LIBRARY

Michigan State
University

This is to certify that the
thesis entitled
METHODS FOR INOCULATION OF SOYBEAN SEEDLINGS WITH
ZOOSPORES AND OOSPORES OF PHYTOPHTHORA MEGASPERMA
VAR. SOJAE FOR PATHOGENICITY AND RACE DETERMINATION

presented by
Linda L. Eye

has been accepted towards fulfillment
of the requirements for

___M.._S_._degree in Want Pathology

 

Major professor

Q/w Z :50 [My g
V»

Date 6 Decgmbe: 1922

0—7639

 

METHODS FOR INOCULATION OF SOYBEAN SEEDLINGS WITH ZOOSPORES AND

OOSPORES OF PHYTOPHTHORA.MEGASPERMA VAR. SOJAE FOR

 

PATHOGENICITY AND RACE DETERMINATION

By

Linda L. Eye

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1978

ABSTRACT

METHODS FOR INOCULATION OF SOYBEAN SEEDLINGS WITH ZOOSPORES AND
OOSPORES OF PHYTOPHTHORA MEGASPERMA VAR. SOJAE FOR
PATHOGENICITY AND RACE DETERMINATION

 

. By

Linda L. Eye

Factors affecting zoospore production by Phytophthora megasperma

 

var. sgiae in culture were studied so that large numbers of zoospores
could be produced for use in soybean seedling inoculation experiments.
Two inoculation methods were developed and evaluated for their
reliability in producing disease and for identifying races of the

pathogen using differential cultivars of soybean (Glycine max (L.)

 

Merr.).

Cultures of g, megasperma var. sojae grown on lima bean agar were

 

flooded with distilled water to produce zoospores. Maximum numbers
of 4 X 105 zoospores/ml were obtained when 6-day-old cultures of race 1
of the pathogen received five water changes (15 ml/plate/change) at 30
minute intervals, followed by incubation in the dark for 8 to 18 hours
at 20°C. Isolates of races 2 to 6 also produced large numbers of
zoospores using this method.

One inoculation procedure involved inoculating soybeans planted
in moist vermiculite with a zoospore suspension when Seedlings were 2
to 6 days old. Disease was most severe when 2-day-old seedlings were
inoculated. Concentrations of 500 zoospores/seedling did not produce

disease; 104 zoospores/seedling differentiated susceptible from

 

Linda L. Eye

resistant cultivars, but 105 zoospores/seedling caused disease in some
cultivars which were resistant when wounded hypocotyls were inoculated
with.mycelium. Disease development differed little when seedlings

were incubated for 5 to 6 days at temperatures of 20 to 30°C from the
time of inoculation. Zoospores from isolates of six races of the
pathogen were used to individually inoculate nine differential cultivars
of soybean. Susceptible cultivars usually rotted before emergence,
whereas resistant cultivars remained healthy. Results corresponded
with those obtained by hypocotyl inoculation.

The second inoculation procedure involved placing 2—day—old
soybean seedlings in petri dishes containing 10 to 20 g steamed soil
that had been flooded with 30 ml distilled water and infested with
zoospores. Some seedlings were infected when soil was infested with as
few as 10 zoospores/petri dish (5 seedlings/petri dish); 5 X 103
zoospores/petri dish caused disease in some cultivars which were re—
sistant by hypocotyl inoculation. Illumination did not affect disease
when zoospores were used as inoculum. However, disease incidence was
higher in naturally infested soil or soil artificially infested with
oospores kept in darkness than under continuous fluorescent light.
Temperatures of 20 to 30°C did not affect disease development. How-
ever, temperatures above or below this range decreased disease inci-
dence. Nine differential cultivars of soybean were inoculated indivi-
dually with zoospores (2 X 102/seedling) of six races of the pathogen
in flooded soil. Results corresponded with those obtained by
hypocotyl inoculation.

Soybean seedlings were evaluated as baits for recovering E.

megasperma var. sojae from naturally infested soil. Two—day-old

 

Linda L. Eye

seedlings were placed in petri dishes containing 20 g soil and 30 ml
distilled water. Seedling infection was evaluated after 3 days
incubation, and sporangia were identified by direct examination with a
dissecting microscope. Using this method, the pathogen was recovered
from several field soils. The pathogen was similarly recovered from

diseased soybean root and stem tissue placed on flooded sterile soil.

To Doug

ii

 

ACKNOWLEDGMENTS

To Dr. John L. Lockwood, I express deep gratitude for his
guidance and patience during my tenure as a graduate student, and
for his encouragement and assistance in the preparation of this
manuscript.

To the members of my guidance committee: Drs. G. Safir and
D.C. Ramsdell, I extend my thanks for their suggestions and evalua-
tion of the manuscript.

To Sue Cohen, I extend appreciation for her friendship and
assistance in statistical evaluation of this manuscript.

Without the understanding and encouragement of my husband

Doug, this study would never have been possible.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES o o o o o o o o o o o o o o o o o o o o o o o o o Vii

LIST OF FIGIJRES o o o o o o o o o o o o o o o o o o o o o o o 0 ix

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 3
The Pathogen . . . . . . . . . . . . . . . . . . . . . . . 3
Inoculation Methods . . . . . . . . . . . . . . . . . . . . 6
Isolation from Soil . . . . . . . . . . . . . . . . . . . . 6

METHODS AND MATERIALS . . . . . . . . . . . . . . . . . . . . . 10

Maintenance of Fungus . . . . . . .'. . . . . . . . . . . . 10
Production, Collection, and Preparation of Inocula . . . . lO
Isolation Methods . . . . . . . . . . . . . . . . . . . . . 12
Source and Preparation of Soils . . . . . . . . . . . . . . 13

Zoospore Inoculation of Soybean Seedlings Growing in
vermiculite . O O 0 O O O O O O O O O O O O O O O O O 14

Zoospore or Oospore Inoculation of Soybean Seedlings
Placed on Soil . . . . . . . . . . . . . . . . . . . . 15

Soybean Seedlings as Baits for Recovering P, megasperma
var. sojae from Soil . . . . . . . . . . . . . . . . . l7

 

Hypocotyl Inculation Method . . . . . . . . . . . . . . . . 18

RESULTS 0 O O O O O O O O O O O O O O O O O O O O 0 O O O O O O 20

Zoospore Production . . . . . . . . . . . . . . . . . . . . 20
Effect of culture age on zoospore production . . . . . 20
Effect of mycelial age on zoospore production . . . . 20

iv

—— —'.-‘-4._- .

Effect of number of water changes on zoospore pro—
duction O O O O O O O O O O O O O O O O O O O O 0

Effect of flooding interval between water changes
on production of zoospores . . . . . . . . . . .

Effect of incubation temperature on production of
zoospores O O O O O O O O O O O O O O O O O O O 0

Effect of light on zoospore production . . . . . . . .
Effect of amount of agar on zoospore production . . .

Effect of different flooding solutions on zoospore
production . . . . . . . . . . . . . . . . . . .

Length of time required for zoospore production . . .

Evaluation of zoospore production method with
P, megasperma var. sojae races 2, 3, 4, 5, and 6

 

Inoculation of Soybean Seedlings Grown in Vermiculite
with Zoospores . . . . . . . . . . . . . . . . . . . .

Effect of seedling age on disease incidence . . . . .
Effect of temperature on disease incidence . o,~ . . .
Effect of zoospore concentration on disease incidence.

Effect of zoospore concentration on disease incidence
in resistant cultivars . . . . . . . . . . . . .

Differential response of soybean cultivars to six
races of P, megasperma_var. sojae . . . . . . . .

 

Differential response of cultivars to six races of
P, megasperma var. sojae in seedlings inoculated
using the hypocotyl inoculation method .. . . . .

 

 

Inoculation of Soybean Seedlings with Zoospores in Soil . .

Effect of amount of soil or of several aqueous media
on disease incidence . . . . . . . . . . . . . .

Effect of temperature on disease incidence . . . . .

Effect of zoospore concentration on disease incidence.

Page

23

23

26
31

31

32

32

33

33
33
35

37

4O

40

42

46

46
48

48

Page

Effect of zoospore concentration on disease incidence
in differential soybean cultivars . . . . . . . . 51

Differential response of soybean cultivars to six

 

races of P, megasperma var. sojae . . . . . . . . 54
Inoculation of Soybean Seedlings with Oospores in Soil . . 56
Effect of different soil media on disease incidence . 56

Effect of temperature on disease incidence using
naturally infested soil . . . . . . . . . . . . . 60

Effect of light on oospore germination and on disease
incidence . . . . . . . . . . . . . . . . . . . . 6O

Soybean seedlings as baits for recovering
P, megasperma var. sojae from soil . . . . . . . 61

 

DISCUSSION 0 O O C O O O O O O O O O O O O O O O O O O O O O O O 64

LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . . . . . 70

vi

LIST OF TABLES

Table Page

1. Differential response of soybean cultivars to races
1 to 6 of Phytophthora megasperma var. sojae . . . . . . . 16

 

2. Effect of incubation period, after the final flooding with
distilled water, on zoospore production by Phytophthora
megasperma var. sojae race 1 . . . . . . . . . . . . . . . 34

 

 

3. Effect of seedling age on disease incidence of soybeans
grown in vermiculite and inoculated with zoospores of
Phytophthora megasperma var. sojae race 1 . . . . . . . . . 36

 

4. Effect of temperature on disease incidence in 'Hark' soy-
beans grown in vermiculite and inoculated with zoospores
of Phytophthora megasperma var. sojae race 1 . . . . . . . 38

 

5. Effect of zoospore concentration on disease incidence of
soybeans grown in vermiculite and inoculated with
zoospores of Phytophthora megasperma var. sojae race 1 . . 39

 

6. Effect of zoospore concentration on disease incidence in
differential soybean cultivars grown in vermiculite and
inoculated with Phytophthora megasperma var. sojae race 1 . 41

 

7. Differential response of selected soybean cultivars to six
physiologic races of Phytophthora megasperma var. sojae
when seedlings were grown in vermiculite and inoculated
with zoospores . . . . . . . . . . . . . . . . . . . . . . 45

 

8. Differential response of selected soybean cultivars to six
physiologic races of Phytophthora megasperma var. sojae
when seedlings were inoculated using the hypocotyl inocu-
lation method . . . . . . . . . . . . . . . . . . . . . . 47

 

9. Effect of amount of soil on disease incidence in 'Hark'
soybeans placed in petri dishes and incubated with
zoospores of Phytophthora megasperma var. sojae race 1 . . 49

 

10. Effect of temperature on disease incidence of 'Hark' soy—
beans incubated on flooded sterilized soil artificially
infested with zoospores of Phytophthora megasperma var.
sojae or on soil naturally infested with the pathogen . . . 50

 

vii

Table Page

11. Effect of zoospore concentration on disease incidence in
'Hark' soybeans placed in petri dishes containing 10 g
autoclaved soil and inoculated with zoospores of
Phytophthora megasperma var. sojae race 1 . . . . . . . . . 52

 

12. Effect of zoospore concentration on disease incidence in
differential soybean cultivars placed in trays containing
sterilized soil and inoculated with Phytophthora
megasperma var. sojae race 1 . . . . . . . . . . . . . . . 53

 

 

13. Effect of zoospore concentration on disease incidence of
differential soybean cultivars 2 and 3 days old placed on
soil and inoculated with Phytophthora megasperma var.
sojae race 1 . . . . . . . . . . . . . . . . . . . . . . . 55

 

14. Differential response of selected soybean cultivars to six
physiologic races of Phytophthora megasperma var. sojae
when seedlings were incubated on flooded soil and
inoculated with zoospores . . . . . . . . . . . . . . . . . 59

 

15. Effect of light on oospore germination and soybean seedling
infection by Phytophthora megasperma var. sojae race 1 in
naturally infested soil and in soil artificially infested
with oospores or zoospores of the pathogen . . . . . . . . 62

 

viii

 

LIST OF FIGURES

Figure Page

1. Effect of culture age on zoospore production by
Phytophthora megasperma var. sojae race 1. Least
significant range by Tukey's iw' procedure is 9.9

(g=o.05)......................... 21

 

2. Effect of the number of times the culture was flooded
with water (30 minutes each) on zoospore production by
Phytophthora megasperma var. sojae race 1. Least
significant range by Tukey's 'w' procedure is 7.3
(P.= 0.05). . . . . . . . . . . . . . . . . . . . . . . . . 24

 

3. Effect of flooding interval between water changes on
production of zoospores by 4- and 6-day-old cultures of
Phytophthora megasperma var. sojae race 1. Least signi-
ficant range by Tukey's 'w' procedure is 4.3 (P_= 0.05)
for 4-day-old cultures and 2.3 (P_= 0.05) for 6-day-old
cultures. . . . . . . . . . . . . . . . . . . . . . . . . . 27

 

4. Effect of incubation temperature on colony growth and
on zoospore production (after the final flooding) by
_Phytophthora megasperma var. sojae race 1. Least
significant range by Tukey's 'w' procedure is 3.1
(P_= 0.05) for zoospore production and 0.4 (P_= 0.05)
for colony diameter . . . . . . . . . . . . . . . . . . . . 29

 

5. Differential soybean cultivars (left to right) Altona,
Hark, Harosoy 63, Higan, Sanga, Toku, and Tra y in
vermiculite and inoculated with zoospores (10 /seed1ing)
of race 4 of Phytophthora megasperma var. sojae. Hark,
Harosoy 63, and Higan are susceptible to race 4 and
failed to emerge after inoculation . . . . . . . . . . . . 43

 

6. Differential soybean cultivars (left to right) Altona,
Hark, Harosoy 63, Higan, Kingwa, Eanga, Toku. and Tracy
inoculated with zoospores (2 X 10 /seedling) of race 5
of Phytophthora megasperma var. sojae in the presence
of steamed soil. Altona, Hark, Harosoy 63, Higan, and
Toku are susceptible to race 5; the hypocotyls turned
brown and failed to develop after inoculation . . . . . . . 57

 

 

ix

INTRODUCTION

Phytophthora root and stem rot, caused by Phytophthora megasperma

 

(Drechs.) var. gglae_Hildeb., is a very widespread and damaging disease
of soybeans in Michigan. Although there are no comprehensive figures
available as to the extent of economic losses, it is considered to be
the major disease problem in the over 600,000 acres under soybean cul-
tivation. It is also an important disease in other parts of the U.S.
and in Ontario.

Phytophthora root and stem rot of soybeans was first observed in
Ohio in 1951 (82). A number of different sources of resistant germ
plasm were found by 1954 (9), and resistant cultivars were developed
that controlled the disease until new races of the pathogen appeared
that caused disease in soybean cultivars which were formerly resistant.
In 1965 a second physiological race of the fungus was identified in
Mississippi (40). Since 1972 races 3, 4, 5, 6, 7, 8, and 9 have been
identified (25, 47, 69, 72). The occurrence of numerous races of the
pathogen has made necessary the development of reliable race-identifica-
tion techniques.

Hypocotyl inoculation is the conventional method of evaluating
resistance in soybeans. A small piece of mycelium is inserted into an
incision in the hypocotyl, and susceptible plants collapse and die
within a few days (37).

However, the hypocotyl inoculation method has shortcomings. The
method is tedious to perform, requires considerable amounts of

1

 

greenhouse space, is susceptible to changes in environmental conditions
such as temperature fluctuations, may yield inconsistent results, and
does not detect types of resistance designated as 'field tolerance'.
Moreover, the hypocotyl-wound technique is of no use for studies of
environmental factors on the infection process, inoculation experiments
using natural propagules, i.e., oospores and zoospores in relation to
the natural infection court, or for investigations of biological
control. An alternative method utilizing spores of the pathogen and
the plant root as the site of penetration and infection would therefore
be desirable.

Phytophthora_spp. can readily be isolated from plant tissue,
but isolation even with the aid of selective media directly from soil
has proven difficult. Susceptible host plants or fruits have been
relied upon to detect and isolate these pathogens directly from natural-
ly infested soils (6, ll, l3, 14, 21, 50, 58, 62, 63, 64, 83). Baiting
methods allow rapid detection and identification of the pathogen, often
without the necessity for isolation and inoculation procedures.

The present investigation was undertaken to i) develop methods
for abundant zoospore production, ii) develop reliable inoculation
methods using natural propagules which could be used for race identifi-
cation, and iii) determine the feasibility of employing soybean seed-

lings as baits to detect and isolate P, megasperma var. sojae from

 

natural soil.

 

LITERATURE REVIEW

The Pathogen

 

Phytophthora root and stem rot, incited by Phytophthora

 

megasperma (Drechs.) var. sojae Hildeb., is a relatively new but

 

important disease of soybeans (Glycine max (L.) Merr.). It attacks

 

soybean plants in all stages of growth, causing pre-emergence and post-
emergence damping-off of seedlings, and a root and stem rot that
results in wilting and death of plants from the primary-leaf stage to
maturity (31, 37). It was first observed in Ohio in 1955 by Suhovecky
and Schmitthenner (82) who reported that the disease was caused by a

species of Phytophthora. Subsequently, the pathogen was referred to,

 

incorrectly, as Phytophthora cactorum (Leb. & Cohn) Schroet. (29).

 

Kaufmann and Gerdemann (37) considered the organism distinct from

described species of Phytophthora and proposed the name Phytophthora

 

 

sojae. The current trinomial, based on morphological similarity to

Phytophthora megasperma and host specificity, was proposed by

 

Hildebrand in 1959 (31).

After the first report of this root rot of soybean in Ohio, the
disease appeared in many other areas throughout the soybean-producing
regions of the Midwest, South, and Ontario (25, 40, 47, 69, 72). In
1954 resistant cultivars were identified, with resistance of the
cultivar Mukden controlled by a single dominant gene (9, 56). The

Mukden—type resistance, assigned the gene symbol Rps, was used to

develop several resistant cultivars for the Midwest. In 1965 a second

physiological race of P, megasperma var. sojae was identified in

 

Mississippi, and an additional allele, Epsg, recessive to Pp§_but
dominant to EEE: was discovered (27, 40). In 1972 a third and more
virulent race was found in Ohio (69). Cultivars with the Mukden-type
resistance were susceptible to race 3, whereas Arksoy and its
derivative, Mack, were resistant to race 3. In 1974 a strain obtained
from Kansas was designated race 4; it was pathogenic to cultivars with
both the Arksoy and Mukden types of resistance (72). Those cultivars
with the.£2§?£2§? allele are resistant to races 1, 3, and 4, but
susceptible to race 2 (57). Cultivars Altona, Kingwa, and Toku are
resistant to races 1, 2, 3, and 4 (57). In 1976 races 5 and 6, to
which Altona is susceptible, were reported from Ontario, Canada (25).
The two races were differentiated by the susceptibility of Mack to
race 5 and its resistance to race 6. In 1977 races 7, 8, and 9 were
reported from Indiana (47). The three races were differentiated as
follows: Harosoy, Harosoy 63, and Altona were susceptible and Sanga
and Mack were resistant to all the Indiana isolates; P.I. 103.091 was
resistant and P.I. 171.442 was susceptible to race 7; but P.I. 103.091
was susceptible and P.I. 171.442 was resistant to race 8; both P.I.
103.091 and P.I. 171.442 were resistant to race 9. Races 5 and 9 were
differentiated by the susceptibility of Mack to race 5 and its
resistance to race 9. The cultivar 'Tracy' is resistant to all nine
races of the pathogen and studies have shown that three different
dominant genes are involved in the resistance (4). In July 1975 at
Harrow, Ontario plant pathologists and plant breeders agreed to adopt

a uniform set of soybean cultivars for differentiating races 1 through

9. These consisted of Harosoy, Sanga, Harosoy 63, Mack, Altona,
P.I. 103.091, and 171.442 (47).

Oospores of Phyt0phthora spp. have a constitutive dormancy

 

period (7, 17, 78) which may be broken by various treatments.
Germinability of P, caCtorum oospores in vitro was increased by prior

incubation in natural soil (78), and that of P, megasperma var. sojae

 

was increased by preincubation in water for 48 hours at 36°C (49).
Water or water agar is usually used as a medium for oospore germina-
tion, but soil extract agar (7) has given improved germination of
oospores of P, cactorum. Germination of oospores in soil has not been
studied.

Visible light has been shown to stimulate oospore germination

of Phytophthora spp. in vitro (7, 8, 17, 49, 68). The blue and far-red

 

regions of the light spectrum have been shown to increase oospore

germination 19.2: cinnamomi, P, megasperma var. sojae, and P, capsici

 

(68). However, the significance of light as a factor in oospore
germination in natural soil apparently has not been studied.

Zoospore production by P, megasperma var. sojae apparently has

 

been studied very little. Previous workers found that sporangia never
developed on solid media, but could be induced to form in variously
treated media, e.g., i) placing mycelial wefts in Petri's solution
(75), ii) treating corn meal culture squares with Petri's solution and
then with distilled water (31), and iii) placing mycelial plugs in
non-sterile stream water (33, 35). However, the factors affecting

zoospore production have not been systematically investigated.

Inoculation Methods

 

Hypocotyl inoculation is the conventional method of testing

pathogenicity of P, megasperma var. sojae, and evaluating resistance

 

in soybeans (4, 27, 31, 37, 40, 45, 47, 56, 69, 72). A small piece of
mycelium is inserted into a longitudinal incision in the hypocotyl
midway between the soil line and the cotyledons. Kaufmann and
Gerdemann (37) were the first to use this method for studies of soy-

bean resistance t°.§: megasperma var. sojae.

 

Some investigators have sought a more natural host-parasite
system for evaluating resistance. Inoculation methods have been worked
out whereby plants were grown in either sand or soil (31, 36, 38, 56,
69) or liquid culture (38, 40) infested with a mycelial suspension.
Studies comparing these methods showed no clear advantage of one
method over another (31, 40, 56). Recently Keeling (38) compared 1)
hypocotyl inoculation, ii) seed planted in sand infested with mycelia,
and iii) plants grown in liquid medium infested with mycelia. He used
plants representing a wide range of germ plasm and two races of the
pathogen. He concluded that the hypocotyl inoculation method was more
reliable and less subject to experimental variation due to inoculum
concentration, environmental influence, and differences in seed or

seedling vigor.

Isolation from Soil

 

Selective media have been developed for isolation of Phytophthora

 

spp. from soils and plant tissue. These media were based mainly on the
selective inhibition of non-pythiaceous fungi by the use of the

tetraene antibiotics, pimaricin and mycostatin (15, 16, 24, 59, 60, 84,

 

85) or gallic acid (18), and of bacteria by penicillin, polymixin,
chloramphenicol, and vancomycin. The literature on this subject was
summarized by Tsao (84). Using these selective media, Phytophthora
colonies from soil usually can be identified under low magnification
after a short incubation period. However, all these media have

allowed the growth of Pythium spp. and most Mortierella spp. which are

 

common in soils and infected tissues, and often interfere with the

direct isolation and enumeration of Phytophthora spp. Sneh (76)

 

reported a selective agar medium containing corn oil which permitted
P, cactorum colonies to become visible macroscopically on plates free
from other organisms. Very recently, HMI (3 - hydroxy - 5 methyli-
soxazole), was reported to greatly inhibit the growth of Pythium spp.

and Mortierella spp., but was virtually noninhibitory to germination of

 

various kinds of spores of selected Phytophthora spp. (52, 86). A

 

selective agar medium supplemented with HMI was used to successfully

isolate Phytophthora spp. from artificially and naturally infested

 

soils containing high populations of Pythium spp. and Mortierella spp.

 

The medium may have potential for the direct isolation and enumeration

of Phytophthora spp. from soil.

 

Because Phytophthora is one of the most difficult genera of
pathogenic fungi to isolate from soil, its detection, isolation, and
enumeration have usually relied upon the use of susceptible host plants

or fruits as baits. Phytophthora cactorum has been isolated from

 

naturally infested soil by baits such as apple fruit (58, 74, 88), pear
fruit (74), and blue lupine seedlings (62). The apple technique is
not specific for P, cactorum and also has been used for isolation of

other Phytophthora species such as P, cinnamomi (ll, 58), P, citricola

 

(58, 74), P, cryptogea (58), and P. syringae (53, 58, 74). Alfalfa

seedlings have been used as baits to detect P, megasperma in infested

 

soils and to study the ecology of the fungus (50, 51, 63, 64). Pine

needles (Pinus radiata and Cedrus deosara) have been used to bait

 

 

P, boehmeriae, P, cactorum, P, cambivora, P, cinnamomi, P. citricola,

 

.P. crytogea, P, drechsleri, P, hibernalis, P, megasperma var.

 

 

 

megasperma, P, nicotianae var. nicotianae, P, nicotianae var.

 

 

 

 

parasitica, and P, rachardiae from naturally infested soils (13, 21).

 

 

Laboratory tests with zoospore suspensions have established that

P, citrophthora, P, heveae, P, megasperma var. sojae, and P, palmivora

 

 

were also able to infest needles of Pinus radiata. Radicles of lupin

 

(Lupinus angustifolius) were shown to have the same spectrum of sus-

 

ceptibility as pine needles (l3).
Tsao (83) described a lemon fruit baiting technique for

Phytophthora citrophthora and P, parasitica which allowed estimation of

 

 

inoculum levels in soils by assaying them in a series of dilutions
with uninfested soil. Marks and Mitchell (50) used the "serial

dilution end-point" method to estimate inoculum levels of P, megasperma

 

in soil by using alfalfa seedlings as baits. Duncan (14) used rooted

runners of a clone of Fragaria vesca as baits to detect P, fragariae

 

in infested soils and assessed inoculum levels using dilutEd soil
suspensions and the 'most probable number' method.

Baiting methods allow detection and enumeration of Phytophthora

 

spp. directly from naturally infested soils. However, identification
of species may require isolation on agar since sexual structures may

not be sufficiently visible (and measurable) on the baits. Race

identification of many Phytophthora spp. may also be possible using

 

differential bait plants without the need for isolation and inoculation

procedures.

 

l“_,. - m

METHODS AND MATERIALS

Maintenance of Fungus

 

Phytophthora megasperma var. sojae, races 1, 2, 3, 4, 5, and 6

 

were used. Isolates of races 1 to 5 were obtained from Dr. A. F.
Schmitthenner (Ohio Agricultural Research and Development Center,
Wooster), and isolates of races 1, 2, and 6 were obtained from Dr.
J. H. Haas (Agriculture Canada Research Station, Ontario, Canada).
Race 1, obtained from Dr. Schmitthenner, was used in all experiments
in which the factors affecting zoospore production were evaluated.
All the isolates were used in experiments to evaluate inoculation
methods. Each race of the fungus was maintained as a stock culture
on V-8 juice agar slants (per liter: 200 ml V—8 juice, 3.0 g CaCO3,
and 20 g agar). New cultures were prepared every 2 to 3 months from
soybean seedlings (cultivar 'Hark') placed on flooded cultures of the

fungus on Difco lima bean agar. Three days later the fungus was

reisolated as described in 'Isolation Methods'.

Production, Collection, and Preparation of Inocula

 

Oospores of the fungus were produced on V-8 juice broth in
petri dishes [per liter: 100 ml V—8 juice, 1.0 g CaCO3, and 30 mg
cholesterol (dissolved in 1.5 ml ethanol over heat)] (5). The plates
were incubated in the dark at room temperature (23 :_2°C). When the
cultures were 1 to 6 weeks old, the mycelial mats containing oospores

were placed on disks of nylon mesh (54 um) and excess moisture was

10

ll

removed by placing the disks on the base of a membrane filter apparatus,
and applying a slight vacuum. The mycelial mats were then air-dried
for 24 to 36 hours to kill the mycelium. When dried material was
placed on V-8 juice agar for four days, there was no evidence of
mycelial growth. Oospore suspensions were prepared by grinding the
dried mycelial mats with a glass tissue homogenizer. Concentrations
were determined using a hemacytometer.

When the effect of light on oospore germination was studied, a
suspension of 5-week—old oospores was applied to Nuclepore (polycar—
bonate) membrane filters (13 mm diameter, 0.4 pm pore size; General
Electric Corp., Pleasantville, California). About 103 oospores were
applied per membrane. The membranes were incubated on flooded sterile
soil for seven days. For determination of germination, the oospores
were transferred to water agar (77).

Zoospores were produced as follows: mycelial disks were cut
with a 5 mm diameter cork borer from the periphery of 4- to 6-day-old
cultures grown on Difco lima bean agar. Individual disks were trans-
ferred to the center of 10 cm diameter petri dishes containing 7 to
16 ml of agar, and incubated at 23 I.2°C for 2 to 10 days. The
plates were then flooded with 15 ml distilled water or other flooding
solutions (See Results) which submerged all mycelia. The water was
changed 0 to 8 times at 2.5 to 60 minute intervals. Incubation
temperatures of 10 to 35°C were used. The plates were left uncovered
.throughout the washing sequence. After the final water change, the
plates were flooded once more, covered, and placed under various light
conditions (See Results) for 4 to 24 hours. The volume of water

recovered from each plate was recorded as the zoospores were collected.

 

12

Volumes varied from 13 to 17 ml. A second and third crop of zoospores
could be harvested from the same plates when flooding was repeated,
once for each crop. Zoospore concentration was determined either by
counting the spores in five random fields of a hemacytometer or by
counting the spores of five different 2 pl aliquots (44). Before
counting, zoospore motility was stopped by adding a drop of 0.1%

cotton blue in lactophenol (w/v) to 1 m1 of zoospore suspension.

Isolation Methods

 

The pathogen was isolated from diseased hypocotyls of soybean
seedlings. The cotyledons were removed, and the hypocotyls were
surface-disinfested by immersion in 0.5% sodium hypochlorite for 10
seconds. The tissue was then washed with water and dried on filter
paper. Small portions of the tissue were excised by making cross-
sectional cuts, and placing them, cut surface down, on a modified

selective medium (76) (per liter: 0.1 g CaSO 0.02 g thiamine-HCl,

4,
5.0 g sucrose, 7 ml corn oil, 0.1 m1 Triton B 1965, 15 g agar, 20 mg
benomyl, 60 mg penicillin G (KI), 60 mg polymixin B sulfate, and 60 mg
chloramphenicol). The plates were incubated for l to 3 days at

23;: 2°C. Mycelial growth from the diseased segments was transferred,
as soon as visible, to V—8 juice agar containing antibiotics (per

liter: 200 ml V—8 juice, 3.0 g CaCO 20 g agar, 60 mg penicillin

39
G (K+), 60 mg polymixin B sulfate, and 60 mg chloramphenicol). These
plates were incubated for 24 hours and then the agar was inverted.

Three to five days later, when the fungus had grown up through the

agar layer, it was transferred to Difco lima bean agar.

13

Source and Preparation of Soils

 

Conover sandy clay loam was collected from the Michigan State
University Botany and Plant Pathology Farm. It possessed the following
characteristics: pH 7.8, organic matter 2.96%, water holding capacity

28%, clay 26%, silt 24%, and sand 50%. This soil was found to be free

 

of the pathogen, P, megasperma var. sojae, and was used in its natural
state or was steamed at 100°C for 30 minutes. Brookston loam soil,
also collected from the Michigan State University Botany and Plant

Pathology Farm, was found to be infested with P, megasperma var. sojae.

 

It possessed the following characteristics: pH 7.5, organic matter
6.46%, water holding capacity 48%, clay 19%, silt 33%, and sand 48%.
The soils were screened through a sieve with 2.5 mm openings, uniformly
mixed, and stored air-dried in polyethylene bags in the laboratory.

In some experiments, steamed Conover sandy clay loam.was
artificially infested with an aqueous suspension of oospores of

P, megasperma var. sojae. The oospores had been concentrated by

 

centrifugation, and the oospore suspension was mixed with the air-
dried soil by hand.

Extract from natural soil was prepared by suspending 1 kg of
air-dried Conover sandy clay loam in 1 liter distilled water for 24
hours. The supernatant soil suspension was coarsely filtered through
3 layers of cheesecloth under a slight vacuum, and was centrifuged at
5000 rpm for 10 minutes. The soil extract, either used as prepared,
diluted, or autoclaved, was employed to test its effect on disease

incidence in soybean seedlings inoculated with zoospores and oospores.

 

l4

Zoospore Inoculation of Soybean Seedlings Growing in Vermiculite

Soybean seeds were planted on the surface of a 4 cm-deep layer
of distilled water-saturated vermiculite in plastic trays 27 X 19 X 6,
19 X 19 X 6, or 19 X 9 X 6 cm. The trays contained varying numbers
of rows at least 2.5 cm apart each with 5 to 10 seeds. The seeds were
covered with a 1.5 cm layer of dry vermiculite and trays were covered
with polyethylene bags to minimize evaporation. After 2 days at
23: 2°C, the polyethylene bags were removed and distilled water was
added to saturate the vermiculite. The seedlings were inoculated by
applying 2 ml of a zoospore suspension, usually containing 104
zoospores, at the base of each seedling using a Cornwall automatic
pipette syringe. The zoospore suspension was constantly agitated with
a magnetic stirrer throughout inoculation of seedlings. After inocu-
lation, the trays were incubated in a growth chamber under light
(17,000 lux) for 12 hour photoperiods at 23 112°C. In controlled
temperature experiments, trays were placed in non-illuminated incuba-
tors. The standing water in the trays evaporated during the first 24
to 36 hours. After that time, water was added as needed to keep the
vermiculite moist. Disease was evaluated 4 to 6 days after inoculation,
when the uninoculated control seedlings had produced their first true
leaves.

Disease was evaluated on the basis of emergence and by examining
the root systems of emerged seedlings. A plant was considered sus-
ceptible if it failed to emerge or if the taproot and lateral roots-
were discolored and flaccid. Roots free of lesions or with small
(<1 mm), localized lesions were considered resistant to the pathogen.

The pathogen could be isolated from these lesions.

15

Races 1, 2, 3, 4, 5, and 6 of P, megasperma var. sojae were used

 

to inoculate the following cultivars of soybean: Altona, Hark,
Harosoy 63, Higan, Kingwa, Mack, Sanga, Toku, and Tracy (Table 1).

In July of 1975 plant pathologists and plant breeders agreed to
adopt a uniform set of soybean cultivars for differentiating races of

P, megasperma var. sojae (47). These are Harosoy, Harosoy 63, Sanga,

 

Mack, Altona, P.I. 103.091, and P.I. 171.442. This author used as
many of these cultivars as were available but substituted Hark for
Harosoy, Higan for Mack in many cases, and Kingwa for P.I. 103.091.
The substituted cultivars had the same differential reactions as the

cultivars they replaced (25, 47).

Zoospore or Oospore Inoculation of Soybean Seedlings Placed on Soil
Weighed amounts of soil were placed into autoclaved 10 cm
diameter glass petri dishes (0.5 to 60 g/petri dish) and 30 ml of
distilled water were added. In some cases plastic trays 27 X 19 X 6
or 19 X 19 X 6 cm were used (100 g soil and 150 ml distilled water, or
50 g soil and 100 ml distilled water, respectively). When soil was
to be artificially infested with zoospores or oospores, the inocula
were added with the water. Soybean seeds were germinated in moist
vermiculite at 23 i_2°C in trays covered with polyethylene bags. When
the seedlings were 2 days old, with hypocotyls 0.5 to 2 cm long, the
seedlings were removed from vermiculite, washed, sorted by length, and
placed on the soil surface with hypocotyls submerged in water (5 seed-
lings/petri dish, 20 to 45 seedlings/tray). The petri dishes were
covered with glass tops or in the case of trays with polyethylene

bags to prevent desiccation, and incubated on the laboratory bench at

 

16

Table 1. Differential response of soybean cultivars
to races 1 to 6 of Phytophthora megasperma

var. sojaea

 

 

 

 

 

Race

Cultivar

l 2 3 4 5 6
Hark S S S S S S
Sanga R S R R R R
Harosoy 63 R R S S S S
Mack R R R S S R
Higan R R R S S R
Altona R R R R S S
Toku R R R R S R
Kingwa R R R R R R
Tracy R R R R R R

 

a . . .
Reactions: S = susceptible; R = re31stant.

 

17

23 112°C, usually under ambient light conditions (130 lux). When the
effect of light on disease incidence was studied, petri dishes were
incubated under fluorescent light (4800 lux provided by 4 Sylvania
Gro-Lux WS 40 watt lamps placed 35 cm above the plates), or incubated
on the laboratory bench under ambient light (130 lux). Petri dishes
for dark treatments were wrapped in aluminum foil.

Seedling infection was evaluated after 3 days' incubation. The
plants were removed from the soil surface and placed in distilled
water. Six to 24 hours later, sporangia of the pathogen were identi-
fied by direct examination with a dissecting microscope. If sporangia
were not present, the presence of the pathogen was confirmed by re-
isolation of the fungus as previously described in 'Isolation Methods'.

Infected seedlings were stunted and failed to develop lateral
roots. The hypocotyls were either totally brown in color and flaccid,
or contained one or two severe localized lesions about 1 cm long.
Cotyledons never showed signs of infection.

Nine differential cultivars of soybean and six races of

P, megasperma var. sojae were used to evaluate this method (Table l).

 

Soybean Seedlings as Baits for Recovering
P. megasperma var. sojae from Soil

 

 

Two-day-old soybean seedlings (cultivar 'Hark'), previously
germinated at 23;: 2°C in moist vermiculite, were placed in petri
dishes (5 seedlings/plate) containing 20 g air—dried natural soil and
30 ml distilled water. Two days later the seedlings were removed
and discarded. A second set of two-day-old seedlings was then placed
on the soil. Disease developed more consistently in the second set of

seedlings than the first. Seedling infection was evaluated after 3

18

days' incubation by direct examination for sporangia of the pathogen
with a dissecting microscope. If sporangia were not observed,
hypocotyl segments were plated on a selective medium (76) to verify the
presence of the pathogen.

Disease symptoms of seedlings incubated on natural soils were
similar to those occurring on seedlings artificially inoculated in
soils. However, a much higher incidence of these seedlings had 1 or
2 individual lesions (0.5 to 1 cm) and fewer had totally brown
hypocotyls. Pythium spp. in the soil could also cause these symptoms
on soybean seedlings, but upon microscopic examination, the sporangia
and mycelia of Pythium spp. could be differentiated from those of

P. megasperma var. sojae.

 

Hypocotyl Inculation Method

 

Soybeans were planted in 10 X 14 cm plastic pots (5 to 8 seeds/
pot) in a potting mix (soilzpeatzsand; 1:1:1; v/v/v). Plants were
inoculated when they were 8 days old or had their first true leaves.

A longitudinal incision was made with a razor blade in the hypocotyl
midway between the soil line and the cotyledons. A 2 X 5 mm mycelial

segment from a 5-day—old culture of P, megasperma var. sojae was

 

placed in the wound. Petroleum jelly was used to seal the wound to
prevent desiccation and each pot was covered with a polyethylene bag
for 24 hours. The pots remained in the greenhouse at 26_: 4°C.
Disease was evaluated after 5 days (37). Plants which had collapsed
hypocotyls both above and below the wound were considered susceptible.
Those which developed restricted lesions around the point of incision

but did not collapse, were considered resistant. In some cases, lesion

 

19

development was severe and caused collapse of the seedling above the
inoculation site. These questionable reactions were considered
resistant reactions, and may have resulted from wounding the hypocotyl
too deeply.

Nine differential cultivars of soybean and six races of

.P. megasperma var. sojae were used (Table l).

 

In all experiments treatments were replicated at least three
times, and experiments were repeated 2 to 5 times with similar results.
Differences between means were detected using Tukey's 'w' procedure

following analysis of variance (81).

 

 

 

RESULTS

Zoospore Production

 

A number of factors affecting zoospore production of

P, megasperma var. sojae in culture was studied in order to optimize

 

conditions for zoospore production for use in inoculation experiments.

Effect of culture age on zoospore production. Cultures 2 to 10
days old were evaluated for zoospore production. Cultures were washed
4 times at half-hour intervals, then reflooded, covered, and incubated
at 23 :;2°C for 20 hours.

Maximum numbers of zoospores were produced from 6-day—old
cultures (Fig. 1). At this age, the culture completely covered the
surface of the agar, and probably contained the greatest amount of
young and vigorous mycelia.. Cultures older than 6 days showed a
decline in zoospore numbers probably due to aging and loss of vigor of
mycelium.

Effect of mycelial age on zoospore production. Three cultures

 

were started by placing a 5 mm diameter disk of agar inoculum in the
center of each agar plate. Each day's growth was measured, and the
growth ring marked with ink on the underside of the petri dishes.
After six days incubation, 7 mm diameter disks ranging from 1 to 5
days old were cut from the rings of mycelium. Five mycelial disks of
each age were transferred to sterile petri dishes and flooded with

15 ml of distilled water for one-half hour, flooded once more with 10
ml, then incubated for 20 hours at 23 j:2°C.

20

 

21

Fig. 1. Effect of culture age on zoospore production by Phytophthora
megasperma var. sojae race 1. Least significant range by
Tukey's 'w' procedure is 9.9 (P_= 0.05).

 

—

 

22

 

 

 

70 -

_
0
5

0
3

no. x 551356.303

'IO-

 

6 8 IO
CULTURE AGE, DAYS

4

 

23

The younger the mycelium, the greater was the zoospore produc-
tion. Oneeday-old mycelia produced 1.4 X 104 zoospores/disk, which
was four times greater than that produced by 5-day—old mycelium (3.7 X
103 zoospores/disk). Two’, 3—, and 4-day—old disks produced 7.5 X
103, 6.1 X 103, and 4.2 X 103 zoospores, respectively.

Effect of number of water changes on zoospore production.

 

Five—day—old cultures were washed at one—half hour intervals. The
effect of zero to 8 water changes on zoospore production was examined.
After 16 hours incubation at 23 : 2°C, zoospores were collected and

concentrations determined.

"

A single flooding, without any water change, was sufficient to
cause sporangial development and release of low numbers of zoospores
(4 X 103/plate, 2.7 X lOz/ml) (Fig. 2). However, many more zoospores
were obtained with additional changes of water until maximum numbers
of zoospores (5.2 X 106/plate, 3.6 X 105/ml) occurred with 5 water
changes. Zoospore production decreased after more than 5 water
changes. Apparently, removal of nutrients from the culture enhances
zoospore production, but excessive washing may impose an excessive
stress on the mycelium.

Effect of flooding interval between water changes on production

 

of zoospores. Since a sequence of washings of the culture promoted
zoospore production, an experiment was designed to determine the opti—
mum time interval between changes of water on zoospore production.
Four—day—old and 6-day—old cultures were flooded for intervals of

2.5 minutes to 60 minutes. Each culture received 5 water changes.
Zoospores were collected and counted after 18 hours incubation. The

times of flooding were so arranged that washing in all treatments was

‘

 

’1

Fig. 2.

24

Effect of the number of times the culture was flooded with
water (30 minutes each) on zoospore production by
Phytophthora megasperma var. sojae race 1. Least signi-
ficant range by Tukey's 'w' procedure is 7.3 (P>= 0.05).

 

25

 

50—

.5
O
I

00
O
I

20-

zoosponrs / PLATE, x105

6'
I

 

0.11
O'ML

0

I I I I I I
2 4 6
NO. OF WATER CHANGES

@—

 

26

terminated at the same time.

The flooding interval had a significant effect on zoospore pro—
duction (Fig. 3). Numbers of zoospores increased as the time interval
between washings lengthened up to 30 minutes. However, a 60 minute
interval did not produce significantly greater numbers of zoospores
than the 30 minute interval. Cultures 4 and 6 days old had similar
trends.

Effect of incubation temperature on production of zoospores.

 

Five—day—old cultures were washed four times at half—hour intervals

with water at room temperature (23 :_2°C). The water used for the

fifth and final flooding was adjusted to the specific incubation I
temperature for each treatment, and the plates were incubated at

these same temperatures in darkness by covering the plates with aluminum

foil. Incubation temperatures were 10, 15, 20, 25, 30, and 35°C.

Eighteen hours later, the zoospores were collected and counted.

Plates incubated at 20°C yielded the greatest numbers of zoo—
spores (2.5 X 106/plate, 1.7 X lOS/ml) (Fig. 4). This treatment also
appeared to result in the highest proportion of actively swimming
zoospores, though precise determinations were not made. Plates in—
cubated at 30°C contained many dehiscent sporangia (35) in which the
zoospores remained trapped. Many zoospores germinated within the
sporangia, and produced germ tubes which penetrated the sporangial
wall. At 35 °C no sporangial production was observed.

The effect of temperature from 10 to 35°C on colony growth was
also examined. The incubation temperature for optimal colony growth
was 25°C. These results are substantiated by other researchers (32,

33, 75). The optimum temperature for colony growth was higher than

27

Fig. 3. Effect of flooding interval between water changes on produc—
tion of zoospores by 4— and 6—day—old cultures of Phytoph-
thora megasperma var. sojae race 1. Least significant range
by Tukey's 'w' procedure is 4.3 (P = 0.05) for 4—day—old
cultures and 2.3 (P_= 0.05) for 6-day-old cultures.

 

ZOOSPORES/ PLATE. x105

25

20

-l
U1

8

28

 

 

 

 

l I

 

 

30 p 60
FLOODING INTERVAL, MIN .

 

 

 

Fig. 4.

 

29

Effect of incubation temperature on colony growth and on
zoospore production (after the final flooding) by Phytoph—
thora megasperma var. sojae race 1. Least significant

 

range by Tukey's 'w' procedure is 3.1 (P = 0.05) for
zoospore production and 0.4 (P_= 0.05) for colony diameter.

 

30

So .mmEzsg >298

 

 

ZOOSPORES

  

 

NCOLONY
DIAMETER

'0
I
O
O
l
0
t
O
U
C
C
O
D
O
0'
O

I
0'.
0
O
O

   

2O 25 3O 35
TEMPERATURE , °C

15

TO

 

 

_
O
2

25-

1

mo; .EfiahuzoamooN

 

 

31

that for sporangial development and zoospore production.

Effect of light on zoospore production. Five—day-old cultures

 

were washed at half—hour intervals four times with water at room
temperature (23 : 2°C). Water, adjusted to 20°C, was used to flood

the plates the fifth and final time. All cultures were then incubated
at 20°C for 20 hours. Treatments included cultures incubated: i) under
fluorescent light (4800 lux) for 20 hours, ii) under fluorescent light
for 10 hours followed by 10 hours in darkness, and iii) in darkness

for 20 hours. Dark treatments were prepared by wrapping the plates in
aluminum foil. The temperature inside foil-covered and uncovered plates
did not differ.

Cultures incubated in the dark produced 10 times more zoospores
than those incubated in the light. The 20-hour dark treatment pro-
duced 2.0 X 106 zoospores/plate (1.3 X lOS/ml), whereas the 20-hour
light treatment only produced 1.8 X 105 zoospores/plate (1.2 X 104/ml).
The sequential light/dark treatment produced 9.4 X 105 zoospores/plate
(6.3 X 104/m1). Dark incubation of flooded plates enhanced zoospore
production, but the length of the dark period did not appear to be
critical since the light/dark treatment produced almost as many zoo-
spores as the totally dark treatment.

Effect of amount of agar on zoospore production. Five—day—old

 

cultures were washed at half—hour intervals five times. The cultures
were incubated on the laboratory bench at 23 : 2°C for 18 hours
before zoospores were harvested. Treatments consisted of plates con—
taining 7, 10, 13, and 16 m1 of lima bean agar.

Before the plates were washed, colony diameters were measured.

The diameters were 7.8 cm for 7 ml agar, 7.5 cm for 10 m1 agar, and

 

32

7.3 cm for both 13 and 16 ml treatments. Zoospore numbers per ml
were 3.0 X 105, 5.6 X 105, 1.5 X 105, and 4.9 X 104 for 7, 10, 13, and
16 ml agar, respectively. Ten ml was chosen for future work since

it gave large numbers of zoospores, was convenient to dispense, and

easily covered the petri dish.

Effect of different flooding solutions on zoospore production.

 

Five-day—old cultures were flooded five times at half—hour intervals.
Tapwater, single distilled water, double distilled water, vermiculite
extract (approximately 1 g vermiculite was shaken in 100 ml distilled

water, then passed through cheesecloth), and a salt solution (per

3

liter: CaCl 1.75 X 10_ M; KCl, 10.3 M; MgSO 10.3 M) were tested 1

2’ 4’
for their effect on zoospore production. It had been observed that
zoospores diluted in a vermiculite extract retained motility longer
than those diluted in distilled water. The salt solution has been

used for zoospore production in Aphanomyces euteiches (55). Fifteen

 

ml of distilled water was used in each of the five washings for each
treatment, but 10 ml of each test solution was used in the final
flooding. The plates were incubated at 23‘: 2°C for 20 hours.
Zoospore numbers were greatest with double and single distilled
water. Double distilled water produced 1.6 X 105, single distilled
water 1.1 X 105, salt solution 3.0 X 104, vermiculite extract 2.4 X 104,

and tapwater 8.0 X 103 zoospores/m1.

Length of time required for zoospore production. Six-day-old

 

cultures were flooded six times at half-hour intervals. Cultures were
examined and zoospores harvested at 2, 4, 6, 8, and 18 hour periods
after the washing procedure was completed. Plates were incubated on

the laboratory bench at 23 i 2°C.

 

 

 

33

After 2 hours incubation in a flooded condition, sporangia had
begun to appear, but no zoospores had been released (Table 2). However,

at 4 hours, 8.6 X 105 zoospores/plate (5.7 X 104/ml) were collected.

Numbers continued to increase with incubations of 6, 8, and 18 hours.
After the supernatant fluid was collected for each treatment (except
the 18 hour treatment), the plates were flooded once more and allowed
to stand overnight. All plates, regardless of treatment, produced a
total of 5.1 to 6.2 X 106 zoospores in the two harvests. In subsequent
experiments, the incubation period was usually 18 to 20 hours.
Evaluation of zoospoge production method with P. megasperma var.

sojae races 2, 3, 4, 5, and 6. Once the optimum conditions for

 

zoospore production were established with an isolate of race 1 of the
pathogen, isolates of races 2, 3, 4, 5, and 6 were used to evaluate

the zoospore production method. Six—day-old cultures of the races
grown on 10 ml agar/plate were flooded six times at half-hour intervals
with 15 ml of distilled water. The plates were then incubated in dark—
ness at 20°C for 18 hours. High zoospore counts were obtained with all
races using the optimum conditions established with race 1 of the
pathogen. Race 2 produced 1.0 X 105; race 3, 1.4 X 105; race 4,

1.2 X 105; race 5, 9 X 104; and race 6, 2.1 X 104 zoospores/m1.

Inoculation of Soybean Seedlings Grown
in Vermiculite with Zoospores

 

 

Several factors were examined in order to develop a reliable
method for the inoculation of soybeans grown in vermiculite with
zoospores of P. megasperma var. sojae.

Effect of seedling age on disease incidence. Seeds of soybean

 

cultivars 'Hark' and 'Harosoy 63' were planted in water—saturated

 

 

 

 

34

 

 

 

 

Table 2. Effect of incubation period, after the final flooding with
distilled water, on zoospore production by Phytophthora
megasperma var. sojae race 1

Zoospores/plateb
Incubation Total production
period, hr First harvest Second harvest 0f two harvests
2 0 5.8 X 106 5.8 X 106
4 8.6 x 105 5.3 x 106 6.2 x 10°
6 1.1 X 106 4.0 X 106 5.1 X 106
8 2.4 X 106 3.3 X 106 5.7 X 106
6
18 5.3 X 10

 

a . . .
Incubat1on period after the washing procedure was completed.

bMean values for three replicate plates. After the initial zoospore
harvest, plates were flooded again and zoospores were collected at

18 hours.

--

 

35

vermiculite in plastic trays 19 X 9 X 6 cm. Each tray contained one
row of each cultivar with 6 seeds/row. Seeds were planted l, 2, 3, 4,
and 6 days prior to inoculation and all seedlings were inoculated on
the same day with zoospores of race 1 of the pathogen. Controls were
uninoculated seedlings of the same ages.

Hark seedlings were most susceptible when inoculated after 2
days incubation when their hypocotyls were 1 to 3 cm long. Those
inoculated after one day, or older than 2 days had consistently less
disease (Table 3). All 1- and 2—day—old seedlings which were diseased
were dead. Over half of the diseased 3-day—old seedlings emerged and
continued to develop after inoculation. and had not died at the time I
of evaluation. The diseased seedlings in 4— and 6~day-old treatments
continued to grow, but the root systems were browning and becoming
flaccid. Harosoy 63, a cultivar resistant to race 1 of the pathogen,
showed no disease when 2- to 6—day—old seedlings were inoculated, but
some of the l—day-old seedlings were infected. Uninoculated seedlings
remained healthy.

Effect of temperature on disease incidence. Seeds of soybean

 

cultivar 'Hark' were planted in water—saturated vermiculite in plastic
trays 19 X 9 X 6 cm. Each tray contained 2 rows of 5 seeds each.

When seedlings were 2 days old, the trays were flooded with distilled
water which had been adjusted to the specific incubation temperature
of each treatment. Inoculum concentrations of 103 and 5 X 103
zoospores in 2 m1 of suspension were then aplied to each seedling.

The trays were incubated in darkness at 15, 20, 25, 30, or 35°C.
Control plants were incubated at the same temperatures but were not

inoculated. Disease was evaluated in the 25, 30, and 35°C treatments

36

Table 3. Effect of seedling age on disease incidence of soybeans
grown in vermiculite and inoculated with zoospores of
Phytophthora megasperma var. sojae race 1

 

 

 

Diseased seedlings (%)a

 

 

Age at ‘
' lation daysb i
1nocu ) Hark Harosoy 63 i,
l 23 13
2 100 0
3 60 0
4 51 0
6 23 0

 

aSeedlings were evaluated for disease 5 days after inoculation.
There were 6 seedlings/age/cultivar/tray and 4 trays/age. Hark was
susceptible and Harosoy 63 resistant to race 1.

bEach seedling was inoculated with 104 zoospores in 2 ml of suspension.

 

 

 

37

after 5 days. Since the uninculated plants incubated at 15 and 20°C
had not emerged in 5 days, the trays were removed from the incubators
and allowed to remain one day at room temperature (23 : 2°C) before
disease evaluation. It was also necessary to decant excess water from
the trays incubated at 15 and 20°C 24 to 36 hours after inoculation.
The greatest numbers of diseased seedlings were obtained at
temperatures of 20, 25, and 30°C (Table 4). All diseased seedlings
failed to emerge at 25°C. Incubation at 20°C slowed development and
neither inoculated or control seedlings emerged. However, at 30°C
many of the seedlings emerged even though they were severely infected.
Uninoculated plants remained healthy. ‘ A

Effect of zoospore concentration on disease incidence. Seeds of

 

soybean cultivars 'Hark', 'Corsoy', 'Wayne', and 'Williams' were planted
in water—saturated vermiculite in plastic trays 17 X 19 X 6 cm, con-
taining one row of each of the four cultivars with 10 seeds/row.

Zoospores of race 1 of the pathogen at concentrations of 102, 5 X 102,

103, 5 X 103, and 104 in 2 ml volumes were applied to each seedling.
Controls consisted of uninoculated plants.

The lowest concentration giving consistent disease was 103
zoospores/seedling (Table 5). At 104 zoospores/seedling all four
cultivars exhibited 100% disease. There was some indication of differ—
ences among the cultivars in susceptibility to the pathogen at an
inoculum level of 103 zoospores. The cultivars ‘Wayne' and 'Williams'
are considered to have field tolerance to race 1 of_P. megasperma var.
sojae, They resist Phytophthora root rot in the field even under

favorable disease conditions. These differences tended to be masked

3 .
at inoculum concentrations above 10 zoospores/seedling. Controls

 

 

38

Table 4. Effect of temperature on disease incidence in 'Hark' soybeans
grown in vermiculite and inoculated with zoospores of
Phytophthora megasperma var. sojae race 13

 

 

 

Diseased seedlings (%) at
indicated zoospore concentration

‘2-

 

Temperature, °C

 

1o3 ‘ 5 x 103
15 16 3o
20 88 88
25 88 100
30 84 88
35 40 56
LSRC 33.6 34.4

 

aTwo—day-old seedlings were inoculated, and disease was evaluated 5
days later for 25, 30, and 35°C treatments, and 6 days later for 15
and 20°C treatments. There wereJO seedlings/tray and 4 trays/
concentration/temperature.

bZoospores/seedling.

CLeast significant range by Tukey's 'w' procedure (P_= 0.05).

 

 

 

Table 5.

Effect of zoospore concentration on disease incidence of
soybeans grown in vermiculite and inoculated with zoospores
of Phytophthora megasperma var. sojae race 1

 

 

 

Diseased seedlings (%)a

 

 

Zoospores/
seedling Corsoy Hark Wayne Williams Meanb
0 0d 0 0 0 0.0 A

102 0 0 0 13 3.3
5 X 102 0 0 0 0 0.0

103 67 83 33 63 61.5
5 X 103 90 100 87 93 92.5

104 100 100 100 100 100.0

MeanC 42.8 47.2 36.7 44.8

 

aTwo—day-old seedlings were inoculated and disease was evaluated 5

days later.
1:.

treatmen

bLSR (g
CLSR (g
dLSR (g
21. 8 .

0.05)
0.05)

0.05)

= 8.9.

= 10.9.

There were 10 seedlings/cultivar/tray and 3 trays/

for interaction of inoculum concentration X cultivar =

 

 

40

remained healthy.

Effect of zoospore concentration on disease incidence in

 

resistant cultivars. Cultivars 'Hark', 'Altona', 'Harosoy 63', and
'Toku' were chosen for this experiment because their susceptibilities
to race 1 of the pathogen differed. Hark is susceptible, Altona is re-
sistant [but some individuals are.susceptible (A.F. Schmitthenner,
personal communication)], and Harosoy 63 and Toku are homogeneously
resistant.

Seeds were planted in water-saturated vermiculite in plastic
trays 19 X 19 X 6 cm containing one row each of the four cultivars

with 5 seeds/row. Seedlings were inoculated with 104, 2.5 X 104, 5.0 X

104, or 105 zoospores in 2 ml of suspension.

The three resistant cultivars Altona, Harosoy 63, and Toku
exhibited different reactions to the various inoculum concentrations
(Table 6). For example when 105 zoospores were applied, 47%, 7%,
and 0%, of the seedlings, respectively, exhibited the typical severe
decay of the hypocotyl. The percentage of severely infected Altona
seedlings tended to increase as inoculum concentrations increased.
Resistance apparently can be overcome with sufficient inoculum. There
was no significant interaction between the cultivars and inoculum
concentrations. In addition to the severe infections, hypocotyls of
some plants of each of these resistant cultivars frequently developed
numerous, tiny (<1mm), brown lesions which remained localized (Table 6).
Control seedlings remained healthy.

Differential response of soybean cultivarsto six races of

 

P. megasperma var. sojae. Nine differential soybean cultivars growing

 

in vermiculite were inoculated with zoospores of races 1 to 6 of the

 

 

Table 6. Effect of zoospore concentration on disease incidence in
differential soybean cultivars grown in vermiculite and
inoculated with Phytophthora megasperma var. sojae race 1

 

 

 

Diseased seedlings (%)b

 

 

Zoospores/ Mean
seedling Hark Altona Harosoy 63 Toku
104 100 13 (7) 0 0 28.3
2.5 x 104 100 13 (40) o (7) o 28.3
5.0 x 104 100 o (27) o (27) o 25.0
105 100 47 (31) 7 (33) o (27) 38.5
Mean 100 18.3 1.8 0

 

aTwo—day-old seedlings were inoculated and disease was evaluated 5
There were 5 seedlings/cultivar/tray and 3 trays/

days later.
treatment.

bHark is susceptible to race 1; the other three cultivars are re—
sistant. Values are percent of seedlings which exhibited severe
Values in parentheses are percentage of addi—
tional seedlings which exhibited only a slight browning of the tap—
root or small localized lesions on the hypocotyls.

susceptible reaction.

cant ranges by Tukey's 'w' procedure (P = 0.05):

Least signifi—
Means, 12.9;
interaction of inoculum concentration X cultivar, 25.8.

 

 

42

pathogen to determine if this inoculation method could be used to
identify races of the pathogen.

Soybeans were planted in trays 27 X 19 X 6 cm containing one row
of each of the nine cultivars with 5 seeds/row. Two days after sowing,
the seedlings were inoculated with 104 zoospores each. Controls were
uninoculated seedlings of each cultivar.

The nine soybean cultivars selected exhibited differential
responses to the six races of P, megasperma var. sojae generally in
accord with published data based on hypocotyl inoculation (Fig. 5,
Table 7) (25, 47). Some individuals of some cultivars showed a
susceptible reaction when a resistant reaction was expected. In Sanga
(races 1, 3, 4, 5, and 6) where the proportion of susceptible reactions
tended to be high, seed contaminated with fungi capable of rotting
seedlings complicated disease evaluation. However, in Altona (races 1,
3, and 4), Higan (races 3 and 6), Kingwa (races 4 and 5), and Toku
(races 3 and 6) the proportion of susceptible reactions was usually
less than one—third, and did not complicate interpretation of
results. False susceptible reactions in the latter three cultivars was
attributed to poor seed quality or moldy seed.

Differential response of cultivars to six races of P. megasperma

 

var. sojae in seedlings inoculated using the hypocotyl inoculation

 

method. Nine differential cultivars of soybean were inoculated by
placing mycelium of races 1 to 6, individually, into an incision in the
hypocotyls. One replication (pot) consisted of 5 seedlings and the
other 8 seedlings. Control plants of each cultivar were wounded but

uninoculated.

 

 

 

43

Fig. 5. Differential soybean cultivars (left to right) Altona, Hark,

Harosoy 63, Higan, Sanga, Toku, and Tracy in vermiculite and
inoculated with zoospores (104/seedling) of race 4 of Phytoph—
thora megasperma var. sojae. Hark, Harosoy 63, and Higan are

 

 

susceptible to race 4 and failed to emerge after inoculation.

 

 

44

 

 

45

Table 7. Differential response of selected soybean cultivars to six
physiologic races of Phytophthora megasperma var. sojae
when seedlings were grown in vermiculite and inoculated
with zoosporesa

 

 

 

Diseased seedlings (%) resulting from
inoculation with the races indicated

 

 

Cultivar
1 2 3 4 5 6

Altona 28 (R) o (R) 32 (R) 40 (R) 96 (S) 68 (S) 1
Hark 100 (S) 76 (s) 100 (S) 100 (s) 84 (S) 100 (S)
Harosoy 63 o (R) o (R) 72 (s) 92 (S) 96 (S) 80 (S)
Higan o (R) 8 (R) 20 (R) 96 (s) 92 (S) 28 (R)
Kingwa 8 (R) o (R) o (R) 20 (R) 28 (R) 92 (s)
Mack 12 (R) o (R) 4 (R) — (S) — (S) — (R)
Sanga 52 (R) 100 (S) 52 (R) 64 (R) 70 (R) 32 (R)
Toku 0 (R) 0 (R) 24 (R) 8 (R) 84 (S) 24 (R)
Tracy o (R) o (R) o (R) — (R) o (R) - (R)

 

aTwo-day—old seedlings were inoculated and disease evaluated 5 days
later. There were 5 seedlings/cultivar/tray and 9 trays/treatment.
R = resistant; S = susceptible, based upon data obtained using
hypocotyl inoculations. Missing data are indicated by —.

46

The soybean cultivars exhibited differential responses to the
six races of the pathogen generally in accord with published data
based on hypocotyl inoculation (Table 8) (25, 47). With this method
also, some individuals of some cultivars showed a susceptible reaction
when a resistant reaction was expected. In almost all cases discre—
pancies with expected results with this method occurred with the same
cultivar-race combinations as was the case with zoospore inoculation.

Mack and Kingwa exhibited poor emergence.

Inoculation of Soybean Seedlings with Zoospores in Soil

 

A number of factors was examined to evaluate the effectiveness
of inoculating soybean seedlings with zoospores in the presence of
sterilized soil.

Effect of amount of soil or of several aqueous media on disease
incidence. In preliminary experiments zoospores produced disease
readily in soil extract or in flooded sterile soil but no disease was
produced in aqueous suspensions of zoospores with no soil. Therefore,
weighed amounts of sterile air-dried Conover sandy clay 10am (0, 0.5,
1.5, 2.5, 5, 10, 20, 30, 40, and 60 g) were placed in petri dishes
and 3 X 103 zoospores of race 1 were added in 30 ml distilled water
to each plate. Several aqueous solutions in the absence of soil also
were tested for their effect on seedling infection. Three thousand
zoospores in 30 ml of tapwater, vermiculite extract, or soil extract
were added to petri dishes. The soil extract was either i) used as
prepared, ii) autoclaved, iii) centrifuged, or iv) centrifuged and
autoclaved. Controls were uninoculated seedlings exposed to the same

conditions.

 

 

47

Table 8. Differential response of selected soybean cultivars to six
physiologic races of Phytophthora megasperma var. sojae when
seedlings were inoculated using the hypocotyl inoculation

 

 

 

 

 

 

methoda .
Diseased seedlings (%) resulting from
Cultivar inoculation with the races indicated
1 2 3 4 5 6

Altona 35 (R) 10 (R) 55 (R) 0 (R) 75 (S) 96 (S)
Hark 100 (S) 100 (S) 70 (S) 90 (S) 100 (S) 100 (S)
Harosoy 63 0 (R) 4 (R) 90 (S) 72 (S) 100 (S) 100 (S)
Higan 0 (R) 0 (R) 0 (R) 100 (S) 83 (S) 0 (R)
Kingwa 0 (R) 0 (R) 0 (R) 67 (R) — (R) 38 (S)
Mack — (R) ~ (R) 0 (R) 100 (S) — (S) - (R)
Sanga 18 (R) 100 (S) 0 (R) 5 (R) 0 (R) 7 (R)
Toku 0 (R) 8 (R) 4 (R) 11 (R) 80 (S) 85 (R)
Tracy 0 (R) 0 (R) 0 (R) O (R) 0 (R) 0 (R)

 

aSeedlings were inoculated at 8 days of age; disease was evaluated 5
days later. There were two replications, one containing 5 plants and
one containing 8 plants. R = resistant; S = susceptible, based upon
published results obtained using hypocotyl inoculations. Missing data
are indicated by —.

 

 

48

Seedling infection was 100% or nearly so using soil in a range
of 0.5 to 30 g soil/petri dish (Table 9). At 40 and 60 g, the soil
was no longer saturated and disease incidence declined. Seedlings
incubated in the variously prepared soil extracts had 50% disease in—
cidence, whereas those incubated in distilled water, tapwater, or
vermiculite suspension did not become infected. Uninoculated seed-
lings remained healthy.

Effect of temperature on disease incidence. Ten grams of sterile

 

air-dried Conover sandy clay loam were placed into petri dishes to
each of which 3 X 103 zoospores of race 1 were added in 30 m1 of
distilled water. Distilled water used to flood the soil was adjusted
to the specific incubation temperature of the treatment. All petri
dishes were wrapped in aluminum foil before being placed in incubators
at temperatures of 10, 15, 20, 25, 30, and 35°C. Controls were un-
inoculated seedlings incubated at the same temperatures. Petri dishes
incubated at temperatures of 10 and 15°C for 3 days were placed at

23 i 2°C for one day before evaluation.

Greatest numbers of diseased seedlings were obtained at
temperatures of 20, 25, and 30°C (Table 10). Temperatures higher or
lower than this range significantly decreased disease incidence. The
appearance of symptoms was delayed at temperatures of 10 and 15°C.

Effect of zoospore concentration on disease incidence. Ten

 

grams of sterile air—dried Conover sandy clay loam were placed into
autoclaved glass petri dishes. Concentrations of l, 3, 10, 30, 100,
500, and 1000 zoospores of race 1 in 30 ml of distilled water were

added to each petri dish. Controls were uninoculated seedlings.

 

 

 

49

Table 9. Effect of amount of soil on disease
incidence in 'Hark' soybeans placed
in petri dishes and incubated with
zoospores of Phytophthora megasperma
var. sojae race ld

 

 

 

Soil, g/plate Diseased seedlings (%)
0 0
0.5 94
1.5 100
2.5 100
5 100

10 100
20 100
30 100
40 80
60 66

 

aTwo—day—old seedlings in petri dishes con—
taining soil and 30 m1 of water were infested
with 3 X 103 zoospores/petri dish. Disease
was evaluated 3 days later. There were 5
seedlings/petri dish and 3 petri dishes/
treatment.

Table 10. Effect of temperature on disease incidence

50

of 'Hark' soybeans incubated on flooded

sterilized soil artificially infested with
zoospores of Phytophthora megasperma var.
sojae or on soil naturally infested with
the pathogena

 

 

 

Diseased seedlings (%)

 

 

Temperature,°C Zoospore Naturally
infested infested

soil soil
10 28 16
15 36 16
20' 80 24
25 100 60
30 88 68
35 12 88

b
LSR 23.3 32.2

 

aTwo-day—old seedlings were placed on 10 g

zoospore infested soil (3 X 103

zoospores/plate) or
20 g naturally infested soil in petri dishes.

Disease was evaluated 4 days later. there were 5

seedlings/petri dish and 5 petri dishes/treatment.

bLeast significant range by Tukey's 'w' procedure

(g,= 0.05).

 

51

This method appeared to be a very sensitive inoculation method
since only 3 zoospores/petri dish caused disease in one of five
seedlings (Table 11). Disease incidence increased as inoculum con—
centrations increased until 100% of the seedlings were infected at
1000 zoospores/petri dish. Localized lesions about 1 to 2 cm in
length were present on the hypocotyl of seedlings exposed to low (3 to
30) numbers of zoospores, whereas concentrations of 100 zoospores and
higher produced the typical uniform browning of the hypocotyls.
Uninoculated control seedlings remained healthy.

Effect of zoospore concentration on disease incidence in

differential soybean cultivars. Fifty grams of steamed Conover sandy

 

clay loam were placed in trays 19 X 19 X 6 cm. Each tray contained
one row each of Hark, Altona, Harosoy 63, and Toku with 5 seedlings/
row. Suspensions containing 4 X 103, 2 X 104, 2 X 105, and 106
zoospores of race 1/100 m1 of distilled water were added to trays
(equivalent of 2 X 102, 103, 104, and 5 X 104 zoospores/seedling,
respectively). Controls were uninoculated seedlings.

Hypocotyls of Hark, a susceptible cultivar, were severely
decayed at all inoculum concentrations (Table 12). The percentage of
infected seedlings of the resistant cultivars Altona, Harosoy 63, and
Toku increased as inoculum concentrations increased. However, the
cultivars exhibited different degrees of resistance as inoculum in—
creased. For example, when 103 zoospores were applied, 35%, 20%, and
5%, of the seedlings, respectively, became diseased, but when 5 X 104
zoospores were applied, 100% of the seedlings of all three cultivars

became diseased. Thus, resistance apparently can be overcome with

sufficient inoculum. In addition to the severe infections, hypocotyls

 

 

 

52

Table 11. Effect of zoospore concentration on
disease incidence in 'Hark' soybeans
placed in petri dishes containing 10
g autoclaved soil and inoculated with
zoospores of Phytophthora megasperma
var. sojae race la

 

 

 

 

Zoospores/plate Diseased seedlings (%)b

0 0
l 0
3 20
10 24
30 36
100 64
500 88
1000 100

LSRC 22.6

 

aTwo-day—old seedlings were inoculated, and
disease was evaluated 3 days later. There were
5 seedlings/petri dish and 5 petri dishes/

tr eatment .

bSymptoms on plants inoculated with 3, 10, and 30
zoospores/plate were restricted to lesions of l to
2 cm in length, whereas those inoculated with
higher concentrations were completely decayed.

CLeast significant range by Tukey's 'w' procedure
(3.: 0.05).

 

 

 

 

53

Table 12. Effect of zoospore concentration on disease incidence in
differential soybean cultivars placed in trays containing
sterilized soil and inoculated with Phytophthora megasperma
var. sojae race la

 

 

Diseased seedlings (%)b

 

 

Zoospores/
seedling Mean
Hark Altona Harosoy 63 Toku
2
2 X 10 100 10 0 0 27.5
103 100 35 20 5 40.0
104 100 50 65 35 62. 5
5 X 104 100 100 100 100 100
Mean 100 48.8 46.3 35.0

 

aTwo—day—old seedlings were incubated on flooded soil for 3 days before
evaluation. There were 5 seedlings/cultivar/tray, and 4 trays/treat—
ment.

bLeast significant ranges by Tukey's 'w' procedure (P = 0.05): Means,
7.2; interactions of inoculum concentration X cultivar, 14.4.

 

 

54

of some plants frequently developed numerous, small (<1 mm), brown
'lesions which remained localized. Some Altona seedlings were diseased
at all inoculum concentrations. Control seedlings remained healthy.

Since it appeared that resistance could be overcome with suffi-
cient inoculum, the experiment was repeated using seedlings of two ages.
One hundred grams of steamed Conover sandy clay loam was placed in
trays 27 X 19 X 6 cm, each containing one row each of 2— and 3-day-old
seedlings of Hark, Altona, Harosoy 63, and Toku with 5 seedlings/row.
Suspensions containing 4 X 103, 2 X 104, end 2 X 105 Zoospores of race
1/150 ml distilled water were added to each tray (equivalent of
2 X 102, 103, and 104 zoospores/seedling, respectively). Controls were
uninoculated seedlings.

The susceptible cultivar, Hark, at both ages, exhibited 100%
disease at all inoculum concentrations (Table 13). Here again, disease
in the resistant cultivars usually increased with inoculum concentra-
tions. However, 3—day—old seedlings of Altona, Harosoy 63, and Toku
exhibited less disease than 2-day—old seedlings at all inoculum
concentrations. Apparently, resistance can be overcome with sufficient
inoculum, and the younger the seedlings are at the time of inoculation,

the greater their susceptibility. Control seedlings remained healthy.

Differential response of soybean cultivars to six races of

 

P. megasperma var. sojae. Nine differential soybean cultivars placed on

 

flooded soil were inoculated with zoospores of isolates of races 1 to 6
of the pathogen to determine if this inoculation method could be used
to identify races of the pathogen.

One hundred grams of steamed Conover sandy clay loam was placed in

trays 27 X 19 X 6 cm. Each tray contained one row each of Altona, Hark,

 

 

55

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cwonmom HmHuamummmHv we munwwHuaH mmmome co GOHumuudmumoo mnoamoow wo acummm .MH mHnma

 

 

 

56

Harosoy 63, Higan, Kingwa, Mack, Sanga, Toku, and Tracy with 5 seed—
lings/row. A suspension containing 9 X 103 zoospores/150 ml of dis-
tilled water was added to each tray (equivalent of 2 X 102 zoospores/
seedling). Controls were uninoculated seedlings.

The nine soybean cultivars selected exhibited differential
responses to the six races of P, megasperma var. sojae in close accord
with published data based on hypocotyl inoculations (Fig. 6, Table 14)
(25, 47). Ten and 50% of Altona seedlings inoculated with race 1 and
race 4, respectively, showed a susceptible reaction when a resistant
reaction was expected. Also, Higan showed 5% (1 out of 20 seedlings)
disease when inoculated with race 6 and Toku showed 5% disease when

inoculated with race 3.

Inoculation of Soybean Seedlings with Oospores in Soil

 

Some factors were examined to evaluate the effectiveness of
inoculating soybean seedlings with oospores in artificially infested
sterilized soil. Attempts also were made to isolate the pathogen
from naturally infested soils using soybean seedlings as baits.

Effect of different soil media on disease incidence. Four soil

 

media were evaluated for their effect on seedling infection by 5—week—
old oospores. Twenty grams of natural Conover sandy clay loam,

steamed Conover loam, potting mix, or sand were placed in petri

dishes. Concentrations of 103 and 104 oospores in 30 ml distilled
water were added to the first three treatments, and the same concen-
trations of oospores in 30 ml of soil extract (conover sandy clay loam)
were added to the sand. All petri dishes were incubated in darkness

at 23 i 2°C. Two—day—old Hark seedlings were incubated on the plates

 

 

 

Fig. 6.

 

57

Differential soybean cultivars (left to right) Altona, Hark,
Harosoy 63, Higan, Kin wa, Sanga, Toku, and Tracy inoculated
with zoospores (2 X 10 /seedling) of race 5 of Phytophthora
megasperma var. sojae in the presence of steamed soil.
Altona, Hark, Harosoy 63, Higan, and Toku are susceptible to
race 5; the hypocotyls turned brown and failed to develop
after inoculation.

 

 

 

 

 

59

Table 14. Differential response of selected soybean cultivars to six
physiologic races of Phytophthora megasperma var. sojae when
seedlings were incubated on flooded soil and inoculated with

 

 

 

 

 

 

zoosporesa
Diseased seedlings (2) resulting from
Cultivar inoculation with the races indicated
1 2 3 4 5 6

Altona 10 (R) 0 (R) 0 (R) 50 (R) 70 (S) 100 (s)
Hark 100 (s) 100 (S) 100 (S) 100 (S) 100 (S) 100 (S)
Harosoy 63 o (R) o (R) 100 (S) 100 (s) 85 (S) 100 (S)
Higan o (R) 0 (R) o (R) 90 (S) 65 (S) 5 (R)
Kingwa 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 75 (S)
Mack 0 (R) o (R) 0 (R) 100 (s) 85 (s) 0 (R)
Sanga 0 (R) 100 (s) 0 (R) o (R) o (R) 0 (R)
Toku 0 (R) 0 (R) 5 (R) o (R) 75 (s) 0 (R)
Tracy 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) o (R)

 

aSeedlings were inoculated at 2 days of age; disease was evaluated
after 3 days incubation on flooded soil. There were 5 seedlings/cul—
tivar/tray and 4 trays/treatment. R = resistant; S = susceptible,
based upon data obtained using hypocotyl inoculations.

 

 

 

60

for 2 days, then replaced with a new set of seedlings. Disease was
evaluated 3 days later.

Disease incidence was 100% at both oospore concentrations in
steamed Conover loam, potting mix, and sand. There was no seedling
infection with 103 oospores/petri dish in the natural Conover loam, and
disease was 80% with 104 oospores/petri dish. All plants in the first
and second set of seedlings were diseased in all treatments except
for natural Conover 10am. Uninoculated seedlings remained healthy.

Effect of temperature on disease incidence using naturally

 

infested soil. Twenty grams of air—dried Brookston loam, naturally
infested with P. megasperma var. soja§_were placed into petri dishes
and 30 ml of distilled water added. Distilled water used to flood
the soil was adjusted to the specific incubation temperature of the
treatment. All plates were wrapped in aluminum foil before being
placed in incubators at temperatures of 10, 15, 20, 25, 30, and 35°C.
In the naturally infested soil, disease incidence increased as
the temperature increased (Table 10). Temperatures of 25 to 35°C
produced significantly more disease than lower temperatures. Seedling
development was retarded at temperatures below 20°C. Attempts to cause
seedling infection using soil artificially infested with oospores
failed.

Effect of light on oospore germination and on disease incidence.

 

Twenty grams of air-dried Brookston loam, naturally infested with_P.
megasperma var. sojae, 20 g sterile Conover loam infested with 103
oospores/petri dish, or 10 g sterile Conover loam artificially infested

with 103 zoospores/petri dish were added in 30 ml of distilled water to

separate petri dishes. Petri dishes were incubated under fluorescent

 

 

61

light (1800 lUX) with either a 24 hour or 12 hour photoperiod or in
continuous darkness. For dark treatments plates were wrapped in aluminum
foil. Two—day-old soybean seedlings were incubated on the flooded soil
for 2 days, then replaced with a new set of seedlings. Disease was
evaluated 3 days later. Controls were uninoculated seedlings exposed

to the same light conditions. To study the effect of light on oospore
germination, a suspension of 5—week old oospores was applied to mem-
brane filters (ca. 103/membrane). The membranes were incubated on
flooded, sterile Conover loam for seven days. For determination of
germination oospores were transferred to water agar (77).

Germination of oospores was greatest in darkness, whereas germi—
nation of those incubated under a 12 hour photoperiod or continuous
light was significantly decreased. Development of mature sporangia
on relatively longer germ tubes was characteristic of oospores incu—
bated in darkness, whereas short germ tubes and only a few sporangia
were characteristic of those incubated under light.

Disease incidence of seedlings in soils naturally or artifi—
cially infested with oospores of the pathogen was significantly greater
in darkness than under continuous light. With a 12 hour photoperiod,
results were intermediate. Apparently, light had no effect on the
susceptibility of the seedlings. since they were uniformly infected at
all photoperiod treatments when inoculated with zoospores. In another
experiment, 500 zoospores/petri dish also gave 100% disease in all
three light conditions. Uninoculated controls remained healthy.

Soybean seedlings as baits for recovering P. megasperma var.
sojae from soil. Two—day-old soybean seedlings (cultivar 'Hark')

were placed in petri dishes (5 seedlings/petri dish) containing 40 g

 

 

62

 

 

 

 

 

 

Table 15. Effect of light on oospore germination and soybean seedling
infection by Phytophthora megasperma var. sojae race 1 in
naturally infested soil and in soil artificially infested
with oospores or zoospores of the pathogen

Diseased seedlings (%)a

Photoperiod, Oospore

hours/dayb germinatlon, Naturally Oospore Zoospore
%C infested infested infested
(103/plate) (103/plate)
24 8 4 8 100
12 23 28 20 100
0 36 60 44 100
LSRd 8 24 31 0

 

aTwo—day-old soybean seedlings were incubated on flooded soil for 2
days, then replaced with new seedlings (5 seedlings/petri dish and

5 petri dishes/treatment).

b

Petri dishes were incubated under fluorescent light providing 4800

lux.

CFive—week—old oospores were incubated on flooded soil for 7 days
before evaluation; 20—36% were infected with hyperparasites, which

were included among those nongerminated.

dLeast significant range by Tukey's 'w' procedure (P>= 0.05).

 

 

 

63

air-dried natural soil and 40 ml distilled water. Seedling infection
was evaluated after 3 days' incubation by direct examination for
sporangia of the pathogen with a dissecting microscope. Segments of
infected tissue were also plated on a selective medium to verify the
presence of the pathogen.

Nine different natural soils were tested with soybean seedling
baits. Five of the soils were taken from fields under soybean cultiva—
tion; the other fOur soils were from fields with crops other than
soybean.

.P. megasperma var. sojae was successfully baited from all nine
natural soils even when they had been stored air-dried. Pythium spp.

were also observed on seedlings in six of the soils.

 

 

DISCUSSION

Methods were described for inoculating soybean seedlings with

zoospores and oospores of Phytophthora megasperma var. sojae, and for

 

detection and isolation of the pathogen in natural soils. These
methods offer advantages over those presently available. The methods
are more rapid and easier to perform, less susceptible to variable
environmental conditions, gave more consistent results, and are less
artificial than the hypocotyl-wound technique. These methods also can
be used for testing pathogenicity and virulence of isolates, and for
studies on field tolerance, biological control, and environmental
factors.

Large numbers of zoospores of the pathogen were readily produced
in flooded cultures on lima bean agar, provided that culture age,
number and frequency of water changes, temperature, and light condi—
tions were appropriately standardized. The optimal temperature in
this study for zoospore production by P, megasperma var. sojae_was
found to be 20°C. Previous workers (35) found 25°C to be better than
15°C. The number of zoospores produced on plates incubated in darkness
was ten times greater than on plates incubated under continuous light.
Light has been variously reported 1) to have no influence on the
production of sporangia (23, 89), ii) to be stimulatory (2, 18), and
iii) to be inhibitory (91) to several Species of Phytophthora. The
effect of light on sporangial development or zoospore production by
P, megasperma var. sojae_has not previously been studied. The method

64

65

yielded high numbers of zoospores (IDS/petri dish) in as few as 4 hours
post—washing; previous methods have required 24 to 72 hours post—
treatment before harvest (31, 32, 33, 75). Further, the new method

may yield a second and third harvest of zoospores from the same

petri dish after reflooding.

Only one isolate of race 1 of the fungus was used in this study,
so it is not known whether the factors tested may affect other isolates
differently. However, the method was verified using isolates of races
2, 3, 4, 5, and 6 each of which produced more than 105 zoospores/
petri dish. Moreover, additional isolates of races 1, 3, 4, 5, and 6
all produced numerous zoospores in further experiments (Lockwood and
Cohen, unpublished results).

Hypocotyl inoculation is the conventional method for determining

pathogenicity of the fungus and evaluating resistance in soybeans.
Two techniques were successfully developed for the inoculation of
soybean seedlings with zoospores of P, megaseprma var. sojae, In
vermiculite, disease could usually be evaluated on a simple emergence
vs. non—emergence basis when inoculation took place before seedlings
were three days old. Results were available within 6 to 8 days after
inoculation. This inoculation method yielded optimal results over a
temperature range of 20 to 30°C. Experiments done on the laboratory
bench under ambient light (130 lux) and fluorescent light (1800 lux),
and in a growth chamber (17,000 lux), for varying photoperiods all
gave similar disease responses.

Inoculation of 2-day—old seedlings of nine cultivars with six

. 4 .
races of P, megasperma var. s03ae at 10 zoospores/seedling gave results

66

corresponding with those obtained using hypocotyl inoculation (25, 47).
Concentrations of 500 zoospores/seedling failed to produce disease in
susceptible cultivars while 105 zoospores/seedling caused disease in
some cultivars which were resistant by hypocotyl inoculation. Seedlings
of "resistant" tobacco cultivars have also been infected when inoculated
with an excessive concentration of zoospores of P, parasitica var.
nicotianae (23, 54). For example, increasing zoospore concentration
from 104/ml to 105/ml resulted in black shank symptoms on seedlings of
resistant cultivars (54).

In some cases, resistant soybean cultivars showed a susceptible
reaction when inoculated with 104 zoospores/seedling. These 'false'
susceptible reactions were usually less than 30%, and did not compli-
cate interpretation of results. The decreased germination was
attributed to poor seed quality. However, in Sanga the proportion of
'false' susceptible reactions tended to be higher and apparently was
due to the seed being contaminated with fungi capable of rotting
the seedlings. Seed quality is an uncontrollable factor in this
inoculation method because germination cannot be checked prior to the
pre—emergence inoculation.

Soybean seedlings were also successfully infected when incubated
on flooded soil infested with zoospores 0f.2- megasperma var. sojae.
Seedlings were selected for use after germination which eliminated
the problem of seed quality control. As with vermiculite inoculations,
disease incidence was optimal in the temperature range of 20 to 30°C.
Variable light conditions did not affect results. The soil system
required fewer zoospores than did vermiculite. As few as three

zoospores were found capable of causing infection in a seedling despite

67

zoospore motility loss in the process of dilution (35).

Differentiation of resistant and susceptible cultivars with
this method was most consistent in 2-day—old seedlings inoculated with
2 X 102 zoospores/seedling. Inoculation of nine cultivars with six
races of P, megasperma var. sojae_very closely corresponded with
results obtained using hypocotyl inoculation. However, resistant
cultivars showed disease symptoms at higher zoospore concentrations.
This susceptibility of 'resistant' cultivars to infection decreased
with increasing age of seedlings.

These inoculation methods may distinguish resistance from
'field tolerance' which cannot be detected using hypocotyl inoculation.
Those seedlings of field tolerant cultivars which did develop hypocotyl
infections tended to be less severely affected than those of
susceptible cultivars.

Oospores of P, megasperma var. sojae have a constitutive dormancy
period (17). Attempts to induce germination in these resting struc—
tures have in large part failed (8, 67, 68). In this study most
attempts to cause seedling infection using oospores produced in culture
and added to soil proved unsuccessful. In other work from our labora-
tory, oospores were found to be parasitized by a diversity of soil
microorganisms which have the potential to reduce populations of the
pathogen in soil (80). It was suggested that culturally produced
oospores may be more susceptible to parasitism than those produced in
and protected by plant tissue (80). Parasitized oospores may explain
my lack of success in a number of experiments to induce disease in

seedlings when oospores were added to natural soil.

 

68

Light has been shown to stimulate oospore germination in vitro
(8, 26, 48, 68). Contrary to these reports, when oospores were
incubated on natural soil, more germination occurred in darkness than
in light. Moreover, in the presence of soil, germ tube elongation and
sporangium formation were greater in darkness than in light.
Additionally, seedling disease incidence was greater in darkness than
under continuous light when seedlings were incubated on naturally in—
fested soil or soil artificially infested with oospores. Since
disease incidence was not affected by varying light conditions when
zoospores were used as the source of inocula, it appears that light
conditions directly affected oospores and their germination.

Isolation of P, megasperma var. sojae directly from soil has

 

proved to be difficult (84), but my results showed that soybean
seedlings could be used as baits to detect the presence of P,

megasperma var. sojae in soil. The fungus was microscopically

 

identified without isolation and reinoculation and pure cultures
were readily obtained from disease tissue. The baiting method was
sensitive enough to detect the pathogen in soils with no disease
history or in soils not under soybean cultivation. In other work
(Cohen and Lockwood, unpublished), several races of the fungus were
identified directly from soil when differential soybean cultivars
were used as baits. The ease with which the pathogen was detected
and isolated directly from soil by means of soybean seedling baits
suggests that the method could be adapted to study the occurrence and

behavior of the fungus in soil as has been done with Phytophthora

 

megasperma using alfalfa seedling baits (50, 51, 63, 64, 65). In

 

69

addition, the method provides a natural system by which the effect of

environmental factors on the infection process can be investigated.

 

 

10.

11.

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