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O rder N um ber 8900101

Cherry tree decline in Michigan: Associated pythiaceous species,
pathogenicity and control
Smither, Margaret Lydia, Ph.D.
Michigan State University, 1988

UMI

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CHERRY

TREE

DECLINE IN

MICHIGAN:

ASSOCIATED

PYTHIACEOUS

SPECIES, PATHOGENICITY AND CONTROL

By
Margaret Lydia Smither

A Dissertation

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1988

ABSTRACT

CHERRY

TREE

DECLINE
IN MICHIGAN;
ASSOCIATED
SPECIES, PATHOGENICITY, AND CONTROL

PYTHIACEOUS

By
Margaret Lydia Smither

Montmorency
those

sour cherry trees in

planted on heavy soils,

poor

growth

The

problem

and discolored and

Michigan,

especially

exhibit decline symptoms
diminished

root

of

systems.

is most severe in orchards with trees

grafted

onto Mahaleb rootstocks.
Isolations
soil.
from

for Pvthium species were made from roots and

Pythium

was commonly associated with

orchards

with heavy clay soils but not with

trees from orchards
in

heavy

soils.

rootsof
roots

of

with light sandy soils, and populations

soils were high compared to populations in

£.

trees

irregulare

was

isolated most

light

frequently

from

discolored and necrotic cherry roots and from cherry orchard
soils.

Other species isolated included: £. svlvaticum. £.

ultimum.

£.

Pythium

rostratum.

spp..

necrosis

and several infrequently

isolated

In greenhouse trials root discoloration

and a reduction in root and shoot growth

occurred

on Mahaleb seedlings inoculated with £. irregulare.
reductions

and

necrosis were greater when

and

seedlings

Growth
were

flooded, but unflooded seedlings exhibited symptoms as well.
None
after

of
3

the seedlings inoculated with £.
months.

£.

irregulare

may

irregulare
contribute

died
to

a

reduction in growth of cherry trees planted on heavy soils.
Orchards
in

and woodlands in

composition

sylvaticum.

of

and

Michigan were found to

Pythium species.

£.

differ

irregulare.

£.

£. ultimum were frequently isolated

orchard

soils

but were not isolated from

Pythium

group

'HS'

was

isolated

most

woodland

from
soils.

frequently

from

woodland soils, followed by Pvthium group ’X ‘, £. rostratum.
and

Pythium group ’Y'.

Populations of £.

irregulare.

E.

sylvaticum. and £. ultimum in two orchards sampled over 2 yr
and one orchard over 1 year, varied independently.
Seven

pythiaceous

species associated with the roots

of

sour cherry in Michigan were tested for their sensitivity to
metalaxyl and phosphorous acid in culture.

All fungi were

inhibited by both fungicides and sensitivity varied
species.
400

and

between

Mahaleb seedlings exhibited phytotoxic symptoms at
was

more

effective than fosetyl-Al in preventing fungus induced

root

necrosis

800

/^g/ml

metalaxyl.

Metalaxyl

and growth reduction to 9-wk-old Mahaleb seedlings

growing in soil infested with £. irregulare. Eh- megasoerma.
and Eh- cactorum.

To my father

ii

ACKNOWLEDGEMENTS

I wish to thank Dr A.
help and
of

L. Jones my major professor for his

financial support.

my guidance committee;

Stephens, Dr G. Adams,

I am grateful to the members
Dr J.

T.

Stephens

latter
for

Lockwood,

R.

encouragement.

T.

for financial and moral support during the
I wish to thank Dr Ed

financial and other assistance.
G.

C.

Especial thanks are due to Dr

part of this project.

Bielenin,

Dr

and Dr R. L. Perry for their faith in

me, support and encouragment.
C.

L.

Ehret

and R.

E.

I also thank

Klos
Dr

Comstock for help

A.
and

I wish to thank my fellow graduate students

for their assistance;

especially Dr.

V. F. Stanis for help

in the initial stages of this work, and Reginald Mandego for
collaborating in the soil compaction experiment.
I wish to thank Laura Wolf for financial help.
my

husband

Ben for his

understanding,

support.

iii

I

encouragement

thank
and

TABLE OF CONTENTS
Page
LIST OF TABLES ......................................

viii

LIST OF FIGURES..........................................

xi

INTRODUCTION AND GENERAL LITERATURE REVIEW.........

1

LITERATURE CITED.....................................

7

PART I

PYTHIUM SPECIES ASSOCIATED WITH THE ROOTS OF SOUR CHERRY AND
THE EFFECT OF £. IRREGULARE ON THE GROWTH OF MAHALEB CHERRY

13

ABSTRACT..............................................
T M w n A T \n n n » T A iT

i t u i v j y u v j i 1WL1............................................................................................................................... .

a

a

14

MATERIALS AND METHODS................................

15

RESULTS...............................................

19

DISCUSSION............................................

30

LITERATURE CITED.....................................

33

iv

PART II
SPECIES AND POPULATIONS OF PYTHIUM ISOLATED FROM SOUR CHERRY
ORCHARD AND WOODLAND SOILS IN MICHIGAN
Page
ABSTRACT..............................................

36

INTRODUCTION..........................................

37

MATERIALS AND METHODS................................

38

RESULTS...............................................

40

DISCUSSION............................................

44

LITERATURE CITED.....................................

50

PART III

EFFECTS

OF

PHYTOPHTHORA

METALAXYL
AND

AND

FOSETYL-AL

ON

GROWTH

PYTHIUM SPECIES ’IN VITRO' AND

ON

OF
THEIR

PATHOGENICITY TO MAHALEB CHERRY

ABSTRACT,,............................................

53

INTRODUCTION.........................................

53

MATERIALS AND METHODS................................

55

RESULTS...............................................

58

DISCUSSION............................................

74

LITERATURE CITED.....................................

76

v

APPENDICES

APPENDIX A

DESCRIPTIONS OF PYTHIUM SPECIES ASSOCIATED WITH MONTMORENCY
SOUR CHERRY IN MICHIGAN............................

79

APPENDIX B
EFFECT

OF PYTHIUM AND PHYTOPHTHORA ISOLATED IN MICHIGAN

ON

GROWTH OF MAHALEB SEEDLINGS
Page
INTRODUCTION..........................................

86

MATERIALS AND METHODS................................

86

RESULTS...............................................

88

DISCUSSION............................................

91

LITERATURE CITED.....................................

92

vi

APPENDIX C

THE EFFECTS OF INCREASED SOIL COMPACTION LEVELS WITH PYTHIUM
IRREGULARE AND PHYTOPHTHORA MEGASPERMA ON GROWTH OF MAHALEB
SEEDLINGS, AND ETHYLENE LEVELS IN SOIL
Page
ABSTRACT..............................................

94

INTRODUCTION..........................................

94

MATERIALS AND METHODS................................

95

RESULTS...............................................

98

DISCUSSION...............................................

101

LITERATURE CITED........................................

102

APPENDIX D

VARIATION IN SUSCEPTIBILITY OF CHERRY ROOTSTOCK CULTIVARS TO
INFECTION BY PHYTOPHTHORA SPECIES AND PYTHIUM IRREGULARE

ABSTRACT..............................................

105

INTRODUCTION..........................................

106

MATERIALS AND METHODS................................

107

RESULTS...............................................

109

DISCUSSION............................................

114

LITERATURE CITED.....................................

115

vii

LIST OF TABLES

PART I

Table 1.
Species of Pythium isolated from the
roots of sour cherry trees with reduced terminal
growth or from the roots of Mahaleb seedlings....
Table 2.
Populations of Pvthium species as
estimated by dilution-plate counts from soil
taken from sour cherry orchards....................
Table 3. Effect of nine isolates of Pythium
irregulare on the growth of Mahaleb seedlings
growing in unflooded soil..........................
Table 4. Analysis of variance for shoot and root
dry weights of Mahaleb seedlings growing in soil
infested with Pythium irregulare and two
Phytoohthora species in single and combined
treatments under flooded and unflooded conditions.
Table 5. Effect of Pythium irregulare (£.) and two
Phytoohthora (Eh-) species on the growth of Mahaleb
seedlings in single and combined treatments under
flooded and unf 1 coded coudibiouis
Table 6. Reisolation of Pythium irregulare and
two Phytoohthora species from Mahaleb seedlings
(experiment 2) grown for 15 weeks with periodic
flooding..........................................

viii

PART II
Page
Table 1.
Populations of Pythium species as
estimated by dilution-plate counts from soil taken
from sour cherry orchards planted on two soil types

42

Table 2. Populations of Pythium species as
estimated by dilution-plate counts from soil taken
from sour cherry orchards..and woodlands..........

43

PART III
Table 1.
EDg0 values for Phytophthora and Pythium
species to metalaxyl and phosphorous acid in
culture...............................................

63

Table 2.
Effect of various concentrations of
metalaxyl on growth of 1-yr-old Mahaleb seedlings
in Phytophthora and Pythium infested soil in the
greenhouse............................................

68

Table 3.
Effect of fosetyl-Al and metalaxyl on
growth of 1-yr-old Mahaleb seedlings in
Phytophthora and Pythium infested soil under
lath..................................................

71

Table 4.
Effect of fungicides metalaxyl and
fosetyl-Al on growth of Mahaleb seedlings in soil
infested with Phytophthora species and Pythium
irregulare............................................

72

APPENDIX B
Table 1.
Virulence of Pythium and Phytophthora
species isolated from Michigan sour cherry
orchards to Mahaleb seedlings.......................

ix

89

APPENDIX C
Page
Table
1.
Effects of soil bulk density and
Pythium irregulare on growth of Mahaleb
seedlings............................................

99

Table 2.
Effects of soil bulk density, Pvthium
irregulare and Phytophthora megasperma on growth
of Mahaleb seedlings................................

100

APPENDIX D
Table 1. Relative susceptibility of dormant excised
shoot tissue of cherry rootstock cultivars to
infection by Phytophthora species and Pythium
irregulare during 1983..............................

Ill

Table 2. Relative susceptibility of dormant excised
shoot tissue of cherry rootstock cultivars to
infection by Phytophthora species and Pythium
irregulare during 1984..............................

112

Table 3. Growth of cherry rootstock cultivars
planted in soil infested with Phytophthora species
and Pythium irregulare under lath..................

113

x

LIST OF FIGURES

PART I
Figure 1. Roots of representative Mahaleb
seedlings grown in a growth chamber for 18 wk in
a soil/sand/peat mixture infested with Pythium
irregulare.
Soil in the pots was a) uninfested,
or infested with p. irregulare isolated from
orchard B, b) root and c) soil;
orchard C, d)
root and e) soil;
orchard J, f) root and g)
soil;
orchard L, h) root and i) soil; and
orchard P, k) soil.
The treatments were not
flooded.............................................
Figure 2.
Shoots (A) and roots (B) of
representative Mahaleb seedlings grown in a
greenhouse for 3 mo in a soil/sand/peat mixture.
Soilin the pots was (a) uninfested and unflooded,
(b) uninfested and flooded, or flooded and
infested with (c) Pythium irregulars C6,
(d )Phytophthora megasperma. avirulent isolate
M224, (e) P. irregulare C6/Ph- megasperma.
avirulent M224,
Ph. megasperma. virulent isolate
M333, and p. irregulare C6/Ph. megasperma.
virulent M 3 3 3
Figure 3.
Shoots (A) and roots (B) of
representative Mahaleb seedlings grown in a
greenhouse for 3 mo in a soil/sand/peat mixture.
Soil in the pots was (a) uninfested and flooded,
or flooded and infested with (b) Pvthium
irregulare C6, (c)Phyt<?phth<?ra cactorum B2, (d)
p. irregulare C6/pfc. cactorum B2 ................

PART II
Page
Figure 1. Fluctuations in population (propagules
per gram of soil) of Pythium species as estimated
by dilution-plate counts for soil samples taken
from sour cherry orchard J in southwest Michigan
during 1984 and 1985.
Species identified were £.
irregulare. £. sylvaticum. £. ultimum. and Pythium
spp ...................................................

45

Figure 2. Fluctuations in population (propagules
per gram of soil) of Pythium species as estimated
by dilution-plate counts for soil samples taken
from sour cherry orchard L in southwest Michigan
during 1984 and 1985.
Species identified were £.
.ijcr.efflUflEg. £• gzlzafcicum. £• u ltimmn. and Pz.th i.um
spp ...................................................

46

Figure 3. Fluctuations in population (propagules
per gram of soil) of Pythium species as estimated
by dilution-plate counts for soil samples taken
from sour cherry orchard P in southwest Michigan
during 1985.
Species identified were I) £.
irreftulare, S) £. sylvaticum. U) £. ultimum. and P)
Pythium s p p .........................................

47

PART III

Figure la. Radial growth of Phytophthora cactorum
in culture amended with metalaxyl at rates of 0.0,
0.01, 0.1, 1, 10, and 100 yUg/ml....................

59

Figure lb.
Radial growth of Phytophthora citricola
in culture amended with metalaxyl at rates of 0.0,
..............
0.01, 0.1, 1, 10, and 100 M S / m l

59

Figure lc. Radial growth of Phytophthora drechsleri
in culture amended with metalaxyl at rates of 0.0,
0.01, 0.1, 1, 10, and 1 0 0 ^ g / m l .....................

60

Figure Id. Radial growth of Phytophthora megasperma
in culture amended with metalaxyl at rates of 0.0,
0.01, 0.1, 1, 10, and 100 ^(g/ml.....................

60

xii

Page
Figure le. Radial growth of Pythium irregulare in
culture amended with metalaxyl at rates of 0.0,
0.01, 0.1, 1, 10, and 100 ^ g / m l .....................

61

Figure If. Radial growth of Pythium sylvaticum in
culture amended with metalaxyl at rates of 0.0,
0.01, 0.1, 1, 10, and 100

61

Figure Ig. Radial growth of Pythium ultimum in
culture amended with metalaxyl at rates of 0.0,
0.01, 0.1, 1, 10, and 100 /Hg/ml.....................

62

Figure 2a. Radial growth of Phytophthora cactorum
in culture amended with phosphorous acid at rates of
0, 10, 25, 50, 75, and 100,tfg/ml....................

64

Figure 2b.
Radial growth of Phytophthora citricola
in culture amended with phosphorous acid at rates of
0, 10, 25, 50, 75, and 100 ^ g / m l ....................

64

Figure 2c. Radial growth of Phytophthora drechsleri
in culture amended with phosphorous acid at rates of
0, 10, 25, 50, 75, and 100 M g / m l ....................

65

Figure 2d. Radial growth of Phytophthora megasperma
in culture amended with phosphorous acid at rates of
0, 10, 25, 50, 75, and 100/>(g/ml....................

65

Figure 2e. Radial growth of Pythium irregulare in
culture amended with phosphorous acid at rates of 0,

Figure 2 f . Radial growth of Pythium sylvaticum in
culture amended with phosphorous acid at rates of 0,
10, 25, 50, 75, and l O O ^ g / m l .......................

66

Figure 2g. Radial growth of Pythium ultimum in
culture amended with phosphorous acid at rates of 0,
10, 25, 50, 75, and 100yttg/ml.......................

67

Figure 3. Phytotoxic symptoms caused by metalaxyl
at rates of (a) 0, (b) 100, and (c) 400 /(g/ml on
leaves of Mahaleb cherry.............................

69

xiii

Page
Figure 4.
Roots of representative Mahaleb
seedlings grown in a) uninfested soil, or soil
infested with b) Phytophthora cactorum. c) Ehmegasperma and d) Pythium irregulare.
Roots in
columns were x) untreated, or treated with y)
metalaxyl, and z) fosetyl-Al........................

73

APPENDIX A
Figure

la. Pythium ixreflfllare sporangia .........

82

Figure
lb. Pythium irregulare irregularly
shaped oogonium and aplerotic oospore..............

82

Figure

83

2a. Pythium sylvaticum hyphal swelling ..

Figure
2b. Pythium sylvaticum oogonium
surrounded by branched antheridial cells...........

83

Figure
3a.
Pythium ultimum oogonium with
paragynous antheridium attached close to the
oogonial stalk.......................................

84

Figure 3b.
Pythium ultimum oogonium with
paragynous antheridia and an aplerotic oospore

84

APPENDIX B
Figure 1. Roots of representative Mahaleb
seedlings grown in a growth chamber for j . o w k _lh
a soil/sand/peat mixture infested with (A)
Phytophthora species and (B) Pythium species.
Soil in the pots was (a) uninfested and non­
flooded, or flooded and infested with (b) P h .
fifls.tgc.mn. (c) £h. citricola, (d) Eh- drechsleri.
(e) £h. mega&perma> (f) £. irregulare. (g) £.
sylvaticum. and (h) £. ultimum......................

xiv

90

INTRODUCTION AND GENERAL LITERATURE REVIEW

Michigan
accounting

is the leading state in sour cherry

for about two thirds of the total production

the United States (27).
1981

was set at

producing
Michigan.

areas

in

The economic value of the crop for

over 40 million dollars (26).

The

are in the west along the shores

Concentrations

northwest

production

of

production

around Traverse City,

cherry
of

occur

Lake

in

the

in the west central region

around Hart and Shelby,

and in the south west around Benton

Harbor.

of

High

resulted
with

prices

cherries

in

the

last

in a 14% increase of land under cherry

a

total of 4.5 million trees planted

However

decade

production

by

1982

(27).

as cherry production increased so did instances

of

poor tree vigor and decreased longevity.
Tree
life

decline is a comprehensive term encompassing

and replant problems (47,55).

short

Tree decline problems

have been identified throughout the world and recognised for
over

two hundred years (42).

The main symptom is

reduced

vigor with a possible reduction in life span.

Root systems

are

Cankers

weak

develop
causal
abiotic

and poorly developed
on the trunk.

or

necrotic.

Often there is no clearly

may

defined

agent but rather a combination of several biotic and
factors

(22,23,47,55).

1

These

may

include;

environmental
drought

factors such as cold injury,

or oxygen defeciency caused

injury

caused by damaging cultural

factors

of

infection

by

mycoplasma-like-organisms,
insect

infestation;

toxicity from plant

waterlogging,

practices;

bacteria,
and

and

by

stresses due to

pathogenic

fungi,

nematodes;

physiological

and

viruses,

injury

factors

from

such

as

and spray residues, and biochemical and

hormonal

imbalances

distinguished

between

specific

replant disorders which occurred when one

species

followed

the

(55).

same

or

a

Savory

closely

related

species

and

nonspecific replant disorders affecting all tree crops (42).
This distinction has also been used to classify other
of tree decline problems (47).
tree

decline

has

been

types

The literature pertaining to

reviewed

in

several

articles

(42,47,55).
Stone

Fruit Decline was the subject of a

Michigan
which

State University in 1982 (46).

were

pplant

selected

as most critical

pathogenic viruses,

root rots,

and improvement,

mechanical harvesting,

structure.

grant

A

conference

Areas of

at

research

included

study

of

rootstock selection
and trunk and tree

was awarded to study

root

rot

of

cherry in Michigan through the Stone Fruit Decline Project.
Root

rot of sour cherry in Michigan caused by Armillaria

occurs on sandy sites from the Hart/Shelby area north to the
Traverse City area.
isolated
from

Three species of Armillaria have been

from these orchard sites which have

woodlands

of

oak and pine on

which

been
the

cleared

Armillaria

species

were

Phytophthora
although
The

endemic

(39,51).

Root

rot

caused

occurs throughout the cherry production

area,

symptoms are most severe on poorly drained

symptoms

by

soils.

include extensive lateral root necrosis

and

development

of a canker on the lower trunk below the

union (6).

A preliminary survey associated Pythium species

with decline only on heavy poorly drained soils.

graft

The root

rot symptoms include extensive necrosis and eventual absence
of

lateral branchlet roots.

closely

related

genera

Pythium and Phytophthora

(13) and may

present under similar conditions.
pathogens

are

most

be

expected

are

to

be

Symptoms for the three

severe when the trees are

planted

on

Mahaleb (Prunus mahaleb L . ) rootstocks although orchards

on

Mazzard (£. avium L . ) rootstocks are also affected.
The

genus

Pythium

which

have

from

diverse

pathogens
economic

a world wide distribution and can

and

habitats.

Many

are

the

closely

genus

isolated
plant

cause

severe

including

strawberry (35,52),
related

species

be

under suitable conditions can

wheat (9),

of

soil-bourne

losses on a wide range of crops,

(1,21,37),
Unlike

contains a large number

beans

and grape (48).

Phytophthora

not

all

species are pathogens.

Pythium species survive in soil by

saprophytic

production

growth

and

structures (13,50).
conditions
competitors

of

resistant

resting

They tend to be more successful under

high soil moisture
with

of

a

well

(29,49),

established

but

soil

are

poor

microflora

(4,13,49). Pythium species have the ability to colonise host

tissue (13,28,50),

and produce a variety of cellulytic

pectolytic enzymes (10,50).

and

The literature about the genus

Pythium has been reviewed (13,50).
The
and

objectives of this study were to

examine

associated

the

pathogenicity

with

decline

on

Pythium

species

cherry

heavy

trees

sites;

of

isolate,

Pvthum

showing

species

poor

woodland

found

growth

to investigate the

in orchard and

identify

and

ecology

soils;

of

and

to

examine possible control measures of rootstock selection and
chemical control.

The literature on root and collar rot of

cherry

deciduous

and

other

fruit tree

crops

caused

by

pythiaceous species will be reviewed in the remainder of the
introduction.
Environmental
levels

influence

pythiaceous
higher

conditions
the

species.

under

particularly

development

of

soil

disease

Populations of Pvthium

conditions of high soil

moisture
caused

species

moisture

by
are

(4,29,49).

Tree loss due to Phytophthora infection is typically greater
in

poorly

mounds,
for

drained

areas

(25,33).

trees

on

thus improving drainage, was reported as beneficial

reducing losses caused by £h.

Subsoiling
reduced

Planting

before
losses

Improvement

was

cactorum on apple

(25).

planting improved peach tree growth
due

to

peach

tree

short

life

(41).

believed due to greater stability in

moisture

levels.

improves

tree vigor will tend to ameliorate disease

by Pythium species.

Any

practice which reduces

and

soil

stress

and

caused

Pythium
cherry

species

trees

isolated from roots of

have been reported as pathogenic

seedlings (12).
diseased

growing

to

Mahaleb

Pythium species isolated from roots of

trees

were

(14,15,28,38).
to

poorly

pathogenic

to

peach

seedlings

However, the importance of Pythium species

peach tree decline has been questioned by some

workers,

as

disease

pathogenicity
which

the

symptoms

tests.

were

Unfortunately

experiments

were

run

not

research

reproduced

in

the temperatures
was

not

at

reported

(20,29,30,36), and pathogenicity of the Pythium species been
shown

to be dependent on temperature

(7).

In

addition

total

Pythium populations were not correlated with

decline

symptoms (29).
Pythium
apple.

species
Several

cause decline and replant
Pythium species including

problems

£.

of

irregulare

Buisman and £. sylvaticum Campbell and Hendrix isolated from
roots

of

declining trees or soil from replant

pathogenic

to

roots

contribute

to

apple replant problems in New York (16)

Europe (34,43).
with

collar

rot

seedlings (17).
was

found

of apple

seedlings

and

sites
thought

Pythium species isolated from apple
in

New York

were

were

pathogenic

to

to
and

trees
apple

Pythium ultimum from apple trees in Poland

to cause a trunk canker

(5).

Pythium

species

also caused a retardation of tree growth in an apple orchard
in Australia (45).
Phytophthora species have been implicated as the
causal agents of root and crown rots of fruit trees.

primary
Root

and

collar

rot of sweet cherry on wet sites in

California

was

first shown to be caused by Phytophthora in 1976

(32).

The following species have since been found to be pathogenic
to

Mahaleb;

CEZPtPgaea

£h-

cambivora (Petri)

Pethyb.

Tucker (32,54),

and

and Ph.

Laff.

Buisman

(6,53,54),

(32,54),
£h-

Ph.

drechsleri

megasperma Drechsler (6,32,53,54),

Eh- cactorum (Leb. and Cohn)Schroet (6,24), and Eh. syringae
(Kleb.)Kleb.

(19).

£h- cambivora. Eh- cryptogaea. and Eh-

megasperma have been isolated from soils in South Australian
cherry orchards (8).
as

Mahaleb rootstocks have been reported

more susceptible to Phytophthora infection than

Mazzard

and other cherry rootstocks (24,33,54).
A root and collar rot of peach was caused by Phytophthora
sumamsmi

(32),

Eh.

cactorum

(11,28) and

£h.

syringae

(19,56).
Apple collar and root rot is caused by £h-

cactorum

and

the disease has a world wide distribution (2,3,8,25,44). P h .
cambivora (18),
and

£h-

Ph. citricola (44), Eh- megagperms (17,40),

syringae

(44)

have also been reported

to

cause

collar rot of apple.
The
stone

role

of Phytophthora species in

rapid

decline

of

fruit trees has recieved more attention than that

of

Pythium species because of the greater relative virulence of
Phytophthora.
tree

In

stone

fruit growing regions with

declines a more thorough investigation of the role

Pythium species should be undertaken.

slow
of

LITERATURE CITED

1.

Adegbola, M. 0. K . , and Hagedorn, D. J.
1969.
Symptomatology and epidemiology of Pythium bean blight.
Phytopathology 59:1113-1118.

2.

Aldwinkle, H. S., Polach, F. J . , Molin, W. T. , and
Pearson, R.C.
1975.
Pathogenicity of Phytophthora
cactorum isolates from New York apple trees and other
sources.
Phytopathology 65:989-994.

3.

Baines, R. C. 1935.
Phytophthora trunk canker of
apple. (Abstr.) Phytopathology 25:5.

4.

Barton, R. H.
1958.
Occurrence and establishment of
Pythium in soils.
Trans. Brit. Mycol. Soc. 41:207-222.

5.

Bielenin, A., Borecki, Z., and Millikan, D. F. 1976.
Identification of Pythium ultimum in the collar rot
complex of apple.
Phytopathology 66:127-129.

6.

Bielenin, A., and Jones, A. L. 1988.
Prevalence and
pathogenicity of Phytophthora spp. from sour cherry
trees in Michigan.
Plant Disease 72:473-476.

7.

Biesbrock, J. A., and Hendrix, F. F . , Jr.
1970.
Influence of soil water and temperature on root necrosis
of peach caused by Pythium spp.
Phytopathology 60:880882.

8.

Bumbieris, M . , Wicks, T. J . , and Windle, B. E.
1982.
Phytophthora species in apple and cherry orchards in
South Australia.
Australasian Plant Path. 11:28-29.

9.

Chamswarng, C. and Cook, R. J.
1985.
Identification
and comparative pathogenicity of Pythium species from
wheat root and wheat-field soils in the Pacific
Northwest. Phytopathology 75:821-827.

10. Deacon, J. W. 1979.
Cellulose decomposition by Pythium
and its relevance to substrate-groups of fungi. Tra n s .
Br. mycol. Soc. 72:469-477.
11. Dunegan, J. C. 1935.
A Phytophthora disease of peach
seedlings.
Phytopathology 25:800-809.
7

8
12. Fliegel, P., Parker, K. G . , and Mai, W. F.
1963.
The
fungous flora of nonsuberized roots of poorly growing
cherry trees.
Phytopathology 53:1368-1369.
13. Hendrix, F.F.Jr., and Campbell, W.A.
1973
Pythiums
as plant pathogens. Ann. Rev. Phytopathology 11:77-98.
14. Hendrix, F. F . , Jr., Powell, W. M . , and Owen, J. H.
1966.
Relation of root necrosis caused by Pythium
species to peach tree decline.
Phytopathology 56:12291232.
15. Hine, R. B. 1961.
replant problem.

The role of fungi in the peach
Plant Dis. Rep. 45:462-465.

16. Jaffee, B. A., Abawi, G. S., and Mai, W. F.
1982.
Fungi associated with roots of apple seedlings grown in
soil from an apple replant site.
Plant Dis. 66:942944.
17. Jeffers, S. N . , Aldwinkle, H. S., Burr, T. J . , and
Arneson, P. A.
1982.
Phytophthora and Pythium species
associated with crown rot in New York apple orchards.
Phytopathology 72:533-538.
18. Julis, A. J . , Clayton, C. N . , and Sutton, T. B. 1978.
Detection and distribution of Phytophthora cactorum and
£. cambivora on apple rootstocks.
Plant Dis. Rep.
62:516-520.
19. Kouyeas, H. 1971.
On the apoplexy of stone fruit trees
caused by Phytophthora spp.
Annls. Inst. Phytopathol.
Benaki. N.S. 10:163-170.
20. Lownsbery, B. F . , English, H . , Moody, E. H . . and Shick.
F. J.
1973.
Criconemoides xenoplax experimentally
associated with a disease of peach.
Phytopathology
63:994-997.
21. Lumsden, R. D . , Ayers, W. A., Adams, P. B . , Dow, R. L . ,
Lewis, J. A . , Papavizas, G. C . , and Kantzes, J. G.
1976.
Ecology and epidemiology of Pythium species in
field soil.
Phytopathology 66:1203-1209.
22. Mai, W. F . , and Abawi, G. S.
1978. Determining the
cause and extent of apple, cherry and pear replant
diseases under controlled conditions.
Phytopathology
68:1540-1544.
23. Mai, W. F . , and Abawi, G. S.
1981. Controlling
replant diseases of pome and stone fruits in
Northeastern United States by preplant fumigation.
Plant Dis. 65:859-864.

9
24. McIntosh, D. L.
1953.
A trunk and crown rot of sweet
cherry in British Columbia.
Phytopathology 43:402403.
25. McIntosh, D. L. (Ed.)
1975.
Proceedings of the 1974
APDW workshop on crown rot of apple trees.
Can. Plant
Dis. Surv. 55:109-106.
26. Michigan Agricultural Statistics.
1982.
Michigan
Agricultural Reporting Service, Lansing. 88 pp.
27. Michigan Orchard and Vineyard Survey.
1982.
Michigan
Agricultural Reporting Service, Lansing. 49 pp.
28. Miller, C. R . , Dowler, W. M . , Petersen, D. H . , and
Ashworth, R. P.
1966.
Observations on the mode of
infection of Pythium ultimum and Phytoohthora cactorum
on young roots of peach.
Phytopathology 56:46-49.
29. Mircetich, S. M.
1971.
The role of Pythium in feeder
roots of diseased and symptomless peach trees and in
orchard soils in peach tree decline.
Phytopathology
61:357-360.
30.

Mircetich, S. M . ,and
Fogle, H. W.
1968.
Occurrence
and pathogenicity of Pvthium in peach orchard soils.
(Abstr.)
Phytopathology 58:886.

31. Mircetich, S. M . , and Keil, H. L.
1970.
Phytoohthora
cinnamomi root rot and stem canker of peach trees.
Phytopathology 60:1376-1382.
32.

Mircetich, S. M . ,and
Matheron, M. E. 1976.
Phytoohthora root and crown rot of cherry trees.
Phytopathology 66:549-558.

33. Mircetich, S. M . , and Matheron, M. E.
1981.
Differential resistance of various cherry rootstocks to
Phytoohthora species.
(Abstr.) Phytopathology 71:243.
34. Mulder, D.
1969.
The pathogenicity of several Pvthium
species to the rootlets of apple seedlings.
Neth. J.
Plant Path. 75:178-181.
35. Nemec, S., and Sanders, H. 1970.
Pvthium species
associated with strawberry root necrosis in southern
Illinois.
Plant Dis, Rep.
54:49-51.
36. Nyczepir, A. P., and Lewis, S. A.
1984.
Incidence of
Fusarium and Pythium spp. in peach feeder roots as
related to dibromochloropropane application for control
of Criconemella xenonlex.
Plant Dis. 68:497-499.

10
37. Pieczarka, D. J. , and Abawi, G. S.
1978.
Populations
and biology of Pythium species associated with snap bean
roots and soils in New York.
Phytopathology
68:409416.
38. Powell, W. M . , Owen, J. H. and Campbell, W. A. 1965.
Association of phycomycetous fungi with peach tree
decline in Georgia.
Plant Dis. Rep.
49:279.
39. Proffer, T. J . , Jones, A. L . , and Ehret, G. R.
1987.
Biological species of Armillaria mellea isolated from
sour cherry orchards in Michigan. Phytopathology 77:941943.
40. Robertson, G. L. and Dance, H. M.
1971.
The
association of Phytophthora megasperma with crown rot of
apple trres.
N. Z. J. Agric. Res. 14:509-514.
41. Savage, E. F . , Hayden, R. A., and Ward, W. E.
1968.
The effect of sub-soiling on the growth and yield of
peach trees.
Ga. Agr. Expt. Sta. Res. Bui. 30.
42. Savory, B. M.
1966.
Specific replant diseases
causing root necrosis and growth depression in perennial
fruit and plantation crops.
Research Rev. 1. Commonw.
Bur. Hort. Plant. Crops.
East Mailing, Maidstone Kent,
England. 64 pp.
43. Sewell, G. W. F. 1981.
Effects of Pythium species on
the growth of apple and their possible causal role in
apple replant disease.
Ann. Appl. Biol.
97:31-42.
44. Sewell, G. W. F . , Wilson, J. F . , and Dakwa J. T.
1974.
Seasonal variations in the activity in soil of
Phytophthora cactorum. P. sxringae. and P. citricola in
relation to collar rot disease of apple.
Ann. Appl.
Biol.
76:179-186.
45. Sitepu, D . , and Wallace, H. R.
1974.
Diagnoses of
retarded growth in an apple orchard.
Austral. J. Expt.
Agr. and Animal Husb.
14:577-584.
46. Stone Fruit Decline Workshop Proceedings.
1982.
Michigan State University, E. Lansing. 128 pp.
47. Traquair, J. A.
1984.
Etiology and control of
orchard replant problems: a review.
Can. J. Plant
Pathol.
6:54-62.
48. Utkhede, R. S., and Vielvoye, E. 1984.
Cause and
control of decline of grapevines in British Columbia.
Plant Dis.
68:820-822.

11
49. Van der Plaats-Niterink, A. J.
1975. Species of Pvthium
in the Netherlands.
Neth. J. PI. Path. 81:22-37.
50. Van der Plaats-Niterink, A. J.
1981.
Monograph of the
genus Pythium. Studies in Mycology No 21 Centraal
Bureau voor Schimmelcultures, Baarn. 242 pp.
51.

Wargo, P. M . , and Shaw, C. G. III.
1985.
Armillaria
root rot: The puzzle is being solved.
Plant Dis.
69:826-832.

52.

Watanabe, T . , K. Hashimoto, amd M. Sato.
1977.
Pythium species associated with strawberry roots in
Japan and the strawberry stunt disease.
Phytopathology
67:1324-1332.

53. Wilcox, W. F . , Jeffers, S. N . , Hayes, J. E. K . , and
Aldwinkle, H. S. 1985.
Phytophthora species causing
root and crown rot of cherry trees in New York.
(Abstr.) Phytopathology 75:1347.
54. Wilcox, W. F . , and Mircetich, S. M. 1985.
Pathogenicity and relative virulence of seven
Phytophthora species on Mahaleb and Mazzard cherry.
Phytopathology 75:221-226.
55. Yadava, U. L . , and Doud, S. L.
1980.
The short life
and replant problems of deciduous fruit trees.
Pages
1-116 in J. Janick, ed. Horticultural Reviews.
Vol 11.
AVA publ. Co., Inc., Westport, Conn.
56. Young, R. A. and Milbraith, J. A.
1959.
A stem
canker disease of fruit tree nursery stock caused by
Phytophthora. syrinaae.
Phytopathology 49:114-115.

PART I

PYTHIUM SPECIES ASSOCIATED WITH THE ROOTS OF SOUR CHERRY AND
THE EFFECT OF £. IRREGULARE ON THE GROWTH OF MAHALEB CHERRY

12

ABSTRACT

Isolations
early

for

Pythium

species were made

during

summer of 1984-1985 from 73 sour cherry trees

Michigan

orchards,

nursery,

and from soil samples from nine orchards.

was

commonly

from Mahaleb seedlings in a

heavy soil but not with roots of trees

with

light soil,

compared

in

Pythium
orchards

from

orchards

and populations in heavy soils were

to populations in light soils.

E.

12

commercial

associated with roots of trees from

with

the

high

irregulare was

isolated most frequently from discolored and necrotic cherry
roots

and

from

cherry

isolated included:
and

several

soils.

infrequently

isolated

Pvthium

discoloration

sp p . .

and necrosis were obtained

on

Growth reductions

seedlings exhibited symptoms as well.

seedlings inoculated with £.

months.
cause

None

irregulare died

after

but
of
3

This suggests that £. irregulare is not a primary
in the decline and death of cherry trees in

orchards,
cherry

In

Mahaleb

necrosis were greater when seedlings were flooded,

unflooded
the

species

a reduction in root and shoot growth and

seedlings inoculated with £. irregulare.
and

Other

£. sy.lvaticm n, E- ultimum. £. rostratum.

greenhouse trials,
root

orchard

but it may contribute to a reduction in growth of

trees

between
seedlings

Michigan

planted

Pvthium
when

on

heavy

soils.

No

and Phytoohthora was detected
£.

irregulare

castorum and Eh. megasoerma.

was

inoculated

interaction
on

Mahaleb
with

Ph.

14

INTRODUCTION

Root

rot

cherry
L.

is

Montmorency

trees ( P r m u s cerasus L . ) grafted on Prunus

(Mahaleb)

species
for

an important problem on

seedling

rootstocks

in

causal
drained

for

soils

soils

(1).

where

identified

as

Phytophthora

Although

damping-off

naturally

with

(11).

be

primary

heavy

poorly-

Populations of Pythium were high

the cause of

unpublished).
a

root rot on sites

agents

soils

of Phytoohthora were reported to

agents

cause

Three

of Armillaria reported to be primary causal

species

many

mahaleb

Michigan.

root rot on sites with light well-drained

Five

sour

root

tree

and

death

crown
(M.

rot

L.

infested orchard soils,

it is not

was

Smither,

Pythium has been reported (2)

of young Mahaleb seedlings

in

grown

considered

to
in
a

pathogen of sour cherry trees and the presence of Pvthium is
usually
Recent

ignored

for

Phytoohthora.

reports suggest Pythium may be involved in root

problems
The

when making isolations

on

role

of

apple (6,12),

particularly in replant

Pythium has not been

defined

and

rot

sites.

therefore

warrants further study.
The

overall objective of this study was to determine

Pythium.
rot

of

in addition to Phytoohthora.
sour

Subobjectives

cherry on sites with
were

to

isolate and

if

was involved in root

poorly

drained

identify

the

soils.
Pvthium

species associated with sour cherry trees and orchard soils,

15
to

test

the effect of the predominant Pvthium

irregulare

on

the

growth

of

Mahaleb

species

cherry,

P.

and

to

investigate the effect of £. irregulare and two Phytoohthora
species combined on the growth of Mahaleb cherry.

MATERIALS AND METHODS

Isolation

of

Pythium from cherry roots.

Root

pieces

were collected from sour cherry trees with reduced growth of
terminal
Kent,

shoots.

Mason,

The orchards were located in

Oceana,

and Van Buren counties.

trees were sampled per orchard.

Berrien,

Four to six

A total of 73 trees

from

12 orchards were sampled during the early summer of 1984 and
1985.

In

addition,

22

Mahaleb seedlings received by

a

local nursery from a supplier on the West Coast were sampled
prior to planting in Michigan.
The

samples

were

transported to

the

laboratory

in

plastic bags in an ice chest, soaked overnight in tap water,
then thoroughly washed
paper towels,
pieces

5

mm

to remove soil particles,

dried

on

and rinsed in sterile distilled water. Rootlet
in length

were pressed into corn

meal

agar

(CMA) (Difco) amended with 10 mg pimaricin (0.4 ml of a 2.5%
suspension),

250 mg ampicillin,

pentachloronitrobenzene
M O ) (6).
PARP),
samples

10 mg rifampicin, and 50 mg

(Sigma Chemical Co.,

Saint

Twenty-40 plates of amended corn meal agar
each

with 10 root pieces,

collected

from

each

were prepared from

tree.

The

plates

Louis,
(CMAroot
were

incubated at 21 C in darkness and examined regularly with

a

16

binocular

microscope for fungal growth.

transferred

from

Hyphal tips were

all Pythium colonies

to

fresh

CMA-PARP

plates and then to CMA prior to identification.
Isolation

Pythium from orchard soil. The population

of

Pythium

in soil from sour cherry orchards was estimated

by

surface

soil dilution plates (9).

taken

of

between the drip line and the trunk from each

trees per orchard.
with

a

were
of

ice

and stored at 4 C

until

processed.

5- and 1.68-mm-mesh sieves.
each bulk sample,
plated at 1:10,

replicate

through

A 1 g subsample was taken from

suspended in 0.2% water agar, and dilution
1:50, and 1:100 on fresh CMA-PARP selective

Diluted
of

a

Soil

samples were air dried and then passed sequentially

surface

in

Samples were transported to the laboratory in

chest

medium.

10

The cores were taken to a depth of 15 cm

Hoffer soil sampler and bulked for each tree

plastic bag.
an

Four cores of soil

the

samples (1 ml) were distributed over the
agar with a

plates

sterile

were made per

glass

dilution.

rod.

Three

After

42

hr

incubation in darkness, the soil was removed by washing each
plate

under

running

tap water and

the

counted. Hyphal tips were transferred
15

colonies

Pvthium

colonies

from the edge of 5 to

per sample to CMA-PARP plates and

then

after

incubation for 48 hr at 21 C to CMA.
Identification

of Pythium species.

Fungal structures were

examined after 1-2 wk and identifications were made based on
colony

morphology,

sporangia,

hyphal

growth rates,
swellings,

and

characteristics

oogonia,

antheridia,

of
and

17

oospores
The

using

the

key of Van der

identification

sylvaticum
each

of

the

Plaats-Niterink

heterothallic

(13).

species

Campbell and Hendrix was confirmed

by

£.

crossing

suspect isolate with strains ATCC 18195 and ATCC 18196

of £. sylvaticum on potato-carrot agar (13).
Effect on

the growth of Mahaleb

virulence
roots
was

cherry seedlings.

of nine isolates of £.

irregulare isolated

and soil in five orchards were compared.
conducted

in a growth chamber

seedlings.

The

basic

prepared

with

procedure

Mircetich and Matheron (10).

The

The

6-wk-old

was

from

as

test

Mahaleb

described

by

Inoculum of £. irregulare was

by growing each isolate at 21 C for 8 days on

200

ml vermiculite and 20 g oat kernels moistened with 100 ml V8

juice

Camden,

solution
NJ),

(200 ml V-8

2 g

CaCO^,

juice

and

(Campbell

800 ml

distilled

sterilized in 500 ml Erlenmeyer flasks.
washed
funnel
the

with

distilled water over cheesecloth in a

to remove unassimilated nutrients,

(1/1/1 by volume).
with

vermiculite/oat

replicates. The

minimum

was 18 C.

daytime

The nighttime

The seedlings were fertilized biweekly

(Peters Professional,
W.

plastic

received

Maximum

temperatures were 21 C with 14 hr of light.

at

soil/sand/peat

controls

mixture without fungus.

was

Buchner

then mixed

Seedlings were planted in 4 L

six

Co.

water)

The inoculum

rate of 20 cc per 1000 cc of sterilized

pots

Soup

20:20:20, Peters Fertilizer Products,

R. Grace and Co., Fogelsville, PA).

terminated after 18 wk.

The experiment was

Isolations from root tissue

were

18

made

on

roots

CMA-PARP selective medium.

and

shoots

repeated.

Dry weights

were recorded.

The

of

the

experiment

was

Data were analyzed by an analysis of variance for

a completely randomized experimental design.
Interaction

of

PzAhlum lr.ggfiVllar.fi and

Phytophthora

spp. .

Four isolates of £. irregulare (isolates B306, C6, J210, and
L215)

were used in single and combined treatments with

cactorum (B2)
M333).
Eh.

and two isolates of Eh.

Ph.

megasperma (M224 and

All isolates were from cherry orchards in Michigan.

megasperma M224 was avirulent and M333 was virulent

Mahaleb

cherry

in

Inoculum

of

irregulare and of

prepared

as described in the preceding section

kernels
grown

£.

a

previous

pathogenicity

test

Phytophthora

to
(1).

spp.

was

except

oat

were omitted from the mixture and the isolates were
for

5

wk.

Inoculum

was

mixed

per

1000

cc

sterilized soil/sand/peat as follows: control treatments,
cc

of

vermiculite

treatments,
without

20

cc

fungus;

and

without
fungal

containers

bench.

inoculum +

combined species

combined fungal inoculum.
L

fungus;

single
20

cc

treatments

40

species

vermiculite

treatments,

40

cc

The seedlings were planted in 1

and arranged in eight blocks on a

All

of

in one half of

each

greenhouse
block

were

flooded for 48 hr every 2 wk by immersing the pots in water.
The

experiment

temperature

was

conducted

during

was maintained at 21 C,

the

winter,

with 16 hr

of

The plants were fertilized as previously described.
experiment

was

terminated after 15 wk.

Isolations

the
light.
The
from

19

root tissue were made on CMA-PARP selective medium and on
Phytoohthora

selective

medium

(3).

£.

a

irregulare

populations in soil were monitored with a soil assay on CMAPARP selective medium.
were recorded.
analyzed

for

randomized
versus

The experiment was repeated.
a

2x8

isolate

split-plot

complete blocks.

nonflooding

involving

Dry weights of the shoots and roots

was

factorial

Data were

design

The main factor of
split

single isolates,

across

four

with

flooding
treatments

three treatments involving

of P.irregulare plus one isolate

of

one

Phytophthora.

plus an uninoculated control.

RESULTS

Pythium

species isolated from roots.

sylvaticum.
Pythium
and

spp.

from

the

irregulare
from
two

£.

ultimum Trow.,

£.

£.

irregulare.

rostratum

roots

of

Mahaleb

liners

was isolated most frequently.

orchards were located on sites with
orchards

and

were isolated from roots of sour cherry trees
(Table

1).

It was

isolated

trees in all orchards except N and U.

other

Butler,

£.

except

orchard

RW where

These

P.

latter

sandy

soil.

All

the

trees

were

declining due to X-disease, were located on sites with heavy
soils.
Pvthium
orchards

species isolated from soil.

The six

sour

cherry

on clay soils had Pythium populations estimated at

54.3 to 746.4 propagules per gram of soil (Table

2).

The

Table 1.

Species of Pvthium isolated from the roots of sour cherry trees with

reduced terminal growth or :?rom the roots of Mahaleb seedlings

Pvthium species isolated3
P. irreaulare
Year
1984

1985

Orchard
code

Fraction
of trees

B
E
J
L
N.
Nd
U

3/4
4/5
3/4
6/6
5/5
0/5
0/5

B
D
F
J
JC
L
P *
RW®
Xe
Ye

6/6
5/5
4/5
3/4
5/5
5/5
4/4
4/5
11/12
10/10

Totals

78/95

V*
1:1

1:1

22
—
36
24
20
1£
IS
72
79
IS
-

-

P. svlvatlcum

P. ultimum

Fraction
of trees

*

Fraction
of trees

1/4
0/5
2/4
2/6
1/5
0/5
0/5

1
4
4
—

0/4
0/5
0/4
1/6
2/5
0/5
0/5

0/6
0/6
2/5
4/4
2/5
2/5
0/4
0/5
10/12
8/10

0
O
9
8
7
19
0
0
-

0/6
0/5
1/5
2/4
0/5
2/5
1/4
0/5
11/12
0/10

34/95

20/95

P. rostratum
%

0

Fraction
of trees

0
1
-

1/4
0/5
0/4
1/6
1/5
0/5
0/5

0
0
7
10
0
7
IS
0
—

1/6
0/5
0/5
0/4
2/5
0/5
1/4
0/5
0/12
2/10

-

10/95

%

1

Pvthium

so d . “

Fraction
of trees

%

0
2
—

0/4
1/5
2/4
0/6
0/5
0/5
0/5

1
0
-

2
0
0
0
2
0
2
0
-

2/6
0/5
1/5
0/4
4/5
0/5
1/4
0/5
2/12
0/10

2
0
0
0
3
0
2
0
-

-

0
-

13/95

aSamples were collected in early summer
Twenty to 40 plates of CMA-■PARP medium (6)
containing ten rootlet or bark pieces were prepared from each tree. Two or more
fungal species were often isolated from the same tree.
“Values for Pvthium incidence are expressed as a percentage of the total number
of plated tissue pieces.
'"Combined total of remaining Pvthium spp.
“Orchards were located on sites with sandy soil.
el8olations were from Mahaleb liners from the Hest Coast.

Table 2.

Populations of P v t h i u m species as esti m a t e d by d i lution-plate counts

from soil taken from sour cher r y orchards

Pvthium p o p u l a t i o n by species

Year:

Orchard

(propagules per gram

1984

code:

(ppg))a

1985

ub

B

E

J

L

N

irreaulare

52.5

56.0

52.5

32.6

74.3

1.0

0.0

52.1

279.2

64.0

1.5

P. svlva t i c u m

6.1

5.4 122.6

12.5 157.4

0.0

1.0

25.2

17. 2

0.0

0.0

P. ultimu m

9.9

6.9

19. 8

3.2

52 .0

0.0

0.0

23. 1

259 .2

11.3

1.8

P . rostr a t u m

6.8

2.3

13. 1

6.0

52.4

0.3

1.0

1.9

18.0

4.2

0.3

Pvthium spp.c

0.8

6.1

10.9

0.0

100.5

1.7

0.6

16.1

0.0

0.0

0.0

54.3 437.0

3.0

2.6

746.4

79.5

3.5

P.

Total

76.1

76.4 219.0

Nb

J

119.3

L

P

RWb

a Samples were c ollected once in early summer.
E a c h v a l u e is the mean of ten
soil samples per orchard. E ach soil sample is the composite of four sub samples
collected a r ound one tree.
“Orchards were located on sites w i t h sandy soil.
c Combi n e d total of rema i n i n g Pvthium spp.

22

three
very

orchards on sandy soil (orchards N,
low

Pythium

populations

propagules per gram of soil.
species
from

estimated

U,

and RW)

had

at

2.6

3.5

£. irregulare was the

isolated most consistently (50% of all

clay soils in both years,

(19%) and by £.

to

followed by

ultimum (15%).

Pythium

propagules)

£.

sylvaticum

Estimates for £. sylvaticum

were high in soils from orchards J and N in 1984, and for £.
ultimum

in

soils from orchard N in 1984 and orchard L

1985. £. rostratum (6%) was found

in

primarily in orchard N in

1984.
Effect of Pythium irregulare on growth of Mahaleb seedlings.
The dry weights of shoots and roots of Mahaleb seedlings
soil

inoculated

(£=0.05)
seedlings

with

were

significantly

reduced over the dry weights of the

noninoculated

(Table

obtained

in

3,

£.

irregulare

Figure

1).

both experiments,

Similar

results

were

although dry

weights

were

lower for all plants in experiment 2.
with

£.

Seedlings inoculated

irregulare exhibited many discolored rootlets

diminished
exhibited

root

systems

while

noninoculated

root

and shoot growth were reduced,

seedlings were killed.

There was no consistent

in

isolates.

virulence

reisolated

between

£.

and

seedlings

healthy roots and a greater density of

Although

in

rootlets.

none of

the

difference

irregulare

from the roots of plants grown in infested

was
soil

but not from roots of plants grown in noninfested soil.
Interaction of
initial

Pythium

analysis

irregulare and Phytophthora.

of variance,

no significant

In

an

differences

Table 3. Effect of nine Isolates of Pvthium i rregulare on the growth of M a h a l e b seedlings

growing in unflooded soil
Experiment 1
Shoots3
■Isolate
number®

Dry
weight
(9)

Relative
growth
<*>

Experiment 2

Roots3
Dry
weight

(g)

Rootsa

Shoots3

Relative
growth
(*)

Dry
weight
(9)

Relative
growth
(«)

Dry
weight

(g)

Relative
growth
(*)

10.4 abc

82

3.0

be

73

2.7 b

45

1.4 b

48

B306

9.0 be

71

2.9

be

71

2.6 b

43

1.5 b

52

C6

6.1 c

48

2.6

be

63

2.5 b

42

1.4 b

48

C304

6.1 c

48

2.7

be

66

3.3 b

55

1.6 b

56

J210

6.0 c

47

2.6

be

63

2.7 b

45

1.2 b

41

J301

6.0 c

47

2.2

c

54

2.3 b

39

1.5 b

52

L215

7.8 be

61

2.7

be

66

2.6 b

43

1. 2 b

41

L304

8.3 be

65

2.9

be

71

4.9 ab

82

2.3 ab

79

P309

8.5 be

67

3.5

ab

85

3.8 ab

63

1.9 ab

66

100

4.1

a

100

6.0 a

100

2.9 a

100

B17

Control

12.7 a

aSix-wk-old Mahaleb seedlings were transplanted into soil infested with 20 cc of inoculum
per 1000 cc of soil. The experiment was conducted in a growth chamber for 3 mo.
There were six replicates.
“The letter preceding each isolate number codes for the orchard of origin.
cValues followed by the same letter do not differ significantly (P=0.05) using Duncan's
multiple range test.

24

Figure 1.
Roots of representative Mahaleb seedlings
grown
in a growth chamber for 18 wk in a soil/sand/peat mixture
artificially infested with Pythium irregulare.
Soil in the
pots was (a) uninfested;
or infested with E.
irregulare
isolated from orchard B,
(b) root and (c) soil;
orchard C,
(d)
root and (e) soil;
orchard J,
(f) root and (g)
soil;
orchard L,
(h) root and (i) soil;
and orchard P, (k) soil.
The treatments were not flooded.

25
were

detected

inoculated

in

the dry

with

the

weights

four

of

isolates

Mahaleb
of

Therefore,

only

irregulare

were presented in Tables 4-5 to

seedlings

P.irregulare.

the results obtained with isolate C6 of p.
illustrate

the

results obtained with all Pvthium isolates.
Dry

weights

(P=0.01)

of

Mahaleb

seedlings

were

significantly

reduced in both experiments by flooding for 48

every 2 wk (Table 4).

hr

In the uninoculated controls flooding

reduced shoot dry weights by 43.1-46.7% and root dry weights
by 20.7-40.7% (Table 5, Figure 2).

Significant differences

(P=0.01)

inoculation

were

in

dry weights among the

detected

interaction
the

in

both

between

flooding

under

but

no

treatments was detected by

the

significantly

seedlings

analysis

reduced shoot and root dry

inoculated

with

P.

Dry weights
i rregyilare

and

were not significantly different from

dry

seedlings inoculated

of

alone,

except

reduce

the

treatments
significantly

P h .megasperma

weights of shoots as

and

it

failed

with

either

to

reduce

the control (Table

much

as

root
5).

the

2-3.

No seedlings inoculated with P.

were killed (Table 5).

a

fungus
not
other

dry

weights

The

reduced

growth from roots and shoots from inoculation is evident
Figures

of

the

M224 in experiment 1 did

dry

from

of

weights

Phytophthora sp.
weights

and

p. irregulare. Ph. megasperma. and Ph.

flooded and unflooded conditions.

Mahaleb

significant

any of the inoculation treatments

variance (Table 4).
cactorum

experiments,

treatments

in

irregulare

In inoculation treatments involving

26

Table

4. Analysis of variance for shoot and root dry weights of Mahaleb

seedlings growing In soil infested with Pvthium irregulare and two
Phytoohthora species in single and combined treatments under flooded and
unflooded conditions

Source of variation

Degrees of
freedom

Shoot dry weight

Root dry weight

Mean
square

Mean
square

F
value

F
value

Experiment 1
Replication
Flooding
Error
Treatments
Treatment x flooding
Error

7
l
7
7
7
98

1.424
131.828
1.713
14.933
0.798
0.935

0.83
76.95** a
15.97**
0.85

0.432
0.79
77.969 142.95**
0.545
2.244 11.16**
2.04
0.411
0.201

Experiment 2
Replication
Flooding
Error
Treatments
Treatment x flooding
Error

7
1
7
7
98

3.526
1.28
124.228 45.04**
2.758
10.735
9.51**
1.196
1.06
1.129

a** indicates that values were significant (P=0.01).

1.443
37.303
0.672
1.940
0.362
0.524

2.15
55.50**
3.70**
0.69

Table 5.

Effect of Pvthium Irregulare (P.) and two Phytophthora (Ph.) species on the growth of Mahaleb seedlings in

single and combined treatments under flooded and unflooded conditions
Shoots
Unflooded
Fungus and isolate
number

Dry
Relative
weight growth*1
(*)
(g)

Roots

Flooded
Dry
weight
(g)

Unflooded

Relative Mean
growth® dry
(*)
wesight

Dry
weight
(g)

Relative
growth®
(X)

2.1
2.7
2.1
2.3
2.5
1.9
2.0
2.7

77
101
93
85
92
70
71
100

Flooded
Relative Mean Dead
Dry
weight growth® dry
weight
(*)
(9)

Experiment 1
P. irreaulare C6
Ph. meaasoerma M224
Isolates C6 + M224
Ph. megasperma M333
Isolates C6 + M333
Ph. cactorum B2
Isolates C6 + B2
Control

3.8
4.6
3.0
3.0
3.5
2.8
2.7
5.8

Flooded means

3.6

65
79
52
. 52
60
48
47
100

1.4
2.8
1.3
1.2
1.0
1.2
1.0
3.3

42
85
39
36
30
36
30
100

2.5 cc
3.7 b
2.1 c
2.1 c
2.2 c
1.9 c
1.8 c
4.5 a

1.6

2.3

1.1
1.3
0.6
0.4
0.3
0.5
0.4
1.6

33
81
37
25
19
31
25
100

1.4 bc
2.0 a
1.3 b
1.4 b
1.4 b
1.1 b
1.2 b
2.2 a

0
0
0
1
3
1
2
0

48
61
48
52
39
48
43
100

1.8 b
1.7 b
1.7 b
1.8 b
1.4 b
1.7 b
1.6 b
2.6 a

0
0
0
3
1
2'
1
0

0.7

Experiment 2
P. irrequlare C6
Ph. meaasoerma M224
Isolates C6 + M224
Ph. megasperma M333
Isolates C6 -1-M333
Ph. cactorum B2
Isolates C6 + B2
Control

3.3
2.9
3.4
3.7
3.0
3.9
3.7
6.2

Flooded means

3.8

53
47
55
60
48
63
60
100

1.4
1.8
1.8
1.7
1.1
1.8
1.3
3.3
1.8

42
54
54
51
33
54
39
100

2.4 b
2.4 b
2.6 b
2.7 b
2.1 b
2.8 b
2.5 b
4.7 a

2.5
2.0
2.3
2.4
1.9
2.3
2.2
2.9
2.3

86
69
79
83
65
79
76
100

1.1
1.4
1.1
1.2
0.9
1.1
1.0
2.3
1.2

aSix-wk-old Mahaleb seedlings were transplanted in soil infested with 20 cc of.inoculum per 1000 cc of soil, flooding
was for 48 hr every 2 wk. The experiment was conducted in a greenhouse for 15 vie. There were eight replicates.
®Values are expressed as a percentage of control.
cValues followed by the same letter do not differ significantly (P=0.05) using Duncans Multiple Range test.

28

gsufl

^^wrTTJrr^'• r

' \ V i r ; ?f '*.iSK\ » '
<

*

«

<

i ft. ■ i

<-

'

,

Figure 2.
Shoots
(A) and roots
(B) of
representative
Mahaleb seedlings grown in
a greenhouse for 3 mo
in a
soil/sand/peat mixture.
Soil
in the
pots was (a) uninfested
and non-flooded,
(b) uninfested and
flooded,or flooded and
infested with (c) pythium.
irregulare C6, (d) Phytoohthora.
megasperma. avirulent isolate M224, (e) £. irregulare C6/Ph.
megasperma. avirulent M224 (f) Ph. megasperma. virulent M333
(S ) P- irregulare C6/£h- megasperma. virulent M333

29

Figure 3.
Shoots
(A) and roots
(B) of representative
Mahaleb seedlings grown in a greenhouse for 3 mo in a
soil/sand/peat mixture.
Soil in the pots was (a) uninfested
and flooded,
or flooded and infested with
(b) Pvthium.
irregulars C6,
(c) Phytophthora. cactorum B2,
(d ) p.
irregulare C6/fh. cactorum B2.

30

a

Phytophthora sp.,

the

experiment,

1-3 seedlings were dead at the end

except

no

seedlings

were

killed

of
in

treatments involving Ph. megasperma M224 (Table 5).
Soil

populations

propagules
270

of £.

irregulare

J210

averaged

per gram of soil for the unflooded treatment and

propagules per gram of soil for the flooded

P.

irregulare

Ph.

treatment.

was not reisolated from soil taken from

uninoculated treatment,
and

250

cactorum

but p.

were

the

irregulare, Ph. megasperma.

reisolated

from

the

single

and

combined treatments (Table 6).

DISCUSSION

There was an universal association of Pvthium species and
particularly of p. irregulare with sour cherry trees located
on

heavy soils but not with trees located on

sandy

soils,

and Pythium populations in heavy soils were high compared to
populations
associated
States

in

sandy soils.

Pythium species have

with peach tree decline in the

(4,8).

One

southern

study correlated Pvthium

been
United

populations

with decline symptoms (4), while another using total Pvthium
found
Mahaleb
trials

no relationship (8).

p.

irregulare failed to

cherry seedlings in greenhouse and

chamber

suggesting that Pythium is not the primary cause

death of cherry trees in Michigan.
consistently

and

However P.

of

irregulare

caused reductions in shoot and root growth

these trials indicating that P.
cherry

growth

kill

in

irregulare is a pathogen of

may contribute to the reduction

in

growth

of

31

Table

6.

Reiso l a t i o n of Pvthium irregulare and

two Phytophthora species from M a h a l e b seedlings
(experiment 2) grown for 15 weeks wit h periodic
flooding

Reisolations3

Fungus and isolate
number

P. irregulare*3

Phytoohthora0

J210

8/8

0/8

P h . megasperma M224

0/8

5/8

P h . megasperma M224/
P. irregulare
J210

7/7

3/7

Ph. megasperma M333

0/5

5/5

P h . megasperma M333/
P. irregulare
J210

6/6

4/6

Ph. cactorum

B2

0/6

6/6

Ph. cactorum
P . irregulare

B2/
J210

6/6

6/6

1/8

0/8

P. irregulare

Control

aFraction of Mahaleb seedlings from which P.
irregulare or Phytophthora were reisolated.
“Reisolations were made on CMA-PARP selective medium.
cReisolations were made on Phytophthora selective
medium.

32

cherry trees.
Recently

Bielenin

and

Jones

(1)

identified

Ph.

megasperma. £h- gflytexvm, Eh- QambiYQKa (Petri) Busiman, £hcryptogea Pethyb.
as

causal

trees

agents in the decline and death of

in

soils.

Michigan

located on sites

with

sour

cherry

poorly

drained

In the present study, infection by £. irregulare in

combination
increase
In

and Laff., and £h. syringae (Kleb.) Kleb.

with Eh-

the

megasperma and Eh-

death of seedlings over

cactorum did

not

Phytophthora

alone.

single and combined treatments reductions in dry

weight

were similar for £•
that

competition

between
trees

for

infection sites

may

indicating

have

the genera in the combined treatments.
£.

occurred
In mature

irregulare appears to be confined to the

roots (7),
(1.14)

irregulare and Phytophthora

while Eh.

and £h.

lateral

cactorum is associated with the crown

megasperma with the lateral and main

roots

(1.11.14).
Pythium has been implicated in the apple replant
(5,12)
and

and has been repeatedly shown to cause discoloration

necrosis

development
trials.

of
of

In

demonstrated

lateral roots,

the

present study,

significant
in

necrosis

and

apple seedlings in

statistically

Root

disease

inhibition

greenhouse

of

inoculation

necrosis of roots

reduction

inoculation trials

with

in

root

and

growth
Mahaleb

were
cherry.

and reduced weights were obtained even

seedlings

were

inoculated but not

flooded,

reduction

in growth and deterioration of young

a

although
roots

when
the
were

33

greater with flooding.

However, there was no indication of

a replant problem in the orchards studied here, as growth of
the

young

Stunting,

trees

was

good

for

the

first

few

years.

decline and death of the trees developed when the

trees came into bearing.

LITERATURE CITED
1.

Bielenin,
A.,
and Jones A.
L.
1988.
Prevalence
pathogenicity of Phytophthora spp. from sour cherry
trees in Michigan.
Plant Dis. 72:473-476.

2.

Fliegel, P., Parker, K. G . , and Mai, W. F.
1963.
The
fungous flora of nonsuberized roots of poorly growing
cherry trees.
Phytopathology 53:1368-1369.

3.

and

Harris, D. C . , and Bielenin, A.
1986.
Evaluation of
selective media and bait methods for estimating
Phytophthora cactorum in apple orchard soils.
Plant
Pathol. 35:365-374.

4.

Hendrix, F. F . , Jr., Powell, W. M . , and Owen, J. H.
1966.
Relation of root necrosis caused by Pythium
species to peach tree decline.
Phytopathology 56:12291232.

5.

Jaffe«; B: A., Abawi, G. S., and Mai, V!. F.
1982.
Fungi associated with roots of apple seedlings grown in
soil from an apple replant site.
Plant Dis. 66:942944.

6.

Jeffers,
S. N . , and Martin, S. B. 1986.
two
media selective for Phytophthora
species.
Plant Dis. 70:1038-1043.

7.

Miller, C. R . , Dowler, W. M . , Petersen, D. H . , and
Ashworth, R. P.
1966.
Observations on the mode of
infection of Pythium ilLtimum and Phytophthora cactorum
on young roots of peach.
Phytopathology
61:357-360.

8.

Mircetich, S. M.
1971.
The role of Pythium in feeder
roots of diseased and symptomless peach trees and in
orchard soils in peach tree decline.
Phytopathology
61:357-360.

Comparison of
and
Pythium

34
9.

Mircetich, S. M . , and Kraft, J. M. 1973.
Efficiency of
various
selective
media
in
determining
Pvthiurn
populations in soil.
Mycopathol. Mycol. Appl. 50:151161.

10. Mircetich, S. M . , and Matheron, M. E. 1976.
Phytophthora root and crown rot of cherry trees.
Phytopathology 66:549-558.
11. Proffer, T. J . , Jones, A. L . , and Ehret, G. R. 1987.
Biological species of Armillaria isolated from sour
cherry orchards in Michigan.
Phytopathology 77:941-943.
12. Sewell, G. W. F. 1981.
Effects of Pythium species on
the growth of apple and their possible causal role in
apple replant disease.
Ann. Appl. Biol.
97:31-42.
13. Van der Plaats-Niterink, A. J.
1981.
Monograph of the
genus Pythium. Studies in Mycology No 21 Centraal
Bureau voor Schimmelcultures, Baarn. 242 pp.
14. Wilcox, W. F . , and Mircetich, S. M. 1985.
Pathogenicity and relative virulence of seven
Phytophthora species on Mahaleb and Mazzard cherry.
Phytopathology 75:221-226.

PART II
SPECIES AND POPULATIONS OF PYTHIUM ISOLATED FROM SOUR CHERRY
ORCHARD AND

WOODLAND SOILS IN MICHIGAN

35

ABSTRACT

Pythium

populations

were

high (54.3 to

746.4

viable

propagules per gram of soil) in eight orchards on clay

soil

and

four

low

orchards
1985.
to

(1.2 to 3.5 propagules per gram of soil)

in

on sandy soil sampled in western Michigan in 1984Populations were also high in two woodlands adjacent

orchards in 1986,

central

Michigan

Michigan
species.

were

in

found

and in six woodlands in
1987.

Orchards

and

to differ in composition

of

in

Pythium

In soil from orchards, £. irregulare was isolated

sylYatis.um (26%),

£.

followed by £.

ultimum (13%), £. rostratum (6%), and

Pythium group 'HS' (8%).

In soil from woodlands,

’HS* was isolated most frequently (60%)

Ezthi.um

and

woodlands

most frequently (47% of Pythium propagules),

group

western

group ’X* (29%),

group

’Y ' (9%).

soils

of all woodlands,

£.

rostratum (2%),

Pythium

followed
and

by

Pythium

The first two species were isolated from
while the latter two species

isolated from two woodland sites.

No

£.

were

irregulare.

£.

sylvaticum. or £. ultimum were isolated from woodland soils.
Populations of £.

irregulars. £. sylvaticum. and £. ultimum

in two orchards sampled over 2 yr and one orchard over 1 yr,
varied independently.

37

INTRODUCTION
cherry ( Prunus cerasus L.

Sour

southwestern

Michigan

are planted on sites

were beech-maple woodlands.
orchards
decline

Montmorency) orchards in
that

formerly

It was observed that trees in

planted on heavy clay soils

frequently

exhibited

symptoms of poor growth and decreased longevity and

had root systems that exhibited extensive discoloration
decay

of

lateral roots.

Trees in orchards on sandy

and
soils

grew well and had root systems with little discoloration
lateral

roots.

isolated

Pythium irregulare (Buisman) was frequently

from roots of trees with discolored

lateral

roots,

growth

of

conducted

and Isolates of £.

Mahaleb
in

of

the

seedlings

greenhouse

necrotic

irregulare reduced

in
and

and

pathogenicity
growth

chamber

the

trials
(17).

Seedlings of Prunus mahaleb L. are the most common rootstock
used for propagation of cherry trees.
Although

Pythium

species are soilborne pathogens

wide range of crops (6,19),
an

soils

undisturbed

are

sites

been

tree decline

(7,12).

One

study

Pythium populations with decline

(11).

from

have

correlated

using

soils

species

with

another

in

Pythium

associated

while

peach

Species common

infrequently present in

(18).

a

they are poor competitors among

established soil microflora (2,6).

cultivated

of

total Pythium found

symptoms
no

(7),

relationship

Pythium species isolated from peach decline orchards

38
in

Georgia

were

also isolated from forest

southeastern United States (4).
of

studies

soils

in

the

There have been no reports

on the composition of

populations

of

Pvthium

species in cherry orchard soils, nor have there been reports
of comparisons in Pythium populations between cherry orchard
soils and woodland soils.
The

objectives

species
from

of this study were

to

investigate

and populations of Pythium in clay and sandy

cherry

orchard

sites

and

in

soil

from

the
soils

woodlands

adjacent to orchards.

MATERIALS AND METHODS
Sampling
clay

sites.

Eight commercial sour cherry

orchards

on

soil exhibiting poor growth and two on sandy soil with

healthy trees were sampled in 1984 and four orchards on clay
soil

and

two on sandy soil were

sampled

orchard was sampled once in early summer.
of

the

orchards

on

in

1985.

Each

In addition, two

clay soil (orchards J

and

were

a

third

sampled

four times both in 1984 and in 1985,

orchard

on clay soil (orchard P) was sampled four times

1985.

and

L)

in

Two sour cherry orchards and adjacent woodlands were

sampled

once in early summer in 1986 and six woodlands were

sampled once in October in 1987. The woodlands were composed
primarily

of

beech (Fagus grandifolia

(Afi££ rubrwn L.),
maple (A-

Ehrh).,

red

maple

silver maple (A- saccharinum L . ) and hard

saccharum Marsh).

All sites were in Berrien and

Van Buren counties of western Michigan except woodland sites

39

MSU F and MSU G were located in East Lansing (Ingham county)
in central Michigan.
Isolation
Pythium

of Pythium species from soil. The populations
species

plates (13).
(CMA)

were estimated by

surface

soil

of

dilution

The isolation medium used was corn meal agar

(Difco)

ampicillin,

amended
10

with

mg

pentachloronitrobenzene

10

mg

pimaricin,

rifampicin,
(CMA-PARP)

and

per liter

250

mg

50

mg

(9).

Four

cores of soil were taken between the drip line and the trunk
from

each

sampled

of 10 trees per orchard.

like orchard sites,

Woodland

sites

were

except 5 rather than 10

per site were sampled in 1987.

trees

The cores were taken to

a

depth of 15 cm with a Hoffer soil sampler and mixed for each
tree in a plastic bag.

The samples were transported to the

laboratory

chest

in

an

ice

stored

at

processed.

Soil

sequentially

through 5- and 1.68-mm-mesh sieves.

subsample

samples

and

until
passed

A

1

g

from each bulk sample was suspended in 0.2% water

selective medium.

distributed
Three

C

were air dried and then

agar and dilution plated at 1:10,
CMA-PARP

4

over

replicate

the
plates

1:50,

and 1:100 on fresh

Diluted samples (1

surface with a
were made per

dilution (nine plates per tree).

sterile

ml)

were

glass

rod.

subsample

and

per

After 42 hr incubation in

darkness at 21 C, the soil was removed by washing each plate
under

running tap water and the number of Pythium

counted.

colonies

40
Identification
15

of Pythium species.

Hyphal tips from 5

colonies per sample were transferred to CMA-PARP

and

then,

after

incubation

for 48 hr at 21

C,

plates
to

Growth rates were recorded after 24 hr incubation on CMA
25

C.

to

CMA.
at

Fungal structures were examined after the cultures

had grown for 1-2 wk.

Identifications of the various Pythium

species were made with the aid of the key of Van der PlaatsNiterink (19).

Identification of the heterothallic species

£. sylvaticum was confirmed by crossing each suspect isolate
with strains ATCC 18195 and ATCC 18196 of £.

sylvaticum

on

potato-carrot agar (19).

RESULTS
Pythium

species identified.

colony

morphology,

sporangia,

£.

Hendrix,
group

irregulare.

oogonia,
£.

were as described by

Two

identified,
were

swellings,

and charachteristics
antheridia,

sylvaticum

Campbell

of
and
and

£. ultimum Trow . , £. rostratum Butler, and Pythium

'HS*

(19).

growth rates,

hyphal

oospores.

Identifications were based on

made

groups

of

Pythium

Van

der

Plaats-Niterink

isolates

could

not

be

as oogonia were not produced, even when crosses
on

potato-carrot agar.

These

Pythium group ’X* and Pythium group ’Y'.

were

designated

Colony morphology

was radiate on CMA although Pythium group ’X' had a variable
rosette

pattern.

Growth

rates of Pythium group

Pythium group *Y' were 20 and 13 mm,
at

25

C on CMA.

Pythium group

respectively,
’X'

produced

’X'

and

in 24 hr
numerous

41

terminal

and

intercalary

hyphal

diameter

on CMA and in water.

swellings

20-25

Pythium group 'Y'

m

in

produced

sparse, ovoid intercalary hyphal swellings on CMA.
Pythium
sour

populations

in soil.

Samples of soil

from

cherry orchards on clay soils had Pvthium

estimated
(Table
very

at

54.3

1).
low

populations

gram

of

soil

Samples from four orchards on sandy soil
Pythium

propagules

to 746.4 propagules per

eight

populations

per gram of soil.

estimated
£.

at

1.2

had

to

irregulare was

3.5

isolated

most consistently (47% of all propagules) from clay soils in
both years,
(13%).

followed by £. sylvaticum (26%) and

Estimates

for

£. ultimum

these latter species were

high

soils of some orchards but not in both years.

£.

(6%)

primarily

and

Pythium group ’HS*

(8%) were found

in

rostratum
in

orchard N in 1984, and in orchard C in 1985.
The

seven

estimated
2).

woodland

sites

had

Pvthium

populations

at 180 to 1153 propagules per gram of soil (Table

Pvthium group ’HS' and Pvthium group ’X' were isolated

from soil taken from each of the sites.
was

isolated

group

’X'

most frequently (60%),

(29%);

£.

rostratum (2%)

woodland J in 1986 and MSU F in 1987;
(9%)

was

Pythium group ’HS'
followed
was

by

Pvthium

isolated

from

and Pvthium group ’Y'

isolated from woodland sites C and J in 1986

and

site B in 1987.
Sequential
over
season

isolations from soil taken from two

two growing seasons and one orchard over
showed

that populations of Pythium

one

species

orchards
growing
varied

42

Table

1.

Populations of Pvthium species as estimated by dilution-plate

counts from soil taken from sour cherry orchards planted on two soil types

Pvthium population by species (propagules per gram (ppg))a
Orchard Soil
£•
E*
E>
Ecode
type irregulars sylvaticum ultimum rostratum

E.

E.

’HS*

spp.

Total
(PPg)

1984 growing season
B

Clay

52.5

6.1

9.9

6.8

0.0

0.8

76.1

C

Clay

26.2

26.7

0.0

0.0

2.8

0.0

55.7

6

Clay

56.0

5.4

6.9

2.3

6.1

0.0

76.4

H

Clay

52.3

17.4

26.2

5.0

23.7

0.0

124.6

J

Clay

52.5

122.6

19.8

13.1

10.9

0.0

219.0

L

Clay

32.6

12.5

3.2

6.0

0.0

0.0

54.3

P

Clay

47.8

0.8

16.4

8.2

0.0

1.5

74.7

N

Clay

74.3

157.4

52.4

52.4

100.5

0.0

437.0

N

Sand

1.0

0.0

0.0

0.3

1.7

0.0

3.0

0

Sand

0.0

1.0

0.0

1.0

0.6

0.0

2.6

1985 growing season
J

Clay

52.1

25.2

23.1

1.9

16.1

0.0

119.3

L

Clay

279.2

17.2

259.2

18.0

0.0

0.0

746.4

P

Clay

64.0

0.0

11.3

4.2

0.0

0.0

79.5

C

Clay

67.1

189.5

0.0

26.1

160.5

0.0

443.2

C

Sand

0.6

0.6

0.0

0.0

0.0

0.0

1.2

RW

Sand

1.5

0.0

1.8

0.3

0.0

0.0

3.5

Each value is the mean of ten soil samples per orchard.
Each soil
sample is the composite of four sub-samples collected around one tree.
Sites were sampled on one occasion.

Table

2.

Populations of Pvthium species as estimated by dilution-plate

counts from soil taken from sou]? cherry orchards and woodlands

Pvthium population bv species (prooaoules per crram fppa))a
Site

Soil
P.
P.
P.
P.
type irrequlare svlvaticum ultimum rostratum

P.
'HS'

P.
'X'

P.
•Y~

■total
(PP9)

Clay
Clay
Clay
Clay

0
238
0
165

0
147
6
258

240
1153
181
900

102
0
0
0
0
0

340
420
260
260
180
560

May 1986
C
C
J
J

orchard
woodland
orchard
woodland

46
0
72
0

60
0
<17
0

0
0
30
0

11
0
0
51

123
768
26
426

0
0
0
0
0
0

0
0
0
0
0
0

0
0
0
0
18
0

204
262
130
88
126
504

October 1987 Woodland sites
Bb

Sand
Clay
H
Sand
Lc
Clay
MSU F Sandy loam
MSU Gb Sandy loam
C°

0
0
0
0
0
0

34
168
130
132
36
56

aEach value is the mean of ten soil samples taken in 1986 and five soil samples
in 1987.
Each soil sample; is the composite of four subsamples collected
around one tree.
PSites were adjacent to sour cherry orchards.
cSite was not close to a cheriy orchard.

44

independently

of

each other (Figure 1-3).

Estimates

total Pythium populations in soil from orchard J
from

a

high

of 313.2 propagules per gram in October

1);

fluctuated

low of 107.0 propagules per gram in July 1984 to

from

1984

orchard L,

1985

and

from 54.3 propagules per gram in

from orchard P,

from

a

(Figure

to 1009.0 propagules per gram in October 1985

2);

of

May

(Figure

79.5 propagules per gram in

May 1985 to 351.1 propagules per gram in August 1985 (Figure
3). .

DISCUSSION

Soils from orchards and from woodlands in Michigan

were

found to differ in their composition of Pythium species.
Pythium group ’HS' were isolated

rostratum

and

habitats,

indicating that these species were competitive in

both cultivated and woodland soils.
the

most

frequent isolate

from

farmland soils in England (1,5),

isolated

worldwide

woodland.

parkland;

and

has

£. rostratum has

been found

to

has
be

pathogenic

or weakly pathogenic on a wide variety of

(19).

irregulare.

£.

isolated
soil

from

£.

distributions

a

nonhosts

sylvaticum. and £. ultimum were

from soil from orchards but were not
woodlands.

and

however its pathogenicity

from a variety of habitats (1,5,18),

distribution,

both

Pythium group ’HS' was

to crop plants has not been investigated.
been

from

£.

All three species

detected

have

in

worldwide

and have been isolated from a wide variety of

45

200

150-

- P.irregulare
- P.sylvaticum
. P.ultimum
- Pvthium spp.

O)
O.

a

100-

M ay

Ju l

Aug

O ct

M ay

Jun

Aug

Oct

TIME
Figure 1. Fluctuations in population (propagules per gram of
soil)
of Pythium species as estimated by dilution-plate
counts for soil samples taken from sour cherry orchard J in
southwest Michigan during 1984 and 1985.
Species identified
were £. irregulars,
£• szlyflticum, £. ultimum. and Pythium
spp. .

46

800

700-

600-

500 -

400 -

200-

io o H

E

"T "
May

T
Jul

— I---------P' "
I
Aug Oct
May

1 ....... 1
Jun
Aug

I 1
Oct

TIME

Figure 2. Fluctuations in population (propagules per gram of
soil)
of Pvthium species as estimated by dilution-plate
counts for soil samples taken from sour cherry orchard L in
southwest Michigan during 1984 and 1985.
Species identified
were £.
irregulare, £. sylvaticum, £. ultimum. and Pythium
spp. .

47

200

150O)
a

a

^

ioo-

X
I—
>Q_
50-

May

Ju l

Aug

O ct

TIME
Figure 3.
Fluctuations in population (propagules per gram
of soil) of Pythium species as estimated by aiiution-plate
counts for soil samples taken from sour cherry orchard P in
southwest Michigan during 1985.
Species identified were I)
£.
irregulars,
S) £.
ay.lgatis.ma.
U) £.
ultimum. and P)
Ezthlum s p p . .

48
hosts (19).

Among deciduous orchard crops,

is reported as pathogenic to apple (8,16),
and

to cherry (17);

£.

P. irregulare

to peach (7,12),

sylvaticum to apple (16);

and

£.

ultimum to apple (3) and to peach (7,12).
The
sites

Pythium species found in soils from
were

seven

similar regardless of soil type,

orchard sites,

and location in Michigan.

woodland

proximity

The

to

predominant

species of trees in all woodland sites were beech and maple,
indicating

that

the areas had previously belonged

to

the

beech-maple climax community, which was common over southern
Michigan.
been

The trees in the sites had obviously previously

clean cut or selectively felled,

varying degrees of disturbance,
orchard

sites.

between

the

There

and the sites showed

but had never been used

appears to be a stable

isolated Pvthium species and

as

association

the

beech/maple

community as indicated by the similarity between the Pvthium
species isolated from diverse counties in Michigan.
Populations

of Pvthium species in soils taken from

cherry

orchards

were high in clay soils and low

soils.

The physical and chemical properties of soil

Pythium populations.

sour

in

sandy
affect

Pythium populations were reported

to

be higher under conditions of high soil moisture (1, 11, 18)
and

high

conditions
and

soil

soil organic content (10),
(5).

and lower under

Clay soils favor retention of

organic

matter

in the

soils

of

acid

moisture,

orchards

was

increased due to mechanical cultivation.
Populations

of

Pythium species from

orchards

on

clay

49
soils varied over time.
bean

field

response

soils

to

Populations of Pythium species in

were reported to vary

temperature

organic matter (10).
parkland,

and

(10,14),

and

independently

in

incorporation

of

The predominant species in woodland,

farmland soils in England were

reported

to

because

£.

show seasonal fluctuations in population (1).
This

study

irregulare

was

seedlings

soil

(17).

found

to

implications

reduce

the

growth

chamber

of

studies.

appears at 10 fold greater population
from

Mahaleb

orchards on clay soils than on

£.

densities

sandy

soils

It may limit the growth of cherry trees planted

sites

with

clay

possibility
sandy,
Pythium

would

well

species.

increase

One

method

for

avoiding

be to establish new cherry

this

orchards

Additionally the

on

on

pathogenic

species were not detected in woodland soils even on
so on newly cleared sites on clay soils it

possible

present

soil.

drained sites.

clay soils,
be

practical

in greenhouse and growth

irregulare
in

has

to prevent the introduction of
However

in

it

woodlands

these

Pythium

is possible that these species
at

under cultivation.

undetectable

levels

may

and

are
could

This is in contrast to

root

rot

caused by Armillaria species which are endemic on

pine

and

oak

and

cause disease in orchards

planted

cleared from these woodlands in Michigan (12).

on

sites

50

LITERATURE CITED

1.

Ali-shtayeh, M. S., Lim-ho, C. L . , and Dick, M. W. 1986.
The phenology of Pythium (Peronosporomycetidae) in soil.
Journal of Ecology 74:823-840.

2.

Barton, R. H.
1958.
Occurrence and establishment of
Pythium in soils.
Trans. Brit, mycol. Soc. 41:207-

222.
3.

Bielenin, A., Borecki, Z., and Millikan, D. F. 1976.
Identification of Pythium ultimum in the collar rot
complex of apple.
Phytopathology 66:127-129.

4.

Campbell, W. A. and Hendrix, F. F. 1967.
Pythium and
Phytophthora species in forest soils in the southeastern
United States.
Plant Dis. Rep. 51:929-932.

5.

Dick, M. W . , and Ali-shtayeh, M. S. 1986.
Distribution
and frequency of Pythium species in parkland and
farmland soils.
Trans. Br. mycol. Soc. 86:49-62.

6.

Hendrix, F.F.Jr., and Campbell, W.A.
1973
Pythiums
as plant pathogens. Ann. Rev. Phytopathology 11:77-98.

7.

Hendrix, F. F . , Jr., Powell, W. M. , and Owen, J. H.
1966.
Relation of root necrosis caused by Pvthium
species to peach tree decline.
Phytopathology 56:12291232.

8.

Jaffee, B. A., Abawi, G. S., and Mai, W. F.
1982.
Fungi associated with roots of apple seedlings grown in
soil from an apple replant site.
Plant Dis. 66:942944.

9.

Jeffers,
S. N . , and Martin, S. B. 1986.
two
media selective for Phytophthora
species.
Plant Dis. 70:1038-1043.

Comparison of
and
Pythium

10. Lumsden, R. D . , Ayers, W. A., Adams, P. B . , Dow, R. L . ,
Lewis, J. A., Papavizas, G. C . , and Kantzes, J. G.
1976.
Ecology and epidemiology of Pvthium species in
field soil.
Phytopathology 66:1203-1209.
11. Mircetich, S. M.
1971.
The role of Pvthium in feeder
roots of diseased and symptomless peach trees and in
orchard soils in peach tree decline.
Phytopathology
61:357-360.

51
12. Mircetich, S. M . , and Fogle, H. W.
1969.
Role of
Pythium in damping-off of peach.
Phytopathology
59:356-358.
13. Mircetich, S. M . , and Kraft, J. M. 1973.
Efficiency of
various selective media in determining Pythium
populations in soil.
Mycopathol. Mycol. Appl. 50:151161.
14.

Piekzarka, D. J . , and Abawi, G. S.
1978.
Influence
of soil water potential and temperature on severity of
Pythium root rot of snap beans.
Phytopathology 68:766772.

15. Proffer, T. J., Jones, A. L. and Ehret, G. R. 1987.
Biological species of Armillaria mellea isolated from
sour cherry orchards in Michigan.
Phytopathology
77:941-943.
16.

Sewell, G. W. F. 1981. Effects of Pythium species on
the growth of apple and their possible causal role in
apple replant disease.
Ann. Appl. Biol.
97:31-42.

17. Smither, M. L . , 1988. Cherry tree decline: associated
Pythiaceous species, pathogenicity and control. Ph.D.
Thesis, Michigan State University.
18. Van der Plaats-Niterink, A. J.
1975. Species of Pvthium
in the Netherlands.
Neth. J. PI. Path. 81:22-37.
19. Van der Plaats-Niterink, A. J.
1981.
Monograph of the
genus Pythium. Studies in Mycology No 21 Centraal
Bureau voor Schimmelcultures, Baarn. 242 pp.

PART III

EFFECTS

OF

PHYTOPHTHORA

METALAXYL
AND

AND

FOSETYL-AL

ON

PYTHIUM SPECIES ’IN VITRO' AND

PATHOGENICITY TO MAHALEB CHERRY

52

GROWTH
ON

OF
THEIR

53

ABSTRACT

Seven
cherry

pythiaceous species associated with roots of
in

metalaxyl

Michigan were tested for

their

and phosphorus acid in culture.

sensitivity

acid,
7.5;

The

EDgQ

respectively

values for metalaxyl
were:

to

All fungi were

inhibited by both fungicides and sensitivity varied
species.

sour

and

between

phosphorus

Phytophthora cactorum.

0.06 and

£h. citricola. 0.17 and <7.5; Eh- drechsleri. 0.26 and

27.0;

£h.

megasperma.

0.49 and 80.0;

£•

sylvaticum. 0.5 and 78.1; and E- ultimum

0.06 and 7 8 . 7 ytig/ml.
1-yr-old

1.52 and 49.5; Pvthium irregulare.

Phytotoxic symptoms were observed

Mahaleb seedlings at 400 and 800 ^g/ m l

on

metalaxyl.

Significant (E=0.05) reductions in shoot and root dry weight
occurred

with

9-wk-old Mahaleb seedlings growing

in

soil

infested with £. irregulare. Ph.megasperma. and £h. cactorum
in

a

growth chamber.

Metalaxyl was more effective

fosetyl-Al in preventing fungus induced necrosis and

than
growth

reduction.

INTRODUCTION
Root

rot of sour cherry trees occurs in orchards planted

on poorly drained soils in Michigan.
are

considered

Phytophthora species

to be the primary causal agents

of

cherry

root and crown rot in Michigan (2), California (22), and New
York

(29).

Pythium

species,

and

£.

irregulare

in

54

particular,

were

found to be associated with lateral

root

necrosis of cherry trees, and caused reductions in growth of
Mahaleb

seedlings

in greenhouse

trials

(M.

L.

Smither,

unpublished).
Metalaxyl
used

and experimentally to

blight

induced

thus

by

Pythium

and

(23,26).

been

diseases

damping-off

Variations

in

to metalaxyl occur between species (6,8,17,21),
variations in the degree of

metalaxyl

has

control

by Phytophthora (1,5,11,13,15,28)

sensitivity
and

an acylalanine fungicide and it

commercially

induced
and

is

can

control

exerted

be expected on the Phytophthora and

species associated with cherry decline.

by

Pythium

The development of

resistance to metalaxyl has occurred (4,10).
Fosetyl-Al (aluminium tris(0-ethyl phosphonate)) has been
used effectively to control diseases induced by Phytophthora
(11,13,16,24).
required
Coffey

to

High

inhibit Phytophthora ’in

convincingly

fungicidal
(18,19).

concentrations

demonstrated

of

fosetyl-Al

v i t r o 1.
that

the

are

Fenn

and

effective

agent is the breakdown product phosphorous

acid

As with metalaxyl variations in sensitivity occur

between species (7,8,18).

The development of resistance to

phosphorus acid has been demonstrated experimentally (3).
The objectives of this study were to test the sensitivity
of

pythiaceous species isolated from the roots of cherry to

metalaxyl and phosphorus acid in culture,
the

effectiveness

control

of

of

metalaxyl

and

and to

fosetyl-Al

determine
for

root rot of Mahaleb cherry seedlings caused

the
by

55

various pythiaceous species.

MATERIALS AND METHODS

Sensitivity
culture.
(Leb.

of

species to metalaxyl

Single

&

PyftMum

(Bl)

Ph. citricola (HI)

Tucker,

Ph. megasperma

irregulars (L215)

Campbell and Hendrix,
from

cherry

sensitivity

orchards
to

fosetyl-Al

Buisman,

and P.

p.

Sawada,

(Bl)

sylvaticum

ultimum (B302) Trow,

in Michigan were

margin

Disks
and

amended

tested

(C201)
isolated

for

phosphorous acid and metalaxyl in

their

culture.

placed in the center of
metalaxyl

replicate

initial experiment.

twice at rates of 0,

10,

plates

phosphorous
0.01,

0.1,

The experiment was repeated.

of phosphorous acid were 0,
an

six

or

Concentrations of metalaxyl were 0,

in

agar

5 mm in diameter were taken from the colony

with

100 ^g/ml.

Ph.

Drechsler,

The fungal isolates were grown for 5 days on corn meal
(CMA).

in

isolates of Phytophthora cactorum (Bl)

Cohn) Schroet.,

drechsleri

and

acid.
1, 10, and

Concentrations

10, 50, 100, 250, and 500 /|g/ml
The

experiment was

repeated

25, 50, 75, and 100/(g/ml.

plates

were

stored in darkness at 21 C and

growth

was measured daily for 7 days.

radial

The
colony

®^50 va-*-ues

were

calculated from linear regression lines obtained by plotting
the

mean percent inhibition of mycelial growth against

the

concentration of metalaxyl and phosphorus acid.
Fungicide

trials.

Fungal

inoculum

for

the

three

56
fungicide trials was prepared by growing the isolates at
C

for 5 wk on 200 cc vermiculite moistened with 100 ml

juice

solution (200 ml V-8 juice,

distilled
(22).

water)
The

in

a Buchner funnel

and

sterilized

in 500 ml

V-8

and 800

Erlenmeyer

mixed

at

to

remove

the rate of 10

soil/sand/peat (1/1/1).

ml

flasks

inoculum was washed with distilled water

cheesecloth
nutrients

sterilized

2 g CaCO^,

21

over

unassimilated

cc/1000

cc

with

Controls recieved

the

vermiculite mixture but no fungus.
Experiment one was conducted in a greenhouse with dormant
1-yr-old

Mahaleb

infested

with isolates of Eh-

(HI),

Eh-

ultimum

megasperma

(G7).

seedlings

planted

(CA),

directly

cactorum (Bl),
£.

soil

P h .citricola

irregulare (C6),

4

L

complete

containers,
blocks with

which
six

were

and

with 14 hr of light.

arranged

nighttime

£.

three

replications.

daytime temperatures were 24 C,

was 18 C,

into

The experimental units consisted of

in

randomised
maximum

seedlings

as
The

minimum

Fungicide solutions

were

applied to the soil after 8 days at rates of 0, 10, 25, 100,
400,
The

800 M g /ml metalaxyl in 250 ml

and

distilled

experiment was terminated after 3 mo.

water.

Dry weights of

roots and shoots were recorded.
Experiment
with

dormant

into

soil

Eh-fiiirisala
(L215),

two was conducted outside,

shaded

1-yr-old Mahaleb seedlings

planted

infested
(HI),

and £.

with isolates of
Eh-

megasperma

sylvaticum (C201).

Eh(CA),

by

directly

cactorum
E.

lath,

(Bl),

irregulare

The experiment was set

57
up as for experiment one.
6 wk after planting.
250

ml

Fungicide treatments were applied

Metalaxyl was applied at 100^g/ml in

distilled water as a soil

drench.

Fosetyl-Al

was

sprayed onto the foliage to run-off with a hand sprayer,
rates

of

1.5

terminated

and

3.0

g

a.i.

after 3 mo.

L.

The

at

experiment

Dry weights of roots and

was

shoots

were recorded.
Experiment three was conducted in a growth chamber

using

9-wk-old

Mahaleb seedlings,

and isolates of £.

irregulare

(C6),

cactorum (B2) and Eh- meaasperma (Bl).

Combined

Phytophthora

and

£.

irregulare treatments used 10

fungal inoculum from each isolate.

Seedlings were

in 1 L pots with eight replicates.
at the rate of 70
soil

drench

Fosetyl-Al
hand

the seedlings

at

had

been

monthly

g

a.i./L.

every
roots

was

intervals.

transplanted.

Maximum

18 C.

shoots

were

recorded

experiment was repeated in a greenhouse.

were

The nighttime

Dry weights
after

a

daytime

The seedlings were flooded for 48

2 wk and fertilised biweekly.
and

was applied

Fungicides

temperatures were 21 C with 14 hr of light.
minimum

planted

sprayed onto the foliage to run-off with

sprayer at a rate of 4

reapplied

of

^ g a.i./ml in 50 ml distilled water, as a

after
was

Metalaxyl

cc

10

wk.

of

hr
the
This

58

RESULTS

Sensitivity
in

of species to metalaxyl and phosphorous

culture.

Rates

increasing

of fungal growth

were

acid

reduced

metalaxyl concentration (Figure 1).

with

Growth

of

P h . cactorum and p. ultimum was completely inhibited above 1
y^g/ml.

Growth of £.

100 M S / m l

irregulare and P. sylvaticum at 10 and

only occurred during the initial 24

hours.

Slow

growth of Ph. drechsleri and Ph. megasperma continued at 100
^g/ml.

The EDg0 values were lowest,

cactorum and P.

ultimum (Table 1).

highest EDgQ value,
Rates

of

phosphorous

fungal

1.52

0.06xxg/ml,

Ph. megasoerma had the

jus/ ml, of the fungi tested.

growth were

reduced

<10 M S / ml.

Ph.

increasing

Growth
Ph.

most sensitive of the species tested,

of

with an ED^ q

(Table 1).

of
49.5

The three Pvthium

were least sensitive with EDgQ values between

and 80 Mg/ml

all

citricola was

megasoerma had the highest ED_~ value.
ou

^g/ml of the Phytoohthora species tested.
species

with

acid concentration (Figure 2)

isolates was prevented with 500 Mg/ral.
the

for P h .

78.1

The experiments were repeated with

similar results.
Fungicide
Mahaleb

trials.
seedlings

In

experiment

one,

for all fungal treatments

weights
were

of

highest

jl{g/ml (Table

2).

symptoms of leaf margin necrosis occured at

400

with metalaxyl concentrations from 10-100
Phytotoxic

dry

and 800 MS/™! (Figure 3).

A factorial analysis showed that

59

40
0.0
0.01

30 ■

-O- 10
-V-

100

o 20 ■

at

10

■

0

2

3

4

5

6

7

8

TIME (d ay s )

Figure la. Radial growth of Phytophthora cactorum in culture
amended with metalaxyl at rates of 0.0,
0.01,
0.1,
1, 10,
and 100 iia /ml.
40

B '■ o.o
- i f

0.01

-V-

100

30 ■

o 20ac

10 ■

0

2

3
TIME

4

5

6

7

a

(days)

Figure lb.
Radial growth of Phytophthora citricola in
culture amended with metalaxyl at rates of 0.0,
0.01,
0.1,
1, 10, and 100 i\g/nl.

60

40

-O - 10
-V -

100

O 20-

10

■

TIME (day s)

Figure lc. Radial growth of Phytophthora drechsleri
in
culture amended with metalax/l at rates of 0.0,
0.01,
0.1,
1, 10, and lOO^g/ml.
40

o.o
0.01
30 ■

-O- 10
100

o 20-

ac

10

•

0

2

3
TIME

4

5

6

7

8

(day s)

Figure Id.
Radial growth of Phytophthora megasperma in
culture amended with metalaxyl at rates of 0.0,
0.01,
0.1,
1, 10, and 100 ng/ml.

61

40

- B " o.o
0.01
30 -

- O - io
- V - 100

10

•

0

2

4
3
TIME (d ay s)

S

6

7

8

Figure le. Radial growth of Pythium irregulare in culture
amended with metalaxyl at rates of 0.0,
0.01,
0.1,
1, 10,
and 100 ^g/ml.
~B ■■ o.o
-A— 0.01
0.1

100
O 20at

10

■

TIME (day s)

Figure If.
Radial growth of Pythium sylvatlcum in
amended with metalaxyl at rates of 0.0,
0.01,
0.1,
and 100 /Mg/ml.

culture
1, 10,

62

40
0.0
0.01
0.1
30 •

-O- 10
-4?- 100

10

'

0

1

2

3

4

5

6

7

8

TIME (d a y s )

Figure lg.
Radial growth of Pythium ultimum in culture
amended with metalaxyl at rates of 0.0,
0.01,
0.1,
1, 10,
and 100 /j(g/ml.

63

Table 1.

ED5Q

values for Phytophthora (Eh.) and

Pythium (£•) species to metalaxyl and phosphorus acid
in culture

E D 50

Fungus

Metalaxyl ^g/ml

values

Phosphorus acid M g / ml

CO
o
o

X

Ph. cactorum

7.5

Ph. citricola

0.17

<10y

Ph. drechsleri

0.26

27.0

Ph. megasperma

1.52

49.5

P. irregulare

0.49

80.0

P. gylyaticum

0.50

78.1

T
5
i, .

A •uu
AA
V

rr r>

„ 1 J.4
U J . V0.4I4LUU

t O •«7t

Values were calculated from means of six
replicate plates. The experiment was repeated with
similar results.
The value was too low to be calculated by this
concentration range.

64

40
'A " 1 10

25

30 •

- G - 75
s?—

10

100

■

a
0

2

3

4

5

6

7

8

TIME ( d a y s )

Figure 2a. Radial growth of Phytophthora cactorum in culture
amended with phosphorous acid at rates of 0, 10, 25, 50, 75,
and 100 yUg/ml.
40

25

30 ■
100

o 20-

10

■

TIME ( d a y s )

Figure 2b.
Radial growth of Phytophthora citricola in
culture amended with phosphorous acid at rates of 0, 10, 25,
50, 75, and 100 ;Ug/ml.

65

40

25
30 ■

-O- 7i
-V -

100

O 20 ■

10

■

TIME ( d a y s )

Figure 2c.
Radial growth of Phytophthora drechaleri
in
culture amended with phosphorous acid at rates of 0, 10, 25,
50, 75, and lOO^g/ml.
40

- A — 10
25
30 •

-V -

100

O 20-

10

■

0

2

4
3
TIME ( d ay s )

5

6

7

3

Figure 2d.
Radial growth of Phytophthora magaaP-SEma in
culture amended with phosphorous acid at rates of 0, 10, 25,
50, 75, and 100 yUg/ml.

66

40

25
—t~ 50
- O - 75

30 •

100

“

10

■

o

2

3

4

5

6

7

a

TIME ( d a y t )

Figure 2e. Radial growth of Pythium irregulare in culture
amended with phosphorous acid at rates of 0, 10, 25, 50, 75,
and 100 /t^g/ml.
40

25
30 ■

50

- O - 75
-^ 7—
100
O 20-

10

■

0

2

3

4

5

6

7

3

TIME ( days )

Figure 2 f . Radial growth of Pythium sylvaticum in culture
amended with phosphorous acid at rates of 0, 10, 25, 50, 75,
and 100 /(g/ml.

67

40

30

RADIAL

GROWTH

( mm)

-o20

10

0
TIME ( d a y s )

Figure 2g.
Radial
growth of Pythium ultimum in culture
amended with phosphorous acid at rates of 0, 10, 25, 50, 75,
and 100 /WS/ml.

Table 2.

Effect of various concentrations of metalaxyl on growth of 1-yr-old Mataleb seedlings in Phytophthora and Pythium

infested soil in the greenhouse

Shoot dry weight (g)a
Root dry weight (g)a
Concentration ___________________________________________________________________________________________________________
of metalaxyl"
Ph.
Ph.
Ph.
L
L
Ph.
Ph.
Ph.
P.
P.
(ng/ml)
Control cactorum c itric o la megasperra irregulare ultimum Control cactoruis c itric o la irsaasperra irregulare u ltim a

0

9.7 abc 7.2 ab

6.8 b

13.2 a

7.7 ab

12.9 a

2.9 abc 1.3 c

2.1 a

3.4 ab

2.6 ab

3.8 a

9.8 ab

13.1 a

3.3 abc 3.1 ab

2.5 a

4.1 a

2.6 ab

3.8 ab

3.5 ab 3.7 a

3.3 a

2.0 a

3.6 ab

3.6 abc

2.1 a

3.0 a

4.1 a

2.2 abc

10

11.0 ab 10.5 a

10.9 {ib

11.9 a

25

13.9 a

11.8 a

13.7 ci

9.9 a

12.0 a

12.4 a

100

13.9 a

8.9 a

10.7 ib

11.5 a

11.7 a

9.7 a

4.9 a

12.6 a

8.6 ab

8.9 b

2.3 be 1.4 c

1.6 a

2.7 a

2.7 ab

1.6 c

9.7 a

6.4 a

8.7 ab

1.4 c

1.5 a

2.4 a

1.6 b

1.7 be

400

9.8 ab

8.8 a

9.3 ab

800

8.0 b

7.9 a

7.1 t

2.2 be

1.2 c

a0ormant 1-yr-old Mahaleb seedlings were transplanted in so il infested with 10 cc of inoculum per 1CC0 cc of so il. The experiment
was conducted in a greenhouse for 3mo. There were six replicates.
^Metalaxyl(250 ml per container) was a l l i e d as a so il drench
cValues in coluire followed by the sama le tte r do not differ significantly according to Ouican'stultlple range te st (P=0.05).

Figure 3. Fhytotoxic symptoms caused by metalaxyl at rates
of (a) 0,
(b) 100,
and (c) 400 M g /ml on leaves of Mahaleb
cherry.

70
there

were no significant variations in shoot and root

weights

due to soil infesting fungi or metalaxyl,

dry

as there

was a high degree of variability between experimental units.
Analyses

of variance calculated separately for each of

soil infesting fungi indicated that metalaxyl
(£=0.05)

increased

root

dry weight of

the

significantly

Mahaleb

seedlings

planted in soil infested with £h. cactorum.
In experiment two,

shoot and root dry weights of Mahaleb

seedlings tended to be lower when grown in infested soil

or

treated

no

with

significant
fungi

or

fosetyl-Al

(Table

3).

There

variations in dry weight due to soil
the

fungicides

metalaxyl

and

were

infesting

fosetyl-Al

as

estimated by a factorial analysis.
In experiment three,
£,

irregulare.

significant
4,

alone

weights

of

combination

all

caused

the

significant

between fungicide treatments.

significant

controlled

in

P h . megasnerma. and

A factorial analysis also found

(£=0.05) differences
no

or

cactorum.

reductions in shoot and root dry weights (Table

Figure 4)

were

Eh-

interaction

effects.

There
Metalaxyl

soil infesting fungi as shoot and root

metalaxyl treated seedlings grown

in

dry

infested

«

soil

were

treated

not

significantly reduced

control.

from

the

Fosetyl-Al reduced shoot and

metalaxyl
root

dry

weights of seedlings growing in uninfested soil. Dry weights
of
not

fosetyl-Al treated seedlings grown in infested soil were
significantly

different

from

the

treated

The experiment was repeated with similar results.

control.

Table 3.

Effect of fosetyl-Al and enatalaxyl on growth of 1-yr-old Mahaleb seedlings in Phvtoortnora and Pvthiun infested

soil under lath

Shoot dry weight (g)a

Treatment
and rate

Root dry weight (g)a

P.
fL
EL
EL
EL
Ei
Ei
EL
EL
EL
Control cactorum citricolci meoaspenra irreoulare svlvaticum Central cactorum c itric o la meoaspenra irreoulare svlvaticum

Control

7.1b

5.2

4.5

5.4

6.7

6.4

3.0

1.8

2.5

2.0

2.5

2.4

Fosetyl-Alc
1.5 g a .i./L

5.2

2.5

3.9

4.1

4.7

4.2

2.8

0.5

1.4

1.7

2.2

2.8

Fosetyl-Alc
3.0 g a .i./L

6.4

2.7

4.5

4.1

4.8

4.8

3.0

1.0

1.7

2.1

2.3

1.8

Metalaxyl1*
ICOjug/mi

6.6

5.1

2.9

4.1

5.6

5.5

2.6

2.0

1.0

1.6

3.0

2.6

a0orrant 1-yr-old Mahaleb seedlings wera transplanted in soil infested with 10 cc of inoculum per 1CGQ cc of so il. The experiment
was conducted outside, shaded by lath, for 3mo. There were six replicates.
^Values in colusns followed by the sate le tte r do not d iffer significantly according to Duncan'smultiple range te s t (P=0.05).
cFosetyl-Al was sprayed onto the foliage to run-off with a hand sprayer a t a
^Metalaxyl(250 ail per container) was applied as a so il drench.

Table 4. Effect of fungicides metalaxyl and fosetyl-Al on growth of Mahaleb seedlings
growing in soil infested with Phytophthora species and

P v th iu e

Irreoulare

Dry weight (g)
Fungicides

Control

Metalaxyl1'

Shoot

Root

Control

2.7

a* 1.4 a X

2.6 a

X

1.2

a

X

2.0

a X

1.0 a X

Ph. cactorum

1.0

b X

0.4

b

X

1.7 a

X

1.0

a

X

1.3

a X

0.6

Ph. meaasnerma

1.5

b X

0.8

b

XY

1.7

a X

1.2 a X

0.8 a

X

P. irreoulare

1.0

b

Y

0.4

b

Y

2.2

a X

1.2

a

X

1.4

y 0.7

Ph. cactorum/

1.1

b

Y

0.5

b

Y

2.8 a

1.4

a

X

1.3 a y 0.7 a Y

Ph. megasperma/
P. irregulare

1.1 b y

1.6 a X

1.3 a y 0.S a Y

Treatment8

a

Shoot

Fosetyl-Al1'

0.3 b Y

Root

X

2.6 a x

Shoot

a

Root

a

X

0.3 a Y
a

Y

aSlx-wk-old Mahaleb seedlings were transplanted in soil infested with 20 cc of
inoculum per 1000 cc of soil. The experiment was conducted in a greenhouse for 2
.mo. There were eight replicates.
Plants were flooded for 48 hr every 2 wk.
“Metalaxyl was applied at the rate of 70 M a a.i./ml in 50 ml distilled
water, as a soil drench after the seedlings had been transplanted.
cFosetyl-Al was sprayed onto the foliage to run-off with a hand sprayer at a
rate of 4 g a.i./L.
“Values in columns (a,b) acid rows (x,y and X.Y) followed by the same letter do
not differ significantly according to Duncan'smultiple range test (P»0.0S).
There
was no interaction between the main effects according to a factorial analysis.

73

Figure 4.
Roots of representative Mahaleb seedlings grown
in a) uninfested soil, or soil infested with b) Phytophthora
.cactorum, c) Eh. megasperma and d) Pythium irregulare.
Roots
in columns were x) untreated,
or treated with y )
metalaxyl, and a) fosetyl-Al.

74

DISCUSSION

The

fungal

cherries

in

metalaxyl
tested

species
Michigan

differed

in

their

Eh.

cyifrhnrnm
The

phosphorous

the

most

confirms another

of

Of the

sensitive

high sensitivity of

acid

Pythium

was

rot

sour

sensitivity

and phosphorous acid in culture.

fungicides.

three

associated with root

Eh.

report

to

fungi

to

citricola
(7).

both
to
The

species showed a similar low sensitivity

phosphorous acid.

£.

metalaxyl,

irregulare and £. svlvaticum were less

while £.

ultimum had a high sensitivity

to
to

sensitive.
The

fungicide

effective

trials

showed that

metalaxyl

was

than fosetyl-Al in increasing shoot and root

more
dry

weights of plants growing in soil infested with Phytophthora
species and £. irregulare. However at high concentrations of
metalaxyl

phytotoxic symptoms ocurred with reduced

Reductions

of

dry weights of plants growing in

growth.

uninfested

soil when treated with fosetyl-Al was probably due to stress
caused
from

by the chemical.
the

first

uncontrolled
experiments,

two

The inconclusive results

experiments

temperatures
and

previous

were

during

probably

the

course

contamination of

the

obtained
due
of

to
the

1-yr-old

Mahaleb seedlings with Phytophthora and Pvthiurn species.
Effective
present

at

disease
the

site

control requires that a fungicide
of

infection

at

a

high

is

enough

75
concentration

to

inhibit or kill

the

invading

organism.

Diseases successfully controlled by metalaxyl include collar
rot of apple (15) and foot rot of citrus (13,16,28,),

where

a canker is produced on the trunk and control is effected by
concentration
Metalaxyl

of

has

applied

as

the

fungicide

controlled

a soil drench

at

the

root rot of

infection

woody

site.

plants

with nursery or container

when
grown

plants (1,5).
Control

of

the

lateral root

necrosis

associated

with

cherry decline requires that the fungicide is present at

an

inhibitory

is

strongly
was

concentration

the

roots.

Metalaxyl

adsorbed to soil organic matter and in loam

found

applied

in

to stay in the top 7.5 cm

as

a

soil drench as

no

(27).

soils

Metalaxyl

basipetal

is

translocation

occurs (14,30), and it is questionable if on the heavy soils
of symptomatic orchards metalaxyl would reach all the feeder
roots.

Fosetyl-Al is applied as a foliar spray as basipetal

translocation occurs (14,24), however it is not known if the
concentrations reaching the feeder roots are high enough

to

prevent infection.
The
species

longterm
with

prospects

these

for

control

of

chemicals are not good

as

pythiaceous
there

several reasons for doubting the long term effectiveness
these chemicals.
fungicides,
together
Resistance

are
of

Species differ in their sensitivity to the

and as more than one species are commonly found

selection of the more tolerant species will occur.
to metalaxyl has already

occured,

particularly

76
with foliar pathogens (4,10),
has

been

demonstrated

reports

of

the

induced

by Eh-

and resistance to

experimentally

failure of metalaxyl to
megasnerma and

Eh-

(3).
control

drechsleri

fosetyl-Al
There

are

diseases
(20),

and

Pythium species (12,25).

LITERATURE CITED

1.

Benson, D. M.
1979.
Efficacy and in vitro activity
of two systemic acylalanines and ethazole for control of
Phytophthora cinnamomi root rot of azealea.
Phytopathology 69:174-178.

2.

Bielenin, A., and Jones, A. L. 1988. Prevalence and
pathogenicity of Phytophthora spp. from sour cherry
trees in Michigan. Plant Dis. 72:473-476.

3.

Bower,
L. A., and Coffey, M. D.
resistance to phosphorus acid in
Phytopathology 74:811.

1984.
Development of
Phytophthora capsici.

4.

Bruin,
G.
C. A. and Edgington,
resistance in Peronosporales to
Plant Path.
3:201-206.

L. V. 1981.
metalaxyl.

5.

Bruck,
R.
metalaxyl
fraseri.

6.

Coffey,
M. D . , and Bower,
L.
A.
1984.
In vitro
variability among isolates of six Phytophthora species
in response to metalaxyl.
Phytopathology 74:502-506.

7.

Coffey,
M. D . , and Bower,
L. A.
1984.
In vitro
variability among isolates of eight Phytophthora species
in reponse to Phosphorous acid.
Phytopathology 74:738742.

8.

Coffey,
M. D . ,
phosphorus acid

9.

Coffey,
M.
Variability

Adaptive
Can.
J.

I. and Kenerley, C. M.
1983.
Effects of
on Phytophthora cinnamomi root rot of Abies
Plant Dis.
67:688-690.

and Joseph, M. C.
1985.
Effects of
and fosetyl-Al on the life cycle of
Phytophthora
cinnamomi
and
Ehcitricola.
Phytopathology 75:1042-1046.

D . , Klure, L. J . , and Bower, L. A.
1984.
in sensitivity to metalaxyl of isolates of
PhztQPhthora
cinnamomi
and Phytophthora
citricola.
Phytopathology 74:417-422.

77
10. Coffey,
M.
D . , and Young, L.H.
1984.
Responses to
metalaxyl
of sensitive and resistant
isolates
of
Phytophthora infestans.
Phytopathology 74:615-620.
11. Cohn, Y. and Coffey, M. D. 1986.
Systemic fungicides
and the control of oomycetes.
Annu. Rev. Phytopathol.
24:311-338.
12. Cook, R. J . , Zhang, B. X., and Doerr, A.
1983.
Failure of metalaxyl to control all of the Pythium
population pathogenic on roots of Pacific Northwest
wheat.
Phytopathology 73:957.
13. Davis, R. M.
1982.
Control of Phytophthora root and
foot rot of citrus with systemic fungicides metalaxyl
and Phosethyl Aluminium.
Plant Dis. 66:218-220.
14. Edgington, L. V.
1981.
Structural requirements of
systemic fungicides.
Ann. Rev.
Phytopathol.
19:107124.
15. Ellis, M. A., Grove, G. C . , and Ferree, D. C.
1982.
Effects of metalaxyl on Phytophthora cactorum and collar
rot of apple.
Phytopathology 72:1431-1434.
16. Farih, A.,
Menge, J. A., Tsao, P. H . , and Ohr, H. D.
1981.
Metalaxyl and efosite aluminium for control of
Phytophthora gummosis and root rot on citrus.
Plant
Dis. 65:654-657.
17. Farih, A., Tsao, P. H . , and Menge, J. A.
1981.
In
vitro effects of metalaxyl on growth, sporulation and
germination of Phytophthora parasitica and £.
citrophthora.
Plant Dis.
65:651-654.
18. Fenn, M. E . , and Coffey, M. D.
1984.
Studies on the
in vitro and in vivo antifungal activity of fosetyl-Al
and phosphorous acid.
Phytopathology 74:606-611.
19. Fenn, M. E . , and Coffey, M. D.
1985.
Further evidence
for the direct mode of action of fosetyl-Al and
phosphorus acid.
Phytopathology 75:1064-1068.
20. Hamm, P. B . , Cooley, S. J . , and Hansen, E. M.
1984.
Response of Phytophthora spp. to metalaxyl in forest
tree nurseries in the Pacific Northwest.
Plant Dis.
68:671-673.
21. Hunger, R. M . , and Hamm, P. B . , Horner, C. E. and
Hansen, E. M. 1982.
Tolerance of Phytophthora
megasperma isolates to metalaxyl.
Plant Dis.
66:645649.

78
22. Mircetich, S. M. , and Matheron, M. E. 1976.
Phytophthora root and crown rot of cherry trees.
Phytopathology. 66:549-558
23. Morton, H.V., Kern, C.L., and Taylor, T. D.
1982.
Control of Pythium damping off and Phytophthora root rot
of soybeans with metalaxyl. (Abstr.) Phytopathology
72:971.
24.

Munnecke, D. E. 1982. Apparent movement of
Aliette
and Ridomil in Persea indica and its effect on root rot.
Phytopathology 72:971.

25.

Sanders, P. L.
1984. Failure of metalaxyl
Pythium blight on turfgrass in Pennsylvania.
68:776-777.

26.

Sanders, P. L . , Burpee, L. L., Cole, H . , Jr., and Duich,
J. M.
1978.
Control of Pythium blight of turf grass
with CGA-48988.
Plant Dis. Rep.
62:663-667.

to control
Plant Dis.

27. Sharom, M. S., and Edgington, L. V.
1982.
The
adsorbtion, mobility, and persistance of metalaxyl in
soil and aqueous systems.
Can. J. Plant Pathol. 4:334340.
28. Timmer, L. W . , and Castle, W. S.
1985. Effectiveness
of metalaxyl and fosetyl-Al against Phytophthora
parasitica on sweet orange.
Plant Dis. 69:741-743.
29. Wilcox, W. F . , Jeffers, S. N . , Hayes, J. E. K . , and
Aldwinkle, H. S. 1985.
Phytophthora species causing
root and crown rot of cherry trees in New York.
(Abstr.) Phytopathology 75:1347.
30. Zaki, A. L . , Zentmeyer, G. A., and-LeBaron, H. M.
1981.
Systemic translocation of
C-labeled metalaxyl
in tomato, avocado, and Persea indica.
Phytopathology
71:509-514.

APPENDICES

APPENDIX A

DESCRIPTIONS OF PYTHIUM SPECIES ASSOCIATED WITH CHERRY TREES

79

80

Colony

morphology and growth rate.

irregulare

Buisman,£.

Colonies of Pythium

svlvaticum Campbell and

£.

ultimum

Trow, were radiate on CMA and produced aerial mycelium.
irregulare had a finer growth pattern with a maximum
diameter

of

6 /<<m.

£.

sylvaticum and £.

maximum hyphal diameter of 11 /^m.
21 C for £.
17,

20,

irregulare.

Growth

hyphal

ultimum had

Butler

lacked

a

rates on CMA at

£. svlvaticum. and £. ultimum were

and 18 mm in 24 hr, respectively. Colonies of

rostratum

aerial

mycelium and

grew

chrysanthemum pattern at a rate of 8 mm in 24 hr.
of

£.

£.
in a

Colonies

Pythium group ’H S ‘ were fine and radiate with no

aerial

mycelium and grew 12 mm in 24 hr.
Sporangia and hyphal swellings.

The structures described

here were produced on solid media and/or in a 1:1 mixture of
sterile

pond

measurements
blue

in

and
were

distilled

water.

Observations

made on structures stained

lactophenol.

Sporangia of

produced terminally or intercalary,

£.

with

barrel

sylvaticum
(Figure

shaped
less

2a).

(Figure la).
than 30 ^(m in

rostratum

Hyphal

intercalary.
intercalary,

produced

only

limoniform

swellings

diameter,

Hyphal swellings of £.

were

were

varied in size from 12-

and

of

intercalary

in

water

Sporangia of
and

Hyphal swellings of Pvthium group ’HS'
numerous

on

CMA,

and

£.

ultimum were globose,

intercalery and less than 30 M.m in diameter.
£.

cotton

irregulare

25 A^m in diameter, and were globose to ellipsoid,
to

and

less than

30 M m

were
were
in

81
diameter.

All

structures germinated directly with one or

more germ tubes or produced zoospores.
Characteristics of oogonia,
irregulare,
£.

£.

liliimum,

sylvaticum

of

and oospores.

£.

and P.rostratum were homothallic.

was heterothallic although some strains were

self-compatible.
Oogonia

antheridia,

Pythium group 'HS' produced no

£.

irregulare were irregular with

a

number of blunt conical projections (Figure lb).

oogonia.
variable
Oogonia

of £. ay-LYfltiqum and £. ulAimum were globose (Figure 2a, 3a,
3b).

The

number

irregulare.P.
1-3,
£.

of

antheridia

per

oogonium

attached

Antheridia

of £.

with

antheridia

irrgfflAlars.P.

Antheridia of

were branched while those of P. ultimum were

typically

1-2

£.

sylvaticum. and £. ultimum were 1-2, 2-4, and

respectively, and all were paragynous.
sylvaticum

for

close

to

the

oogonial

rostratum were hypogynous and
per

sylvaticum.

Oospores of £. rostratum

oogonium.
and

£.

stalk.
paragynous

Oospores

of

£.

ultimum were aplerotic.

were plerotic.

82

Figure la.

gy_thium irregulare sporangia

Figure lb.
Pythium irregulare irregularly shaped oogonium
with aplerotic oospore.

83

Figure 2a.

Pvthium sylvaticum hyphal swelling.

Figure 2b.
Pythium sylvaticum oogonium
branched antheridial cells.

surrounded

by

84

Figure 3a.
Pythium ultimum oogonium with
antheridium attached close to the oogonial stalk.

paragynous

Figure 3b.
Pythium ultimum oogonium
antheridia and an aplerotic oospore.

paragynous

with

APPENDIX B
EFFECT OF PYTHIUM AND PHYTOPHTHORA ISOLATED IN MICHIGAN ON
GROWTH OF MAHALEB CHERRY SEEDLINGS

85

86

INTRODUCTION

Root rot of sour cherry trees (Prunus cerasus L . )
in

orchards

planted on poorly-drained soils

Phytoohthora

species

are considered to be

in

Recently,

Ph.

megasperma

Schroet.,

£fc.

Bielenin

causal

and New York

and Jones reported isolation of

Drechsler,
cryptogea

Michigan.

primary

agents of cherry root rot in California (5,9),
(8).

occurs

Ph.

cactorum

Pethyb.

&

(Leb.

Laff.,

£h.

&

Cohn)

syrinfffle

(Kleb.) Kleb., and £h- cambivora (Petri) Buisman from cherry
orchards in Michigan (1).

Pythium species, particularly £.

irregulare Buisman, were found to be associated with lateral
necrosis

of cherry trees,

and were pathogenic

to

Mahaleb

cherry (M. L. Smither, unpublished).
The
of

objective of this study was to determine the

effect

Pythium and Phytoohthora species isolated in Michigan on

the growth of Mahaleb cherry seedlings.

MATERIALS AND METHODS

Isolation and identification of species

£.

irregulare was

isolated from roots, and £. sylvaticum Campbell and Hendrix,
and £.
L.

ultimum Trow.

Smither,

Sawada

were

bait (4).
sieves,

from soil as previously described (M.

unpublished).

£h. cactorum and £h. citricola

isolated from soil using apple

cotyledons

as

Soil samples were sieved through 5 and 2 mm mesh
and

a

30 ml sub-sample was placed in

a 250

ml

87
beaker

to air dry.

After 3 days the soil in each

was

moistened

The

top of each beaker was covered with clear plastic

in

place

chamber

with a rubber band.
at 21 C,

distilled
Apple

by adding 5 ml of sterile

beaker

distilled

After 3 days in

water.

a

held
growth

the soil in each beaker was flooded

water to a depth of 2 cm above the soil

cotyledons

(variety

surface of the water.

with

surface.

McIntosh) were floated

on

the

Starting daily after the third day,

any plant tissue with necrosis was removed and examined with
a binocular microscope for Phytophthora.
made

Isolations were

from infected tissue using CMA-PARP

(3).

Identifications

Ribeiro

(6),

and

were

made

Waterhouse

selective

based on

(7).

the

medium
keys

Isolates

of

of
Ph.

meaasperma. and Eh. drechsleri Tucker from cherry roots were
supplied by G. C. Adams.
Pathogenicity
chamber

test.

using

The

test was conducted

6-wk-old

Mahaleb

procedure was as described

in

seedlings.

by Mircetich

a

growth

The

basic

and Matheron

(5).

Inoculum of Pythium and Phytoohthora species was prepared by
growing

each isolate at

and

g

20

oat

21

C for 5 wk on 200

kernels moistened with

100 ml

ccvermiculite
V-8

juice

solution (200 ml V-8 juice,

2 g CaCC>3 , and 800 ml distilled

water)

ml

sterilized

in

500

Erlenmeyer

flasks.

The

inoculum was washed with distilled water over cheesecloth in
a

Buchner funnel to remove unassimilated

mixed

at

the

soil/sand/peat

rate of 20

cc per

(1/1/1 by volume).

nutrients,

1000 cc of

then

sterilized

Seedlings were planted

88
in

4

L

received

plastic pots with

six

vermiculite/oat mixture but no

daytime

The

control treatment,

controls

fungus.

temperatures were 21 C with 14 hr of

nighttime minimum was 18 C.

were

replicates.

Maximum

light.

The

The seedlings, apart from one

were flooded for 48 hr every 2 wk.

fertilized biweekly.

All

Dry weights of the roots

shoots were recorded after 18 wk.
on CMA-PARP selective medium.

and

Reisolations were

made

This experiment was repeated

twice in the greenhouse and twice in the growth chamber with
other isolates of these fungi.

RESULTS
Pathogenicity

qaS-tarum,

test.

E.

irregulare.

£.

drechsleri

and

shoots

significantly reduced dry weights of

of Mahaleb seedlings grown in a growth

(Table 1,

Figure 1).

citricola

caused the greatest reduction and

£.

soil

drechsleri.

dry

£.

sylvaticum

weights.

One

and E. sylvaticum isolated from

All

flooded treatments had significantly

growth

as compared to the non-flooded control.

by

irregulare resulted in extensive root

£.

decay,

Phytophthora
and

chamber

did not significantly reduce dry weights of roots

shoots.

and

roots

Eh- cactorum. and £h.

least reduction in shoot and root

isolate of Eh-

Eh-

.ci£riqp.la, Eh- megasperma and one isolate of

Eh-

£h-

the

ultimum.

similar to symptoms observed in

and

reduced

Infections
discoloration

the

orchards.

infections caused cankers on the collar region

main tap root.

Pythium and Phytophthora species

were

89
Table 1.

Virulence of Pythium and Phytophthora species

isolated from Michigan sour cherry orchards to Mahaleb
seedlings

Shootsx

Fungus and isolate
number

Dry
weight

(g)

Relative
growth Y
(*)

Rootsx

Dry
weight

(g)

Relative
growth Y
(*>

Pvthium irreaulare
L215

2.0 cdz

41

1.0 de

42

Pvthium sylvaticum
J310

3.2 be

65

1.9 be

79

Pvthium ultimum
B302

2.0 cd

41

1.4 cde

58

Phytoohthora cactorum
B1

0.7 d

14

0.6 e

25

Phytoohthora citricola
H2

1.5 cd

31

0.6 e

25

Phytophthora drechsleri
B1
JC16

3.3 be
o c ^

67

1.6 cd

66

X • *0

r? a
sJ\J

Phytoohthora meaasoerma
B1

2.4 cd

49

Control
noninoculated flooded
noninoculated nonflooded

4.9 b
7.3 a

M •V

CO

<
■
!
■
»
■
+

100
149

^UC

1.3 cde

2.4 ab
2.8 a

54

100
116

^ix-wk-old Mahaleb seedlings were transplanted in soil infested
with 20 cc of inoculum per 1000 cc of soil. The seedlings apart
fran the non-flooded control were flooded for 48 hr every 2 wk.
The experiment was conducted in a growth chamber for 3 mo.
There were six replicates.
^Values are expressed as a percentage of flooded control.
zValues followed by the same letter do not differ significantly
(P=0.05) using Duncans Multiple Flange test.

90

Figure 1.
Roots of representative Mahaleb seedlings grown
in a growth chamber for 18 wk in a soil/sand/peat mixture
infested with
(A) Phytoohthora species and
(B) Pythium
species.
Soil
in the pots was (a) uninfested and non­
flooded,
or flooded and infested with (b) Ph. cactorum. (c)
£h- sitrisolfl, (d) Ph. drechsleri, (e) Ph- megasperma. (f)
p. irregulare, (g) £. gylvaticum, and (h) P. ultimum.

91
routinely reisolated from seedlings growing in infested soil
but not from control plants.

These results were similar to

those from two experiments conducted in a greenhouse and two
experiments conducted in a growth chamber.

DISCUSSION

All

the species tested caused reductions in dry

of Mahaleb seedlings.
considered
and

to

Although Phytophthora species

be the primary causal agents of cherry

crown rot (1,5,8,9),

with

Pythium

indicates

weight

the consistent

species,

that

these

over
fungi

a

results

series

should

of

also

are
root

obtained

experiments,

be

considered

pathogens of cherry.
The

large

isolated

number

from

of Phytoohthora and

Pythium

cherry orchards in Michigan may

retard

development of a suitable control stratagy using
or

host resistance.

active

against

Phytophthora

all

the

species

which

fungicides

may

be

present.

root and crown rot is associated with

soils,

and

is

Pythium

species

are

also associated

L.

the

A suitable method of control must be

drained

soils (M.

species

Smither,

intensified

unpublished).

by

flooding

with

poorly(10).

poorly-drained

Therefore the most

effective means of control maybe to improve soil

conditions

by

and

selecting

sites

drainage systems.

with

well-drained

soils,

using

92

LITERATURE CITED

1.

Bielenin,
A.,
and Jones A.
L.
1988.
Prevalence and
pathogenicity of Phytophthora spp.
from sour cherry
trees in Michigan. Plant Dis. 72:473-476.

2.

Fliegel, P., Parker, K. G . , and Mai, W. F.
1963.
The
fungous flora of nonsuberized roots of poorly growing
cherry trees. Phytopathology 53:1368-1369.

3.

Jeffers,
S. N. , and Martin, S. B. 1986.
two
media selective for Phytoohthora
species.
Plant Dis. 70:1038-1043.

4.

Jeffers, S. N . , and Aldwinkle, H. S. 1987.
Enhancing
detection of Phytoohthora cactorum in naturally infested
soil.
Phytopathology 77:1475-1482.

5.

Mircetich, S. M . , and Matheron, M. E. 1976.
Phytoohthora root and crown rot of cherry trees.
Phytopathology 66:549-558.

6.

Ribeiro, 0. K. 1978.
A source book of the genus
Phytoohthora. J. Cramer, Lehre, Germany. 420 pp.

7.

Waterhouse, G. M. 1970.
The genus Phytoohthora de Bary.
Diagnosis (or descriptions and figures) of the original
papers.
Commonw. Mycol. Inst., Mycol. Pap. 122. 59 pp.

8.

Wilcox, W. F . , Jeffers, S. N . , Hayes, J. E. K . , and
Aldwinkle, H. S. 1985.
Phytoohthora species causing
root and crown rot of cherry trees in New York.
(Abstr.) Phytopathology 75:1347.

9.

Wilcox, W. F . , and Mircetich, S. M. 1985.
Pathogenicity and relative virulence of seven
Phytoohthora species on Mahaleb and Mazzard cherry.
Phytopathology 75:221-226.

Comparison of
and
Pvthium

10. Wilcox, W. F., and Mircetich, S. M. 1985.
Effects of
flooding duration on the development of root and crown
rot of cherry.
Phytopathology 77:1132-1137.

APPENDIX C

THE EFFECTS OF INCREASED SOIL COMPACTION LEVELS WITH PYTHIUM
IBBEOTLABE

AND PHYTOPHTHORA MEGASPERMA ON GROWTH OF MAHALEB
SEEDLINGS AND ETHYLENE LEVELS IN SOIL

93

94

ABSTRACT

Sectioned pipes were used to study the effects of soil
compaction

levels

of

1.0,

1.3,

and

1.7

g/cc,

and

PhytOBhthaca megasoerma and Pvthium irregulare on growth
Mahaleb

seedlings.

(£=0.05)in
bulk

were

significant

reductions

dry weights of Mahaleb seedlings with

density

reductions

There

levels.

in

£.

irregulare caused

dry weight in one of

two

experiments.

with

seedlings.

£.

sections

aerograph

of

with

irregulare populations were higher

increasing bulk densities.

basal

£.

(propagules

gram) ppg with no Mahaleb seedlings and 248.7 ppg

Mahaleb

ppm

increased
significant

irregulare populations were an average of 104.3
per

of

Ethylene levels

the pipe was measured

1400 gas chromatograph.

using

in
a

the

Varian

Ethylene levels over 1

were detected in treatments with Mahaleb

seedlings

at

planted on clay soils in Michigan tend

to

1.7 g/cc soil bulk density.

INTRODUCTION
Cherry
exhibit

trees

poor growth and

pathogenic
(3,12).
to

Phytoohthora

species

to cherry have been isolated from trees and soil
Pythium irregulare has been found to be pathogenic

cherry,

associated

survival.

and
with

unpublished).

high populations of
heavy

clay

soils

Pvthium
(M.

species
L.

are

Smither,

95

Soil compaction occurs frequently on clay soils.
compacted

zones

impede root growth resulting in a

restricted root system (5).
reduced

water

movement

The
shallow,

Reduced diffusion of gases and

cause waterlogging

and

sites

anaerobiosis

which may predispose trees to infection

Phytophthora

species

Pythium

species

germinating

(17).

which infect by motile zoospores

which

sporangia

infect

by zoospores

are stimulated

by

or

of

and

directly

fluctuations

in

matric potentials which occur near the surface (6).
Ethylene

is

a growth regulator produced by

microorganisms

including

concentrations

fungi

(8,18).

and may directly affect plants causing

in

extension

senescence
the

host

(7).
to

and

Ethylene

increase in soils under anaerobic conditions

(9,15),
root

plants

(16) and leaf chlorosis

and

It is not known if ethylene

infection

(1,2,13,14),

although

reductions
premature
predisposes
increased

ethylene concentrations often occur after infection (1,4).
The

objectives

were

to

study

the

effects

of

soil

compaction levels and the pathogens, Phytoohthora megasoerma
and Pythium irregulare.
to

investigate

on growth of Mahaleb seedlings, and

variations in soil ethylene

concentrations

caused by these factors.

MATERIALS AND METHODS
Pasteurized sandy loam orchard soil was sieved through

10

and 30 mesh sieves to retain soil particles between 0.05 and

96
2

mm in

2.5,

diameter.

5.0,

and 7.5

PVC pipe of 7.5 cm diameter was cut into
cm sections.

A 5 mm hole drilled 4.0 cm

from the

base of the 7.5 cm section was closed with a rubber

septum.

The 7.5 cm sections were covered at the base

piece

of

Whatman

No 1 filter paper under

two

with a

layers

of

cheesecloth held in place with a rubber band.

The sections

were

filled

trays

were

half

with soil and placed in aluminum
filled

with water.

More soil

was

which

added

maintain the level with the top of the sections as the
subsided upon wetting.
g/cc were obtained
soil
the

by placing 135.5,

densities

soil

Soil densities of 1.0, 1.3 and 1.7
176.2, and 230.4 g of

at 18% moisture in the 2.5 cm section and
higher

to

with a flat

weight.

compressing
The

2.5

cm

and the 5.0

cm

section

was taped over the saturated soil,

section

was taped in place over the compressed section

and

filled to the surface with soil.

The completed cores were

left

surface

until water had risen

to the

through

the

COmpciG'uOcL x a.yS2T .

Roots
from

of four-wk-old Mahaleb seedlings were washed

free

soil and cut back to 1.5 cm before planting in the top

section.
Inoculum of Pythium and Phytophthora species was prepared
by

growing

each

isolate

at

21 C for 5

wk

on

200

cc

vermiculite moistened with 100 ml V-8 juice solution (200 ml
V-8

juice,

2g

CaCOg,

and

800

ml

distilled

sterilized in 500 ml Erlenmeyer flasks (12).
was

washed with

distilled water

water)

The inoculum

over cheesecloth

in

a

97
Buchner funnel to remove unassimilated nutrients.
(2cc)

was

Inoculum

placed in a 1 cm furrow in the soil

around

the

foil

pie

periphery of the core.
The
pans

cores
and

light.

were placed in individual aluminum

kept
The

in a growth chamber at
cores

were

watered by

65 C with 14 hr

of

applying

of

50 ml

distilled water to the top of the cores as required.
Ethylene

samples

(1 cc) were taken each week

from

the

basal portion of the cores through the rubber septum.
samples

The

were tested for ethylene concentration (ppm) with a

Varian aerograph 1400 gas chromatograph.
After

8 wk the experiment was terminated.

were made of root and shoot dry weights.
Pythium

propagules

The population of

in soil was determined with

soil

dilution plate assay (11).

with

a 5 mm diameter cork borer.

taken

Measurements

Samples

a

were

prepared

in

collected

Three sub samples

from the top and bottom sections of each

each soil sample,

surface

d i l u t i o n s of 1:10,

1:50,

were

core.

From

and 1:100 were

molten sterile 0.2% water agar in test

tubes.

Diluted samples (1 ml) were distributed over the surface
fresh
(0.4

corn meal agar (Difco) amended with 10 mg
ml

of a 2.5% suspension),

rifampicin,
Chemical
sterile
each

and

Co.,

Saint

glass rod.

dilution.

plastic

50

mg

of

pimaricin

250 mg ampicillin,

10 mg

pentachloronitrobenzene,

(Sigma

Louis,

MO) (CMA-PARP)

(10)

with

a

There were three replicate plates for
The

plates were

bags in the dark at 21 C.

incubated

inverted

After 42 hr,

in

the soil

98
was

removed by washing each plate under running tap

A

colony count was made and the number of

water.

propagules

per

gram of dried soil was determined.
An
with

initial
3

Mahaleb

experiment was set up as a

levels

of compacted soil;

seedlings;

and

no

no fungus or

3x2x2

plant

factorial

or

Pythium

irregulare.

There were five replications for each treatment.
experiment

was

Phytophthora

conducted

as

a

3x2x3

megasperma as the additional

3-wk-old

A second

factorial

with

treatment.

The

procedures followed were those previously described.

RESULTS

Increasing
(£=0.05)in
initial

bulk

root

density caused

dry

experiment

significant

significant

weight of Mahaleb
(Table

1).

£.

reductions in dry weight.

were not significant.

£.

reductions

seedlings

in

irregulare

the

caused

Interaction effects

irregulare populations in

the

top sections at 1.0, 1.3, and 1.7 g/cc bulk density were 93,
250

and 217 ppg respectively.

the

basal

detected

The population was 3 ppg in

section at 1.0 g/cc but £.

irregulare

in the basal sections from higher bulk

Populations

was

not

densities.

were not determined in treatments with

Mahaleb

seedlings.
Increasing bulk density caused significant reductions
dry

weight

of Mahaleb seedlings in the

second

in

experiment

(Table 2).

In this experiment neither £h. megasperma nor P.

irregulare

caused

reductions

significantly

greater

than

99

Table 1.

Effects of soil bulk density and Pvthium irregulare on

growth of Mahaleb seedlings

Treatment

Root

Dry
Dry weight
weight reduction
(*)
(g)

Dry
Dry weight
weight reduction
(*)
(g)

1.0

0.31z

0.51

1.3

0.24

23

0.38

25

1.7

0.22

29

0.20

61

Mean

0.26

1.0

0.22

29

0.39

24

1.3

0.23

25

0.28

45

1.7

0.18

42

0.19

63

Mean

0.21

0.29

1.0

0.26

0.45

1.3

0.24

0.36

1.7

0.20

0.20

LSD (P=0.05)

0.06

0.02

(g/cc)

Control

P .irregulare^

Mean

Shoot*

Bulk density

0.36

xFour-wk-old Mahaleb seedlings were transplanted into pasteurized
soil in 7.5 cm diameter PVC pipe. The experiment was conducted
in a grcwth chamber for 8 weeks at 21 C.
^Inoculum of P. irregulare was applied in a 1 cm furrow around
the periphery of the core.
ZA factorial analysis showed significant differences (P=0.05)
between the main effects and no significant interaction.

100
Table 2.

Effects of soil bulk density, Pythium irregulare and

Phytophthora megasperma on growth of Mahaleb seedlings

Treatment

Shoot*

Bulk density
(g/cc)

Root

Dry
Dry weight
weight reduction

(g)

(*)

Dry weight
Dry
weight reduction
{*)
(g)

1.0

1.52

1.3

1.2

20

0.7

30

1.7

1.3

13

0.3

70

Mean

1.3

1.0

1.5

0

0.8

20

1.3

1.2

20

0.6

40

1.7

1.2

20

0.3

70

Mean

1.3

1.0

1.2

20

0.7

30

Ph. megasperma^ 1.3

0.8

47

0.7

30

1.7

1.1

27

0.4

60

Control

P .irregulare^

i
* 4 W W M *

Mean

1.0

0.7

0.6

r\

U

•

1.0

1.4

0.8

1.3

1.1

0.7

1.7

1.0

0.3

LSD (P=0.05)

0.3

•

-

0.2

xFour*-wk-old Mahaleb seedlings were transplanted into pasteurized
soil in 7.5 an diameter PVC pipe. The experiment was conducted
in a growth chamber for 8 weeks at 21 C.
YInoculum of P. irregulare and Ph.meqasperma were
applied in a 1 cm furrow around the periphery of the core.
ZA factorial analysis showed significant differences (P=0.05)
with bulk density for both shoot and root dry weights. Fungus
treatments did not cause significant differences, nor were
there interaction effects.

101

those

without

effects.
1.0,
87,

the pathogens.

There were

no

interaction

E- irregulare populations in the top sections

1.3,

at

and 1.7 g/cc with no Mahaleb seedlings were 100,

and 126 ppg;

and with Mahaleb seedlings were 183,

and 367 ppg.

E-

basal

of the non- seedling treatment at 1.0

section

196,

irregulare at 26 ppg was detected in the
g/cc.

£. irregulare was not detected in the other treatments.
Ethylene

was

concentrations
treatments

not
were

detected at 1.0
<0.01

in

the

at 1.3 g/cc bulk density;

density were <0.01 ppm,
2.02 ppm,

ppm

g/cc

bulk

density;

Mahaleb

seedling

and at 1.7 g/cc

bulk

control; 0.04 ppm .Pvthium control;

Mahaleb control;

1.13 ppm,

Mahaleb and Pythium;

and 1.1 ppm Mahaleb and Phytophthora.

DISCUSSION
Increased soil compaction levels were shown to reduce dry
weights

of Mahaleb seedlings.

There was

no

interaction

effect

between soil compaction and reduction in dry

caused

by

increased

£.
with

significantly
but

neither

significant
experiment.
megasperma

irregulare.
soil

Populations of

compaction

levels.

£.

irregulare

£.

irregulare

reduced dry weights in the first
£.

irEfigyilars

reductions

in

nor
dry

Eft.
weight

The reason for this is not clear,
requires

periodic

flooding

(19), and none of the seedlings grew well.

experiment,

megasperma
in

for

weight

the

caused
second

although P h .
pathogenicity

102
These

findings can be correlated with instances of

growth

in

Several

factors

which

occurs

orchards

with

high

may be involved

soil

compaction

in the poor

under such conditions.

Root

levels.

root

growth

penetrance

reduced through zones of compacted soil (5).
may predispose plants to infection (17,19).

poor

is

Anaerobiosis
Root growth is

decreased upon exposure to increased ethylene concentrations
(7,16).
ethylene

However there is no clear evidence that increased
concentrations

predispose

plants

to

infection

(1,2,13,14).

LITERATURE CITED
1.

2.

Abbatista, G. I., and Matta, A.
1975.
Production of
and some effects of ethylene in relation to Fusarium
wilt of tomato.
Physiol. Plant Pathol. 5:27-35.
Archer, S. A., and Hislop, E. C.
1975.
Ethylene in
host-pathogen relationships.
Ann. Appl. Bio. 81:95126.

3.

Bielenin, A., and Jones, A. L. 1988. Prevalence and
pathogenicity of rhytopht hora spp. from sour cherry
trees in Michigan.
Plant Disease 72:473-476.

4.

Cohn, R . , Riov, J . , Lisker, N . , and Katen, J.
1986.
Involvement of ethylene in herbicide-induced resistance
to Fuaarium oxysporum f . sp. melonis.
Phytopathology
76:1281-1285.

5.

Drew, M. C . , and Lynch, J. M.
1980. Soil anaerobiosis,
microorganisms and root function.
Ann. Rev.
Phytopathol. 18:37-66

6.

Duniway, J. M.
1975.
Limiting influence of low water
potential on the formation of sporangia by Phytophthora
drechsleri in soil.
Phytopathology 65:1089-1093,

7.

El-Beltagy, A. S., and Hall, M. A.
1974.
Effect of
water stress upon endogenous ethylene levels in Vlcia
fa b a . New Pytol.
73:47-60.

103
8.

H a g , L . , and Curtis, R.W.
1968. Production of
ethylene by fungi.
Science 159:1357-1358.

9.

Jackson, M. B . , Gales, K . , and Campbell, D. J.
1978.
Effect of waterlogged soil conditions on the production
of ethylene and on water relationships in tomato plants,
J. Exp. Bot.
29:183-189.

10. Jeffers, S. N . , and Martin, S. B.
1986.
Comparison of
two media selective for Phytophthora and Pythium
species.
Plant Disease 70:1038-1043.
11. Mircetich, S. M . , and Kraft, J. M. 1973.
Efficiency of
various selective media in determining Pythium popula­
tions in soil.
Mycopathol. Mycol. Appl. 50:151-161.
12. Mircetich, S. M . , and Matheron, M. E. 1976.
Phytophthora root and crown rot of cherry trees.
Phytopathology 66:549-558.
13. Pegg, G. F.
1976.
The response of ethylene-treated
tomato plants to infection by Verticillium albo-atrum.
Physiol. Plant Pathol. 9:215-226
14. Pegg, G. F . , and Cronshaw, D. K.
1976.
Ethylene
production in tomato plants infected with Verticillium
albo-atrum.
Physiol. Plant Pathol.
8:279-295.
15. Smith, A. M . , and Cook, R. J.
1974.
Implications of
ethylene production for biological balance of soil.
Nature
252:703-705.
16. Smith, K. A., and Robertson, P. D. 1971.
Effect of
ethylene on root extension in cereals. Nature 234:148-

17. Stolzy, L. H . , Zentmyer, G. A., Klotz, L. J . , and
Labanauskas, C. K.
1965.
Water and aeration as
factors in root decay of Citrus sinensis.
Phytopathology
55:270-275.
18. Walther, H. F . , Hoffman, H. F . , and Elstner, E. F. 1981.
Ethylene formation by germinating Drechslera gramineainfected barley (Hordeum sativum) grains:
A simple test
for fungicides.
Planta 151:251-255.
19. Wilcox, W. F . , and Mircetich, S. M.
1985.
Effects of
flooding duration on the development of Phytophthora
root and crown rots of cherry.
Phytopathology 75:14511455.
20. Yang, S. F . , and Hoffman, N. E. 1984.
Ethylene
biosynthesis and its regulation in higher plants.
Annu. Rev. Plant Physiol. 35:155-189.

APPENDIX D
VARIATION IN SUSCEPTIBILITY OF CHERRY ROOTSTOCK CULTIVARS TO
INFECTION BY PHYTOPHTHORA SPECIES AND PYTHIUM IRREGULARE

104

105

ABSTRACT

A

dormant

excised twig assay was used to determine

relative

resistance

infection

by Phytophthora species and

Twigs
The

of

cherry

rootstock

cultivars

Pvthium

resulting necrosis above the agar surface was

163091,

to

irregulare.

were placed base down in previously inoculated

after 10 days.

the

agar.

recorded

During 1983 dormant shoots of Mahaleb clones

193688,

193693,

194096, Mazzard, MxM #2, MxM #14,

MxM #39,

MxM #60, MxM #97, Meteor, and Vladimir were tested

with Eh-

ga.Ct.or.um,

irregulare.

MxM

cactorum,

drechsleri.

Eh- megasperma and £.

During 1984 dormant shoots of Mahaleb seedlings

and clone 163091,
#60,

Eh.

#97,

Mazzard,
Vladimir,

MxM #2,
and

MxM #14,

MxM #39, MxM

Colt were tested

with

Ph.

£h- citricola, £h. drechsleri. Eh. megasperma and

£. irregulare. The assays showed that rootstocks differed in
relative resistance to infection by Phytophthora species and
£.

irregulare.

significantly

-C.actgr.um,
but

Dry

weights

of Mahaleb and MxM #60

reduced when grown in soil infested with

were
Ph.

Eh. drechsleri, Eh. megasperma and £. irregulare.

dry weights of MxM #2,

significantly
correlations

reduced.

MxM #39,
There were

and MxM #46 were
significant

(E=0.05)

of 0.463 and 0.521 between the rootsock

and twigs assays of 1983 and 1984.

not

trial

106

INTRODUCTION

Sour cherry production is of major economic importance to
Michigan.
root

In

and

orchards planted on heavy soils losses

collar

rot

are

often

severe.

from

Phvtophthora

species cause root and collar rot of cherry in Michigan (4),
California

(10,17),

and New York (16).

Pvthlum irregulare

Buisman has been associated with poorly growing cherry trees
in Michigan (M. L. Smither, unpublished).
Mahaleb

(Prunus

frequently
L.).
is

used

mahaleb

L.)

is

the

with Montmorency sour cherry

Mahaleb is preferred to Mazzard (£.
more resistant to crown gall and root

(7,12).

rootstock

However

(£.

most
cerasus

avium L . ) as it
lesion

nematodes

Mahaleb grows poorly on heavy soils (7),

and is more susceptible than Mazzard to diseases induced
Phytophthora
both

species (10,11,17).

seedling

rootstocks

and

by

Mahaleb and Mazzard are
therefore

differences

in

resistance to infection can be expected between individuals.
Several

other

resistance

rootstocks

have been

developed

and

to diseases induced by Phvtophthora and

their
Pvthium

needs to be evaluated.
Excised
examine

twig assays have been used as a rapid method

variations in pathogenicity and virulence of fungal

isolates (2,9,13),
to

infection

seasonal

to

to investigate variations in

associated

differences (3).

with host

genotype

In one assay bark

resistance
(1,14),
discs

and
were

107
removed

from

the mid-way point of

inoculum

was applied,

measured

after

placed

excised

fungal

and the resulting necrotic area

several days (6).

A second assay

twigs with the bark stripped from the base

inoculated with a fungal pathogen.
length

twigs,

was

method
in

agar

The resulting necrosis

above the agar was measured after several days

(8).

The second assay method was used in this work.
The objectives were to examine the relative resistance of
cherry

rootstock

irregulare

cultivars to Phytophthora species and

infection

with

an

excised

twig

assay

P.
and

rootstock trial.

MATERIALS AND METHODS

Excised
rootstock

dormant

twig assay

Dormant shoots

of

cherry

cultivars were tested for relative resistance

to

infection by Pythophthora species and p. irregulare using an
excised

twig assay (8).

Fungi were grown on 30

ml

corn

meal agar (CMA) plus agar ( 17 g CMA,

5 g Bacto agar, and 1

L

mg

distilled

Chemical

water

Co.,

beakers.
beakers

amended

Saint

Control
were

Greenwich,

Louis,
beakers

covered

with

6-10

MO])

in

pimaricin
sterilized

contained
Parafilm

no

[Sigma
250

fungus.

(American

ml
The

Can

Co.,

Ct 06830) over aluminum foil and maintained at 21

C in darkness for 7 days.
from

with 10

mm

in

Dormant cherry rootstock

diameter were cut into

disinfested in 0.6% NaOCl for 5 min,

7

cm

shoots

segments,

rinsed three times

distilled water and dried on paper towels.

in

Five mm of the

108

base

of

each

twig was removed

with

disinfested

pruning

shears and a cut 1 cm in length by 2 mm in depth was made in
each

side at the base to expose the cambium.

Single twigs

from each cultivar were placed base down into the medium
the

edge

of

the fungal colony,

so that the

cambium

at
was

exposed to the fungus. There were fifteen replicate beakers.
The

beakers

were covered again and maintained

as

before.

After 10 days the twigs were stripped of bark and the length
of

necrosis

in

the

periderm above

the

agar

level

was

recorded.
Rootstock
(Route

#

material was obtained from

2,

Hartford,

Hilltop

Nurseries

MI) in February of 1983 and

During 1983 dormant shoots of Mahaleb clones 163091,
193693,
MxM

193688,

194096, Mazzard, MxM #2, MxM #14, MxM #39, MxM #60,

#97,

Meteor,

resistance

£.

to

Eh-

Schroet.,
and

1984.

and Vladimir were assayed

infection

by

£h.

drechsleri Tucker,

irregulare.

cactorum

Eh-

for
(Leb

relative
&

Cohn)

megasnerma Drechsler

During 1984 dormant shoots of Mahaleb

seedlings and clone 163091,

Mazzard,

MxM #2,

MxM #14, MxM

#39, MxM #60, MxM #97, Vladimir, and Colt (provided by R. L.
Perry, Horticulture) were assayed for relative resistance to
infection

£h.

by

saS-tOEUm,

d rach sleri, Eh- megasperma
Rootstock trial
with
soil.
and

Eh-

citricola

Sawada,

Eh.

and £. irregulare.

The experiments were conducted under

lath

rootstock cultivars planted in uninfested and infested
The

basic proceedure was as described by

Matheron (10).

Inoculum of Phytophthora and

Mircetich
Pvthium

109

species
wk

on

was prepared by growing each isolate at 21 C for
200 cc vermiculite moistened with 100 ml

solution (200 ml 7-8 juice,
water)

in

washed

with

of

500 ml Erlenmeyer

10

flasks.

The

pots

with

vermiculite

inoculum

distilled water over cheesecloth in a

cc per 1000 cc

(1/1/1 by volume).
six

of

was

Buchner

then mixed at the

sterilized

soil/sand/peat

Rootstocks were planted in 4 L plastic

replicates.

mixture

juice

2 g CaCOg, and 800 ml distilled

funnel to remove unassimilated nutrients,
rate

V-8

5

but

The

controls

no fungus.

The

received

the

rootstocks

were

flooded for 48 hr every 2 wk and fertilized biweekly.

Dry

weights

wk.

In

an

of the roots and shoots were recorded after 15
initial

experiment dormant

1-yr-old

seedlings

Mahaleb and Mazzard and dormant rooted cuttings of
MxM #14,

MxM

of
#2,

MxM #39, MxM #60, MxM #97, and Meteor were planted

in uninfested soil and soil infested with Eh-

sitc is-sla, Eh- megaspsrma. E- Irre g u la rs,

cactorum. P h .

and £. sylvaticum.

A second experiment used actively growing 1-yr-old seedlings
of Mahaleb and Mazzard,
#39,

MxM

uninfested

#39,

MxM #46,

soil and soil

drftc h sle ri, Eh-

and rooted cuttings of

MxM #2, MxM

and MxM #60 which were planted in
infested with

megasperma. and

E-

Eh-

cactorum. P h .

irregulare.

RESULTS
Excised twig assay.

Necrosis developed on twigs of

all

rootstock cultivars inoculated with Phvtophthora species and

110
£.

irregulare.

rootstock

No

necrosis

cultivars.

The

developed

length

of

on

uninoculated

necrosis

produced

differed significantly between cultivars (Table 1-2).
results

varied

between the two years,

but

a

The

correlation

coefficient of 0.396 gave a significant (P=0.05) correlation
between

the

greater

during

necrotic

two assays.

Necrosis length

1984 than 1983.

area

was

produced

cas.t,<?rum> Eh’

drechsleri. and

194096 by p.

irregulare.

area

was produced

MxM

#60

During 1983
on

Eh-

Mahaleb

generally

the

longest

193688

by

Ph.

megasperma. and on Mahaleb

A significantly longer necrotic

by the

than on the other

was

Phvtophthora species on MxM #2 and
MxM

cultivars.

The

shortest

necrotic area was produced on Vladimir by Ph. drechsleri and
Ph.

megasperma. During 1984, the longest necrotic area was

produced on MxM #2 by Ph. cactorum and Ph. citricola. and on
Mahaleb 163091 by Phirregulare.
Colt by Ph.

The

drechsleri.

shortest

cactorum and Ph.

drechsleri

and

P.

Ph.

megasperma.

and p.

necrotic area was produced
citricola.

irregulare.

and

on

on

on Mazzard by P h .
Vladimir

by

Ph.

megasperma.
Rootstock

trial

Results from the initial experiment

were

not significant due to poor establishment of the rootstocks.
In the second experiment shoot dry weights of Mahaleb
reduced in soil infested with

Ph.

cas.torum. Ph-

megasperma. and p. irregulare. and root

soil infested with
3).

Ph.

Shoot

Ph.

cactorum.

and

Ph.

were

drechsleri.

dry weights in

megasperma (Table

dry weights of Mazzard were not

significantly

Ill

Table 1. Relative susceptibility of dormant excised shoot tissue
of cherry rootstock cultivars to infection by Phvtophthora
species and Pvthium irregulare during 1983

Necrosis length on shoot (mm)^

Cultivar

Ph. cactorum

Ph. drechsleri

Ph. megasperma

P. irregulare

Mahaleb
163091

11.2 cz

11.1 e

12.5 be

193688

23.8 a

24.6 a

19.2 a

11.6 b

193693

11.0 c

7.9 e

7.3 d

10.6 b

194096

16.0 b

18.1 be

17.2 ab

18.8 a

Mazzard

18.5 b

9.6 e

7.1 d

4.6 c

MxM #2

16.5 b

18.9 b

15.6 ab

4.1 c

MxM #14

1.1 d

10.3 e

4.5 de

4.0 c

MxM #39

9.2 c

8.1 e

6.2 d

3.6 c

MxM #60

19.6 ab

13.8 cd

16.9 ab

3.5 c

MxM #97

6.4 c

Meteor

1.3 d

6.8 e

3.2 e .

2.9 c

Vladimir

7.5 c

1.6 f

0.1 e

7.8 be

8.2 be

7.9 cd

^Mean length of necrosis above agar level from 15 replicate shoot
pieces.
V a l ue s followed by the same letter are not significantly different
using Duncan's multiple range test (P=0.05)..

Table 2. Relative susceptibility of dormant excised shoot tissue of cherry rootstock
cultivars to infection by Phvtophthora species and Pythium irregulare during 1904

Necrosis length on shoot (tnn)^

Cultivar

Mahaleb

Ph. cactorum Ph. citricola Ph. drechsleri

Ph. meqaspeima

Pv. irrequlane

26.9 afc?

25.3 ab

16.5 a

16.4 a

12.1 be

163091

22.3 be

25.9 ab

15.5 a

16.1 a

17.7 a

Mazzard

17.3 cd

18.3 c

3.5 d

9.5 b

5.2 d

MxM #2

29.0 a

30.1 a

12.1 abc

11.8 ab

17.3 ab

MxM #14

14.8 d

24.9 ab

7.7 cd

9.5 b

11.9 be

MxM #39

17.3 cd

21.9 be

9.2 be

11.7 ab

MxM #60

24.4 ab

28.7 a

13.5 ab

11.7 ab

11.3 c
\
14.3 abc

MxM #97

17.7 cd

22.1 be

11.3 abc

12.9 ab

13.3 abc

Vladimir

17.7 cd

27.9 a

9.0 d

9.7 cd

9.3 e

17.0 c

11.4 ab

13.4 abc

Colt

11.8 abc

yMean length of necrosis above agar level from 15 replicate shoot pieces
Values followed by the same letter are not significantly different Duncan's
multiple range test (P=0.05).

Table 3.

Growth of cherry rootstock cultivars planted In soil Infested with Phvtophthora species and Pvthium

Irregulare under lath

Shoot dry weight (g)Y

Root dry weight (g)

Cultivar

Ph.
Ph.
Ph.
Ph.
Ph.
P.
Ph.
P.
Control cactorum drechsleri meoasDerma irregulare Control cactorum drechsleri megasperma irregulare

Mahaleb

17.2 a2

9.8 b

14.0 be

14.0 be

12.3 be

8.9 a

4.0 b

7.4 a

5.0 b

8.0 a

Mazzard

13.8 b

13.5 b

17.0 a

13.1 b

14.3 ab

9.9 a

3.7 b

11.7 a

4.5 b

12.3 a

MxM #2

5.1 a

4.6 a

4.6 a

6.4 a

4.7 a

3.8 a

3.0 a

3.9 a

4.7 a

3.6 a

MxM #39

4.3 a

3.3 a

4.9 a

5.3 a

4.7 a

4.5 a

2.7 a

4.5 a

4.3 a

3.4 a

MxM #46

7.7 a

4.8 a

6.7 a

5.3 a

7.2 a

6.3 a

4.3 a

4.7 a

5.1 a

6.2 a

MxM #60

6.4 a

3.7 b

3.1 b

3.6 b

7.9 a

5.6 a

3.0 b

2.6 b

3.5 ab

5.5 a

^Actively growing rootstocks were transplanted in soil Infested with 10 cc of inoculum per 1000 cc of soil. The
experiment was conducted under lath. There were six replicates.
zValues across raws followed by the sane letter are not significantly different using Duncan's multiple range
test <P=O.05).

114
reduced

by soil infested by any isolate,

weights were reduced with P h .

with

megasperma.

Ph.

Ph-

cactorum. and

Shoot and root dry weights of MxM #60
infested

although root dry

were reduced in

Ph-

cactorum.

megasperma.

dr-gfihslsri,

soil

Ph.

and

Shoot and root dry weights of MxM #2, MxM #39,

and MxM #46 were not significantly reduced in infested soil.
Correlation
results
and

coefficients

were

calculated

to

from the rootstock trial with those from

1984

twig assays.

compare
the

1983

Results from the rootstock

trial

were expressed as percent loss in dry weight as compared

to

the control.

Correlation coefficients were 0.463 for 1983,

and

for

0.521

1984;

both

figures

show

significant

correlation (P=0.05).

DISCUSSION

Cherry rootstock cultivars in the twig assay differed
relative

resistance

irregulare.
colonization

As
is

to
the

only

resistance,

rootstock

evaluation.

Mahaleb

Phytophthora

resistance of the host
one
trials
clones

infection

in the twig assay,

resistant

than Mahaleb.

were
those

of

several

are

and
tissue

components

required

varied

in

for

p.
to
of

complete

resistance

to

but overall Mazzard was

more

Dry weights of Mahaleb seedlings

significantly reduced in the
of

species

in

rootstock

Mazzard were less effected.

trial,

Mahaleb

has

while
been

reported to be more susceptible than Mazzard to Phvtophthora
infection

(10,11,17).

The vertically oriented root system

115

of

Mahaleb

problems

is

believed to predispose

on heavy soils (7).

it

to

’wet

feet'

Dry weights of MxM #2,

MxM

#39, and MxM 46 were not reduced by growth in infested soil,
while dry weights of MxM #60 were significantly reduced.
The
1983

variation

and

1984

in results for the twig

is

believed to be

environmental conditions.
during

February

of

due

assays

to

between

variations

in

An extremely cold period occured

1984,

and

extreme

environmental

conditions , including low temeratures, predispose plants to
infection (15).
£. irregulare produced necrosis on rootstock cultivars in
the twig assay although to a lesser extent than Phvtophthora
species.
significant

In

the

rootstock trial £.

reduction in shoot dry weight with Mahaleb

not the other cultivars.
most

pathogenic

rootstock
by

lath

irregulare caused

at

temperatures

the

but

£. irregulare has been shown to be
below

21

C

trial was conducted outside under shade
during

a

summer

with

no

(5).

The

produced

regulation

of

temperatures.

LITERATURE CITED

1.

Alston, F. H. 1970.
Resistance to collar rot,
Phvtophthora cactorum (Leb and Cohn) Schroet., in apple.
Pages 143-145 in: East Mailing Res. Stn. Rep. for 1969.

2.

Bielenin, A.
1977.
Collar rot of
apple trees.
I
Pathogenicity of isolates of Phvtophthora cactorum (Leb
and Cohn) Schroet.
Fruit Sci. Rep. 4:15-26.

116
3.

Bielenin, A.
1977.
Collar rot of apple trees.
II
Seasonal fluctuations in susceptibility of apple trees
to Phytophthora cactorum (Leb and Cohn) Schroet.
Fruit
Sci. Rep. 4:27-39.

4.

Bielenin, A., and Jones, A. L. 1988.
Prevalence and
pathogenicity of Phytophthora spp.
from sour cherry
trees in Michigan. Plant Dis. 72:473-476.

5.

Biesbrock, J. A., and Hendrix, F. F . , Jr.
1970.
Influence of soil water and temperature on root necrosis
of peach caused by Pythium spp.
Phytopathology 60:880882.

6.

Borecki, Z . , and Millikan, D. F.
1969.
A rapid
method for determining the pathogenicity and factors
associated with the pathogenicity of Phytophthora
cactorum.
Phytopathology 59:247-248.

7.

Cummins, J. N.
1979.
Interspecific hybrids as
rootstocks for cherries.
Fruit Var. J. 33:85-89.

8.

Jeffers, S. N . , and Aldwinkle, H. S.
1986.
Seasonal
variation in extent of colonization of two apple
rootstocks by five species of Phytophthora.
Plant
Disease 70:941-945.

9.

Jeffers, S. N . , Aldwinkle, H. S., Burr, T. J., and
Arneson, P. A.
1981.
Excised twig assay for the study
of apple tree crown rot pathogens in vitro.
Plant Dis.
65:823-825.

10. Jeffers, S. N . , Aldwinkle, H. S., Burr, T. J . , and
Arneson^ P.
1982.
Phytophthora^ and Pythium species
associated with crown rot in Hew York apple orchards.
Phytopathology 72:533-538.
11. Mircetich, S. M . , and Matheron, M. E. 1976.
Phytophthora root and crown rot of cherry trees.
Phytopathology 66:549-558.
12. Mircetich, S. M . , and Matheron, M. E.
1981.
Differential resistance of various cherry rootstocks to
Phytophthora species.
(Abstr.) Phytopathology 71:243.
13. Perry, R. L.
1986.
A simple method for field
identification of Mahaleb (Prunus mahaleb L.) cherry
rootstock.
Fruit Var. J. 40:68-70.
14. Robertson, G. L. and Dance, H. M.
1971.
The
association of Phytophthora megasperma with crown rot of
apple trres.
N. Z. J. Agric. Res. 14:509-514.

117
15. Sewell, G. W. F. , and Wilson, J. F. 1959.
Resistance
trials of some apple rootstock varieties to Phvtophthora
cactorum (Leb and Cohn) Schroet. J. Hortic. Sci. 34:5158.
16. Schoenweiss, D. F. 1975.
Predisposition, stress, and
plant disease.
Ann. Rev. Plant Pathol.
13:193-211.
17. Wilcox, W. F . , Jeffers, S. N . , Hayes, J. E. K . , and
Aldwinkle, H. S. 1985.
Phytophthora species causing
root and crown rot of cherry trees in New York.
(Abstr.) Phytopathology 75:1347.
18. Wilcox, W. F . , and Mircetich, S. M. 1985.
Pathogenicity
and relative virulence of seven Phytophthora species on
Mahaleb and Mazzard cherry.
Phytopathology 75:221-226.