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THESIS

LIBRARY

Michlgan State
Universxty

 

 

ROOM USE ONLY

, «.12. . ’

 

ABSTRACT

FARASITIC POTENTIAL CF VERTICILLIUM ALBO-ATRUM
FROM CULTIVATED AND UNCULTIVATED AREAS

 

by Joseph A. Ignatoski

The origin of Verticillium albo-atrum on strawberry

 

was investigated by conducting a survey of the host range
and parasitic potentials of l3 isolates from cultivated
crops and 7 from uncultivated areas on 20 different
plants normally growing in these two areas.

All isolates were parasitic on each test plant with
the exception of the peppermint isolate on violet. Isolates
from the uncultivated areas were parasitic on cultivated
strawberry and thus are potential pathogens.

Three isolates from soil in uncultivated areas and
u isolates from crop plants in cultivated areas were similar
in parasitic potential and host range, suggesting that the
M isolates from the cultivated areas could have originated
in an uncultivated area.

Some plants from both uncultivated and from cul—
tivated areas were as susceptible to invasion by

Verticillium isolates as susceptible cultivated plants

 

and thus could serve as multiplication sites for the

fungus.

PARASITIC POTENTIAL OF VERTICILLIUM ALBO-ATRUM

 

FROM CULTIVATED AND UNCULTIVATED AREAS

By

Joseph A. Ignatoski

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

1965

ACKNOWLEDGMENTS

The author would like to express his deepest
appreciation and thanks to Dr. E. H. Barnes for his
advice and guidance throughout the duration of this
work, and for his suggestions during the writing of
this theses.

He is also grateful to Dr. J. E. Cantlon, Dr. J.
L. Lockwood and Dr. M. L. Lacy for their careful

reading and criticisms of this thesis.

ii

TABLE OF CONTENTS

Page
INTRODUCTION 0 O O O O 0 O O O 0 0 0 O O O O O O O 1
MATERIALS AND METHODS . . . . . . . . . . . . . .

Fungus isolates .

oooouu: 43

Plants 0 O O 0 O O O 0 0 O O 0 O O O 0 O
Inoculation . . . . . . . . . . . . . .
Isolation . . . . . . . . .

RESULTS 0 O O I O O O O O O O O O O O O O O O O 0 13
DISCUSSION . . . . . . . . . . . . . . . . . . . . 26
SUMMARY . . . . . . . . . . . . . . . . . . . . . 28

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . 29

iii

Table

LIST OF TABLES

Page

Isolates of Verticillium from
Michigan and their source . . . . . . . . . . 5

Plants used to determine host range
and parasitic potentials . . . . . . . . . . 7

Average distance (cm) 20 isolates of
:1. albo-atrum moved upward from level
of inoculation in 20 test plants.
(Average for A replications, ground
line 6 cm). Coding of plants
corresponds to Table l: Coding of
isolates corresponds to Table 2. The
least significant differences between
means was calculated according to the
Tukey method for multiple Comparisons:

d.OS = 1.84; d.Ol = 2.03 . . . . . . . . . . 12

 

iv

LIST OF FIGURES
Page
Figure
l. Dissection of plants for isolation . . . . . 9

2. Frequency distribution of correlation
for all 400 possible pairs of 20
isolates when inoculated into 20 hosts
and plotted in intervals of 0.1 for
correlation coefficients . . . . . . . . . . l5

3. Frequency distribution of parasitic
potentials for 20 isolates as shown
by the number of isolates plotted
against the average distance they
moved (cm) from the level of
inoculation in 20 test plants.
Isolates from cultivated crops-—

[ Earlidawn (Ear.) Surecrop (Sur.)

and Midway (mid.) moving 6.0 to 6.8

cm] and from uncultivated areas———

[Two soil isolates moving 5.6 to

5.9 cm and one moving 6.5 cm] are

significantly similar at the .05

level of probability for their

correlation coefficients . . . . . . . . . . 15

4. Frequency distribution of parasitic
potentials for 7 isolates from virgin
areas as shown by the average number of
these isolates per plant of each
respective group plotted against the
average distance they moved (cm) from
the level of inoculation . . . . . . . . . . 18

5. Frequency distribution of parasitic
potentials for 13 isolates from
cultivated crops as shown by the average
number of these isolates per plant of each
respective group plotted against the
average distance they moved (cm) from
the level of inoculation . . . . . . . . . 20

6. Frequency distribution of 400 host—parasite
(isolate) combinations shown by the
frequency of these combinations plotted
against the average distance moved (cm)
by the fungus from the level of
inoculation . . . . . . . . . . . . . . . . 21

Figure

7.

Page

Frequency distribution of test plants
as shown by the number of test plants
in each respective group plotted against
the average distance moved (cm) by 20
isolates in A replications. . .

a and b. Frequency distribution of
the 20 isolates on Dock, Potentilla,_§.
virginiana etc., as shown by the number

 

of isolates plotted against the average
distance they moved (cm) from the level
of inoculation . . . . . . . . . . . . . . . 23-24

Frequency distribution of the 20 isolates

on these groups of plants, as shown by

the average number of isolates_per plant

species of each respective group plotted

against the average distance they moved

(cm) from the level of inoculation . . . . . 25

vi

INTRODUCTION

The form genus Verticillium was established by Ness

 

VenEsenbeck (19) in the year 1816. In 1879 a pathogenic

form was observed on potato (Solanum tuberosum L.) by

 

Reinke and Berthold (21) and given the species epithet

of albo-atrum. Since this initial discovery of path-

 

ogenicity, numerous hosts for this organism have been
reported (6).

A review of the literature from 1879-1928 was
published by Rudolph in 1931 (22) including a host range
which consisted of trees, shrubs and weeds as well as
cultivated plants. This host range was determined by
observing the death of the plant as a result of infection
and hence was a measurement of pathogenicity. From
experiments cited in this review and others 3, 29;

33) it can be concluded that Verticillium attacks

 

and kills plants in many Speices and families and that
no one isolate is specific to one family or species to
the exclusion of all others.

Others, however, have reported specificity (9).

Verticillium isolates which were pathogenic on peppermint

 

appeared to be pathogenic only on peppermint, (i.e.
killing it), but parasitic on a number of hosts (i.e.

present in the hosts tissue without the plant showing

The purpose of this study was to compare the host
range and parasitic potential (average distance moved by
each isolate above level of inoculation in each test plant)

of Verticillium found in cultivated areas with those of

 

isolates from uncultivated sites with the hope that the
comparison would provide clues that would be useful in
reconstructing the origin of this parasite on cultivated

strawberry.

MATERIALS AND METHODS

Fungus Isolates: Twenty isolates of Verticillium

 

 

were obtained for comparison, 13 from cultivated fields
and 7 from uncultivated more natural plant arrays.

Thirteen isolates of X. albo-atrum were obtained with

 

sterile techniques from cultivated plants exhibiting

symptoms of Verticillium wilt (Table 1). Aerial portions

 

were surface sterilized with 50% Clorox (2.6% sodium
hypochlorite) for 1 — 3 min, cut into 3 cm pieces with
a sterile scalpel and plated on potato dextrose agar.

In addition seven isolates of y, albo—atrum, were

 

obtained from borders of seven out of ten woodlots, in
which wild strawberries were found. This was accom—
plished in two ways: (1) by isolation from aerial portions
of plants as described above; and (2) by isolation from
the soil in each woodlot by a modification of an
a1cohol-agar-streptomycin technique (16). A solution
was prepared containing 95 ml sterile distilled water,

1 g of agar, 5 ml of absolute ethyl alcohol and 1000 ppm
streptomycin or 0.25 ml of 10% lactic acid. To this
solution 1 g of soil was added, swirled, and poured into
5 petri dishes. After 5 - 7 days the plates were

examined for the presence of Verticillium.

 

Cultures were maintained by single spore transfer

on potato-dextrose agar. Inoculum consisting of a

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suspension of Spores and mycelial fragments was prepared
by homogenization of 10 day old cultures (which were
prepared by mass spore transfer) with 100 ml sterile
distilled water in a Waring Blendor.

Plants: The following three categories of plants
were selected to classify the 20 isolates as to host
range and parasitic potential. Those from the cultivated
areas were Tomato var. Bonny Best, representing a
susceptible solanaceous crop, cultivated strawberries,
var. Earlidawn (very susceptible) and Robinson (tolerant),
and five species of plants commonly found as weeds in
cultivated strawberry fields.(Table 2).

Plants from the uncultivated areas were 11 species
commonly found in proximity to wild strawberries (E.

virginiana L.) including wild strawberries and the Del

 

Norte clone of F. chiloensis from California (Table 2).

 

Plants were maintained in the greenhouse. Straw-
berry plants from both uncultivated and cultivated sources
were propagated with supplementary illumination for runner
production. Isolations were attempted from runners and
petioles of the mother plants to confirm that the

daughter plants were free of Verticillium. These Ver—

 

ticillium-free daughter plants were then used for

 

further propagation.

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All other plants were propagated from seed collected
in the field. Seeds*which did not germinate promptly were
placed between layers of moist, sterile vermiculite in
wax milk cartons and stored at 3°C for three months. The
cartons were then placed at room temperature and the seeds
allowed to germinate. The seedlings were tranSplanted into
sterile 4 inch pots containing sterile soil mixed with
unsterile peat and sand (2:1:1).

Inoculation: Once the plants had reached the desired

 

stage of growth (Table 2), four plants of each species or
clone were inoculated with each isolate and ten plants

were used as uninoculated controls. Plants were inoculated
by washing the roots free of soil and dipping them into

a suspension of spores and mycelial fragments up to a

point 7 cm below ground line. The plants were then

placed in sterile pots and then the soil added to avoid
contaminating the roots above the level of inoculation.

Isolation: After 28 days the plants were uprooted

 

and soil removed with running water. The root systems
were completely immersed in 10% Clorox for 3 minutes to
eliminate shallow, superficial infections and to destroy
any remaining inoculum. Aerial portions were immersed in
50% Clorox for l — 3 minutes, depending on their thick-
ness, and then cut into successive 3 cm sections. The
first sections consisted of 2 cm of the inoculated

area and 1 cm above (Fig. 1). Sections were placed into

 

 

 

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10

a petri dish of 1% water agar containing 250 ppm terramycin.

After lO-lA days.the presence of Verticillium was determined.

 

Similar procedures were followed for each of the 10 control

plants.

II

Table 3.--Average distance (cm) 20 isolates of K.

albo-atrum moved upward from level of inoculation in 20

 

test plants. (Average for 4 replications. Ground line is

at 6 cm). Coding of plants corresponds to Table l; Coding

l

of isolates corresponds to Table 2. The least significant
differences between means was calculated according to the

Tukey method for multiple comparisons: d 05 = 1.843
d = 2.03.
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RESULTS

V. albo—atrum was isolated from 7 of the 10 uncul—

 

tivated areas; 5 isolates were directly from soil and one
each from symptomless wild rose and symptomless wild straw—
berry (Table l). Thirteen isolates were obtained from
cultivated crops (Table 1). To determine whether or not
there were differences between these isolates, 20 test plants
(Table 2) were inoculated with each of the 20 isolates
(Table l) and the distance the isolates grew within the
plant determined (Table 3). The distance an isolate grows
in a particular host was considered a measurement of the
parasitic potential of the parasite. All 20 isolates were
parasitic to some degree on every test plant with the
exception of the peppermint isolate on violet. Thus no
host specificity was observed. Only purslane, when ino—
culated with isolate No. 12 from cultivated strawberry or
No. 18 from peppermint, died before the end of 28 days.

No Verticillium was recovered from any control plant. All

 

7 isolates from the uncultivated areas were parasitic on
cultivated strawberry since each isolate grew above ground
line (Table 3).

In order to compare the parasitic potentials of the

13 isolates obtained from cultivated areas with the 7 from

13

IA

uncultivated areas, it was necessary to determine whether
the isolates acted differently or similarly on different
test plants. The parasitic potential of the isolates was
measured by the distance grown above the level of inoculation
for each isolate in every test plant. In order to compare
the parasitic potentials on the 20 test plants, correlation
coefficients were obtained for all 1KX) possible pairs of
isolates. If the isolates from both areas formed two
homogenous but distinct groups a large number of high
positive and negative correlation coefficients would be
obtained. A frequency distribution of these values would
result in a bimodal curve. If the isolates from both areas
are similar and form not two but one homogenous group, a
normal unimodal frequency distribution of these values
would be obtained.

The A00 correlation coefficients obtained (Fig. 2)
ranged from -0.6 to +0.6 and had a unimodal frequency
distribution which appeared to be normally distributed.
Similar frequency distributions were obtained for cor-
relation coefficients among isolates from cultivated areas
and among these.from uncultivated areas. Thus the isolates
from both areas are not two distinct groups but are very
similar and appear to be a part of the same population.

The parasitic potentials, or the average distance moved
by the 20 isolates on each test plant, was skewed with the

majority of isolates moving 5 to 8 cm (Fig. 3). The isolates

N
U!

N
0

NUMBER OF PAIRS
G

d
O

 

    
  

All ISOLATES —
ISOrCROPS -"-
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CORRElAflON coemcusms or ISOLATES

Figure 2.-—Frequency distribution of correlation
coefficients for all 400 possible pairs
of 20 isolates when inoculated into
20 hosts and plotted in intervals of
0.1 for correlation coefficients.

16

 

d

 

NUMBER (ISOLATES)
&

u

  

All. lSOlATES

    
  

 
  

 

 

 

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AVERAGE DISTANCE ABOVE lEVEI. OF INOC.(CM.)

Figure 3.--Frequency distribution of parasitic
potentials for 20 isolates as shown
by the number of isolates plotted
against (cm) from the cultivated crops--
[Earlidawn (ear.) Surecrop (sur.) and
Midway (Mid.) moving 6.0 to 6.8 cm]
and from uncultivated areas-- [Two
soil isolates moving 5.6 to 5.9 cm and
one moving 6.5 cm] are significantly
similar at the .05 level of probability
for their correlation coefficients.

 

17

moving further than 12 cm were the two sclerotia formers.
Similar distributions were obtained for isolates from both
cultivated crops and uncultivated areas (Fig. 3). The points
between 5 and 7 cm on the abscissa corresponding to the
peaks on both curves are not significantly different at the
.05 level of probability. There were high positive cor—
relation coefficients (.05) in regards to similarity of
distance grown above level of inoculation in each of the
20 test plants for 7 of 14 isolates that grew an average
of 5-7 cm from the point of inoculation (3 isolates were
from cultivated crops and A were from soil from the un—
cultivated sites). Thus these isolates are very similar in
host range and parasitic potential.

0n the basis of where the plants came from they can
be grouped into 3 categories for comparison of parasitic
potentials of 7 isolates from the uncultivated areas:
(1) cultivated susceptible crops, (2) plants found growing
in the proximity of wild strawberries, and (3) weeds
commonly found in cultivated strawberry fields (Fig. A).
All 7 isolates were parasitic on the 1st category of plants
with 6 isolates moving farther than 6 cm (ground line)
from the level of inoculation. The same isolates when
inoculated into the second and third category of plants
formed two groups in regards to parasitic potential.
In the first group, isolates did not grow above ground

line (growing 3 to 4 cm) and in the other group the isolates

NE. WMBER OF ISOLATES/ PlANT

18

 

 

 

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z 4 I 8 10 12 14 16 ll 20 22
AVE. DISTANCE nova LEVEL OF moc. (cm)

Figure A.-—Frequency distribution of parasitic
potentials for 7 isolates from virgin
areas as shown by the average number
of these isolates per plant of each
respective group plotted against the
average distance they moved (cm) from
the level of inoculation.

 

l9

grew above ground line (6 cm) and were as parasitic as the
6 isolates were on susceptible crops. The same was ob—
served for isolates from the cultivated crops (Fig. 5).

Many degrees of susceptibility (cm invasion above
inoculation level) were observed between the 20 isolates
and the 20 test plants, and these degrees of susceptibility
appear to exhibit a normal distribution (Fig. 6.) The
test plants formed two groups when the average distance
all isolates moved in each test plant was compared (Fig. 7):
(l) slightly susceptible, the isolates moving 3 to A cm,
and 2) the moderately susceptible to very susceptible, tte
isolates moving farther than 6 cm or above ground line.

The same was true for the plants from the uncultivated areas
and for weeds from cultivated strawberry fields. Straw-
berry plants did not form a homogenous group but reacted
from slightly to very susceptible. Earlidawn was the

most susceptable (the 20 isolates moving an average of
13.20n0 and Robinson was next (the 20 isolates moving

an average of 8.8 cm).

When the test plants are compared as to the distance
each of the 20 isolates moved, four groups are obtained:
(1) variable (Fig. 8 a and b) resistant (Fig. 9, purslane
etc.), (3) slightly resistant (Fig. 9, chickweed etc.),
and (A) susceptible (Fig. 9, strawberry var. Earlidawn

etc.).

AVE. NUMBER OF ISOlA‘I'ESI PLANT

-

 

2O

 

EKIAMESOUROPNNKN5 I3

  
  
  

PlAN‘I’S-WCUL'I’. AREA 8 —

  
 

 

     

 

 

1. nuns-mm FIELD WEEDS s
A 1 L 1 i A n A A /\
0 2 4 6 B Io 12 M 16 IS 20 fl .

AVE. DISTANCE ABOVE LEVEL or moc. (CM)

Figure 5.-—Frequency distribution of parasitic
potentials for 13 isolates from
cultivated crops as shown by the
average number of these isolates per
plant of each reSpective group plotted
against the average distance they moved
(cm) from the level of inoculation.

HHKWENCY

8

21

 

 

8

N
C

 

A. .

GROUND LINE 1;

 

 

l

I 1 M
8 lo 12 I: '6 [B 20 22

’booooooooooo

2 4
AVEDIsrANCE ABOVE LEVEL OF moc. (CM)

Figure 6.--Frequency distribution of A00 host-
parasite (isolate) combinations
shown by the frequency of these
combinations plotted against the
average distance moved (cm) by the
fungus from the level of inoculation.

NUMBER PLANTS

6-

”I
I

22

 

 

GROUND llNE L

  

AII PLANTS 20

M PLANTS-UNCULT. AREA 3

  

PLANTS-STRAWBERRY FIELD WEEDS 5

A

STRAWBERRY PLANTS 6

.1.£.\..A ..

 

 

2 4 a a 10 :2 14 16 18 20
AVERAGE DISTANCE ABOVE LEVEL OF INOCULATION (CM)

Figure 7.-—Frequency distribution of test plants as
shown by the number of test plants in
each respective group plotted against the
average distance moved (cm) by 20 isolates
in A replications.

 

NUMBER ISOLATES

23

 

GROUND LINE 2,:
L :

 
  
  

AVERAGE
DOCK

_ POTENTILLA

EVIRGINIANA

ECHILOENSIS

MEADOW RUE

   

 
  

POTENTILLA

 

 

 

o 2 4 6 8 IO 12 14
AVE. DISTANCE ABOVE LEVEL OF INOC. (CM)

 

Figure 8a.--Frequency distribution of the 20
isolates on Dock, Potentilla, F.
virgininiana etc., as shown by_the
average distance they moved (cm) from
the level of inoculation.

NUMBER ISOLATES

GROUND LINE 2.5
6 - :
AVERAGE
_ DOCK
4 . POTENTILLA
; E VIRGINIANA
2 _ 5 ECHILOENSIS
; MEADOW RUE
6 L 5
4 I—
2 I—
o —- :
6 L E
5 POTENTILLA
4 I—
2 I—
o 2 4 6 B IO I2 I4

2A

 

 
   
   
   
       
     
   

 

 

 

 

 

AVE. DISTANCE ABOVE LEVEL OF INoc. (CM.)

Figure 8b.—-Frequency distribution of the 20 isolates
on dock, potentilla, F. virginiana, etc.,
as shown by the average distance they
moved (cm) from the level of inoculation.

25

 

GROUND LINE 15

d
O

O

 

NUMBER - ISOLATES] PLANT
‘ b

N

 

PURSELANE
NIGHTSHADE

CHICKWEED ’

_ WILD CARROT
TOMATO var. BONNY BEST
DANDELION

STRAW.VOr. EARLIDAWN
STRAW.VOr. ROBINSON

WILD ROSE

‘---\

\
\
\

 

Ef‘ooooo.. fish 1

 

 

a Io 12 I4

AVE. DISTANCE ABOVE LEVEL OF mm. (C M.)

Figure 9.—-Frequency distribution of the 20 isolates on
these groups of plants, as shown by the

average number of isolates per plant species
of each respective group plotted against the
average distance they moved (cm) from the
level of inoculation.

 

-_I_ ‘
*

 

_ Z FF'm

I
I I
i.
9 .

 

 

 

DISCUSSION

The similarity of A isolates from soil in uncultivated
sites and 3 isolates from crop plants in cultivated areas
with regards to host range and parasitic potential sug—
gests that isolates from cultivated strawberry could have
originated in formerly uncultivated areas and are indigenous
to this area. The parasitic potentials of isolates from
uncultivated areas on cultivated strawberry supports this.

Resistance to Verticillium wilt of strawberry has been
found in wild strawberries of North and South America
(20, 28, 30, 31) and presumably is the result of a pro-
longed interaction between host and parasite. This resis—

tance also indicates that Verticillium is indigenous to

this continent.

The susceptible wild rose, Potentilla and wild straw—
berries found in the uncultivated areas presumably could
enable the fungus to persist and multiply in these un—
cultivated areas even in the absence of strawberry.

Similarly, weeds commonly found in cultivated
strawberry fields as Pigweed, chickweed and white
cockle may serve as hosts for multiplication of Verticil—
112s (32). Verticillium is also reported to multiply in
the rhizosphere and infect roots of susceptible or resistant

peppermint, tomato and corn (10)(l3). Thus good weed

26

27

control or crop alternation with non-multiplication hosts if

possible would reduce the incidence of Verticillium wilt.

 

SUMMARY

The origin of Verticillium_albg-atrum on strawberry
was investigated by conducting a survey of the host range
and parasitic potentials of 13 isolates from cultivated
crops and 7 from uncultivated areas on 20 different plants
normally growing in these two areas. I

All isolates were parasitic on each teSt plant with
the exception of the peppermint isolate on violet. Isolates
from the uncultivated areas were parasitic on cultivated
strawberry and thus are potential pathogens.

Three isolates from soil in uncultivated areas and
A isolates from crop plants in cultivated areas were
similar in parasitic potential and host range, suggesting
that the A isolates from the cultivated areas could have
originated in an uncultivated area.

Some plants from both uncultivated and from cultivated

areas were as susceptible to invasion by Verticillium

 

isolates as susceptible cultivated plants and thus could

serve as multiplication sites for the fungus.

28

10.

ll.

12.

BIBLIOGRAPHY

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