EFFECTS OF NEAR -- ULTRAVIOLET
RADIATION AND CHOLESTEROL ON
GROWTH AND SPORULATION 0F
CYTOSPORA .CINCTA AND C. LEUCOSTOMA

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
RICHARD E. STUCKEY
1970

 

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ABSTRACT

EFFECTS OF NEAR-ULTRAVIOLET RADIATION AND
CHOLESTEROL ON GROWTH AND SPORULATION OF

CYTOSPORA CINCTA AND C. LEUCOSTOMA

BY

Richard E. Stuckey

Radiation significantly increased the number of

pycnidia formed and sporulation of isolates of Cytospora

 

cincta and g; leucostoma. Cultures were grown on potato

 

maltose agar (PMA) and potato maltose broth (PMB) adjusted
to pH 6 and held at 25 C in a growth chamber. Pycnidial
numbers in cultures irradiated continuously with near-
ultraviolet (UV) radiation at 2.0 to 7.0 x 102

ergs/cmzsec were increased over those irradiated with
cool—white flourescent lamps at an intensity of 1.0 to
1.3 X 104 ergs/cmzsec. Cultures not receiving radiation
failed to produce pycnidia. The numbers of pycnidia ex-
uding spores from near-UV radiated cultures was increased
from double to 130 fold depending on the isolate when
compared to cultures not receiving near-UV radiation.
Moreover, the percentage of sporulating pycnidia was in—

creased nearly 2—fold in cultures irradiated with near-UV.

Richard E. Stuckey

Near-UV radiation did not significantly decrease dry
weight of cultures grown in PMB suggesting that its af—
fect on sporulation may act in a stimulatory rather than
injurious manner.

Addition of 100 ppm cholesterol to PMB increased
dry weight but had little or no effect on sporulation.
Cultures of one isolate (Ma 4) produced conidia in 4 days
from conidiophores arising directly from the mycelium.
This type of conidial formation appeared to be favored by
addition of cholesterol.

Although large variations exist among isolates
they can generally be grouped in either of two species
based on cultural differences in optimum temperatures for
growth, colony morphology, pigmentation, and fruiting

structure formation and their distribution.

EFFECTS OF NEAR-ULTRAVIOLET RADIATION AND
CHOLESTEROL ON GROWTH AND SPORULATION OF

CYTOSPORA CINCTA AND C. LEUCOSTOMA

BY

Richard E. Stuckey

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

1970

Dedicated to

Judith Ann

ii

ACKNOWLEDGMENTS

I wish to express my sincere appreciation and
gratitude to Dr. A. L. Jones for his counsel and assist-
ance throughout the period of this investigation and
during the preparation of the manuscript.

Drs. W. G. Fields and J. M. Vargas, the other
members of my guidance committee, provided helpful sug-
gestions and assisted in the preparation of this manu-
script. Appreciation is also expressed to other faculty
members for advice freely given and use of laboratory
facilities. Departmental provision of growth chambers
for this study was much appreciated.

To my wife, Judy, for her understanding, encourage-
ment, and patience, I am deeply indebted.

The work was supported in part by Agriculture
Experiment Station project no. 921.

Use of the Michigan State University computing
facilities for data analysis was made possible through

support, in part, from the National Science Foundation.

iii

TABLE OF CONTENTS

Page
DEDICATION . . . . . . . . . . . . . . . . . . . . . ii
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . iii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . vii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
MATERIALS AND METHODS . . . . . . . . . . . . . . . 5

A. ISOlates I O I O O O O O O O O O O O O O O O
B. Preparation of Media . . . . . . . . . . . .
C. Conditions . . . . . . . . . . . . . . . . .
D

. Sporulation, Pycnidial Production, and
Colony Diameter . . . . . . . . . . . . . . .

E. Dry Weight Measurements . . . . . . . . . . .
F. Data Analysis . . . . . . . . . . . . . . . .

RESULTS 0 O O O O O O O O O O O O O O I O O O O O O 10

A. Pycnidial Production and Sporulation . . . . 10
B. Dry Weight and Colony Diameter . . . . . . . 19

DISCUSSION I O O O O O O O O O O O O O O O C O 0 O O 27

LITERATURE CITED . . . . . . . . . . . . . . . . . . 32

APPENDIX 0 O O O O O O O O O O O O O O O O O O O O O 35

Materials and Methods . . . . . . . . . . . . . . 35
(a) Temperature . . . . . . . . . . . . . . . 35
(b) Sterols . . . . . . . . . . . . . . .
(c) Photoperiodicity and thermoperiodicity . 36

(d) Continuous and alternating near-
ultraviolet radiation (UV) . . . . . . . 36

iv

Page

(e) Media . . . . . . . . . . . . . . . . . . 36
(f) Growth chamber studies . . . . . . . . . 37
Results and Discussion . . . . . . . . . . . . . 37
(a) Temperature . . . . . . . . . . . . . . . 37
(b) Sterols . . . . . . . . . . . . . . . . . 38
(c) Photoperiodicity and thermoperiodicity . 38

(d) Continuous and alternating near-UV
radiation . . . . . . . . . . . . . . . . 39

(e) Media . . . . . . . . . . . . . . . . . . 39
(f) Growth chamber studies . . . . . . . . . 40

LIST OF TABLES

Table Page

1.

Effect of near—UV radiation and cholesterol
on pycnidial production of three isolates

Of cytOSBora O O O O O O O O O O O O O O I O O O 12

 

Orthogonal contrast of treatment effects for
pycnidial production of three isolates of
Cytospora . . . . . . . . . . . . . . . . . . . 13

The relationship of sampling time of three
isolates to pycnidial production . . . . . . . . 14
Effect of near-UV radiation and cholesterol on
numbers of pycnidia exuding spores and per-

centage of pycnidia sporulating for three

isolates of Cytospora . . . . . . . . . . . . . 15

 

The relationship of sampling time of three
Cytospora isolates to number of pycnidia
exuding spores . . . . . . . . . . . . . . . . . 17

Effect of near-UV radiation and cholesterol on
mycelial dry weight production by four
isolates of Cytospora . . . . . . . . . . . . . 22

 

Dry weight comparisons of four CytOSpora
isolates using a single degree of freedom
orthogonal contrast test . . . . . . . . . . . . 23

 

Effect of near-UV radiation and cholesterol
on colony diameter of four isolates of
CthSLOI-a O I O O O O O O O O O C D I O O O O O 26

vi

LIST OF FIGURES

Figure Page

1.

Colony diameter rating scale from 1
(smallest) to 5 for estimating growth

of Cytospora . . . . . . . . . . . . . . . . . . 8

 

Cytospora cincta cultures grown for 35 days

under continuous near-ultraviolet radiation

(A), cool-white fluorescent radiation (B),

and darkness (C) . . . . . . . . . . . . . . . . ll

Exudation of orange spore masses from
submerged (A) and superficial (B) pycnidia
of isolate In 5 at 3 weeks . . . . . . . . . . . l8
Sporulation of Cytospora leucostoma isolate

Ma 4 at day 21: (A) orange and cream colored

Spore exudate from pycnidia; (B) spores

arising directly from the mycelia . . . . . . . 20

Microsc0pic view of conidia of Ma 4 at day 5.
Conidia are borne singly (A) or in short

chains (B) from conidiophores (C). 400x
magnification, lactol phenol cotton blue

stain . . . . . . . . . . . . . . . . . . . . . 21

Dry weights at days 3, 6, 9, 12, and 15 for

four Cytospora isolates grown on potato

maltose broth. Each value is the sum of 36

samples. The LSD (.01) for the mean of all

isolates at different days is 0.42 gram . . . . 24

 

vii

INTRODUCTION

Perennial canker has been of major economic im-
portance to Michigan fruit growers ever since Taft (23)
in 1898 called attention to a "gum disease" of peach

(Prunus persica (L.) Batsch.). He attributed the branch

 

swellings and gummosis associated with the disease to the
effects of freezing and thawing, and not to microorganisms.
Two years later, Steward et al. (22) discovered a fungus,

later identified as Cytospora leucostoma (Pers.) Sacc.,

 

intimately associated with dead and dying peach trees.
Later 9; cincta Sacc. was also found to produce cankers

on peach and other stone fruit crops (4). The Cytospora

 

fungi have been reported to cause cankers in the fruit

growing regions of the Eastern (22) and Western (17)

United States, except for the Pacific Northwest; Ontario,

Canada (7); Europe (21); South Africa (19); and Japan (24).
The severity of damage inflicted by perennial

canker depends upon the location of the infection. The

most critical locations are the trunk and scaffold branches.

A peach tree generally has 3 or 4 scaffold branches, and

complete girdling of one branch reduces the yield by

approximately 30%. In addition, this canker provides a

source of inoculum from which new infection can arise.

Pruning wounds and cold injury wound which occur on the
trunk of the tree are critical infection courts since
from this location the canker fungi can cause extensive
girdling. The trees are not usually killed outright, but
productivity and longevity are reduced as cankers enlarge
(13) .

The identification of the two Cytospora species

 

has traditionally been based on cultural characteristics
because the perfect stage is difficult to find in nature.
The following differences predominate for the two species.

9; cincta is a more virulent pathogen than 9; leucostoma

 

(13, 25). The pycnidia of g; leucostoma are black with

 

dark red cirri, while those of g; cincta are brown with

amber-pink cirri (4). Q; leucostoma is hair brown in

 

culture with small dark pycnidia exuding cirri when

mature. g; cincta, on the other hand, is whitish to olive
buff in culture and has large light-colored pycnidia con-
taining, though rarely exuding, spores (25). Helton and
Konicek (9) reported an average optimum temperature of 30 C

and 25 C for g; cincta and g; leucostoma respectively.

 

However, the optimum temperatures generally accepted are

21 C and 30 C for g; cincta and C; leucostoma respectively

 

as found in Hildebrand (13).
Recently, Kern (l4) split the traditional genus
Valsa on the basis that if the fruitification has a con—

ceptacle it belongs to the genus Leucostoma, if the

 

conceptacle is absent it is known as Valsa. Kern (14) in
his taxonomic studies of perennial cankers in Michigan

refers to the two species as Leucostoma persoonii (Nit.)

 

v. H., imperfect stage Cytospora leucostoma Sacc.; and

 

L; cincta (Fr.) v. H., imperfect stage C; cincta Sacc.

He states that although Cytospora species show morphologi-

 

cal differences, they can not be split up on the basis of
morphological characteristics due to the range of vari-
ability. Lukezic, DeVay and English (18) further expressed
this variability observing that monoasosporic isolates

from a single ascus of L; persoonii represented colony

 

types characteristic of both C; cincta and g; leucostoma.

 

The success of future laboratory and field studies,
directed toward the control of these pathogens, may be
dependent on the ability to produce inoculum. Techniques
using sterilized split peach twigs (20) or a pearl barley
honey-peptone mixture (5) are not practical when large
amounts of inoculum are desired. However, sporulation of

Cytospora isolates on common laboratory media is slow and

 

some isolates produce only a few pycnidia even after
several months. In other cases, pycnidia may be formed
in abundance but never produce spores. Calpouzos and

Stallknecht (1) working with Cercospora beticola and

 

Leach (16) using many diverse species reported increased
sporulation with near-ultraviolet radiation. Hendrix (12)

reported that for some fungi, but not others, growth and

reproduction were increased by cholesterol. The purpose
of this research was to determine if near-ultraviolet
irradiation and/or cholesterol would enhance sporulation
and growth of isolates cultured on ordinary laboratory

media.

MATERIALS AND METHODS

A. Isolates

 

Four Cytospora isolates were selected as repre-

 

sentative of the range of isolates found in Michigan
orchards. Two cultures designated as 0c 6 and Ja 17 were

isolated from apricot (Prunnus armeniaca L.) and peach

 

respectively. Their cultural characteristics and tem-
perature requirements (see appendix) corresponded to those
of g; cincta. The other cultures, Ma 4 and In 5, were
isolated from peach and resembled the characteristics of

E; leucostoma. Single spore subcultures of each isolate

 

were obtained by transferring spores from pycnidia to
sterile distilled water and plating a dilution series of
the spore suspension onto potato maltose agar. One to
two days later germinated single spores were transferred
to fresh potato maltose agar and maintained thereafter
through mycelial plug transfers. All isolates were

pathogenic to peach in greenhouse tests.

B. Preparation of Media

 

The procedure of Lacy and Bridgman (15) for pre-
paring potato dextrose agar from dehydrated potatoes was

followed except maltose was substituted for dextrose as

5

suggested by the studies of Helton and Konicek (10). The
media, potato maltose agar (PMA) was adjusted to pH 6
(Beckman Zeromatic II pH meter) and sterilized. Final pH
following sterilization was 5.8. Cholesterol was ground
using a mortar and pestle with 0.5 ml distilled water and
one drop of Triton B - 1956. It was added to the media
at the rate of 100 mg/liter before autoclaving. Twenty
to 25 ml of medium was poured into 9 cm plastic petri
plates to reduce drying out of the cultures. Media for
the growth studies were made using the same procedure
except omitting the agar. The resulting potato maltose
broth (PMB) was pipetted into each dish at the rate of

20 ml/petri plate. One plug (cork borer #3) from 5-7

day old cultures of mycelia was placed centrally and in-

verted in each PMA petri dish or upright in PMB dishes.

C. Conditions

4
Control cultures were grown under 1.0 to 1.3 X 10

ergs/cmzsec of continuous cool-white fluorescent lamps

(15 watt General Electric F15T8/CW lamps). Other treat—
ments (see appendix) were: (a) addition of 100 ppm cho-
lesterol to the media, (b) continuous irradiation with

2.0 to 7.0 X 102 ergs/cmzsec near-ultraviolet (UV) lamps
(15 watt General Electric F15T8/BLB lamps emitting from
320 nm to 460 nm, max. at 365 nm), and (c) a combination

of treatments (a) and (b). Radiation intensity

measurements were conducted with a YSI Kettering model 65
radiometer. Desiccation of cultures was reduced by plac-
ing the petri dishes at a single depth in plastic bags
and closing the bags with twistums.. All experiments were
carried out in two growth chambers maintained at 25 C i l.
Temperatures within the media and inside and outside the
plastic bags were checked periodically with a tele-
thermometer and found to be equivalent for both cool-white
fluorescent lamps and black lamps.
D. Sporulation, Pycnidial Production,
and Colony Diameter

At days 7, 14, 21, 28, and 35 observations were
made in two ways: all plates were (1) judged on a rating
scale from l-5 (see Figure l) for diameter increase; and
(2) viewed under a microsc0pe to determine the develop-
ment and number of pycnidia. Observations from a field
of vision (16 mm2 each) were selected at random in each

plate quadrant and recorded. All studies were replicated

nine times.

E. Dry Weight Measurements

Sampling dates were 3, 6, 9, 12, and 15 days
after inoculation. At every time period 9 plates (repli-
cations) were sacrificed for each treatment-~isolate
combination. A table of random numbers was used to deter-

mine plates chosen. Samples were taken by filtering and

 

Colony diameter rating scale from 1 (smallest)

Figure 1.
to 5 for estimating growth of Cytospora.

rinsing the contents of each petri dish on pre-weighed
filter papers using an Erlenmeyer filter flask, and
placing the resulting residue in drying ovens at 65 C for
48 hours. Filter papers were then removed from ovens,

allowed to cool and weighed.

F. Data Analysis

 

The experimental design was a split-split plot.
The treatments, isolates, and sampling periods constituted
the main unit, sub-unit, and sub-sub-unit respectively.
Least significant differences (LSD) were computed using
the appropriate mean square to compare means. Orthogonal
contrasts using single degree of freedom comparisons and

F-tests were used to compare combinations of treatments

and isolates.

RESULTS

The diameter of cultures grown in total darkness
for 35 days was comparable to cultures irradiated with
either near-UV radiation or fluorescent radiation (Figure
2 and appendix). However, there was an obvious reduction
in pigmentation and an absence of pycnidial formation for

all isolates when radiation was excluded.

A. Pycnidial Production and Sporulation

 

Analysis of the data for isolates 0c 6, Ja l7,
and In 5 indicate that cultures treated with near-UV
radiation plus cholesterol produced more pycnidia than
either of the non UV radiated treatments (Table l).
Pycnidial production of Ja 17 with the near-UV radiation
plus cholesterol treatment contributed heavily in the
significance of the treatment effects. Isolate In 5
produced more pycnidia than either 0c 6 or Ja 17. This
was due largely to the near-UV radiation and fluorescent
control treatments which were significant at the 1% and
5% level respectively. Further analysis of treatment
effects show that a comparison of near-UV radiated treat-
ments with those not receiving near-UV radiation indicate

a highly significant difference in numbers of pycnidia

lO

-ll

 

 

Figure 2. Cytospora cincta cultures grown for 35 days
under continuous near-ultraviolet radiation
(A), cool—white fluorescent radiation (B),
and darkness (C).

12

Table 1. Effect of near-UV radiation and cholesterol on
pycnidial production of three isolates of

 

 

 

 

 

Cytospora.a
Isolates Main
Treatment Effect
Oc6 Ja17 In5 Means
Controlb 1.13 0.00 5.47* 2.20
UV 2.33 1.00 8.42** 3.92
Cholesterolb 2.38 0.13 3.82 2.11
UV + Cholesterol 3.51 6.84** 5.44 5.27**
Main Effect Means 2.34 1.99 5.79**

 

aEach value represents the mean number of pycnidia
for 45 observations of 5 sampling periods.

bIrradiated with fluorescent light.
*LSD values significant at 5% and 1% levels for:
Treatment 1.99, 2.70; isolates 1.95, 2.60; between treat-

ments same isolate 3.44, 4.67; between isolates same
treatment 3.91, 5.19 respectively.

produced between the two sets of treatments (Table 2).
No differences were observed when cholesterol treatments
were paired against non cholesterol treatments. The re—
sponse of the near-UV radiated treatments did not appear
to depend on the presence of cholesterol. An additional
comparison of near-UV radiated treatments vs non UV

radiated treatments shows increased pycnidial production

13

Table 2. Orthogonal contrast of treatment effects for
pycnidial production of three isolates of
Cytospora.

 

 

Anova Table

 

 

Source of variation df Mean square F ratio
Treatments 3 308.30 4.91**
UV vs non UV 1 801.79 12.76**
Cholesterol vs non Cholesterol 1 53.52 0.85ns
Interaction UV and Cholesterol 1 69.70 l.llns
Error 24 62.82

 

**Significant at 1% level.

by 50 and 65% for In 5 and 0c 6 respectively and by 60
fold for Ja 17.

Numbers of pycnidia for the main effect means in-
creased throughout the time of the experiment. However,
when comparing each time period to the preceding one,
only days 14 and 35 were significant (Table 3). Increases
of pycnidial numbers on day 14 are particularly true for
Ja l7 and In 5 while on day 35, 0c 6 was markedly in-
creased. Pycnidial numbers observed were greater for
In 5 compared to 0c 6 on days 14 and 21, In 5 compared to
Ja 17 on day 28, and both In 5 and 0c 6 compared to Ja 17

on day 35.

14

Table 3. The relationship of sampling time of three
Cytospora isolates to pycnidial production.

 

 

Numbers of Pycnidia

 

 

 

Time _
in Isolates Main
Days Effect
Co 6 Ja 17 In 5 Means
7 0.14 0.00 2.86 1.00
14 0.81 3.08 5.25 3.05**
21 1.19 3.47 5.56 3.41
28 2.67 1.75 6.97 3.80
35 6.89 1.67 8.31 5.62**
Main Effect
Means 2.34 1.99 5.79

 

aEach value is the mean of 36 observations regard—
less of treatment.

**LSD values, significant at 5% and 1% levels for:
Time 1.21, 1.60; same time between isolate 4.37, 5.81;
same isolate between time 2.10, 2.77 respectively.

The effects of the near-UV radiated and cholesterol
treatments on the number of pycnidia sporulating paralleled
those for pycnidial production. The near-UV radiated and
near-UV radiated plus cholesterol treatments had greater
sporulation than the fluorescent irradiated control
(Table 4). This was particularly true for In 5 under the
near—UV radiation treatment and Ja 17 under the near-UV
radiation plus cholesterol treatment. Sporulation with

the cholesterol treatment had no detectable effect.

15

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Sporulation of cultures receiving near-UV radiation in-
creased by 220 and 270% for CC 6 and In 5 respectively and
by 130 fold for Ja 17 when compared to similar non UV
radiated cultures. The percentage of pycnidia sporulat-
ing was also doubled with near-UV radiated treatments
compared to those not treated with near-UV radiation
(Table 4).

Differences in the mean number of pycnidia sporu-
lating between isolates were not significant. However,
sporulation was greater with near-UV radiation alone for
In 5 compared to Ja 17 while the reverse existed for the
near-UV radiation plus cholesterol treatment (Table 4).

Although the mean number of pycnidia sporulating
increased at each time period, only day 35 was significant
when compared to each preceding time period (Table 5).
This result is attributed to CC 6 producing 90% of its
total sporulation at day 35. On day 35, 0c 6 and In 5
produced greater sporulation numbers than Ja 17.

Cultural fruiting comparisons of isolates revealed
pycnidia of In 5 were of uniform size, globose, black,
submerged or superficial, and frequently covered with
white mycelial growth. Conversely, 0c 6 and Ja 17 pycni-
dia were of two forms: large, globose, light-colored,
superficial structures or small, embedded or erumpent

structures within a dark stroma. Developing pycnidia

17

Table 5. The relationship of sampling time of three

Cytospora isolates to number of pycnidia exud-
ing spores.a

 

Number of Pycnidia Exuding Spores

 

Time

 

 

1n Isolates- Main
D Effect
ays 0c 6 Ja 17 In 5 Means
7 0.00 0.00 0.00 0.00
14 0.03 1.08 0.08 .40
21 0.08 1.14 0.39 .54
28 0.25 0.53 1.56' .78
35 3.44 0.58 2.78 .27**
”a1“ EffeCt 0.76 0.67 0.96

Means

 

aEach value is the mean of 36 observations regard-
less of treatments.

**LSD values significant at 5% and 1% levels for:

Time 0.73, 0.96; same time between isolates 1.85, 2.45;
same isolate between time 1.26, 1.66 respectively.

not exuding spores were frequently found to contain spores
when sliced or squashed.

The conidia were normally exuded from the pycnidia
in a colored fluid (Figure 3). Generally pycnidia of 0c 6
and Ja l7 exuded cream colored spore masses in contrast
to In 5 which produced an amber to orange exudate and
upon drying formed a cirrus. However, In 5 occasionally
produced cream colored exudate while 0c 6 and Ja 17 pro-

duced orange exudate, especially from stroma-formed

l8

 

 

Figure 3. Exudation of orange spore masses from submerged
(A) and superficial (B) pycnidia of isolate In
5 at 3 weeks.

l9

pycnidia. Another isolate, Ma 4, exudated both cream
colored and orange exudate (Figure 4).

Isolate Ma 4 not only produced pycnidia, but also
formed conidia arising directly from the mycelium (Figure
4). These conidia were produced singly, or in some cases
in chains, on short conidiophores (Figure 5). They ap—
peared to be slightly larger than conidia produced in the
usual manner. Spore production in Ma 4 was visible after
about 4 days and increased rapidly thereafter. Plates
with cholesterol appeared to have a higher number of
spores than the other treatments.

Neither near-UV radiation nor cholesterol treat-
ments appeared to affect spore germination. Spores of
each isolate including Ma 4, germinated on PMA when held

at room temperature for 24 hours.
B. Dry Weight and Colony Diameter

Dry weights of cultures treated with cholesterol
and near-UV radiation plus cholesterol were significant
at the 5% and 1% level respectively over treatments not
containing cholesterol (Table 6). These differences are

attributed to high dry weight increase of In 5 with cho—

lesterol and near-UV radiation plus cholesterol treatments

and dry weight increases of Oc 6 and Ma 4 with the cho-
lesterol treatment. Only the dry weight of 0c 6 was

decreased by near-UV radiation.

20

 

 

Figure 4. Sporulation of Cytospora leucostoma isolate
Ma 4 at day 21: (A) orange and cream colored
spore exudate from pycnidia; (B) spores aris-
ing directly from the mycelia.

 

 

Figure 5.

 

21

 

-.__-__._'— ----

Microscopic view of conidia of Ma 4 at day 5.
Conidia are borne singly (A) or in short chains
(B) from conidiophores (C). 400x magnifica-
tion, lactol phenol cotton blue stain.

22

Table 6. Effect of near-UV radiation and cholesterol on

mycelial dry weight production by four isolates
of Cytospora.a

 

TT 1
‘J; T—

 

 

Treatments
Isolate ' .
b b - Main
Control UV Cholesterol UV-Chol. Effect
Means
0c 6 95.08 76.72 114.60 99.57 96.49
Ja 17 55.44 53.99 66.78 56.14 58.09**
In 5 133.88 138.26 162.60 161.44 149.05**
Ma 4 82.96 85.06 101.07 85.84 88.73

Main Effect

** *
Means 91.84 88.51 111.26 100.75

 

aEach value represents an average dry weight (mg)
of 45 observations of combined time recordings at 3, 6,
9, 12, and 15 days.

bIrradiation with fluorescent light.
*LSD values significant at 5% and 1% levels for:
Treatment 8.81, 11.96; isolate 9.22, 12.20; same treat-

ment different isolate 18.44, 24.41; same isolate dif-
ferent treatment 17.61, 23.91 respectively.

Analysis of isolates showed In 5 had the highest
mean dry weight while Ja 17 had the lowest (Table 6).
These deviations from DC 6 and Ma 4 were significant at
the 1% level and were found to be consistent regardless
of treatment . The mean dry weight of In 5 was greater
than 2.5 times that of Ja 17. Comparison of dry weight

differences between presumed Q; cincta isolates Oc 6 and

23

Ja l7, and g; leucostoma isolates In 5 and Ma 4 were

 

highly significant (Table 7). Dry weight differences
between 0c 6 and Ja 17 and between In 5 and Ma 4 also

existed.

Table 7. Dry weight comparisons of four Cytospora isolates

using a single degree of freedom orthogonal con-
trast test.

 

 

II

Anova Table

 

Source of Variation df Mean Square F ratio

 

Isolates 3 0.25720 133.16**

0c 6 and Ja 17 vs.

In 5 and Ma 4 1 0.31146 l6l.25**

0c 6 vs. Ja 17 1 0.13273 68.72**

In 5 vs. Ma 4 1 0.32742 l69.51**
Error 96 0.00193

 

**Significant at 1% level.

A highly significant dry weight mean difference
at day 6 compared to day 3, existed as all isolates
showed significant increases (Figure 6). Also, the mean
increase of dry weight was increased at day 9 due to high
increases of 0c 6, In 5 and Ma 4. The mean at day 12 and
day 15 were not significant, part of which was due to a
decrease of dry weight at day 12 for CC 6 and In 5. The
dry weight of In 5 was superior to other isolates at all

times. 0c 6 was slightly less than Ma 4 and Ja 17 at day

24

Figure 6. Dry weights at days 3, 6, 9, 12, and 15 for four
Cytospora isolates grown on potato maltose
broth. Each value is the sum of 36 samples.

The LSD (.01) for the mean of all isolates at
different days is 0.42 gram.

26

3 but was much greater at day 6 and day 9. Ma 4 and Ja l7
separated sharply after day 6. Between day 9 and day 12
the dry weight of Ma 4 surpassed 0c 6.

Colony diameters of the near-UV radiation plus
cholesterol treatment were significantly less than other
treatments due to reduced colony diameter of 0c 6 and Ja
17 (Figure 1 and Table 8). The colony diameter of Ja 17
was also less with the cholesterol treatment. Isolates
In 5 and Ma 4 were different from Co 6 and Ja 17 and also
different from each other. This phenomenon existed for
all treatments. Colony diameters of In 5 and Ma 4 in-
creased rapidly covering the entire plate while those of

0c 6 and Ja 17 were slow and had an average rating of 4

at the end of the experiment (Figure 1).

Table 8. Effect of near-UV radiation and cholesterol on
colony diameter of four isolates of Cytospora.

 

 

 

Treatments
Isolate b b UV- Main
Control UV Cholesterol Choles- Effect
terol Means
0c 6 3.13 3.18 3.18 2.84 3.08
Ja 17 3.33 3.16 2.91 2.93 3.08
In 5 4.80 4.69 4.80 4.71 4.75**
Ma 4 4.40 4.24 4.33 4.22 4.30**
Mal“ EffeCt 3.92 3.82 3.81 3.68**
Means

 

aEach value represents the mean of 45 observations
on a rating scale of 1—5.

bIrradiated with fluorescent light.
**LSD values significant at the 1% level for:

Treatment .112,
isolate .277,

isolate

.139,

same treatment different

same isolate different treatment .224.

DISCUSSION

The mechanisms involved in pycnidial formation
were not examined but rather an attempt was made to
determine if near—UV radiation or cholesterol affected
pycnidial production and sporulation. The result that
near-UV irradiation increased the number of pycnidia
formed and their subsequent sporulation did not signifi-
cantly decrease growth as measured by mycelial dry weight
deserves attention. Perhaps the often proposed mechanism
discussed by Cochrane (2) of injured or death of cells
followed by a releasing of substances into the medium
which acts on surviving cells, diverting them into new
develOpmental pathways, namely that of favoring fruiting

structure development, does not hold true for Cytospora.

 

Favored rather is a contrary hypothesis (2) that sporula-

tion of Cytospora requires specific positive stimuli.

 

Near-UV radiation may initiate or stimulate the synthesis
of factors for reproductive activity. It appears that
this stimulus influences the number of pycnidia formed
and the proportion which produce spores. The idea that
near-UV radiation results in earlier sporulation is dis-
missed by the failure of treatments to affect differences

in time. Failure of cholesterol to affect the

27

28

reproductive process via pycnidial formation suggests
cholesterol acts independent of reproduction. The rela-
tionship between cholesterol visually producing more
"free" conidia with Ma 4 and the results of other treat-
ments on pycnidial sporulation are not fully understood.
Perhaps increased growth of Ma 4 with cholesterol (Table
6) naturally led to increased production of conidia.

Growth studies suggest that cholesterol increased
metabolic activity either by being incorporated or stimu—
lating additional growth as measured by mycelial dry
weight. Although cholesterol increased growth it did not
adversely affect pycnidial formation. Also the consistent
dry weight increase of 20-22% among isolates treated with
cholesterol indicate that the effect of cholesterol is
independent of the isolate. The absence of significant
time differences after day 9 (Figure 6) suggest that
recommended data recording be at closer intervals up to
10 days.

Colony diameter was less with the near-UV radia-
tion plus cholesterol treatment, demonstrating that growth
as measured by diameter and dry weight are not equivalent.
Dry weight data are considered better measures of growth
(2). Therefore, differences in colony diameter were only
recognized at the 1% level. Failure of near-UV radiation
to significantly inhibit dry weight may have been due to

improper radiation dosage or intensity. This again

29

suggests that for Cytospora there is, and possibly for

 

other fungi there may be, a critical dosage of near-UV
radiation that is not inhibitory to growth yet stimulatory
for reproduction.

Significant differences among replications occurred
in several growth cases. This does not invalidate the
significance of other tests but suggests there was an
unknown factor involved. Perhaps the method of inoculat-
ing plates could have been improved to achieve more uni-
formity. Frequently mycelial plugs were slow or failed
to initiate rapid growth resulting in large variations.
Ellingboe (6) found germinated single spore inoculations

quite suitable for growth studies of Phoma herbarum, a

 

technique that may in the future alleviate significance
among replications. Single spore inoculations were not
feasible at the start of this study due to scarcity of

inoculum.

Large differences between presumed species exist;
however, isolates within species also differed supporting
the literature (3, 8, ll, 14) and making interpretation
of information difficult. Excessive variability among
isolates lend some reservations as to whether these re-

sults are expressive of all Cytospora canker isolates.

 

The four isolates can, however, be separated into two
loosely defined species based on several trends where

greater variations between groups than among isolates

30

existed. Temperature studies (appendix) found Optimum
growing temperatures at 21 C for DC 6 and Ja 17 and 27-30 C
for In 5 and Ma 4. Pigmentation of 0c 6 and Ja l7 ranged
anywhere from white, yellow, or brown while In 5 and Ma 4
were black to olive green. In 5 and Ma 4 pycnidia were
evenly distributed and sporulated uniformly on plates.
Variations existed in density and distribution of repro-
ductive structures among and within plates of 0c 6 and

Ja 17. 0c 6 and Ja 17 had a higher sporulating percentage
of pycnidia formed and produced many of their pycnidia
within a stroma in a circular ring at varying distances
from mycelial plugs. The number of "ring" observations
greatly influenced the number of fruiting structures re-
corded. Only Ma 4 produced conidia and conidiophores
arising directly from the mycelium, confirming results of
Hildebrand (13) who noted this same striking characteris-

tic for some of his 9; leucostoma isolates. Colony dia-

 

meters Of In 5 and Ma 4 were much superior to CC 6 and
Ja 17. Growth as measured by mycelial dry weight was
least with Ja l7 and greatest with In 5, however, a sharp
distinction between species does not exist as dry weight
of Co 6 and Ma 4 were not different.

This study indicates that for the four isolates
herein tested: (a) radiation is necessary for fruiting
structure formation, (b) it is the near-UV radiation

which is responsible for fruiting structure formation,

31

confirming studies by Leach (16), (c) near-UV radiation
increases numbers of pycnidia, sporulation, and percent-
age of pycnidia sporulating but has no affect on the time
of occurrence, (d) near-UV radiation does not significantly
decrease dry weight, (e) cholesterol increases dry weight
but not pycnidial formation and sporulation, and (f)

large variations exist among isolates yet they can gener-
ally be grouped in either of two species.

For sporulation of stone fruit Cytospora isolates
cultures should be grown on PMA, pH 5-6, at 25 C under
continuous long wave UV irradiation. Isolates sporulat-
ing directly from the mycelium may be increased by

addition of cholesterol to the media.

LITERATURE CITED

10.

LITERATURE CITED

Calpouzos, L. and G. F. Stallknecht. 1967. Effects
of light on sporulation of Cercospora beticola.
PhytOpathology 57:679-681.

 

Cochrane, V. W. 1958. Physiology of fungi. John
Wiley and Sons, Inc., New York. 485p.

Daines, R. H., and Teresa Mrozowska. 1966. Cytospora
canker (gummosis) of peach. Hort. News, New
Brunsw. 47(2):6-12.

 

Defago, G. 1935. (On certain Valseae von Hohnel
parasitic on dying-off stone fruit trees.) Thesis,
Ecole Polytechnique Federale Zurich, 111p.

(Abst. Rev. Appl. Mycol. 15:447. 1936).

Dhanvantari, B. N. 1968. A culture medium for
pycnidial formation and conidial production of
Cytospora cincta. Phytopathology 58:1040.

 

Ellingoe, A. H. 1959. Studies on the growth of
Phoma herbarium var. medicaginis in culture.
Phytopathology 49:773-776.

 

 

Gussow, H. T. 1912. A new disease of peaches.
Canad. Exp. Farm. Rept. (1910-1911): 251.

Helton, A. W. and D. E. Konicek. 1961. Effects of
selected Cytospora isolates from stone fruits on
certain stone fruit varieties. Phytopathology
51:152-157.

 

Helton, A. W. and D. E. Konicek. 1962. An optimum
environment for the culturing of Cytospora
isolates from stone fruits. I. Temperature.
Mycopathol. Mycol. Appl. 16:19-26.

 

Helton, A. W. and D. E. Konicek. 1962. An Optimum
environment for the culturing of Cytospora
isolates from stone fruits. II. Carbon sources.
Mycopathol. Mycol. Appl. 16:27-34.

 

32

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

33

Helton, A. W. and D. E. Konicek. 1962. An Optimum
environment for the culturing of Cytospora
isolates from stone fruits. III. Nitrogen
sources. Mycopathol. Mycol. Appl. 16:125-132.

 

Hendrix, J. W. 1965. Influence of sterols on growth
and reproduction of Pythium and Phytophthora spp.
Phytopathology 55:790-797.

 

Hildebrand, E. M. 1947. Perennial peach canker and
the canker complex in New York, with methods of
control. Cornell Agr. Exp. Sta. Mem. 276.
61p.

Kern, H. 1955. Taxonomic studies in the genus
Leucostoma. Pap. Mich. Acad. Sci. 40:9-22.

 

Lacy, M. L. and G. H. Bridgman. 1962. Potato-
dextrose agar prepared from dehydrated mashed
potatoes. Phytopathology 52:173.

Leach, C. M. 1962. Sporulation of diverse species
of fungi under near-ultraviolet radiation. Can.
J. Botany 40:150-161.

Leonian, L. H. 1921. Studies on the Valsa apple
canker in New Mexico. Phytopathology 11:236-243.

Lukezic, F. L., J. E. DeVay, and H. English. 1965.
Comparative physiology and pathogenicity of
Leucostoma persoonii and Rhodosticta quercina.
Phytopathology 55:511-518.

 

 

Reinecke, O. S. 1931. Dieback of fruit trees in
the Western Cape Province. S. African Dept. Agr.
Bull. 97. 16p. (Rev. Appl. Mycol. 19:677.
1931).

Rohrback, K. G. and N. S. Leupschen. 1968. Environ-
mental and nutritional factors affecting pycnidio-

spore germination of Cytospora leucostoma.
Phytopathology 58:1134-1I38.

 

Rolfs, F. M. 1910. Winter killing of twigs, can—
kers, and sun scald of peach trees. Missouri
State Fruit Exp. Sta. Bull. 17. 101p.

Stewart, F. C., F. M. Rolfs, and F. H. Hall. 1900.
A fruit-disease survey of Western New York in
1900. N. Y. Agr. Expt. Sta. Bull. 191:291-331.

34

23. Taft, L. R. 1898. Spraying calendar for 1898.
Gum-disease of the peach. Michigan Agr. Exp.
Sta. Bull. 155:304.

24. Togaski, K. 1931. Studies on the pathology of
peach canker. Imp. Coll. Agr. Forest. (Morioka,
Japan). Bull. 16. 178p.

25. Willison, R. S. 1936. Peach canker investigations.
II. Infection studies. Can. J. Res., C-D, 14:
27-44.

APPENDIX

APPENDIX

Materials and Methods

 

Preliminary studies:

 

(a) Temperature: Cultures of isolates 0c 6, Ja 17,

 

Ma 4, and In 5 were grown on potato maltose agar (PMA) in
oven incubators with temperatures adjusted to 15, 18, 21,
24, 27, and 30 C. Colony diameters of the isolates were
compared every 4 days for 3 weeks. The relative growth
of the isolates was recorded.

(b) Sterols: Cultures subjected to sterols in
several cases showed favorable growth and sporulation.
Differing concentrations of two sterols, ergosterol and
cholesterol, were tested using isolates Ja 17 and In 5.
Concentrations tested were: cholesterol 100 ppm, ergos-
terol 100 ppm, and a 50:50 combination of cholesterol
and ergosterol totaling 0, 50, 100, and 1000 ppm, of
sterol per treatment, respectively. In a second test,
cholesterol at 0, 50, 100, 250, and 500 ppm was tested
using all four isolates. Three replications were used.
Every 3 days for 3 weeks the colony diameters for each
treatment were compared. Concentrations producing the
largest colony diameters were selected for use in later

studies.
35

 

 

36

(c) Photoperiodicity and thermoperiodicity:
Many fungi require alternating periods of light and dark
or of high and low temperatures for Optimum growth and
sporulation (2). All four isolates were treated with
five combinations of light and temperature as follows:
(1) Light 12 hours 26 C, dark 12 hours 26 C; (2) light 12
hours 26 C, dark 12 hours, 12 C; (3) dark 10 days 26 C,
light 10 days 12 C, light 10 days 26 C; (4) light 24
hours 26 C; and (5) dark 24 hours 26 C. Three replicates
were used. Observations of the number of pycnidia and
the number of pycnidia exuding spores were taken periodi-
cally at 7 day intervals to day 35.

(d) Continuous and alternating near-ultraviolet

(UV) radiation: Isolates of 0c 6, Ja 17, In 5 and Ma 4

 

were placed in incubators at 26 C with continuous near-
UV radiation or alternating 12 hour periods of near-UV
radiation and darkness. Colony diameter, number of
pycnidia and number of pycnidia exuding spores were ob-
served at 4 weeks.

(e) Media: Cultures were grown on chemically
defined media (18) with the following ingredients:
MgSO4-7H20 3g, NH4C1 3g, KH2PO4 3g, Dextrose anhydrous

209, FeCl ~6H o 0.24 mg, ZnCl 0.15 mg, H BO

2 3 3
°2H20 0.04 mg, NaMOO4°H20 0.03 mg,

3 2 0.06 mg,

CuCl °H20 0.05 mg, MnCl

2 2
Thiamin 100 ug, Biotin 0.05 ug, Choline 100 mg, and

enough distilled H20 to make one liter of media. Failure

37

of cultures to produce fruiting structures or any sub-
stantial growth led to a reversion to the PMA media.

PMA at 2X, X, 1/2X, and 1/4X concentration was tested to
determine if nutrient levels had an effect on growth and
sporulation. Colony diameter and numbers Of pycnidia and
pycnidia sporulating were the criteria used to judge the
suitability of the various substrate. Observations were
made periodically at 4 day intervals for 4 weeks. In a
third experiment concentrations of 0.0, 0.01, 0.1, 1.0 %
NaCl were added to PMA plates. Numbers of pycnidia and
pycnidia exuding spores were compared for each treatment
after 4 weeks.

(f) Growth chamber studies: In some of the pre-

 

vious studies the fluorescent or UV lamps had been in-
stalled in cupboards where temperature could not be
controlled nor outside light sources excluded. This
resulted in considerable variation in the growth and
sporulation Observed within treatments. Two growth
chambers were secured and additional experiments varying
the relative humidity and radiation intensity were done
to select Optimum relative humidity and radiation inten-

sities.

Results and Discussion

 

(a) Temperature: Isolates 0c 6 and Ja l7 grew

 

best at 21 C, isolates Ma 4 and In 5 at 27 to 30 c, At 24 c

38

both species were capable of good growth, thus 25 i 1 C
was selected for growing cultures of both species.

(b) Sterols: Colony diameter, pycnidial number
and sporulation of In 5 with 100 ppm cholesterol were
visually superior to ergosterol at 100 ppm and to the
combinations of cholesterol and ergosterol. Sporulating
Of Ja 17 was unaffected by concentrations of sterols;
however, colony diameters were increased with 100 ppm
cholesterol. Since Ja l7 and In 5 selectively preferred
cholesterol to ergosterol, differing concentrations of
cholesterol were tested using all four isolates. Varia-
tions among isolates for most favorable response to in-
creased colony diameters and sporulation ranged from 50
ppm to 250 ppm with 100 ppm rated as the best overall.

(c) Photoperiodicity and thermoperiodicity:

 

Although there were slight variations among isolates,
continuous radiation at 26 C was considered best overall
for future studies. No sporulating pycnidia occurred on
any cultures subjected to continuous darkness at 26 C;

however, a few pycnidia were observed on g; leucostoma

 

isolates. Their appearance is believed to be due to
brief weekly exposures to the radiation as subsequent 35
day dark treatments revealed no fruiting structures.
Treatment 3 with 10 day photoperiods was less favorable

than daily photoperiods (treatments 1, 2, 4). Continuous

39

radiation at 26 C was the only treatment resulting in
sporulation on all cultures except Dc 6.

(d) Continuous and alternating near-UV radiation:

 

Colony diameters of Ja l7 and 0c 6 were greater with al-
ternating near-UV radiation than with continuous near-UV
radiation. However, sporulation of Ja l7 and 0c 6 was
much greater under continuous near-UV radiation than under
alternating near-UV radiation. Ma 4 and In 5 sporulated
and grew well in both conditions.

(e) Media; Cultures grown on chemically defined
media had greatly inhibited colony diameters and failed
to produce fruiting structures even under the influence
of sterols or near-UV radiation. Colony diameters and
pycnidial numbers were greatest with PMA concentrations
of 2X and X for all isolates. In contrast to some fungi,
lower concentrations of nutrients did not increase repro-

duction (2). Apparently stone fruit Cytospora isolates

 

are not stimulated to sporulate by starvation. Addition
of varying concentrations of 0.01 - 1.0 % NaCl on PMA did

not increase pycnidial formation as suggested by Hamptom.1

 

1M. C. Hampson, 1969. Valsa canker investigations.
Part I. Pathogenesis by CytOSpora leucostoma, and mechanism
of wilt induction in peach. II. Effects of water stress
on disease development and fungal behavior, and a role
for wound gum. Ph.D. Thesis, Cornell University, 89 p.

 

40

(f) Growth chamber studies: Petri dishes had a

 

tendency to dry out at relative humidities of 30 to 50%;
therefore, dishes were placed at single depths in plastic
bags (6 per bag) and tied with twistums to reduce moisture
loss. Cool—white fluorescent lamps producing energy at a

4

rate from 1.00 to 1.32 X 10 erg/cmzsec were considered

best for growth and sporulation of Cytospora isolates.

 

The near-UV radiation used ranged from 2.0 to 7.0 X 102

ergs/cmzsec.