liter. Controls con-
sisting of the basal medium without a carbon source and

the basal medium plus the two concentrations of phytone

and peptone without additional carbon source were included.

22

The data show that in all cases 1.0 g phytone/liter added
to a simple sugar significantly increased growth. Peptone
at 1.0 g/liter gave significantly better growth when added
to simple sugars except in the case of snicime. Growth
with malt extract was not improved by addition of phytone
or peptone.

Table 2.—-Effect of simple sugars with malt extract on
growth of Urocystis colchici.

 

 

 

Dry weight, mg

 

 

Carbohydrate, Plus malt extract
10 g/liter Alone at 1.0 g/liter
Malt extract 24.0 22.5
Maltose 9.0 9.0
Sucrose 8.3 46.5
Glucose 10.0 42.5
Mannose 9.0 26.6

 

Effect of sucrose concentration ongrowth.--Sucrose
at 0.1, 5.0, 10.0, 20.0, 30.0, or 50.0 g/liter was added to
the basal medium containing 2 g NaNOB/liter. Fig. 3 shows
that dry weight increased as the concentration increased up
to 30.0 g/liter, but growth with 50.0 g/liter was not sig-

nificantly better.

23

.OODMOHHQOH xwm mo usmflm3 hue OE cmmfim

 

n.4m m.mm m.ea H.HG s.ms ~.¢H o.mm e.m umuaaxm o.H .mcoumam
a.m H.m~ m.ma N.om H.mm e.m A.AH e.m umuflaxm H.o .maousam
N.Hm k.mm N.OH H.mm m.mm m.HH m.aH a.m neuflaxm o.H .mcoummm
m.ma m.mm m.sH 9.0m s.m~ o.h m.e~ a.m umufla\m H.o .mcoummm
m.» m.¢H o.m m.HH m.ma m.a ~.o~ we.~ pamsmammsm oz

 

omouosnm Omoscmz OmODOMHmw Omoosao Omouosm mmouamz uomuuxm Aconnmo
paws ocv x0

 

AHODAH\O oav OOMDOm mpmupmnonumu

 

 

.Osoumnm Ho
mcoummm spas Use macaw mmOHDOm oumnpmnonumo so HOHQOHOO mflummooun mo nuzouwll.m OHQMB

 

24

 

 

MG DRY WEIGHT PER FLASK

 

 

; n l A L l A I n 1

 

 

O 5 IO I5 20 25 30 35 40 45 50
GRAMS SUCROSE PER LITER

Figure 3.——Effect of concentration of sucrose on Urocystis
colchici growing on a basal mineral salts-NaNO3
medium.

25

Effect of phytone concentration on growth.--Phytone
at six concentrations (0.1, 5.0, 10.0, 20.0, or 30.0 g/
liter) was added to the basal medium. The data of Fig. 4
show a direct correlation between increase in growth and
increase in phytone concentration. The best growth was

obtained with 30.0 g/liter.

Effect of concentration of phytone on growth in
media containing the optimum sucrose concentration.--Phytone
at 0.1, 5.0, 10.0, 20.0, or 30.0 g/liter was added to the
basal medium containing 30.0 g sucrose/liter. Controls
containing 30.0 g sucrose/liter without phytone, and the
basal medium without a carbon or nitrogen source were in-
cluded. Fig. 5 shows that 5.0, 10.0, and 20.0 g phytone/
liter greatly increased growth. The control containing
sucrose without phytone was not significantly different
from the control without carbon and nitrogen. Sucrose plus
0.1 g phytone supported poor growth but it was significantly

better than the control.

Effect of various nitrogen sources on growth.--Nine
amino acids were added at a concentration calculated to
give 0.3 g N/liter. This amount of nitrogen was selected
since it corresponds with that contained in 2.0 g NaNO3/
liter which was used in previous experiments. Sucrose at
10.0 g/liter was added as the carbohydrate source (at this
time it was not known that the optimum concentration of

sucrose was 30.0 g/liter).

26

 

I20

100

80

60

40

MG DRY WEIGHT

20

 

 

 

J l I 1 I I

 

0 5 I0 I5 20 25 30
PH YTONE (93/ liter)

Figure 4.--Effect of concentration of phytone on Urocystis

 

colchici growing in a basal mineral salts-NaNO3
medium.

27

 

 

 

 

100 - .
80 - .
.—
I:
2
uJ 6CI- .
3
>.
a: 40 -
o .
o
2 20 - _
O 1 1 1 l 1 1 I
0 5 I0 15 20 25 30

PHYTONE SUPPLEMENT (9/ liter)

 

Figure 5.--Effect of concentration of phytone on Urocystis
colchici growing in a sucrose (30 g/liter)-NaNO3

(2 g7Iiter) medium.

28

The best growth was obtained with L-aspartic acid
and L-asparagine (Fig. 6). L-glutamine and L-glutamic acid
also gave good growth. L-histidine gave the least amount
of growth but this was still significantly greater than the
sucrose alone.

Four inorganic nitrogen sources were added at a
concentration of 0.3 g N/liter to the basal medium con-
taining sucrose at 10.0 g/liter. All the inorganic ni-
trogen sources were inferior to the best organic nitrogen
sources (Figs. 6 and 7). Potassium nitrate and NH4NO3 were
significantly better than the others. There was no differ-
ence between NaNO3 and (NH4)2
the control without nitrogen (Fig. 7). The pH of the

$04 but they were better than

NH4NO3 medium dropped to 5.7 and that of (NH4)ZSO4 dropped

to 5.9 while the others remained at 6.4.

Effect of temperature on growth.--Three media were

 

used in separate experiments to test the effect of temper-
ature on growth. They were: 1) phytone, 10 g/liter; 2)

malt extract, 10 g/liter, plus NaNO 2 g/liter; and 3)

3!
sucrose, 10 g/liter, plus L-asparagine, 0.3 g N/liter.
Flasks were placed in unlighted incubators set at 12, 16,
20, 24, and 28°C. Fig. 8 shows that 24°C was optimum with
all media except phytone which had maximum growth at 20°C.
The fungus grew very poorly in all media at 28°C. No sig-

nificant difference was found in growth on malt extract

media at 10, 16, or 24°C.

Ck
Hi:
Lou
Gly
Ara
Ala

GIuNH2

29

 

 

 

L
4 8 I2 I6 20 24 28 32

MG DRY WEIGHT

Figure 6.--Effect of amino acids on growth of Urocystis

 

colchici. Amino acids were added at a concen—
tration of .3 g N/liter to the basal mineral
salts medium containing 10.0 g sucrose/liter.

Ck = no nitrogen source; His = L—histidine;

Leu = L-liucine; Gly = glycine; Arg = L—arginine;
Ala = L-alanine; Glu NH = L—glutamine; Glu =
L—glutamic acid; Asp = E-aspartic acid; and
AspNH = L-asparagine. Bars joined by a common
line aid not differ significantly at the 5%
level.

30

 

 

C k
(NH,),so,
NaNC)3
N H‘NO
KNO3

 

i l l l L l l l l L l

0 4 8 I2 16 20
MG DRY WEIGHT

Figure 7.—-Effect of inorganic nitrogen sources on growth
of Urocystis colchici. Nitrogen sources were
added at 0.3 g N/liter to the basal mineral
salts medium containing 10 g sucrose/liter.
Bars joined by a common line did not differ
significantly at the 5% level.

 

31

 

 

 

 

 

40 sucaose ° ‘
:2 ASPARAGINV
U)
4 I
E:35 ",rl/:;:>L _
33 3o - / ‘
a. MALT EXT.

(+NON03)

r- 25 .
I a
0 .
E 20 - , PHYTONE
3 (-NONO3) }
>.l5'
M
0

IO - ‘
0 .
2 A

5 - _
0 I I 1 I I
12 16 20 24 28
T EMPERATURE

Figure 8.--Effect of temperature on growth of Urocystis
colchici in three different media. Malt ex-
tract, sucrose, and phytone were used at 10

g/liter. Asparagine and NaNO3 were used at
0.3 g N/liter.

 

32

Effect of initial pH on growth.--Two buffer systems

 

were used to span the range of pH 3 to 8. The initial pH
was measured after autoclaving to insure that it was un-
altered by heat. Final pH measurements were made after
three weeks growth.

Citrate buffers of pH 3 to 6.4 were made using
combinations of citric acid and sodium citrate to give a
final concentration in the medium of 0.01 M (12). Sucrose,
10 g/liter, and L-asparagine, 0.3 g N/liter, were added to
the buffered basal medium. Growth at initial pH values of
6.0 to 6.4 were best (Fig. 9). Growth at pH 5.4 was less
than at either 6.0 or 6.4 but was significantly more than
at any of the lower pH values. There was no significant
difference between the effect of initial pH 3.0 through 5.0.
The final pH of all treatments varied less than 0.2 pH units
from the original with the exception of pH 3.0 and 3.4
which changed to 3.4 and 3.9 respectively.

Monobasic potassium phosphate and dibasic potassium
phosphate were used in different combinations to obtain pH
values of 5.8 to 8.0 (12). Increments of 0.2 pH units were
used in one set of experiments while 0.4 and 0.6 unit in-
crements were used in another. The basal medium contained
the buffer at 0.01 M concentration. Ten grams sucrose/liter
and 2 g NaNO3/liter were added to the buffered basal medium
in one experiment while 30 g sucrose/liter and 0.3 g N/liter

as asparagine were added to the buffered basal medium in

33

 

  
 
  
   
  
 
   

N
C)
l

PHOSPHATE
Sucrose , 30g/liter

AspNH ..3 N I. ‘
PHOSPHATE Z 9 /'°'
SucroseJOg/liter ( )

NON03,.3gN/liter

(e)

1
N
C)

8
(8)1H9l3M MIG ow

MG DRY WEIGHT (A)
I. on
o o

    
  

CITRATE
Suc roseJOg/Iiter
AspNH2 ..3g/Iiter .I
P (A)

  

 

 

 

 

o .J—r l I 'I 1 _4L .1 I I L I 0
3 4 5 6 7 8
INITIAL pH

Figure 9.—-Effect of initial pH on growth of Urocystis
colchici. Carbon, nitrogen, and citrate or
phosphate buffer were added to the basal
mineral salts medium as indicated. The curves
designated (A) are graphed according to the
scale at the left while the curve designated
(B) follows the scale at the right.

 

34

another experiment. Growth at an initial pH of 6.0 was
found to be best in experiments using sucrose and asparagine
(Fig. 9). A pH of 6.4 gave significantly better growth

than higher pH values. Growth did not differ significantly
at pH 5.8 to 6.4 in media containing sucrose and NaNO3.
Final pH values were within 0.2 units of the original pH
value with the exception of 7.4, 7.6, 7.8, and 8.0 which
were lowered by 0.4, 0.4, 0.6, and 0.6 of a pH unit,

respectively.

Effect of concentration of two amino acids on

 

growth.--L-asparagine and glycine were added at concentra-
tions of 0.1, 0.3, 0.6, 1.0, or 3.0 g N/liter to the basal
medium containing 30 g sucrose. Although the media were
buffered at pH 6.4 it was necessary to adjust the pH with
NaOH. At a concentration of 3.0 g N/liter as asparagine,
a large amount of crystaline precipitation occurred after
autoclaving. Four replicates with the least amount of
precipitation were selected for inoculation. Crystals that
were collected on the filter paper when harvesting these
replicates were removed with forceps. The filtered mycelium
was thoroughly rinsed with distilled water to dissolve any
'small crystals that might not have been observed.

No significant difference was found between the
control and media containing 0.1, 0.3, 0.6, or 1.0 g N/

liter as glycine (Fig. 10). The best growth using glycine,

35

 

 

 

 

 

 

 

200
AspNH2 )
.— .I
I:
9150 "
LIJ .
3
100 -
>-
a:
D -
O 50 -
2 y
1
O I I l 4"
2 3

(QN/ liter)

Figure 10.-—Effect of concentration of two amino acids
on growth of Urocystis colchici. Amino acids
were added at concentrations of 0.1, 0.3, 0.6,
1.0, or 3.0 g N/liter to the basal mineral
salts medium. AspNH2 = L-asparagine and Gly =
glycine.

 

36

although relatively low, was obtained using 3.0 g N/liter.
The pH of all concentrations except 3.0 g N/liter drOpped
from 6.4 to 5.6.

L-asparagine, as shown earlier, was far superior
to glycine at all concentrations (Fig. 10). Each increas-
ing level of nitrogen gave a significantly greater amount
of growth until an optimum concentration of 1.0 g N/liter
was reached. A concentration of 3.0 g N/liter was not

significantly better than 1.0 g N/liter.

DISCUSSION

Malt extract and phytone yielded more growth than
any other single source of nutrients tested. One cannot,
however, attribute the growth obtained with these two media
to their superiority as carbon sources. Malt extract is
not only an excellent source of carbohydrates, primarily
maltose and glucose, but also contains 16 amino acids,
riboflavin, nicotinic acid, pantothenic acid, ascorbic
acid, and pyridoxal (18). Similarly, phytone consists of
carbohydrates, 18 amino acids, and 9 vitamins (15).

D-glucose, a constituent of both malt extract and
phytone, was one of the best of the simple sugars tested.
It is utilized by virtually all fungi (11). Growth with
sucrose and D-mannose were as good as with D-glucose.
These results correspond with those of Wolf (56) who found
Ustilago maydis grew best with these two simple sugars.

Of 57 fungi studied by Lilly and Barnett (30), only four
were able to utilize sucrose. Zscheile (57) also found
that galactose and fructose were poor substitutes for su-
crose, and that neither glucose nor fructose, nor the two
combined in concentrations equal to that of sucrose, pro-

duced growth equal to that obtained with sucrose.

37

38

Possibly only the glucose moiety of sucrose was
utilized by Urocystis colchici in the experiments reported
here. This was suggested by the lack of growth with D-
fructose, the other member of the disaccharide. D-fructose
was no better than the control without a carbon source.

Ustilago heufleri was also reported by Sartoris (39)
to be unable to utilize fructose, and fructose inhibited

Tilletia tritici. Concentrations of 0.02 M to 0.50 M were

 

used with poor growth at all concentrations. With the ex-
ception of these two organisms, most workers have reported
good growth of smut fungi with fructose. According to
Cochrane (ll), D-fructose is generally equivalent to D-
glucose. Fructose is known to be more rapidly degraded by
autoclaving in the presence of minerals than are the other
sugars (33). Since the carbon sources were autoclaved with

the basal medium, the poor growth of Urocystis colchici on

 

fructose may reflect its inability to utilize the breakdown
product, 5-hydroxymethylfurfura1. In experiments conducted
by Chung and Trione (10) the sugars were autoclaved sepa-
rately from the basal medium. They obtained excellent
growth of Tilletia controversa with fructose. Ustilago
maydis was reported by Wolf (56) to grow well on fructose
but no mention was made of the sterilization method.
Maltose, like fructose, supported no more growth of
g. colchici than the control. This contrasts with the many

reports of maltose being one of the best carbon sources for

39

the smut fungi (37, 39, 40, 56). However, two other smut
organisms, Tilletia controversa (10) and T. tritici (25)
have been shown to grow poorly with maltose. The failure

of maltose to be utilized by Urocystis colchici is espec-

 

ially surprising since malt extract, which contains large
amounts of maltose (18), yields such excellent growth.
This phenomenon may be explained in several ways. Other
sugars in malt extract such as sucrose and glucose may
support enough growth to induce production of maltase,
allowing utilization of maltose. Formation of adaptive

enzymes have been reported in Ustilago striiformis (8).

 

Alternatively, the vitamins present in malt extract may
make utilization of maltose possible. Schopfer and Blumer

(41) found that thiamine moieties allowed Ustilago violaceae

 

to grow well on poor or seemingly unutilizable nitrogen
sources. A third possibility is that other carbohydrates
of malt extract are utilized, and maltose simply cannot be
absorbed.

Disaccharides can be utilized by fungal cells in
one of two ways: by exoenzymes cleaving the compound into
monosaccharides which are then transported across the cell
membrane by monosaccharide carrier systems, or by movement
of disaccharides by specific membrane transport systems
directly into the cell (38). The inability to utilize
maltose may be due either to the lack of exoenzymes (mal-

tases) or the lack of a maltose membrane transport system.

40

Indirect evidence from this study suggest that sucrose is
acted upon by an exoenzyme and only glucose is transported
into the cell. Most fungi, however, move the entire mol-
ecule of sucrose across the membrane (38).

Peptone, a carbohydrate-free protein hydrolysate,

supported poor growth of Urocystis colchici. Peptone con-

 

sists of peptides, amino acids and vitamins (15). Growth
with this as a carbon source indicates that U. colchici is
capable of utilizing amino acids, although poorly, as an
energy source. Amino acids have generally been found to
be poor carbon sources for fungi (ll, 29).

Comparing the effect of carbon sources on growth
in this investigation with the effect on spore germination
as reported by Lacy (23), it generally appears that similar
metabolic mechanisms are involved. Malt extract and phy-
tone provided the greatest amount of both growth and spore
germination. Neither growth nor-germination occurred with
maltose. Sucrose gave the greatest amount of both growth
and germination among the simple sugars tested. D-fructose
supported less germination than sucrose but more than glu-
cose, in contrast with the results of growth studies. Pep-
tone was able to support a low level of germination as well
as a small amount of growth.

Oxygen did not play an important role in the growth
of Urocystis colchici under the conditions of this exper-

iment. Aeration of the liquid medium by shaking did not

41

increase growth. Generally aeration increases the amount
of growth in liquid culture (11). A few fungi, however,
appear to have low oxygen requirements. Growth of Diplo-
carbon Eggag, causal organism of black spot of roses, was
not increased by shaking (43).

Growth curves for liquid-cultured fungi typically
show three phases: 1) a phase of no apparent growth; 2) a
phase of rapid, approximately linear growth; and 3) a phase
of no growth or autolysis and decline in dry weight (11).
To insure harvest as close as possible to the end of phase
two but prior to phase three it was necessary to conduct
time course growth experiments. The lag phase in these
experiments was very short since fragmented mycelium was
used as inoculum rather than spores. Optimum growth was
obtained with three media which contained organic nitrogen
and vitamins. The growth phase was lengthened when these
nutrient factors were not present (Fig. 2). This was sug-
gested also by growth experiments not reported here.

Malt extract was found to yield maximum growth at
10 g/liter. These results are similar to those of Lacy
(23) who showed that 5 or 10 g malt extract was superior
to 20 g/liter for spore germination. In the experiments
reported here, malt extract medium appears to be saturated
with nutrients at 10 g/liter and further increases in ini-
tial concentration do not increase growth. At very high
concentrations one would expect the occurrence of inhibition

due to toxic materials.

42

Growth factors and/or organic nitrogen sources in

peptone were found to stimulate growth of Urocystis colchici

 

in the presence of simple sugars. Improved utilization of
nutrient sources by addition of vitamins has been noted by

Schopfer and Blumer (41) with Ustilago violaceae. Peptone

 

or phytone (0.1 g/liter) greatly stimulated growth in the
presence of sucrose, mannose, and glucose, but results with
fructose, maltose, and galactose (three unutilized or poor-
ly utilized carbon sources) varied. Maltose showed no
stimulation with this concentration, fructose showed some
stimulation, and galactose showed more stimulation than the
other two sugars.

Interestingly, no stimulation of Urocystis colchici
was achieved when phytone or peptone were added to malt
extract, in contrast to the results of spore germination
studies (23). This suggests that different requirements
for nutrients (qualitative or quantitative) occur during
spore germination and somatic growth.

Thirty grams sucrose per liter (approximately .088M)
was found to yield maximum growth of any concentration
tested. The 10 g sucrose/liter used in previous carbon
source experiments, on the basis of results with malt ex—
tract, was probably limiting. It appears that Optimum
carbohydrate concentration varies greatly between species

of fungi (10, 16, 39, 41, 57).

43

Studies on the effect of concentration of phytone
demonstrated a direct correlation between increases in
phytone concentration and increases in growth up to the
highest concentration used. Carbohydrate was possibly the
limiting factor in these experiments since phytone is no-
tably high in amino acids and other growth factors. In
media containing an Optimum sucrose concentration an Op-
timum phytone level of 10 g/liter was reached.

Nitrogen studies showed aminoacids were superior to
inorganic nitrogen sources. This had been found to be true
with many fungi (10, ll, 16, 41). L-asparagine was found
to be one of the best nitrogen sources for growth of
Urocystis colchici. This corresponded with the results of
Halbsguth (16) for Tilletia tritici, Zscheile (57) for T.

caries, Chung and Trione (10) for T, controversa, Wolf (56)

 

for Ustilago maydis. Schopfer and Blumer (41) for U.

violaceae, and Ling (31) for Urocystis occulta. L-aspartic

 

acid, which was as good as L-asparagine in the results
reported here, has not been tested as often as asparagine
in nutritional studies of smut fungi. Ustilago maydis (56)
and U. violaceae (41) were found to grow as well with as-
partic acid as with asparagine. L-glutamic acid and L-
glutamine were shown to be good nitrogen sources for

Urocystis colchici. Ustilago violaceae also grew well with

 

glutamic acid (41).

44

Potassium nitrate and NH4NO3 were the best of the

four inorganic nitrogen sources tested. Ammonia of NH4NO3
is believed to be utilized first (34). This is presumed on
the basis of the drOp in pH generally indicating ammonia
utilization. The pH of the NH4NO3 media dropped to 5.7 and
that of (NH4)ZSO4 to 5.9. There was no pH change in the
media containing KNO3 and NaNO3.
Growth was obtained over the range of pH 3 to 8.
At low initial pH values the organism was able to raise the

pH, and at high pH levels it acidified the medium. Thus,

it was demonstrated that Urocystis colchici has the ability

 

to alter its environment by changing the reaction of the
medium. The Optimum pH range appears to be quite narrow,
i.e. pH 5.8 to 6.4, which nearly duplicates the optima of
teliospore germination, i.e. pH 5.5 to 6.5 (23). The pH
Optimum of Urocystis occulta corresponds to that of g.
colchici with the peak amount of growth at pH 6.2 (31).
g. colchici appears to have a narrower pH optimum than

Tilletia controversa which grew best between pH 6.0 and 8.0

 

(10) or.T. caries which favored 6.0 to 8.6 (57).

The optimum temperature for growth of Urocystis

 

colchici depends upon the culture medium used. Sucrose
and asparagine provided the best growth at 24°C while phy-
tone media supported better growth at 20°C than at 24°C.
Malt extract media was able to support similar amounts of

growth at 16, 20, and 24°C. Walker and Wellman (53) found

45

maximum growth of U. colchici to occur at 18°C on onion
decoction agar. Cochrane (11) stated that "there is no
single temperature optimum of growth. Temperature optima
and ranges reported are valid only under specified condi-
tions of time, medium, and method of measurement."

Maximum germination of teliospores of U, colchici
on malt extract-phytone agar obtained by Lacy (23) occurred
at 24°C while Walker and Wellman (53) report maximum ger-
mination at 15°C on onion decoction agar. Togashi (49)
reports that from studies of several plant pathogens Op-
timum temperature for spore germination is close to that
for in vitrg growth.

It has been determined that: 1) sucrose was one of
the best simple carbon sources for growth of U. colchici;
2) thirty grams sucrose per liter was the Optimum concen-
tration; 3) L-asparagine was an excellent nitrogen source;
4) oxygen was not an important factor when using 40 ml of
liquid medium in 125 ml flasks; 5) approximately three
weeks growth gave maximum dry weight of the fungus; 6) the
Optimum temperature for growth was 20 to 24°C; 7) the op-
timum pH range was 5.8 to 6.4; and 8) one 9 N/liter as
asparagine when used with 30 g sucrose/liter gave the best
growth.

For the first time a defined medium had been devel-
Oped for this organism. This defined medium yielded the

greatest amount of growth so far obtained with U. colchici.

46

Information was obtained on the nutritional and environ-
mental growth requirements of U. colchici. Comparisons
were made between the requirements for somatic growth and
Spore germination, thus expanding our knowledge on the

biology of this organism.

LITERATURE CITED

Anderson, P. J. 1921. Development and pathogenesis
of the onion smut fungus. Mass. Agr. Exp. Stat.
Bull. 4: 99-133.

Bauch, R. 1925. Untersuchungen fiber die Entwichlungs
Geschichte und Sexualphysiologie der Ustila o
bromivora und Ustila o grandis. Zeitschrift ffir
Botanik. 17: 9- 7.

Blizzard, A. W. 1926. The nuclear phenomena and life

history of Urocystis cepulae. Bull. Torrey Bot.
Club. 53: 77-117.

Blumer, S. 1937. Untersuchungen fiber die Biologie von

Ustilago violaceae (Pers.) Fuckel. Arch. Mikrobiol.

Brefeld, O. 1883. Fortsetzung der Schimmelpilze.
Botanische Untersuchungen fiber Hefenpilze in
Untersuchungen aus dem Gesammtgebiet der Mykologie.
5: 1-220. Die Brand Pilze I. Verlag von Arthur
Felix, Leipzig.

Brefeld, O. 1895. Fortsetzung der Schimmel-und Hefen-
pilze. Hemibasidii. Brandpilze III in Untersuchung-
en aus dem Gesammtgebiete der Mykologie. 12: 99-
226. Commissions-Verlag von Heinrich SchOningh,
Mfinster.

Brefeld, O. 1908. Die Kultur der Pilze und die Anwendung
der Kulturmethoden ffir die Verschiedenen Formen der
Pilze nebst Beitragen zur vergleichen Morphologie
der Pilze und der natfiralichen Wertschatzung ihrer
zugehOrigen Fruchtformen in Untersuchungen aus dem
Gesammtgebiete der Mykologie. 14: 1-255. Kommis-
sions-Verlag von Heinrich SchOningh, Mfinster.

Cheo, P. C. 1949. Stripe smut of blue grass (Ustilago
striiformis forma poaepratensis): Spore germination,
artificial inoculation, pathological history and
growth in artificial media. Ph.D. Thesis. West
Virginia University. (Original not seen. Reported
in Lilly and Barnett(39) and Cochrane (ll).

47

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

48

Christensen, J. J. 1963. Corn smut caused by Ustilago

ma dis. Monograph No. 2.
Soc1ety. 51 p.

American PhytOpatho ogical

Chung, Chuen-Shang and E. J. Trione.
and inorganic nutrition of Tilletia controversa.
PhytOpathology. 57: 315-319.

1967. Organic

Cochrane, V. W. 1958. Physiology of Fungi. John

Wiley and Sons, Inc., New York.

524 p.

Colowick, S. P., and N. 0. Kaplan (Editors). 1957.
Methods in Enzymology, Academic Press, New York.

Vol. 1. pp. 140, 143.

Evans, R. I. 1933. Cytological studies on the para-
sitic relationship of Uroc stis ce ulae in the
onion. Amer. J. Bot. 20: 255-268.

Fischer, G. W., and C. G. Shaw.

1953.

A proposed

species concept in the smut fungi, with application
Phytopathology 43: 181-

to North American species.
188.

Fisher Bacteriological Culture Media. 1967. Fisher

Scientific Co., Philadelphia.

200 p.

Halbsguth, W. 1949. Uber die Bedingungen der Kultur
von Tilletia tritici haplonten auf 'definiertem'
Substrat und das Verhalten verschiedener Klone
gegenfiber einzelnen Faktoren in Hinblick auf
Wachstum. Konidienbildung and Konidien Keimung.

Planta 36: 551-634.

Hanna, W. F. 1929. Studies in the physiology and
cytology of Ustilago zea and Sorosporium reilianum.

PhytOpathology 9: 415-442.

Harris, G. 1962. The structural chemistry of barley
and malt. p. 431-582. In Cook, A. H. (Ed.).
Barley and Malt: 310109;? Biochemistry and Tech-
nology. Academic Press, New York.

740 p.

Haskins, R. H. 1950. Biochemistry of the Ustilanales.
I. Preliminary cultural studies of Ustilago zea.

 

Herzberg, P. P. 1895. Vergleichende Untersuchungen
fiber landwirtschaftlichwichtige Flugbrandarten.

Beitr. Physiol. Morph. 5:

1-36 0

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

49

Kniep, H. 1921. Urocystis anemones (Pers.) Winter.
Zeitschrift ffir Botanik 13: 289—311.

 

Kreitlow, K. W. 1943. Ustilago striaeformis I.
Germination of chlamydospores and culture of forma

a rostidis on artificial media. PhytOpathology
33: 707-712.

 

Lacy, M. L. 1968. Optimum conditions for germination
of Urocystis colchici teliospores. PhytOpathology
(in pressT.

Lange de la Camp, M. 1936. Gewinnung und Kultur der
Haplonten von Ustila o tritici. Phytopath.
Zeitschrift. 9: 455-477.

Lange de la Camp, M. 1939. Ernfihrungsversuche mit
Haplonten von Tilletia tritici. Kfihns Arch. 48.

Large, E. C. 1940. Advance of the Fungi, Cape, London.
488 p. '

Larson, B. M., and J. C. Walker. 1953. Thiram for
smut control in onion set plantings. J. Agr. Res.
43: 596-597.

Leach, J. G., C. V. Lowther, and M. A. Ryan. 1946.
Stripe smut (Ustilago striaeformis) in relation to

blue grass improvement. Phytopathology 36: 57-72.

Lilly, V. G., and H. L. Barnett. 1951. Physiology of
the fungi. 'McGraw-Hill, New York. 464 p.

Lilly, V. G., and H. L. Barnett. 1953. The utilization
of sugars by fungi. West Va. Univ. Agr. Exp. Sta.
Bull. 362 T: 5-58.

Ling, L. 1940. Factors affecting spore germination
and growth of Urocystis occulta in culture. Phyto-
pathology 30: - .

Maire, R. 1898. Note sur 1e developpement saprophy-
tique et sur la structure cytologique des sporidies-

levures chez Ustila o maydis. Bull. Soc. Mycol.
France. 14: 161-173.

Newth, F. H. 1951. The formation of furan compounds
from hexoses. Advances in carbohydrate Chemistry
6: 83-106.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

50

Nicholas, D. J. D. 1965. Utilization of inorganic
nitrogen compounds and amino acids by fungi. p.
349~376. £2.A' S. Sussman, and G. C. Ainsworth
(Ed.). The Fungi, Vol. I.

Potter, A. A. 1914. Head smut of sorghum and maize.

Prevost, B. 1807. Memoire sur la cause immediate de
la carie ou charbon des bies, et sur 1es preser-
vatifs de la carie, Paris. (Engl. transl. by G. W.
Keitt in PhytOpathological Classics No. 6, American
PhytOpathological Society, Menasha 1939).

Ranker, E. R. 1930. Synthetic nutrient solutions for
culturing Ustilago zea. J. Agr. Res. 41: 435—443.

 

Rothstein, A. 1965. Uptake and translocation. p.
429-456. £2.A° S. Sussman, and G. C. Ainsworth
(Ed.). The Fungi, Vol. I.

Sartoris, G. E. 1924. Studies in the life history and

physiology or certain smuts. Amer. J. Bot. 11:
617-647.

Schaffnit, E. 1926. Zur Physiologie von Ustilago
hordei Kell. u. Sw. Berichte der Deutschen
Botanischen Gesellschaft. 44: 151-156.

SchOpfer, W. H., and S. Blumer. 1938. Untersuchungen
fiber die Biologie von Ustilago violaceae (Pers.)
Fuckel. II Mitteilung. Arch. MikroBIOl. 9: 305-
367.

 

Selby, A. D. 1902. The prevention of onion smut.
Ohio Agr. Expt. Sta. Bull. 122.

Shirakawa, H. S. 1955. The nutrition of Diolocarpon
rosae, of roses pathogen. Amer. J. Bot. 42: 379-
384.

 

Tachibana, H. 1962. A method for maintaining cultures
and producing inoculum of Urocystis colchici.
Phytopathology 52: 754 (Abst.).

 

Thaxter, R. 1890. The smut of onions (Urocystis
cepulae Frost). Ann. Rep. Conn. Exp. Sta. 1889:
129-153.

 

 

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

51

Thirumalachar, M. J., and J. G. Dickson. 1949.
Chlamydospore germination, nuclear cycle, and

artificial culture of Uroc stis agropyri on red
tOp. PhytOpathology 39: 333-339.

Thren, R. 1937. Gewinnung und Kultur von monokary-
otischen und dikaryotischen Myzel. Ein Beitrag
zur Physiologie und Genetik des Gerstenflugbrandes
(Ustilago nuda [Jens.] Kellern, et Sw.).
Zeitschrift ffir Botanik 31: 337-391.

 

Tillet, M. 1755. Dissertation sur la cause qui
corrompt et noircit 1es grains de bled dans les
epis; et sur 1es moyens de prevenir ces accidens,
Bordeus, (Engl. transl. by H. B. Humphrey in Phyto-
pathological Classics No. 5, American Phytopath-
ological Society, Ithaca).

Togashi, K. 1949. Biological Characters of Plant
Pathogens. Temperature Relations. Tokyo:
Meibundo. 478 p.

Trione, E. J. 1964. Isolation and in vitro culture of
the wheat bunt fungi Tilletia caEIes and Tilletia
controversa: PhytopathoIogy 54: 592-596.

 

Volkonsky, M. 1934. Sur la nutrition de quelques
champignon saprophytes et parasites. Ann. Inst.
Pasteur 52: 76-101.

Walker, J. C. 1957. Plant Pathology. McGraw-Hill
Book Co., New York, 707 p.

Walker, J. C., and F. L. Wellman. 1926. Relation of
temperature to spore germination and growth of
Urogystis cepulae. J. Agr. Res. 32: 133-146.

 

Walker, J. C., and L. R. Jones. 1921. Relation of
soil temperature and other factors to onion smut
infection. J. Agr. Res. 22: 235-262.

Whitehead, T. 1921. On the life history and morphology
of Uroc stis cepulae. British Mycol. Soc. Trans.
7: -7 0

Wolf, F. T. 1953. The utilization of carbon and ni-

trogen compounds by Ustilago zeae. Mycologia 45:
516-522.

Zscheile, F. P. 1951. Nutrient studies with the wheat

bunt fungus Tilletia caries. PhytOpathology 41:
1115-1124.

 

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