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RACES OF THE PATHOGEN PH YT OPHT HORA SOJAE FOUND IN MICHIGAN
AND FACTORS AFFECTING ROOT ROT OF SOYBEAN

By

Richard Chemjor Kaitany

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

2000

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ABSTRACT

RACEsiCHP'THE PATHOGEN PHYTOPHTHORA SOJAE FOUND IN MICHIGAN
AND FACTORS AFFECTING ROOT ROT OF SOYBEANS

By

Richard Chemjor Kaitany

A study was conducted in order to establish what races
of P. sojae are currently present in Michigan soybean fields,
and.tx> estimate yield loss tx> P. sojae, and determine the
effect of the isoflavone genistein on the infection of
soybeans by zoospores of P. sojae. Also, a possible
relationship between fluorescence values of root exudates (as
indicators of the amounts of genistein in exudates) and field
tolerance levels of soybean varieties was examined.
Additionally, a soybean field naturally infested with the
soybean cyst nematode (SCN) was surveyed for possible
increased infection by P. sojae.

Ninety isolates of P. sojae collected from Michigan
fields (1993-1997) were tested for virulence and race status.
Races 2, 3, 4, and 27 were identified, and the virulence
formulae of most isolates did not match those of known races
of P. sojae. Fifty five percent (55%) of the isolates tested
were highly virulent defeating 7 or more Rps genes while 13%
showed intermediate virulence defeating 4—7 genes each. Twenty

percent were mildly virulent (defeating 1-3 genes) while 11%

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were avirulent.

Genistein (5 ppm) significantly reduced disease levels
in soybean seedlings inoculated with zoospores of P. sojae.
However, there was no correlation between tolerance levels
of soybean cultivars to the pathogen and fluorescence values
of root exudates from the cultivars. There was significant
correlation between nematode cyst counts and the presence of
P. sojae in the non—fumigated field plots. However, no

correlation was observed between the two pathogens in the

non-fumigated plots.

iniis dissertation is dedicated to my parents who, though not
schooled, made the education of their children the center of

their lives.

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Acknowledgment

I[ am indebted to my advisor, Dr. Gene R. Safir , whose
guidance, patience and support made this work possible. My
thanks also go to the other members of my guidance committee
including Dr. L. Patrick Hart for his patient reading of the
drafts and revisions, and his insightful suggestions, Dr.
Dennis Fulbright for his mentoring friendship and
encouragement. Special thanks to Dr. Ken Poff whose tact and

concern often revived my spirits.

My family provided the necessary springboard for continued
enthusiasnu My children , KipChumba, Kibor, and Jerotich, were
a source of unfailing inspiration. It not possible to express
enough gratitudetxanwrwife, Andrea, without whose contanstant

love and encouragement this work would not have been possible.

List of
List Fig

Chaptc~
In‘

ChaF

TABLE OF CONTENTS

List of Tables ........................................................................................... Page vii
List Figures .............................................................................................. Page viii
Chapter 1: Soybeans in industry and the economy .......................................... Page 1
Introduction . ...................................................................................... Page 1
History of the root rot disease in soybeans ........................................... Page 5
Symptoms ...................................................................................... Page 6
Favorable field conditions ................................................................ Page 8
Disease cycle .................................................................................. Page 9
Taxonomy of Phytophthora .............................................................. Page 11
Ecology and Epidemiology ............................................................... Page 12
Physiological races and resistance in soybeans ...................................... Page 13
Disease Management . ........... . ........................................................... Page 14
Isoflavonoids and field tolerance in soybeans ....................................... Page 15
Bibliography . ..................................................................................... Page 20

Chapter 2: Virulence and race determination of isolates of Phytophthora from
Michigan soybean fields and estimation of impact of PR on yield.

Abstract ............................................................................................... Page 24
Introduction ........................................................................................ Page 25
Materials and Methods .......................................................................... Page 27
Collection of sample ........................................................................... Page 27
Media ................................................................................................ Page 28
Isolation of P. sojae from soybean plant samples . .................................. Page 28
Isolation of P. sojae from soil samples .................................................. Page 29
Production of single zoospore cultures ................................................... Page 30
Identification of isolates ....................................................................... Page 31
Tests for Virulence and race determination ........................................... Page 31
Estimation of impact of P. sojae on yield ............................................... Page 32
Results ................................................................................................ Page 34
Virulence and race identity of isolates .................................................. Page 34
Impact of P. sojae on yield ................................................................. Page 35
Discussion ........................................................................................... Page 58
Bibliography ........................................................................................ Page 62

Chapter 3: Effects of the isoflavonoid genistein on the infection of soybean

seedlings by zoospores of Phytophthora sojae, and the fluorescence of root
exudates ans field tolerance in soybeans.
Abstract ............................................................................................. Page 64

Introduction ....................................................................................... Page 65

vi

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Materials and Methods .............................................................................. Page 68

Influence of genistein on the infection of soybean seedlings by zoospores of

P. sojae ............................................................................................... Page 69
Collection of root exudates .................................................................... Page 70
Results ..................................................................................................... Page 71
Discussion ........................................................................................... Page 80
Bibliography ............................................................................................. Page 83

Chapter 4: Survey of P. sojae in soybeans infested with soybean cyst nematode
Heterodera glycines..

Abstract ............................................................................................. Page 85
Introduction ........................................................................................ Page 85
Materials and Methods ............................................................................. Page 90
Results .................................................................................................. Page 91
Discussion .............................................................................................. Page 96
Bibliography ........................................................................................... Page 99
Chapter 5: Summary and Conclusions ........................................................... Page103

vii

Table

Table

Table

Table I

Table I

Table 3

LIST OF TABLES

Table 2.1. Reaction of soybean resistance genes to isolates of P. sojae ................. Page36
Table 2.2. Virulence levels of isolates of Psojae ............................................... Page 40
Table 2.3. Ranked performance of Rps genes against isolates of P. sojae . ............ Page42
Table 2.4. Year, county of origin and virulence formulae of P. sojae isolates ........ Page 52
Table 2.5. Estimated impact of P. sojae on yield ................................................. Page 56

Table 3.1. Percent increase in root mass (dry weights) of soybean seedlings in the
presence of genistein ...................................................................... Page 73

viii

Figure I
11

Figure 2

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LIST OF FIGURES
Figure 2.1. Typical empty patch in a depressed area of a field affected by P. sojae
near St. Charles in Saginaw county .......................................................... Page 45
Figure 2.2. Host range comparison of P. sojae isolates and P. megasperma. . ....... Page 47

Figure 2.3. Comparisons of grth rates of field isolates of P. sojae and

P. megasperma ..................................................................................... Page 49
Figure 2.4. Increase in the number of highly virulent isolates obtained over the

sampling period .................................................................................... Page 51
Figure 3.1. Dry weights of roots in the presence and absence of genistein ............. Page 75
Figure 3.2. Effect of genistein on disease levels in soybean seedlings .................. Page 77

Figure 3.3. Fluorescence of root exudates and field tolerance values of soybeans ..Page 79

Figure 4.1. Number of cysts of Heterodera glycines in the rhizosphere soil volumes
of soybean varieties, and presence of P. sojae in the soybeans from
non-fumigated plot ............................................................................ . Page 93

Figure 4.2. Number of cysts of Heterodera glycines in the rhizosphere soil volumes

of soybean varieties, and presence of P. sojae in the soybeans from fumigated
plots ..................................................................................................... Page 95

ix

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Chapter 1

INTRODUCT ION

Soybeans in industry and the economy

The soybean (Glycine max L.) was introduced to North
America in 1765 (Hymowitz and Harlan, 1983) but it remained a
minor hay crop until it was deveIOped as an oil seed crop.
Processing of soybeans for oil and meal began in the 1930's
and in 1938, production for processing exceeded production for
hay (Thatcher,1947). Soybean seed is high in protein, making
it highly adaptable to the nourishment of both man and animal.
Soybean meal is a major source of protein in animal feed while
soybean oil is used in cooking oil, margerine, paints,
.varnishes, adhesives and many other products. Soybean oil is
also highly valued because it contains no cholesterol and has
the lowest levels of saturated fats of nearly all vegetable
oils. It may also serve as a source of energy in the future.
Some soybeans are not crushed and are used for human
consumption in such products as full-fat soy flour, soy milk,
tofu, miso and temphe (Scott and Aldrich, 1970). There is

great potential for expanding further the use of soybeans in

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2

food and industrial products which could improve the
profitabilityrof soybeans. Future researchcnisoybean includes
finding new uses for presently grown soybean varieties,
developing new varieties that have altered levels of important
components such as the major fatty acids that comprise the
soybean oil (for specific markets), and protection against
disease. Also environmental concerns for chemical
contamination of surface and ground water from agricultural
practices, may in future, place soybeans at an advantage over
other field crops due to its low-input status. Thus soybean is
expected to remain an important crop in the United States for
many decades to come.

In Michigan, production of soybeans started in the early
19005. By 1930, 1,000 acres were grown in the state. In 1997,
Michigan farmers produced a record yield of 42 bushels per
acre on 1.15 million acres with a market value of $249
million. Owing to market projections, acreage under soybeans
continues to increase and Michigan now ranks eleventh among
producing states with 2.3 percent of the 0.5. production.

As Michigan farmers adapt new soybean varieties for both the
traditional and product specific markets, disease problems may
pose new challenges that will require attention and more
research efforts. Although most soybeans grown in Michigan

have been relatively free of disease and insect problems,

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Phytophthora root rot, white mold, and the soybean cyst
nematode (Heterodera glycines Inchinohe) have been identified
in the state and can be yield-limiting factors in any one
season or on any one field/farm.

Phytophthora root and stem rot of soybean (PRR) caused by
Phytophthora sojae is one of the most important diseases of
soybean in the North Central region of the Midwest“ According
Us the North Central Soybean Disease Committee (NCR-137),
losses due to PRR represent roughly 15% of the total soybean
disease losses in the region (Approximately 192 million
dollars/annum) (Doupnik,B.1993). Nationwide, approximately 16
million hectres are infested (Schmitthenner., 1985). The
Soybean-Phytophthora disease interaction is believed to be a
very young symbiosis with many possibilities for genetic
manipulation through cultural practices enmi host genetics
(Schmitthenner. 1985)

Genetic control of Phytophthora root and stem rot in
soybean has been classified in four (not necessary exlusive),
ways including: 1) resistance-conferring whole—plant immunity
to incompatible races (compatible races cause disease)
(Kaufman and Gardemman,1958); 2) tolerance (schmitthenner and
Walker, 1979); 3) rate reducing resistance (slow rotting)
(Tooley and Grau, 1982); and 4) root resistance conferring low

infection but not immunity to incompatible races in the root

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as indicated by hydroponic inoculation procedure (Kilen et al
1974; Kilen, 1986).
A compatible race can infect, colonize and possibly kill
a susceptible host plant. The same race is incompatible with
a host which has either whole plant or root resistance
specific for that race. In the case of whole plant resistance,
the plant is immune to incompatible races because it has a
hypersensitive response which restricts pathogen growth and
kills plant tissue around the infection site (Kaufman and
Gardemman, 1958). Resistance to root rot in the inoculum layer
technique (Walker and Schmitthenner, 1984c) results in lesion
formation but very limited growth of the pathogen compared to
more extensive rot for soybeans with vflmflra plant or root
resistance (Schmitthenner' and. Kilien 1986). Rate-reducing
resistance is used almost exclusively in epidemiology to
describe reduction in rate and amount of sporulation and
lesion size in pathogenesis. In Phytophthora root rot, 'slow
rotting' is a more appropriate characterization of slow rate
of rotting and does not indicate reduced colonization or
reproduction (Walker and Schmitthenner, 1984c).
Whole plant resistance to Phytopthora sojae was first
identified in 1955 (Suhovecky and Schmitthenner, 1955), and
easily recognized by hypersensitive reaction to infection

upon inoculation of sensitive hypocotyl tissue (Kaufmann and

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Gerdemman, 1958). Thirty nine races of Phytopthora sojae and
13 genes (some are alleles) at seven loci (Rpsla, Rpslb,
Rpslc, Rpsld, Rpslk, Rpsz, RpsBa, RpsBo, RpsBc, Rps,, Rps5 Rps6
and Rps,) which condition differential resistance to the
physiological races in soybean, are known (Athow, 1987; Ploper

et a1, 1985; Leyton et al, 1986).

History of the Disease.

Phytophthora root and stem rot (PRR) of soybean was first
observed as a disease of unknown etiology in Indiana in 1948
and in northwestern Ohio in 1951. The disease was originally
thought to be caused by species of Evsarium or LUaporthe.
Phytophthora was first associated with root and stem rot of
soybean in North Carolina and Ohio in 1954 (Schmitthenner,
1985). The first reports on the disease in the United States
were published in 1955 (Scotland, 1955; Suhovecky, 1955;
Suhovecky et a1, 1955), and identified as a disease caused by
Phytophthora coctorum (LebauKiCohn) Schroeter. Later, Kaufman
et al (1958), found an undescribed species of Phytophthora
associated with root and stem rot of soybean in Illinois.
They published a comprehensive report of the disease and
proposed the name Emytophthora sojae for the causal agent.
PRR has been reported in most parts of the North central

region of the United States and is a limiting factor in

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Symptoms

Phytophthora root and stem rot may be found in soybeans
at any stage of plant development. Seed rot and pre-emergence
damping-off can occur in flooded fields, and is often
misidentified as water damage (Anderson, 1986) . When conditions
are favorable, seed rot, damping-off, and seedling rot and
stem rot may cause losses and yield reductions of up to 100%
in susceptible soybean cultivars.

Under flooded conditions, seeds often rot before
emergence ‘thereby' reducing' stands. After' emergence, young
plants are very susceptible to infection and often wilt and
die. Symptoms on rolder seedlings depend on ‘the relative
susceptibility or tolerance of the cultivar. In low tolerance-
cultivars, at the primary leaf stage, affected plants turn
yellow and wilt, and seedlings are killed gradually. On the
other hand in high tolerance cultivars, the damage may be
restricted to roots and seedlings appear only stunted
(Anderson, 1986).

In older plants of low tolerance cultivars, symptoms
consist of yellowing between veins and along margins of lower
leaves. Upper leaves become chlorotic and the plant wilts

completely. Wilted leaves commonly remain attached tx> the

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plant. In the field, affected plants usually occur in groups
in a row rather than singly. Wilting symptoms occur when the
lateral roots and the taproot are destroyed (Anderson,1986).
A dark brown discoloration progresses up the stem, often as
high as ten nodes before the plant wilts, and internally the
cortex and the vascular tissues are discolored. In older
plants of high tolerance cultivars, symptoms are confined to
the lateral roots. Plants are not killed by the pathogen but
are stunted with mild chlorotic symptoms similar to those
associated with nitrogen deficiency or severe flooding.
Occasionally these symptoms are accompanied by long, narrow,
sunken brown lesions that progress up one side of the stem.
These mild symptoms are referred to as hidden damage and may
reduce yield 'by as much as 40%. Hidden damage is readily
evident if plants with isogenic resistance are subject to
disease control treatments for comparison in the same field.
Progressive light brown lesions with yellowish margins,
characterize the leaflets of young susceptible plants. In
older plants, lesions are severely restricted, a phenomenon

referred to as age-related resistance.

Favourable environmental conditions

Environmental factors greatly influence the infection and

disease severity of Phytophthora root and stem rot of soybean.

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The most important of these factors are soil type, soil
compaction, soil moisture and soil temperature. Conditions
favourable for infection occur most often in heavy, compacted
clay soils with poor drainage. Disease incidence and the
number of dead plants increases with soil compaction while the
total number emerged is reduced (lkxflxset al, 1988). Extended
periods of high soil moisture, rainfall or standing water
highly favour disease development. The disease is most severe
in years with heavy rainfall early in the growing season, and
is most destructive in low, poorly drained portions of the
field (Kittle et al, 1979). In greenhouse experiments (Klein,
1959), the percentage of diseased plants increased with the
length of soil wetness before planting. The optimum soil
temperature for infection ranges from. 27°C to 339C for
seedlings and young plants, and 25%:to 30%:for older plants,
but infection can occur at soil temperatures as low as 15%:
(Kittle et al, 1979). Greatest root loss by soybean plants in
soils infested with P. sojae occurred at lower temperatures
than at optimum. At low temperatures, P. sojae may have
greater metabolic activity and hence the ability to attack and
destroy roots than the plant is able to regenerate them. As
soil temperature increases above 15%» growth.of soybean roots
out-phase disease development or P. sojae becomes less active

or both.

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Disease cycle
Phytophthora sojae (Kaufmann and Gardemman) is
homothallic; sexual reproduction takes place in a single
thallus, and therefore mycelium and sporangia are diploid.
Meiosis occurs in antheridia and oogonia and karyogamy takes
place in the oogonium, which forms a diploid oospore. Oospores
germinate by germ tubes which result in the formation of
single terminal sporangia (conidia)(Ribeiro, 1983). Sporangia
are simple and indeterminate. Typically, sporangia are
obipyriform (42-65 x 32-53 um) and non papillate. Sporangia
extrude zoospores into a thin, delicate membranous vesicle
which quickly expands and raptures. Zoospores sometimes remain
trapped in the sporangiunrand germinate from within. Sporangia
may also germinate directly thus functioning as conidia. Empty
sporangia commonly proliferate internally, forming new
sporangia within the old. Sporangia readily develop in<dilute
extract of lima bean agar or other media. They can also be
induced to form on solid medium if washed repeatedly with
water or Chen-Zentmyer salt solution.
The optimal temperature for zoospore production is 20%:
(the minimum is 5%». Zoospores which form the primary
inoculum are bluntly pointed at both ends and biflagellate.

One of the flagella (tinsel) is short and directed anteriorly

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while the other (whiplash) which is four to five times larger
is directed posteriorly. After a motility period which may
last for several days, zoospores encyst and germinate directly
by producing germ tubes which usually swell to form appresoria
when they come in contact with a solid surface or, rarely,
miniature sporangia form at their tips.

Sexual organs (antheridia and oogonia) which develop
abundantly on a single thallus, are produced in cornmeal, lima
bean and V—8 agars. The antheridia are mostly paragynous with
occasional amphigyny. The oogonia (about 36.9 pm in
diameter) are thin-walled spherical structures in which
oospores are formed. Thick—walled dormant oospores develop
when antheridia fertilize the oogonia. At the euui of the
dormant stage, the smooth inner wall of the oospore erodes and
the central refractive body is absorbed. When oospores
germinate, the inner walls are completely absorbed, and germ
tubes are pmoduced, which develop into hyphae or terminal

sporangia, depending on the availability of moisture.

Taxonomy

Phytophthora sojae is placed in the phythiaceae, a family
of the Oomycetes, a group of organisms that were for a long
time identified with fungi. The Oomycetes have been recognized

as being significantly different from fungi (Pringsheim, 1958,

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Kreisel, 1969, and Shaffer,1975). However due to a number of
factors (Barr, 1992), including traditional as well as
practical considerations, there has lunar a tendency for
mycologists to classify the Oomycetes as true fungi. Kreisel
(1969) and Shaffer (1975) were the first to exclude Oomycetes
from the true fungi. In recent years, more workers have
adopted this approach, as it has become increasingly apparent
that while these cmganisms are nmmphologically similar to
fungi, and exhibit absorptive nutrition, they do not have
close phylogenetic relationship with fungi. The Oomycetes have
been grouped with heterokont organisms such as the
subdivision, Pseudofungi, phylum Heterokonta of the kingdom
Chromista (Cavalier-Smith, 1986, 1987), or the subdivision
Heterokontimycotina of the kingdom Heterokonta (Dick, 1976,
1990a). Another approach has lxxur to include them ix) the
protoctista (Margulis et al. 1989), a group composed of
organisms that are not monophyletic. Patterson and Sogin
(1992) placed the phylum Oomycota in a new kingdom,
Stramenopila. The characteristics that set the Oomycota
organisms apart from the true fungi and also delineate the
phylum, include among others, asexual reproduction by means of
biflagellate zoospores, diploid thallus in which meiosis
occurs in the gametangia, oogamous reproduction.by gametangial

contact, cell wall composition (mostly B-glugans), and.various

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12
ultra-structural features of the oospore (Sogin, 1992). In
modern botanical works, the Oomycetes and other organisms that
are linked by their basic eukaryotic cell structure and their
lack of the distinguishing features of plants, animals, or
fungi are placed in the Kingdom Protista. This is due to
differences in morphology, pathogenicity, and growth rate with
related species of P. coctorum and P. megasperma. In 1959,
Hildebrand changed the name of the soybean pathogen to [K
megasperma Drechs. var. sojae Hildeb. This name remained valid
until 1980, when Kuan T. and Erwin ELC. reclassified the
fungus as P. megasperma f. sp. glycinea, following extensive
studies of the host range and oospore size. They concluded
that oogonial size of isolates of P. megasperma from various
hosts overlap and was, therefore, not a suitable trait for
varietal separation. However, isolates of P. megasperma from
soybean and alfalfa had sufficiently distinct host range to
place them into two forma specialae (P. megasperma f. sp.
glycinea and P. megasperma f. Sp. medigaginis) and to separate
them from P. megasperma found in other hosts. Later, Kaufman
et a1 (1958), found an undescribed species of Phytophthora
associated with. root and stem rot of soybeans in Illinois.
They published a comprehensive report on the disease and

proposed the name Phytophthora sojae for the causal agent.

PU

13

Ecology and epidemiology

P. sojae survives as oospores in crop residue and soil
for many years without growing competetively enough to
colonize soil debris (Schmittenner, 1985). The fungus cannot
be demonstrated in soil immediately after freezing or storage
for long periods at 3%» indicating that mycelium, sporangia
and zoospores do not survive cold temperatures. If
overwintered soil is incubated for one week at 25°C under
suitable .moisture conditions, the fungus can be readily
demonstrated using the leaf disk bait technique
(Schmitthenner, 1985). This demonstration indicates that P.
sojae survives as resistant oospores that germinate when
dormancy is broken and when temperature and moisture are
suitable. It.:hs not known exactly what factors break the
dormancy of oospores or what minimum soil saturation times and
temperatures favour the germination of oospores. It is known
that extended rotations with non-host crops does not eliminate
the pathogen (Schmitthenner, 1985). Work by Stella Avila (MS
Thesis, 1992) showed ability by P. sojae to infect non-host
crops without the manifestation of disease. This may further
explain the failure of crop rotation as'a strategy in the
management of the disease. The association and interaction of

P. sojae with other fungi may increase or decrease the

Cri‘grhnfi
\v AlovhUVv

Physiolo

 

 

l4
severity of phytophthora root rot. Mycorrhizal fungi decrease
the infection of soybean roots by increasing plant vigor and
competing for site while Phythium, Rhizoctonia, and Fusarium
may increase the severity of root rot. Infection of soybean
plants by the northern root-knot nematode (Meloidogyne hapla

Chitwood) also increases the severity of root rot.

Physiologic races and reistance

Two types of susceptible reactions to Phytophthora are
found iri soybeans; race-specifh: and nonrace—specific
(general) reactions. Race-specific reactions are controlled by
14 (Rps) genes at seven loci. More than thirty races of P.
sojae have been described, and isolates have been found that
do not fit any of the described races. The races can be
distinguished on eight soybean cultivars. Among susceptible
cultivars, many quantitative differences in disease reaction
can be found. The least susceptible reactions are referred to
as field resistance, tolerance, or rate reducing resistance.
Tolerance levels can be evaluated by differential plant loss,
vigor and yield in infested soils or in greenhouse and growth

chamber environments.

Disease

— Q—i
.w

a
p

.935). :
increas:
utilize
urderst:

deployrx

 

15

Disease Management

Control of Phytophthora stem and root rot initially
utilized cnflg/ race specific resistance. However, continued
appearance of new virulent races in response to continued
deployment of resistant host cultivars has led to several
documented failures (ME specific resistance (Schmitthenner,
1985). Because of the potential for race shifts, it has become
increasingly apparent that control of PRR cannot continue to
utilize race-specific resistance effectively without a better
understanding for the P. sojae-soybean interaction and
deployment of other forms of host resistance to the pathogen.

Other methods important in controlling PRR include
tolerance‘, fungicides, (primarily metalaxyl), cultural
practices such as tile drainage, moldboard ploughing, but none
of these have singly proven to be entirely effective. Based on
the above observations, an integrated pest management (IPM)
approach has been advocated in the control of PRR
(Schmitthenner, 1985). Under this IPM strategy, it has been
demonstrated that various combinations of the methods stated
above can lead to effective control of PRR. Currently,

breeders try to incorporate resistance to the most important

 

1 Tolerance denotes slow-rotting and root resistance of
soybeans to compatible races of P. sojae (Mussel, 1980 and
Schmittenner and Walker, 1979).

that a:

attract

sojae

er:

dEUCE

 

16

races in their areas, if that knowledge and resistant

germplasm are available.

Isoflavonoids and Field Tolerance

Isoflavonoids (compounds produced and exuded by plant
roots) have been shown to affect the infection characteristics
of soybean roots by non pathogens (Nair et a1., 1991, Siqueira
et a1., 1991). The production of these compounds varies with
plant age and variety (Osman and Pett, 1983) Siqueira et al.,
1991b) and .may 1x2 an important factor in early season
infection. There is also evidence that certain isoflavonoids
that are found in and exuded by soybean roots are capable of
attracting the zoospores of P. sojae and inducing their
encystment and germination in vitro (Morris and Ward, 1992).
Research by Graham (1989) and Reviera-Vargas, et al. (1993)
suggested that certain isoflavonoids may reduce P. sojae
hyphal growth at low concentrations. It is likely that
isoflavonoid stimulation and or attraction of P. sojae
operates independently of race specific gene resistance by
acting as a prerequisite for germination and initial
colonization. Possibly, these compounds may be directly
related to differences in susceptibility and tolerance to P.
sojae among soybean varieties. In this regard, there is

evidence that susceptible alfalfa roots attract P. sojae more

others
have be
sojae°

;“
.so.i3‘

 

.axis <
siich ‘
demons:
and Se

Qfisis '
che l
c-c:
VG..r a
genlSte
SOIU“

17
than do resistant roots (Chi and Sabo, 1978).

There is an increasing evidence of pathogen specificity in
relation to tolerance of soybean cultivars to P. sojae
(Thomson et a1., 1988). In addition, these authors reported
that some P. sojae isolates may differ in virulence whereas
others differ in tolerance. Tolerant soybean cultivars that
have been selected are not equally tolerant to all races of P.
sojae. Research. by Graham (1989) suggested that certain
isoflavonoids such as genistein may have activity against P.
sojae hyphal growth in vivo when glyceollin production is low.
Taxis of zoospores (HE P. sojae to soybean isoflavones, to
which other Phytophthora spp. did not respond, has been
demonstrated (Rivera-Vergas, 1993). Conjugates of genistein
and several iother aromatic metabolites are selectively
secreted into root and seed exudates (Graham, 1991). Morris
and Ward (1992) suggested that P. sojae has developed a
mechanism for recognition of the same chemical signals as B.
japonicum, although not for the purpose of symbiosis. At 10
ug/ml, the isoflavonoid genistein inhibited radial (hyphal)
growth and reduced asexual reproduction of isolates of 1%
sojae in culture (Vedenyapina et a1., 1996). Work by G. R.
Safir and. T. L. Wacker (unpublished) found that adding
genistein at concentrations as low as 5 ppb to a plant growth

solution can reduce infection of soybean seedlings by

(Kansas
Nebrask

specifi

 

18
zoospores of P.sojae. Thus it this possible that field
tolerance of soybean to P. sojae may be controlled to a large
extend by root isoflavonoid exudation characteristics and by
the presence of certain isoflavonoids within the roots.

The research being reported here is part of multi-state
(Kansas, Illinois, Indiana, Iowa, Michigan, Minnesota,
Nebraska, North Dakota, Ohio, and South Dakota) effort whose
specific objective is to assess the population and structure
of P. sojae, using classical, molecular, and biochemical
techniques. The information resulting from this research will
greatly increase our understanding of the basis for race-
specific resistance as well as enhance our ability to identify
races rapidly and enable growers to manage the disease more
efficiently.

The specific objectives for this part of the project are:
1) Monitor the races or virulence structure of P. sojae
populations in Michigan soybean fields. Information on the
variability of P. sojae as it relates to race and virulence
structure would be a key component of any IPM program which
utilizes plant resistance. This information would be valuable
to soybean breeders as well as agronomists and farmers who
must make practical decisions concerning the management of
PRR. 2)Characterize the nature of field resistance (tolerance)

to P. sojae. The specific goal is to determine the effect of

Arv per.
u ... «iv v e

on Ph‘ft
l‘

 

In

line
of

5,.

V
a

19

exogenously applied genistein (4 ' , 5, 7-trihydroxy isoflavonoid)
on Phytophthora root rot in soybean seedlings, and compare the
fluorescence levels of soybean root exudates to field
tolerance. Given the effects genistein has on the zoospores
of P. sojae (Vedenyapina et al. 1996), it is possible that
there is correlation between field tolerance and the
fluorescence levels of root exudates. 3) Investigate possible
synergistic relationship between soybean cyst nematode and P.
sojae in soybeans. Since P.sojae is essentially a stress
pathogen, it is possible that nematode activity in soybeans
increases susceptibility to infection.

In Chapter 2, race determination and virulence structure
of P. sojae isolates were determined, and the effect of
exogenously applied genistein on the infection and development
of Phytophthora root rot in soybean seedlings was evaluated.
This work reports the first information on the effect of
exogenously applied genistein on the soybean disease. In
Chapter 3, findings on the fluorescence levels of root
exudates from soybean varieties containing both race specific
resistance and various levels of field tolerance to P. sojae
are reported. Also included in this chapter is the effect of
exogenously applied genistein on the development of PPR. In
Chapter 4, possible correlation between soybean cyst nematode

infestation and infection by P.sojae on soybeans is examined.

Athcw,

P
‘CY'Va ‘-

(I)

(5
LOVa‘i;

n u
sick, R

 

20

Literature cited

Anderson, T.R. 1986. Plant losses and yield responses to
monoculture of soybean cultivars susceptible,
tolerant , and resistant to Phytophthora megasperma
f. sp. glycinia. Plant Disease 70:468-471.

Athow, K.L. 1987. Fungal diseases. In: Soybeans: Improvement
production and uses. 2nd Ed.J.R. Wilcox (ed). Amer.
Soc. of Agronomy No. 16 p 167-727.

Barr, D.J.S. 1992. Evolution and Kingdoms of Organisms from
the perspective of a mycologist. Mycologia 84:1-11.

Chi, C.C. and Sabo, F.E. 1978. Chemotaxis of zoospores of
Phytophthora megasperma to primary roots of alfalfa
seedlings. Can. J. Botany 56: 795-800.

Covalier-Smith, T. 1986. The Kingdom Chromista: Origin and
systematics. Pp. 309-347. In: Progress in
physiological Research, Vol. 4. Eds. F. E. Round and
D.J. Chapman. Biopress, Bristol, United Kingdom.

Covalier-Smith, T. 1987. The Origin of Fungi and
Pseudofungi. Pp. 339-353. In: Evolutionary Biology
of the Fungi. Eds. A.D.M. Rayner, C. M. Brasier, and
D. Moore. Cambridge University Press, Cambridge,
United Kingdom.

Dick, M.W. 1990a. Oomycota. Pp. 661-685. In: Hanbook of
Protoctista. Eds. L. Margulis, J.O. Corliss, M.
Melkonian, and D.J. Chapman. Jones and Barlett,
Boston.

Dick, M.W. 1976. The Ecology of Aquatic Phycomycetes. Pp. 513-
542. In: Recent Adavances in Aquatic Mycology. Ed.
E.B. Gareth Jones. Halted, Wiley, New York.

Graaam,

u ‘
7.3213411

I,.‘ ~ 7*
r1:J&;~aH

 

21

Graham, T.L. 1991. Flavonoid and isoflanoid distribution in
develOping soybean seedling tissues and in seed and
root exudates. Plant physiology 95:549-603.

Graham, T.L. 1989. Constitutive conjugates of daidzein and
genistein may play multiple roles in early race
specific antibiotic resistance in soybean. (Abstr.)
Phtopathology 79: 1199.

Hildebrand, A.A. 1959. A root and stolk rot of soybeans caused '
by Phytophthora megasperma Drscchler var. sojae.
Canadian Journal of Botany 37:927-957.

Hymowitz, T. and J.R. Haralan. 1983. The introduction of the
soybean to North America by Samuel Bowen in 1765.
Economic Botany 37:372-379.

Kaufmann, M.J. and J.W. Gerdemann. 1958. Root and stem rot
of soybean caused by Phytophthora sojae n. sp.
Phytopathology 48:201-208.

Kilen T.C. 1986. Relationships between Rps2 and other genes
controlling resistance to phytohthora rot in
soybean. Crop. Sci. 26:711-712.

Kilen, T.C., E.E. Hartwig, and B.L. Keeling. 1974.
Inheritance of second major gene for resistance to
Phytophthora rot in soybean. Crop. Sci. 14:260-262.

Kittle, D.R. and Gray, L.E. 1979. The influence of soil
temperature, moisture, porosity, and bulk density on
the pathenicity of Phyotophthora megasperma var.
sojae. Plant Disease 63:231-234.

Kuan. T. and Erwin, D.C. 1980. Formae speciales
differentiation of Phytophthora megasperma isolates
from soybean and alfalfa. Phytopathology 70:333-338.

Layton, L.C., K.L. Athow, and F.A. Laviolette. 1986. New
physiological race of Phytophthora megasperma f. sp.
sojae. Plant Disease. 70:500-501.

Moots, C.K. Nickell, C.D. and Gray, L.E. 1988. Effects of
soil compaction on the incidence of Phytophthora
megasperma f. sp. glycinea in soybean. Plant
Disease. 72:896-900.

VAVY“<
A.Vlb‘-~

h'
iTClr’

A". ,.
Vilma”,

Ribeir

P
P
P

Rivera

Snn~s
GVLH

s~z~ .

. YV-

Vutl‘l t "
h

 

 

22

Morris, P.F. and Ward, E.W.B. 1992. Chemoattraction of
zoospores of the soybean pathogen , Phtophthora
sojae, by isoflavones. Physiological and MOlecular
Plant Pathology. 40:17-22.

Nair, M.G. G.R. Safir, and J.O. Siquera. 1991. Isolation and
identification of vesicular-arbuscular mycorrhiza-
stimulatary compounds from clover (Trifolium repens)
roots. Appl. Environ. Microbiol. 57: 434-439.

Osman, S.F. and W.P. Fett. 1983. Isoflavone glucoside stress
metabolites of soybean leaves. Pytochemistry 22:
1921-1923.

Ribeiro, O.K. 1983. Physiology of Asexual Sporulation and
Spore Germination in Phytophthora. Pp. 55-70. In:
Phytophthora: Its Biology: Taxonomy, Ecology and
Pathology by D.C. Arwin, S. Bartnicki-Garcia, and
P.H. Tsao (eds).

Rivera-Vargas, L.T., A.P. Schmittenner, and T.L. Graham.
1993. Soybean flavonoid effects on metabolism of
Phytophthora sojae. Phytochemistry 32: 851-857.
Schmittenner, A.F. 1985. Problems and progress in
control of Phytophthora root rot in soybean. Plant

Disease 69: 362-368.

Scott, W.O. and Aldrich, S.R. 1970. Modern Soybean Production.
Pp. 167-172. S and A publications, P.O. Box 2660,
Station A, Champaign, Illinois, 161820.

Shaffer, R.L. 1975. The major Groups of Basidiomycetes.
.Mycologia. 67:1-18

Schmitthenner, A.F. and D.M. Van Doren, Jr. 1985. Integrated
control of root rot of soybean caused by Phytophthora
megasperma f2 sp. glycinea. In Ecology and Management
of Soilborne Plant Pathogens. Eds. Parker, C.A.,
A.D. Rovira, K.J. Morre and P.T.W. Wong.

Schmitthenner, A.F. and A.K. Walker. 1979. Tolerance versus
resistance for the control of phytophthora root rot
in soybeans. In: H.D. Loden and D. Wilkenson (eds),
Proc.19th Soybean Seed Research Conf., Chicago, 111.,
13-14 Dec. 1979. Am. Seed Trade Assoc., Washington
D.C.

aa1k9r‘

23

Siqueira, J.O., Nair, M.G., Hammerschmidt, R., and G.R. Safir.
1991. significance of phenolic compounds in Plant-
soil-microbiol systems. CRC Crit. Rev. in Plant
Science 10: 63-121.

Sogin, M.L. 1992. Comments on Genome Sequencing. Pp. 387-389.
In: The Origin and Evolution of Prokaryotic and
Eukaryotic Cells. Eds. Hartman and K. Matsuno. World
Scientific, Singapore.

Suhovecky, A.J. and Schmitthenner, A.F. 1955. Soybeans
affected by early root rot. Ohio Farm and Home
Research, Sept.- Oct., pp. 85-86.

Thatcher, L.L. 1947. Soybeans now a major crop in Ohio. In:
Welcome to Ohio. American Soybean Association 1947
Annual Exposition Booklet, Amer. Soybean Assoc.
St. Louis, MO. P 7-8.

Thomison, P.R., C.A. Thomas, W.J. Kenworthy, and M.S.
McIntosh. 1988. Evidence of pathogen specificity in
tolerance of soybean cultivars to phytophthora rot.
Crop Science 28:714-715.

Tooley, P.W. and C.R. Grau. 1982. Identification and
quantitative characterization of rate-reducing
resistance to Phytophthora megasperma f. sp.
glycinea in soybean seedlings. Phytopathology 72:
727-733.

Vedenyapina, E.G., Gene R. Safir, Brendan A. Niemira, and
Thomas E. Chase. 1996. Low Concentrations of the
Isoflavone Genistein Influence in vitro Asexual
Reproduction and Growth of Phytophthora sojae.
Phytopathology 86:144-148.

Walker, A.K. and A.F. Schmitthenner, 1984c. Comparison of
field and greenhouse evaluations for tolerance to
phytopthora rot in soybean. erp Sci. 24:487-489.

RACE

MICHI

Abstr

in .

w u. at
2.. v a
‘ v ...~.y
«D ..A

.3.
at

it
shit“

24

CHAPTER 2

RACE DETERMINATION OF ISOLATES OF PHYTOPHTHORA SOJAE FROM
MICHIGAN FIELDS AND ESTIMATION OF ECONOMIC IMPACT OF PRR ON

YIELD

Abstract

Knowledge on the race composition of P. sojae (Kaufmann
and Gerdemann) that occur in any one field or soybean growing
area is important in the management of Phytophthora root and
stem rot of soybeans. In this study, plant and soil samples
were obtained from Michigan soybean fields through scouting
and in collaboration with growers and extension service
agents. A total of one hundred and fifty field isolates of P.
sojae were obtained, and ninety were evaluated for virulence
using differential soybean cultivars.

Fifty five per cent (55%) of the isolates tested were
highly virulent, and defeated seven or more Rps genes each.
Ten (13%) defeated four to six genes each while 20% or eihteen
defeated one to four genes each. Eleven of the isolates were

avirulent. as they' did run: attack any (M5 the Rps genes

25
including the susceptible variety Williams (rps). These
results show the potential of P. sojae to impact yield in

soybean production in the state.

Introduction
Monitoring of race or virulence structure of a pathogen

population is a key component in any management program which
utilizes specific resistance in the control of disease. Race
specific genetic resistance has been a major element in the
control of PRR. However, the appearance of new virulent races
in response to continued deployment of resistance host
cultivars has led to several documented failures of specific
resistance (Schmitthenner, 1985). Race characterization of P.
sojae is based on its differential reaction to 13 genes (in 7
loci) using' the ihypocotyl inoculation test (Ward, 1990).
Currently, there are 39 races of P. sojae known. Breeders try
to incorporate resistance to the most important races in their
areas, if that knowledge and resistant germplasm are
available. But in many cases, growers are forced to rely on
varieties in which resistance does not exist or inadequate
knowledge on P. sojae races exist to allow informed choices of
resistant germplasm (Schmittenner, 1985).

Other methods important in controlling PRR have included

tolerance (field resistance), fungicides (primarily

26

metalaxyl), and cultural practices such as moldboard plowing,
and tile drainage, but none of these have by themselves proven
to be entirely effective (Schmittenner, 1985). Based on the
above observations, Schmitthenner (1985) has advocated an
integrated pest management (IPM) approach to control of PRR.
Under this IPM strategy it has been demonstrated that various
combinations of the methods stated above can lead to effective
control of PRR. In order to make host resistance an effective
component oftflmaIPM program, knowledgecflfthe availability of
resistant germplasm and the virulence structure of the
pathogen in target area is vital.

Although Michigan soybeans have been relatively free from
PRR, potential for economic impact does exist. Occasionally,
Phytophthora is identified in plant samples sent to the MSU
plant, diagnostic: clinic tux growers enmi extension service
agents. Lockwood (Lockwood et al, 1985) isolated races 1, 3,
4, and 6, and involvement of P. sojae in the root rot complex
of soybeans in the state has been proven. Thus, there is need
for current information on the occurrence and virulence
structure of PH sojae in the soybean growing areas of the
state. Presently, information on the occurrence or virulence
structue of P. sojae and its impact on yield in Michigan
fields is not sufficient enough for farmers to make informed

decisions in the selection of available resistant varieties.

. . Y .

c. u e . . E

e rt. .. I .. l . t

..r u r n . . LN.» r U «H»
m. . Ci V. C C. no.

 

 

I
.J iGI ark. we. , _ :9 Tu rk ._
r e .. i t t ,3 .. r 31 c, I
e l m.“ «.s FF» a? 4 x \: le Wins
t l firv all; .7. W .. 2; Win ..r I. p r.»
o v. «C . i . v. .. a Q. ..ri. ..l
C c r C .T. .5 C. t r; 3.. C I

27
The first objective of this project was to obtain isolates of
P. sojae ifixxn Michigan soybean fields euui determine their
virulence characteristics and race structure. The second
objective was to locate a soybean field which contains
Phytophthora root/or stem rot and then to estimate the
potential economic impact of P. sojae on yield.

Results from this project will increase our knowledge on
the status of P. sojae and the occurrence of PRR in Michigan.
It is hoped that information from this project will enable
breeders and growers make informed choices in the control of
PRR and thus enhance its efficacy as an IPM component in low

input production systems.

Materials and.methods
Collection of samples

A total of 260 soybean and 20 soil samples were obtained
from Michigan soybean fields through scouting and
collaboration with extension service agents over a period of
five growing seasons (1993—96). Plants with well defined stem
symptoms were collected mostly from depressed areas in the
fields. In a rainy spring, these depressions collect water and
remain wet for long periods, a condition that is conducive to
Phytophthora root and stem rot. Other samples were obtained

from farmers through the MSU plant disease clinic. Plants were

v

\A.

 

5,-
hfir‘ «
vb’n‘.»

5

Media

p-

: s
AU

28
placed in plastic bags and stored at 40c to maintain pathogen
viability as samples were being processed. In 1995, soil
samples were also collected in Eaton and Monroe counties. Each
soil sample consisted.aa composite CH? soils collected from

various parts of a field.

Media

The Canaday — Schmitthenner Medium (Canaday and
Schmitthenner, 1982) was used to isolate Phytophthora from
plant samples. The seedling bioassay method(Canaday and
Schmitthenner, 1982), a technique that minimizes Pythium
contamination, was used to isolate Phytophthora from the soil
samples. The isolation medium consist 40 ml of V-8 juice, 2000
ppm CaCo“ 20 ppm pentachloronitrobenzine, 10 ppm benomyl, 100
ppm neomycin sulfate, and 9 ppm rifampicin per one liter. Two
grammes of CaCo3 was added to one liter of V-8 juice and
heated to 80oc, and allowed to cool to room temperature, and
then centrifuged tx> clarify prior tx> incorporation iji the
medium. With the exception of rifampicin, all ingredients were
added prior tx> autoclaving. After autoclaving the medium,
rifampicin was added in 5 ml of 95% ethanol. Hymexizol was
excluded.because<flfits potential to inhibit sensitive strains
of Phytophthora. All chemicals were obtained from Sigma

Chemical Corporation; St. Louis Missouri.

15018

n‘ ‘
AH»

-.nu

aim:
rats:
Isola‘

v
6
b

-
area» ‘; -

3 to
{155‘

:ainf
mi

29

Isolation from diseased soybean plant samples

Stems and roots of plants with well defined symptoms were
disinfected with 10% bleach for ten min. and thoroughly rinsed
3 to 4 times with sterile distilled water. Small sections of
tissue were taken from the edges of advancing lesions and
placed on the isolation medium. Necrotic tissue was also
removed from plants with severe flood damage (following heavy
rainfall) and placed on the medium after thoroughly surface
sterilizing and washing as described above. In all cases, the
plant tissues were placed under the mediunt in order to
minimize bacterial contamination by limiting oxygen
availability. As soon as hyphae of Phytophthora appeared in
the medium, they were hyphal-tipped in clean areas and

transferred tormnrmedium-platestx>avoid contaminating fungi.

Isolation from soil with the soybean seedling bioassay'method

The Soybean Seedling Bioassay technique (Canaday, and
Schmittenner, 1982) was used to isolate P. sojae from soil.
Each soil sample was air-dried and passed through a 3 mm mesh
sieve. Approximately 800 g of air-dried soil from each soil
sample was placed in plastic pots (3 pots/soil sample). Pots
were flooded overnight, then drained and allowed to air-dry
until the moisture content approached -300 mb matrix potential

(soil cracks or pulls away from side of container although it

cult"

place:

 

30
is still damp). After draining, pots were sealed in plastic
bags and incubated in dark at room temperature to induce
oospore germination. Two weeks after flooding, the surface 1
cm of soil in each pot was tilled. Twenty seeds of the
cultivar sloan (rps), susceptible to all races of P sojae were
placed in the surface of 1 cm of soil and covered with
polyethylene bags to prevent drying during germination. Three
days after germination, the pots were again flooded for 24
hrs. Pots were then drained and incubated for 10 days. During
this period, seedlings emerged. and 'were damped-off when
Phytophthora was present. Phytophthora could be readily
isolated from collapsed seedling hypocotyls using procedures

described earlier.

Production of single zoospore cultures

Fifteen pieces (1 mm diameter) of culture from the edges
of actively growing colonies of P. sojae on dilute V-8 juice
agar were placed into 25 m1 of quarter strength V-8 liquid
medium (50 ml V-8/L water). After 48 hrs., the culture medium
was poured off and replaced with. 25 ml of Aphanomyces
Replacement Solution (2.94 g CaClzJMyO, 2.47 g MgSO4.7HfiL
and 0.75 g KCL/1000 ml of distilled deionized water) at Ph
7.0. The solution was changed 4 times at 5 min. intervals. The

final salt solution was replaced with 20 ml of sterile de-

zoos;
last:

with

 

Ident

 

 

31
ionized distilled water and cultures were incubated under cool
white lights at room temperature. Sporangia formed 12 hrs
after cultures were flooded. Cultures were placed in the
refrigerator (4°C) for 4 hours and then incubated on the bench
at room temperature. Large numbers of zoospores were seen
swimming freely within an hour.

Zoospore concentration was estimated by placing a 50 ul
zoospore suspension on a slide and staining with 25 ul of
lacto-phenol Tryphan blue solution. The suspension was covered
with a 22 X 22 mm cover-slip and zoospores were counted.
Through dilution series, the zoospore concentration was
adjusted to 1x10'2 and plated onto a 1/4 strength V-8 agar

medium (200 ml/l liter) to generate single zoospore cultures.

Identification

Two keys, one that groups species of Phytophthora by
Sporangial characteristics (Waterhouse, 1963) and one that
Used other characteristics were used in the identification of
phytophthora isolates. Host range, growth rate and oogonal
Size were used to delineate P. sojae from P. megasperma. D.H.
Scott of Purdue University supplied the isolate (1819B-type

Culture) of P. megasperma used in the host range study.

32

Test for virulence and race determination

Isolates of P. sojae were tested for virulence and race
determination by inoculating seedlings of a differential set
of soybean cultivars basedcxitheir reactionstx>the pathogen,
using the hypocotyl injection method (Hass and Buzell, 1976).
P. sojae cultures were grown on soft (12 g agar/L) dilute (40
ml V-8 juice/L) V-8 juice agar until the mycelium covered the
plate. Strips of the cultures were cut and placed in a 10 ml
syringe and forced through to make a slurry of the culture.
The syringe was then reloaded with the slurried culture and a
# 18 needle was put on it. Six-day' old seedlings were
inoculated by making a slit about one 1 cm long in the
hypocotyl of the seedling just below the cotyledonary node
with the needle tip. Approximately 0.2 to 0.4 ml (200 to 400
cfu/ml) of the culture slurry was deposited in the slit with
the syringe. Plants were then covered with clear plastic bags
for 12 hrs to prevent drying of the agar slurry before
infection could take place.

The plants were incubated at 25%: in 14 hrs of light for
one week. Soybean seedlings with specific resistance developed
characteristic hypersensitive response which hinders growth of
the pathogen by killing the plant tissue around the infection

site and creating a necrotic fleck. The susceptible varieties

33

died or manifested distinctive lesions during this period.

Estimation of the impact of P.sojae on yield

P. sojae was isolated from all soybean samples from a
field in Saginaw county that were provided by Dr. L. P. Hart.
Initial survey showed random distribution of disease in the
field, and a systematic zigzag approach was used in the
collection. of samples. Samples yielded highly virulent
isolates from the field. Due to the high incidents of PRR in
the field, it was decided that impact on yield be estimated.
In 1997, Grower Service Corporation (St. Charles MI), provided
stand. counts (made (N1 Sept.4) and yield. estimates for
selected strips of seven rows ( 20' combine head) within the
diseased field and also for strips within an adjacent field
containing the same soybean cultivar and grown under the same
cultural and pest management conditions. The two fields (60
acres each) were separated by a narrow strip of corn. The
adjacent field which appeared healthy throughout the growing
season was not sampled extensively for Phytophthora, however
P.sojae was not found in it (table 2.5). The soybean fields in
this study were planted with soybean variety Golden Harvest
1271 (which does not have Rps genes) at a rate of 190,000-
200,000 seeds /acre. The diseased field was planted on June 3

and sprayed on Jul.3 with 2.7 oz Cobra, 1/4 pinnacle, Choice

and AC

ci'fiié

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34
and Act 90. The non-diseased field was planted and sprayed in
similar fashion except for the Cobra application of 2.0 oz.
It is difficult to say whether or not the rate difference in
the application of Cobra was a factor in disease. But unlike
the healthy field, the diseased field had depressed areas
which had high moisture content earlier in the season, and
there was a higher frequency of symptomatic plants around the

depressions. The fields were harvested on Oct. 25.

Results
Isolation and identification of P. sojae

A total of 150 field isolates of P. sojae were obtained
from diseased soybean plant samples from Barrien, Clinton,
Eaton, Ingham, Ionia, Jackson, Lenawee, Monroe, Oakland,
Saginaw, and Shiawassee counties in the 1993-96 growing
seasons (Table 2.1). Isolates were also obtained from soil
samples from Eaton and Monroe counties.

The oogonial size of isolates on V—8 agar ranged from 26-
44 um and were similar to those described for P. sojae (Kuan
and Erwin 1980). The sporangia were pyriform and nonpapillate
and virulent isolates infected only susceptible soybean
seedlings but did not cause disease in alfalfa and dry beans
{(variety black magic) (figure)). Average radial growth rate

for isolate cultures was 3.6 mm/day and was less than that of

Table 3.1 Re:
Sitday (
hypocot}
P.sojae.
susceptil

R: resis

35

Table 2.] Reaction of soybean resistance genes to the Michigan field isolates of P. sojae.
Six-day old soybean seedlings were inoculated by making a 1 cm long slit in the
hypocotyls and depositing 0.2 to 0.4 ml. of agar slurry containing 200 to 400 cfu/ml of
P. sojae. Resistant varieties developed characteristic hypersensitive response while
susceptible lines died or manifested distinctive lesions within one week.

R= resistant; S= susceptible

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m m m m m m m m m m m m m m m m m swam Gmroefi xmm
m a m m m m m m m m m m m m m m m swam oemrmmq
m m m m m m m m m m m m m m m m m dem mamarmeq
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m m m m m m m m m m m m m m m m m Emma MOH Ha
m m m m m m m m m m m m m m m m m swam me mamaaaaz
m m m m m m m m m m m m m m m m m amdm xxmfi NAomoumm
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m m m m m m m m m m m m m m m m m .mom mmmmrmmq
m m m m m m m m m m m m m m m m m amdm marmaa xmm
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m m m m m m m m m m m m m m m m m amom oemrmma
m m m m m m m m m m m m m m m m m dem mamarmeq
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m m m m m m m m m m m m m m m m m .mdm mmmmimma
m m m m m m m m m m m m m m m m m swam marmefi xmm
m m m m m m m m m m m m m m m m m swam mmrmefi xmm
m m m m m m m a m m m m m m m m m amdm oemrmmq
m m m m m m a m m m m m m m m m m dem mmmflrmeq
m m m m m m m m m m m m m m m m m imam mm mamaaaaz
m m m m m m m m m m m m m m m m m Emma mofi Hm
m m m m m m m m m m m m m m m m m amdm me mamflflaaz
m m m m m m m m m m m m m m m m m amdm xxma somoomz
m m m m m m m m m m m m m m m m m smmm :oHumz
m m m m m m m m m m m m m m m m m may mamaafiaz
mm we mm mm em mm mm Hm om mm mm em mm mm em mm mm
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m m m m m m m m m m m m m m m m m .mmm somoum:
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m m m m a m m m m m m m m m m m m .mdm mmomrmmq
m m m m a m m m m m m m m m m m m .mdm mmmmrmmn
m m m m m m m m m m m m m m m m m amdm marmafi xmm
m m m m m m m m m m m m m m m m m emdm mmrmefl xmm
m m m m m m m m m m m m m m m m m amdm oemrmma
m m m m m m m m m m m m m m m m m Nmum mmaHrmea
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m m m m m m m m m m smdm mermefi xmm
m m m m m m m m m m amdm omroefi xmm
m m m m m m m m m m amdm oemrmmo
m m m m m m m m m m dem mmmfiroeo
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m m m m m m m m m m m m m m m m m .mom mmmmrmmo
m m m m m m m m m m m m m m m m m amdm mermaa xmm
m m m m m m m m m m m m m m m m m smdm omroee xmm
m m m m m m m m m m m m m m m m m amdm oemrmmo
m m m m m m m m m m m m m m m m m dem mmmfiroea
m m m m m m m m m m m m m m m m m :mdm mm mamaaaflz
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39

Table 2.2 Virulence levels of field isolates of P.sojae..
Isolates were ranked according to the number of Rps
genes defeated :

Low virulence.................1—4 Rps genes.
Intermediate virulence........4-6 Rps genes.
High virulence...... ..... .....7-12 Rps genes.

* Percent of the 90 isolates tested.

 

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Low virulence Intermediate virulence High virulence
Number of Number of Number of

Isolate genes def. Isolate genes def. Isolate genes def.
MSU 01 2 MSU 78 5 MSU 57 7
MSU 02 2 MSU 81 5 MSU 58 7
MSU 14 3 MSU 85 5 MSU 59 9
MSU 16 3 MSU 89 5 MSU 62 12
MSU 31 4 MSU 64 7
MSU 34 4 Percentage 13% MSU 69 10
MSU 54 3 MSU 70 7
MSU 55 4 High virulence MSU 72 7
MSU 56 3 MSU O8 9 MSU 73 11
MSU 60 4 MSU 10 9 MSU 76 7
MSU 61 2 MSU 15 8 MSU 77 11
MSU 63 3 MSU 17 7 MSU 80 10
MSU 65 4 MSU 22 8 MSU 82 11
MSU 68 3 MSU 23 12 MSU 86 8
MSU 71 3 MSU 24 12 MSU 87 11
MSU 79 4 MSU 25 12 MSU 88 10
MSU 83 4 MSU 26 12 MSU 9O 9
MSU 84 3 MSU 27 13

MSU 28 12 Percentage 55%
Percentage 20%* MSU 29 9

MSU 30 11
High virulence MSU 32 12
MSU O3 5 MSU 33 10
MSU 09 5 MSU 35 9
MSU 21 5 MSU 36 10
MSU 47 6 MSU 37 8
MSU 50 5 MSU 48 7
MSU 51 5 MSU 49 7
MSU 66 6 MSU 52 8
MSU 75 6 MSU 53 8

41

Table 2.3 Ranked performance of Rps genes against isolates
of P. sojae.

* Following hypocotyl inoculations, gene is defeated
when 50% or more of inoculated material are killed.

 

42

 

Percent of isolates*

 

Source Gene defeating the gene
PRX 146-36 Rps3n 20
L83-57O Rps3a 24
Harosoy 13xx Rpslb 27
Williams 82 Rpsl.K 32
Harosoy 62xx RPS6 35
L76-1988 Rps2 39
L85-2352 RPS4 40
L85-3059 RPSS 45
Williams 79 Rpslc 46
PI 103 Rpsld 46
PRX 145-48 Rps3C 55
Harlon Rpsla 78
Harosoy RPS7 79

 

43

Figure 2.1 Typical empty parch in a depressed area of a field affected by P. sojae near St.
Charles in Saginaw county. Plants with fully manifested symptoms of
Phytophthora root rot were concentrated around the empty spot.

Images in this dissertation are presented in color.

44

 

45

Figure 2.2 Field isolates of P. sojae were evaluated for host range by comparing them to
P. megasperma in soybeans, dry beans (black magic) and alfalfa seedlings.
P. sojae isolates killed only the soybean seedlings while P. megsperma did not
attack any of the plants as it was found to be avirulent.

Images are presented in color.

46

 

47

Figure 2.3. For identification, growth rates of field isolates of P. sojae were compared to
that of P. megasperma. P. megasperma (center)covered the plate in six days while
it took P. sojae isolates ten to twelve days to cover plates.

Images are presented in color.

4s

 

49

Figure 2.4 Number of highly virulent isolates obtained increased over the sampling
seasons. In 1993 an average of only 3 Rps genes were defeated by an isolate. In
1997 an average of seven Rps genes were defeated per isolate. This may be
attributed to wider area covered in subsequent years of sampling.

50

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0
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1995 1996 1997
Year

1994

1993

51

Table 2.4 Year, county of origin and virulence formulae of P. sojae isolates.

* Virulence formulae = list of the Rps genes defeated by isolate.
@ NM = virulence formulae do not match those of the known races of P. sojae

52

 

1993

Mite Countv Source Virulence formulae“ Race
MSU Ol Eaton plant 1b , 7 2
MSU 02 Ionia plant 1a, 7 3
MSU O3 Shiawassee plant 1a, 1b, 1c, 1k, 7 25
me

MSU O4 Eaton plant Avirulent

MSU 05 Eaton plant Avirulent

MSU O6 Eaton plant A virulent

MSU O7 Ingham plant Avirulent

MSU 08 Barrien plant 1a, lb, 1c, 1d, 1k, 2. 3a, 3b, 7 NM @
MSU 09 Barrien plant 1a, 2, 3c, 5, 7 NM
MSU IO Saginaw plant la, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 11 Saginaw plant Avirulent

MSU 12 Saginaw plant Avirulent

MSU 13 Oakland plant Avirulent

MSU l4 Eaton plant 1a, 1c, 7 4
19.9.5

MSU 15 Eaton plant 1a, 10, 1k, 2, 3a, 4, 5, 7 NM
MSU 16 Eaton soil 1a, 1c, 7 4
MSU 17 Ionia plant 1a, 10, 2, 3a, 3b, 5, 7 NM
MSU l8 Ionia plant Avirulent

MSU 19 Ionia plant Avirulent

MSU 20 Monroe soil Avirulent

MSU 21 Monroe plant 1a, 1b, 1c, 1k, 7 25
MSU 22 Monroe soil 1a, lb, 1c, 1d, 1k, 3a, 3b, 7 NM
w

MSU 23 Ingham plant 1a, 1b, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 24 Ingham plant 1a, 1b, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 25 Clinton plant 1a, 1b, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 26 Monroe plant 1a, 1b, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 27 Jackson plant 1a, 1b, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 28 Monroe plant 1a, 1b, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 6, 7 NM
MSU 29 Ingham plant 1a, 1b, 1d, 1k, 3a, 3c, 5, 6, 7 NM
MSU 30 Monroe plant 1a, 1b, 1d, 1k, 2, 3b, 3c, 4, 5, 6, 7 NM
MSU 31 Shiawassee plant 1a, 2, 3c, 7 NM
MSU 32 Ingham plant la, lb, 1c, 1d, 1k, 2, 3a, 3c, 4, 5, 6, 7 NM
MSU 33 Lenawee plant 1a, 1b, 1d. 1k, 2, 3c, 4, 5, 6, 7 NM
MSU 34 Lenawee plant la, 1b, 3c, 7 NM
MSU 35 Monroe plant la, 1c, 1d, lk, 3c, 4, 5, 6, 7 NM
MSU 36 Monroe plant 1a, 1c, 1d, 1k, 2, 3c, 4,5,6,7 NM

53

 

 

Race

 

l 97
Isolate Com Sogrce Virulence formme
MSU 37 Saginaw plant la, 1c, 1d, 2, 3c, 4, 5, 7
MSU 38 Saginaw plant 1a, 1c, 1d, 1k, 2, 3a, 3b, 3c, 4, 5, 7
MSU 39 Saginaw plant la, 1c, 1d, 1k, 2, 3c, 4, 5, 7,
MSU 40 Saginaw plant 1a, 1c, 1d, 2, 3c, 4, 5, 7
MSU 41 Saginaw plant la, 1c, 1d, 1k, 2, 3c, 4, 5, 7
MSU 42 Saginaw plant la, 1c, Id, 2, 3c, 4, 5, 7
MSU 43 Saginaw plant 1a, 1c, 1d, 2, 3c, 4, 5, 6, 7
MSU 44 Saginaw plant la, 1c, 1d, 2, 3c, 4, 5, 6, 7
MSU 45 Saginaw plant 1a, 1c, 2, 3c, 4, 5, 6, 7
MSU 46 Saginaw plant 1a, 1c, 1d, 3c, 4, 5, 6, 7
MSU 47 Saginaw plant la, 1c, 1d, 3a, 3c, 7
MSU 48 Saginaw plant 1a, 1c, 1d, 2, 3c, 4, 7
MSU 49 Saginaw plant In, 1c, 1d, 2, 3c, 4, 7
MSU 50 Saginaw plant la, 1c, 1d, 3c, 7
MSU 51 Saginaw plant 1a, 2, 3c, 4, 7
NM
MSU 52 Saginaw plant 1a, 2, 33, 3c, 4, 5, 6, 7
MSU 53 Saginaw plant la, 1c, 1d, 2, 3c, 5, 6, 7
MSU 54 Saginaw plant 1a, 3c, 7
MSU 55 Saginaw plant la, 1c, 3c, 7
MSU 56 Saginaw plant la, 1d, 7
MSU 57 Saginaw plant 1a, 1c, 1d, 3c, 4, 5, 7
MSU 58 Saginaw plant 2, 3a, 3c, 4, 5, 6, 7
MSU 59 Saginaw plant la, 1d, 2, 3a, 3c, 4, 5, 6, 7
MSU 60 Saginaw plant 1a, 1c, 3c, 7
MSU 61 Saginaw plant la, 7
MSU 62 Saginaw plant 1a, lb, 1c, 1d, 1k, 2, 1k, 3b, 3c, 4, 5, 6, 7
MSU 63 Saginaw plant la, 5, 7
MSU 64 Saginaw pant la, 1d, 1k, 3c, 4, 5, 6
MSU 65 Saginaw plant 1a, 1c, 3c, 7
MSU 66 Saginaw plant la, 1b, 1c,_3b, 6, 7
MSU 67 Saginaw plant la, 1c, 1d, 1k, 3c, 5, 6, 7
NM
MSU 68 Saginaw plant 1a, 3c, 7
MSU 69 Saginaw plant la, 1c, 1d, 3a, 3b, 3c, 4, 5, 6, 7
NM
MSU 70 Saginaw plant la, lb, 2, 3a, 5, 6, 7
MSU 71 Saginaw plant la, lb, 1k

MSU 72 Saginaw

plant

la, lb, 1c, 1d, 1k, 5, 7

NM
NM
NM
NM

NM
NM
NM
NM
NM
NM
NM
NM
NM
NM

NM
NM
NM
NM
NM
NM
NM
NM

NM
NM
NM
NM

NM
NM

NM

NM
NM

54

 

NM

NM
MSU 73 Saginaw plant la, 1b, 1d, 2, 3a, 3c, 5, 6, 7
MSU 74 Saginaw plant 1a, 1c, 1d, 2, 3a, 3c, 5, 6, 7
MSU 75 Saginaw plant 1a, 1c, 1k, 3c, 6, 7
MSU 76 Saginaw plant 1a, 1b, 1c, 2, 3b, 5, 7
MSU 77 Saginaw plant la, lb, 1c, 1d, 2, 3a, 3b, 4, 5, 6, 7
NM
MSU 78 Saginaw plant la 3c, 5, 6, 7
1997
[Siam Countv Source Virulence formulae
m
MSU 79 Saginaw plant la, 1c, 7
MSU 80 Saginaw plant la, 1b, 1c, 1d, 1k, 3c, 4, 5, 6, 7
NM
MSU 81 Saginaw plant la, 2, 4, 5, 7
MSU 82 Saginaw plant 1a, 1c, 1d, 1k, 2, 3a, 3c, 4, 5, 6, 7
MSU 83 Saginaw plant la, 1c, 3c, 7
MSU 84 Saginaw plant 1a, 4, 7
NM
MSU 85 Saginaw plant 1a, lb, 1d, 1k, 7
MSU 86 Saginaw plant 1a, lb, 1d, 1k, 3a, 3b, 3c, 7
MSU 87 Saginaw plant 1a, 1b, 1c, 1d, 1k, 3b, 3c, 4, 5, 6, 7
MSU 88 Saginaw plant la, 1b, 1c, 1d, 1k, 2, 3a, 3c, 6, 7
MSU 89 Saginaw plant 1a, 3c, 5, 6, 7
MSU 90 Saginaw plant 1a, 1d, 1k, 2, 3c, 4, 5, 7

NM
NM
NM
NM

NM

NM

NM
NM
NM

NM
NM
NM
NM
NM

55

Table. 2.5. Estimated impact of P. sojae on yield in a field near St. Charles in Saginaw
county. Stand counts and yield data were supplied by Growers service Corporation,
St. Charles, MI.

* Percent reduction in stand count and yield of non-diseased field.

56

 

Di_se_ase field Non disease field Percent reduction”

 

Planting rate 190,000 - 200,000 190,000 - 200,000

Pest mgt. 2.7 oz Cobra, 2.0 oz Cobra,
0.25 oz Pinnacle, 0.25 oz Pinnacle,
Choice and Act 90. Choice and Act 90.

Stand count 123,000 180,000 32

Yield (bu/strip) 37.9 57.5 34

57
P. megasperma (6.8 mm/day). Hypocotyl inoculation of soybean
varieties with different resistance (RPS) genes to P. sojae
resulted in avirulent to highly virulent reactions with
formulae that do not match those of currently known races of
the pathogen (Table 2.4). These traits delineate the isolates
from P..megasperma which has wider host range, larger oogonia

(>45 um) and a faster growth rate (Figure 2.3).

Tests for virulence and race determination
Ninety (90) (ME the field isolates were tested for
virulence and race determination. Based on reactions to genes
(RPS) for resistance to P. sojae, isolates were placed on 4
categories of virulence‘. Fifty or 55% of the isolates tested
defeated more than 7 RPS genes each and were categorized as
highly virulent (table 1.2). Ten isolates (13%) showed
intermediate virulence defeating 4-6 RPS genes while 20%
showed low virulence levels defeating 1-4 genes. Eleven
percent(11%) of the isolates were avirulent as they did not
attack any (M5 the genes including the susceptible variety
Williams (rps).
Performance of the RPS genes in their respective soybean

varieties showed lb, 3,, and 3b with the best performance,

 

1 Degree of virulence is based on number of RPS genes
attacked.

58

resisting 70-78% of the isolates while 1,, and 7 had the

lowest performance, resisting only 12-13% of the isolates

(Table 1.3).

Impact of P. sojae on yield

The average stand count of 10 samples from the diseased
field was 123,000 plants/acre. The health field had
approximately 180,000 plants/acre (Table 2.5). This translates
to 32% reduction in stand count. Yield estimates from four
strips within the diseased field were 35.08, 39.9, 43.8, and
32.8 Bu/strip for an average of 37.9 Bu/strip whereas the
yield for the.healthy field yum; 47.8 Bu/strip. This amounts

to an approximate 21% reduction in yield.

Discussion

This study brings up to date information on the status of
P. sojae and occurrence of PRR in soybean growing areas of
Michigan. The isolates obtained include some (Hf the races
(1,3, and 4) identified by Lockwood et al (1985). Most were
highly virulent (defeat most of the RPS genes) and thus show
the potential to reduce yield when and where environmental
conditions are favourable. The development of PRR is highly

dependent on the environment, particularly moisture and

59
temperature. The empty patches (Figure 1) in the field were
wet spots in the early part of the growing season, and disease
was more intense around these areas at the time of sample
collection.

In light of the highly virulent levels exhibited by the
isolates, it is noteworthy that the hypocotyl injection test
is a wound-inoculation technique and may bypass some natural.
defense mechanisms. It has also been noted (Schmittenner and
Walker, 1979) that some cultivars which are killed by the
hypocotyl inoculation are not severely damaged in the field
and show little yield loss. As such, the technique does not
provide information substantial enoughtx>estimaterperformance
of soybean lines under field conditions. A well-devised non-
wounding inoculation method would therefore be appropriate in
the deduction of such information.

According tx> the 1996/97 Michigan Soybean Performance
Report (B.W. Diers and J.F. Boyse. Department of Crop and Soil
Sciences, Michigan State University, East Lansing, MI), RPS
genes 1,,Lu1w, 3, 6 and 7 are incorporated either singly or in
combinations (1,,+3, lc+3 and 1,+6) in varieties that are
currently planted or being developed in Michigan. Previously,
these genes have been reported to be resistant to most of the
currently known races of P. sojae. In this study, RPS genes

1, and 7 had the lowest resistance levels, each being

6O
susceptible to at least 80% or more of the isolates tested
(Figure 2.1). RPS genes 3,,, 3,, 1b, 1k and 6 were (in that
order) the most resistant. These genes resisted most of the
races including the highly virulent (those defeating more than
8 genes) among them, and only MSU 23 and 27(note that M8023,
24, 25, 26 and 28 have similar virulence formulae) out of the
90 that were tested for virulence defeated all the 5 genes
(Table 2.1). Incorporating these genes singly or in
combinations in soybean varieties should provide improved
genetic protection against most of the races and minimize risk
of yield loss. However, due to race shift and the presence of
rare but compatible races of the pathogen, virulence has been
known to increase and result in disease within eight years of
continued deployment of varieties with a narrow line of
genetic defense (Schmitthenner, 1991. Ohio Agricultural
Research and DeveIOpment Center, Woster, Ohio. Research
Bulletin No. 1187). In recognition of this risk, more enduring
non-race specific genetic protection in soybeans against P.
sojae>has become more attractive particularly when deployed as
part of an IPM program. In some states, growers have their
popular soybean lines screened for field tolerance to virulent
races of P. sojae that are common in their state or growing
areas. In light of the results obtained in this study,

Michigan farmers may benefit from similar program if

61
implemented in the state.

A number of methods to screen soybeans for tolerance to
phytophthora rot have been reported in the literature. The
inoculum-layer method (Walker and Schmitthenner, 1984) allows
plants to be screened quickly (14-28 days) in controlled
environment, and allows the control of race composition. The
slant-board test (Olah and Schmitthenner, 1985) allows the
measurement of tolerance relatively quickly in a controlled
environment. It also allows for the screened plants to be
rescued and regenerated where necessary. Either of these two
methods could be useful in screening soybeans grown in
Michigan for tolerance to the races of P. sojae that are found
in the state. However these methods allow inoculations with a
single race of P.sojae’per treatment and.may be inefficient as
they require much space, time and test material as was
observed in the course of this study.

The objective of screening soybean varieties for field
tolerance, is to provide information to growers on the
tolerance of their selected soybean lines to the races of P.
sojae that occur in their growing areas. Since P. sojae may
not occur in pure race forms in the field, a screening method
that uses a cocktail of inoculum of P. sojae races instead of
a single race may be more beneficial as it would require less

space, time and material; less plant material and time would

62

be required to test soybeans against a number of P.sojae
races. It may be possible to modify the inoculum-layer
technique such that a slurry of agar containing a cocktail of
inoculum is used instead of a culture of a single race of
P. sojae

Virulent races of P. sojae have been identified in'
Michigan and, show the potential to impact on yield in soybean
production as evidenced by an approximate 34% yield reduction
in a field near St. Charles in Saginaw county. Incorporating
RPS genes 1b, LU 3a,.3w and 6 in soybean varieties with good
field tolerance in conjunction with other control measures
should offer more improved protection for PRR. The information
obtained from this study will enable growers rand plant
breeders to better identify and deploy non-race-specific
genetic resistance as part of an IPM program in the protection

of soybeans from Phytophthora root and stem rot in the state.

63

Literature cited

Canaday, C.H. and A.F. Schmittenner. 1982. Isolating
Phytophthora megasperma f. sp. glycinea from soil
with a baiting method that minimizes Pythium
contamination. Soil Biology and Biochemistry 14:67-
68.

Haas, J.H. and Buzzell, R.I. 1976. New races 5 and 6 of
Phyotophthora megasperma var. sojae and different
reactions of soybean cultivars for races 1 to 6.
Phytopathology 66:1361-1362.

Klein, H.H. 1959. Etiology of the Phytohthora disease of
soybeans. Phytopathology 49:380-383.

Lockwod, J.L. and Chen, 8.0. 1978. Race determination of
Phytophthora megasperma var. sojae, using
differential soybean varieties inoculated with
zoospores or incubated on flooded soil samples.
Plant Disease. 62:1687-1690.

Margulis, L., J.O. Corliss, M. Melkonian ,and D.J. Chapman.
1989. Handbook of Protoctista. Jones and Barlett,
Boston.

Mussell, H. 1980. Tolerance to disease. In: J.G. Horsfall and
E.B Cowling(eds.), Plant Disease: An Advance
Treatise, Vol. V, Academic Press, New York.

NewhooK F.J., Waterhouse, G.M. and Stamps, D.J. 1978. Tabular
key to the species of Phytophthora. Mycological
paper 143. Commonwealth Mycological Institute, Kew,
Surrey, England. 20 PP.

Olah, A.F. and Schmittenner, A.F. 1985. Glyceollin
accumulation in soybean lines tolerant to
Phytophthora megasperma f. sp. glycinea.
Phytopathology 75: 542-546

64

Patterson, D.J., and M.L. Sogin. 1992. Eukaryotic Origins and
Protistan Diversity. Pp. 13-46. In: The Origin and
Evolution of the Cell. Eds. H. Harman and K.
Matsuno. World Scientific, Singapore.

Ploper, L.D. K.L. Athow, and F.A. Laviolette. 1985. A new
allele at the Rps3 locus for resistance to
Phytophthora megasperma f. sp. glycinea in soybean.
Phytopathology 75: 690-694.

Waterhouse, G.M. 1963. Key to the species of Phytophthora de
Bary. Mycological papers No. 92. Commonwealth
Mycological Institute, Kew, Surrey, England. 22 PP.

Chapter 3

Effects of the isoflavonoid genistein on the infection of
soybean seedlings by zoospores of Phytophthora sojae, and the

fluorescence of root exudates and field tolerance in soybeans.

Abstract

Compounds exuded by roots of plants have been shown to be
important in plant-microbe interactions. In the case of plant
pathogens, detection of specific plant molecules may' be
critical in the recognition and subsequent infection of the
potential host , or the suppression of pathogen populations.
In this study, the effects of low concentrations of the
genistein on the ability of zoospores of P. sojae to infect
seedlings of soybeans was investigated. Root exudates of
soybeans of various field tolerance levels were also
characterized for exuded levels of genistein.

One hundred milliliters of a 5 ppm genistein solution was
added to half liter Styrofoam cups containing 500 g of wetted
soil and two-day (days after emergence) old soybean seedlings.
Plants were placed in the growth chamber at ZOKL 70% relative
humidity and 14 hours of light. Two weeks after planting,
plants were evaluated for disease severity levels.

Diphenylboricacid (DPBA) was added to the samples of root

65

66

exudates and directly subjected to fluorometric analysis.

Significant differences (Ps0.05) in field tolerance ratings,
which varied with varieties, were observed between treatments
(with and without genistein). Thererwas no correlation between
fluorescence of root exudates and the tolerance values of
soybeans. These results suggest that genistein, when applied
exogenously, does have an effect on the infection of soybeans
by zoospores but the significance of exuded genistein in
Phytophthora root rot is not clear. Differential reduction in
root rot among soybean varieties can be attributed to
differential interaction between individual isolates of 1K
sojae and soybean varieties, and possible differential impact

of genistein on the zoospores of isolates.

Introduction;

The exchange of molecular signals represents the earliest
step in plant-microbe interaction (Bauer, W.D. and G. Caetano-
Anolles.1990). In a complex environment such as in the soil,
the detection of specific plant molecules by microbes may be
critical to recognition and subsequent colonization of the
potential host. In Bradyrhizobiwn and Rhizobium species of
bacteria, expression of nodulation (nod) genes is induced by
flavonoids or isoflavonoids specific to the particular hosts
(Banfalvic et al. 1980; Verma, D.P.S. 1992). The induction of
the nod genes leads to production of a lipo-polysaccharide (by

the bacterium) which initiates formation of the nodule

67

structure by the plant (Lerouge et al. 1990; Verma D.P.S. 1992
). The virulence (vir) genes of Agrobacterium tumefaciens,
which mediate the transfer of DNA to the cells of the plant
symbiont, are specifically induced by phenolic compounds such
as acetosyringnone which are released from a wounded plant
tissue (Bauer, ill). and Caetano-Anollesl990; Zambryski, P.
1988). The response of AgrobaCterium and Rhizobium species to
plant signals also include chemotaxis in which the bacteria
swim to towards potential colonization sites (Bauer, W.D. and
G. Caetano-Anolles. 1990). It has also been suggested that the
isoflavonoids formononetin (7-hydroxy,4'-methoxy isoflavone)
and biochanin A (5,7-dihydroxy, 4'methoxy isoflavone) may act
as signal molecules in vesicular arbuscular mycorrhiza
symbiosis. (Nair et al,1991).

The zoospores of plant pathogenic Oomycetes also exhibit
chemotaxis in response to certain plant compounds (Carlile,
M.J. 1983; Horio et al. 1992; Morris, P.F. and E.W.B. Ward.
1992; Sekizaki, H., and R. Yokosawa. 1988; Sekizaki, H., R.
Yokasawa, C. Chinen, H. Adachi, and Y. Yomane. 1993).
Zoospores, motile unicellular structures that are generally
released under flooded conditions and nutrient deprivation,
form the predominant means by which pathogenic Oomycetes
spread throughout the soil and infect plants (Carlile, M.J.
1983). Zoospores of Oomycetes achieve chemotaxis by the same
strategy as bacteria ; they swim steadily by means of flagella

propulsion in the presence of an attractant, but turn more

68

frequently in the presence of repellent compound (Carlile,
M.J. 1983). Zoospores of most Phytophthora species are
attracted to a variety bf sugars and amino acids, particularly
aspartate, glutamate, arginine and methionine (Carlile, M.J.
1983). Several oomycetes are attracted tx> specific plant
signals. Isovaleraldehide, valeraldehide and anti-
isovareldehide attract zoospores of Phytophthora palmivora at
concentrations as low as 1 pM (Cameron, J.N., and M.J.
Carlile. 1981; Carlile, M.J. 1983). Prunetin (4',5-aldehyde-7-
methoxyisoflavone) and related compounds are potent
attractants (at concentrations as low as 10 nM) of.Aphanomyces
enteiches zoospores (Sezaki, H., and EL. Yokosawa. 1988;
Sezaki, H., R. Yokasawa, C. Chinen, H. Adachi, and Y. Yomane.
1993), and the zoospores of Aphanomyces cochoides are
attracted to cohliophilin A [5-hydroxy-6,7-(methylenedioxy)
flavone] from the roots of its host, the spinach plant at 1 nM
(Horio et al. 1992).

The zoospores of the soybean pathogen Phytophthora sojae
(syn. P. megasperma f.sp. Glycinea) are attracted to the
isoflavone genistein (4',5,7-trihydroxy isoflavone), which is
present in soybean seeds, and is exuded by the roots of the
plant (Morris, P.F. and E.W.B. Ward. 1992). This compound
attracted zoospores of P. sojae and one spp. of Pythium but
those of six other species of Phytophthora 'were not attracted
with concentrations as high as 30 uM ( Morris, P.F. and E.W.B.

Ward. 1992). Apart from chemotaxis, daidzein and genistein

69

also cause rapid encystment and germination of zoospores of P.
sojae. Therefore, Morris and Ward (Morris, P.F. and E.W.B.
Ward. 1992) suggested that sensitive attractions of P. sojae
zoospores to soybean isoflavones may be part of the mechanism
which determine host range. Wacker and Safir (unpublished)
found that genistein at concentrations as low as Eifxxn in
plant growth solution can reduce infection of soybean
seedlings by zoospores of P. sojae. At 10 ug/ml, genistein
inhibited radial (hyphal) growth and reduced asexual
reproduction of P. sojae in culture (Vedenyapina et al. 1996).
Thus, it; is possible that field tolerance (resistance) of
soybeans to P. sojae may be controlled to a large extent by
root isoflavonoid exudation characteristics or the properties
of certain specific isoflavonoids within the roots.

Currently, little information exists (N1 the mechanism
behind the effects of isoflavonoids on microbial activity but
environmental factors are believed to have a significant role
(Zhang anui Donald, 11996). Effects CH? genistein 1J1 plant-
microbe interactions vary with specific organism. Sub-optimal
root zone temperature (RZT) ranging from 13 to 17%: delays
infection and early nodule development in R. japonicum, and
addition of genistein overcomes some of these effects (Zhang
and Donald, 1996). Also, soybeans germinated and maintained at
sub-optimal RTZs have lower root genistein concentrations than
those germinated and maintained an: RZTs above sub-optimal

(Zhang enmi Donald, 1996). Therefore, it; is possible that

70

reduction of genistein concentrations in soybeans due to cool
field conditions result in increased infection by P. sojae.

There is indication that the phenolic 4'—and 7-hydroxyl
group on the aromatic rings of the isoflavone play a crucial
role in chemotaxis (Tyler et al. 1996). Only isoflavones with
a 4'-hydroxyl or methoxyl group attracted zoospores at
concentrations below 20 rfld while methylated flavones with
hydrophobic 13 rings acted an; repellants tx> zoospores of
P.sojae. (Tyler et al. 1996). The process of encystment in
zoospores is associated with both eflux and the uptake of
calcium (Irving et al., 1984, Iser et al., 1989). This process
results in substantial loss of CaW-reserves as zoospores were
reported t1) release Lu) to 30% CH? total cellular CR¥+ at
encystment (Irving et al., 1984). The addition of daidzein and
Ca“+ at LMNV levels (Ps0.05 mM) tx> the media (H? P. sojae
triggered transient increase of calcium in the hyphae, and
caused zoospores to encyst and germinate (Mary et al., 1999
unpublished?). Similar levels of daidzein or Ca1+ alone did
not significantly alter the fate of zoospores (Mary at al.,
1999 unpublished). The interaction between isoflavones and
Ca1+ may also account for changes in hyphal morphology as
observed by Rivera-Vargas et al. (Rivera-Vargas et al.1993),
and Vedenyapina et al. (Vedenyapina et al.1996). The two
studies reported hyphal swellings, increased branching, and
twisting of hyphea of P. sojae grown on media containing less

than 1 uM of genistein.

71

In this study, the effects of low concentration of
genistein on the ability of zoospores of P. sojae to infect
seedlings of soybeans of various field tolerance levels was
investigated. Also the fluorescence levels of root exudates of
soybeans of various field tolerance levels were studied. It is
possible that 5N: certain concentrations, genistein causes
zoospores to encyst away from soybean roots and thus reduce
the inoculum potential. The fluorescence characteristics of
root exudates may' be <directly related to <iifferences in
susceptibility' and ‘tolerance to .P. SOjaE? among soybean
varieties. This work reports what may be first information on
the effects of genistein on the infection of soybean seedlings

by zoospores of P. sojae.

Materials and.Methods

The effects of the isoflavone genistein on the infection
of soybean seedlings by zoospores of P. sojae was studied in
the first experiment . In the second experiment, fluorescence
readings (which served as indicators of the levels of exuded
genistein) of soybean root exudates were compared to field
tolerance values of respective soybean varieties. Selected
isolates of P. sojae from soybean plants were evaluated for
their ability to infect soybean seedlings in the absence and
presence of genistein 01' 5, 7-trihydroxyisoflavone). The
isolates MSU 23, lfifll 25 and DMMJ 32 are single zoospore

cultures of P. sojae obtained from Michigan soybean fields in

72

the 1996 growing season. The isolates were maintained on V8-
juice agar (200 m1 of clarified V8-juice , 2 g of CaCO3 per
liter, and 1.5 % Bacto agar) at 15 0C in the dark. Genistein,
synthesized by and obtained from Dr. M. Nair (Michigan State
University), and was tested at 0 and 5 ppm. The soybean
varieties Chapman (rps), Felix (rps), Sundusky (rps), Conrad
(rps), Repley (rps) and Colfax (rps) were obtained from
Michigan Foundation seeds (Okemos MI, 48864) and do not have
known resistance (Rps) genes to P. sojae. Varieties Williams
82 (Rps 1,), Harosoy (Rps 7), PI 103 (Rps 1,) Pella (rps),
Harlon (Rps 1,), and Sloan (rps) were provided by A. F.
Schimitthenner (Ohio Agricultural Research and Cmvelopment
Center, Wooster,).

Experiments were repeated 3 times.

Influence of genistein on the infection of soybean seedlings
by zoospores of P.sojae

To induce zoospore production in the P. sojae , ten
mycilial plugs (7 mm in diameter) were transferred from the
edges of actively growing cultures to sterile petri dishes
and flooded with 20% clarified V8-juice agar. After 48 hours
of incubation, at room temperature, the broth was removed and
plugs were rinsed 5 times with distilled de-ionized H53 and
re-flooded with 10 ml of ARS (2.94 g CaC12.2H20, 2.47
MgSO4.7HgO, and 0.75 g KCl in 1000 ml of distilled de-ionized
water ) salt at Ph 6.5. Cultures were further incubated at

room temperature and zoosporangia formed in 12 hrs.

73

To accelerate and synchronize the release of zoospores by
sporangia, cultures were incubated at 5%: for 30 min.
Zoospores were released when cultures were returned to room
temperature (24°C). One milliliter aliquots of each zoospore
suspension was transferred into micropreparation wells, and 2
drops of 0.1% trypan blue solution :hi lactoglyceral were
added to each well. The number of zoospores in the wells were
counted under a light microscope at 250x. This was repeated
10X for each isolate. Final inoculum concentrations were
prepared by adjusting the number of zoospores in each culture
to 3 x103 /ml.

Soybean seeds were surface-sterilized with 10% bleach
for 10 min. and rinsed 3 to 4 times with distilled de-ionized
water. The seeds were germinated in germination paper.
Genistein solutions were prepared by dissolving 3 mg of the
isoflavone in 3ml of methanol and added to 997 ml of
distilled de-ionized water to bring the volume to 1000 ml.

Two-day old seedlings were transplanted to B liter pots
containing 5009 wetted soil. 50 ml of the zoospore suspension
were added to the pots and followed by 100 ml the of
genistein solution. The seedlings were placed in the growth
chamber at 20°C, 70 % relative humidity and 14 hours of
light. Plants were watered daily with 50 ml of distilled de-
ionized water. Two weeks after inoculation, data on tolerance
to disease levels (1 = no root rot; 2 = trace of root rot; 3

== bottom third of root mass rotted; 4 = bottom 2/3 of root,

74
mass rotted; 5 = all roots rotted, 10% of seedling kill,
slight stunting of tops of plants; 6 = 50% seedling kill,
moderate stunting of tops of plants; 7 = 75% seedling kill,
severe stunting of tops of plants; 8 = 90% seedling kill; 9 =
all seedlings dead; 10 = all seedlings killed before

emergence) were collected and the dry weights of roots were

determined.

Collection of root exudates

Root exudates of soybean varieties were collected using
the method of T.L. Graham (1990). Surface-sterilized soybean
seeds were germinated and grown in specifically designed
growth chambers, which allowed the seed to imbibe slowly and
the roots to be suspended in air at 100 % RH. Black
polystyrine plant growing trays with wells measuring 2.0 X 2.5
cm (A.H. Hermmert Seed Co., St. Louis, MO) were cut to form
grids of 5 X 6 well rectangles. Trays were sterilized by
soaking' in 70% (ethanol for 20 - 25 min prior' to use.
Individual soybean seeds were loosely wrapped in moistened
sterile germination paper and placed in the individual wells
in trays. The seeds were oriented such that their radicles
would extend out through the drainage holes at the bottom of
the wells. Trays were then suspended in sterilized closed
polypropylene containers (19 cm square X 21 cm deep) lined
\Nith absorbent tissue thoroughly soaked in sterile water, and

:incubated at room temperature for 24 hrs. Pre-washed sterile

75

water-soaked cotton (2 mm diameter) wicks were placed at the
lower sections of actively growing roots suspended in the
growth chamber. After 30 min, wicks from 30 replicate seeds
were collected and centrifuged in modified centrifuge tubes
that allowed the collection of exudate at a lower chamber and
retention of wicks in an upper one. Samples of root exudates
were subjected directly to fluorometric analysis in the
presence of DPBA (diphenylboricacid) using a Sequoia Turner
(model 450) fluorometer. Fluorometer filters were set at 309
nm (excitation) and 520 nm (emission) for maximum.detection of
genistein. Calibration was achieved with standard genistein
(WR Scientific, St. Louis, MO) dissolved in methanol in the
presence of DPBA.

A correlation analysis of the fluorescence levels of root
exudates and the field tolerance values of soybean varieties

was performed using the statistical package Minitab.

Results

Influence of genistein on the infection of soybean seedlings
by zoospores of P. sojae

Genistein significantly increased the percentages of dry
weights of roots of most of the soybean varieties across the
isolates (Table 3.1). Conrad had the highest overall increase
in root mass (63.0%) followed by Colfax at 56.4 %. Felix had
the lowest increase of 29.2 %. The isolate M8025 yielded the
highest increase (83.7%) in combined root mass of soybean

varieties. Isolates M8023 and M8032 yielded lower increases of

76

Table 3.1. Percent ("/o) increase in root mass (dry weights) of soybean seedling roots in
the presence of genistein.

* Total increase in dry weights of all soybean varieties subject to one isolate in the
presence of genistein.

@ Total increase in dry weights of individual soybean varieties subject to all three
isolates of P. sojae in the presence of genistein.

77

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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~ @moua_o£ =< Nmsz 3sz MNDm—z

 

 

78

Figure 3.1. Dry weights of roots in the presence and absence of genistein. Roots of
soybean seedlings were clipped off and dried at 80 ° C for 24 hrs before weighing.
Significant(Ps0.05) increases in the root mass of soybean seedlings were
recorded in the presence of genistein.

79

0.7 " ” ” c i W" ’ ' ’ " "’ ii” :1.

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99° 09“ 99° ‘<°\ ‘3} 4°“ g?" 605‘" 60‘3“ 08" 08" 008 00*“ 00% Dow”

Treatments

80

Figure 3.2. Effect of genistein on field tolerance levels in soybean seedlings. Significant
(Ps0.05)increase in tolerance in the presence of genistein was observed.
Tolerance evaluation was based on the extend of lesions and amount of rotted root
tissue.

Field tolerance values

 

10 -

81

 

   

 

 

 

 

f3 Chapman
I Felix
D Sundusky

 

D Conrad
I Colfax

 

 

 

 

 

 

 

 

MSU23 (-G)

 

 

 

 

 

 

 

 

MSU 23 (+6)

 

 

 

 

 

 

MSU 25 (-G) M80 25 (+G)
Treatments

 

 

Msuazoo)

 

 

 

 

MSU 32 (+6)

 

82

Figure 3.3. Fluorescence of root exudates and field tolerance values of soybeans.
Soybean plants were inoculated with zoospores (3000/ml) of P. sojae isolates.
Fluorescence values were obtained by subjecting root exudates to fluorometric
analysis in the presence of DPBA using a Sequoia Turner fluorometer.

Field tolerance values

83

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1° 120° + mom
9 i +Msu23
\ W —‘— M8025
8 f“ ‘ +MSU32
7 \ A ' +Race 25
~- 800 igmmscenc
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/4 / "
5 11 v
4 V” \<\L E
3 h, /‘.‘ l l 4°°
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2 - 1 200
1
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Resnik Williams Repley Harosoy PI103 Conrad Pella Harlon Slaon
82

 

84
41.1 % and 29.5 % respectively. Colfax had the highest
increase of 136.4 % against M8025 while Felix had the lowest
root mass increase of 2L2? % against M8023. Increases for
individual varieties and isolates were variable, with low
tolerance lines yielding higher increases in dry weights of
roots.

Significant differences (P $0.05) in field tolerance
values were observed between treatments (+G-P, -G+P and +6-
P) iji all soybean ‘varieties across isolates <of ll sojae
(Figure 3.1). Genistein. had time greatest impact (M1 the
isolate M8023 which resulted in increased tolerance by 3.3
points, followed by M8025 at 2.5 points. Variety Conrad had
the highest (3.7 points) overall (across isolates) increase in
tolerance to P. sojae (Table 3.1). The greatest single
increase was with M8025 (4.5points) and the lowest was 2.9
with M8032. Chapman had the lowest overall tolerance increase
of lufl points, but .achieved the highest single isolate

increase in tolerance of 2.6 points against M8023.

Fluorescence of root exudates and field tolerance levels of
soybeans

A negative correlation between fluorescence levels of
root exudates and field tolerance was observed for all the
soybean varieties in the study (Figure 3.3). Resnik , Williams
and Pl 103 which had high tolerance levels, showed high

exudate fluorescence. Conrad, Pella . Harlon and Sloan, which

85
had low or no tolerance to the P. sojae isolates, gave low
fluorescence ‘values. Varieties Repley' and. Harosoy luui no
tolerance to the isolates M8010 and M8032. Resnik , Williams
and PI103 demonstrated high tolerance across isolates while
other varieties gave highly variable results. These results

varied with variety—isolate interactions.

Discussion

The results presented in this study represent what may be
the first report on the reduction of P. sojae zoospore
infection of soybean seedlings by the isoflavonoid genistein.
Decreased infection of roots of soybean seedlings (recorded as
increased dry weights of roots and lower disease ratings)
resulted from exogenous application of genistein (5 ppm) in
gxfl: cultures (1f soybeans inoculated vfljji zoospores (1x103
cfu/ml) of P. sojae. Genistein has previously been shown to
attract zoospores of P.sojae (which swim against increasing
concentration) and hasten their encystment and germination
(Morris and Ward.1992), and may be important in the soybean-P.
sojae interaction. Vedenyapina et al (1996) reported reduced
hyphal growth enui asexual reproduction kn/ genistein at IUD
Mg/ml. fflua results <5f experiments VH3 conducted 1I1 growth
chambers show that at 5ppm, genistein can reduce the infection
of soybean seedlings by zoospores of P. sojae, and
significantly increase the chqx weights CH? roots (P20.05)

Although disease rating values (obtained through visual

86
examination of plant tissue) make the data rather subjective,
they provide reasonable estimates on disease levels. Field
tolerance values of 4.0 and below are considered good by
breeders and growers (Schmitthenner, personal communication)
and genistein did reduce disease to this level in all
varieties that were tested.

Although there appeared to 1x3 a. general inverse
correlation between fluorescence of root exudates and field
tolerance values of soybean varieties, high variability among
soybean varieties—isolate interactions did run: enable
conclusive observation (Ml the role (Hf exuded genistein in
field tolerance. However, differential reduction in root rot
among soybean varieties (INI be attributed ix) differential
interaction between individual isolates of P.sojaezand soybean
varieties, and possible differential impact of genistein on
the zoospores of isolates. Vedenyapina et al (1996) observed
strong intraspecific variation Lu) P. sojae in response to
genistein at concentrations as low as 0.01 to 1 ug/ml. This
adaptive variation to genistein by zoospores may have a role
in field tolerance. It is possible that aggressive isolates of
P. sojaewmay have evolved a fitness trait to concentrations of
genistein that limit the development of unadapted individuals
(Vedenyapina.et al). The possible trait would enable infective
structures to maintain their zoosporic form and swim long
enough to come in contact with plant roots. Differential

interaction between soybean varieties and P. sojae may

87

explain the large variability in disease levels on Harosoy,
Conrad and Pella. These varieties were also defeated by a
large number of field isolates of P.sojae in pathogenicity
tests as reported elsewhere in this study. Resnik, Williams,
and P1103 displayed high tolerance and fluorescence of root
exudates. These varieties were also resistant to most of the
field isolates of P. sojae (see page 42). Results obtained
from this study suggest that the composition of soybean root
exudates may have an impact on Phytophthora root and stem rot
of soybean.

Among the reported effects of genistein on zoospores are
chemotaxis (zoospores swim against increasing concentration)
and rapid encystment and germination of cystospores
(Vedanyapina et al, 1996; Tyler et al, 1996; Irving et al,
1984, and Iser et al., 1989). By swimming in the direction of
increasing isoflavanoid.concentration, zoosporesanxaguided to
the roots of .actively growing seedlings where they' will
aggregate, encyst and germinate. Germ tubes form appresoria
and penetrate the roots at appropriate sites. It is possible
that where sufficient amounts of genistein are exuded and well
dispersed, most zoospores are induced to encyst and germinate
away from roots and thus result in the reduction of inoculum
potential. It is also probable that once germinated, zoospores
CH? P. sojae which are snmflj. amd delicate structures, may
immediately exhaust food reserves and fail to develop in the

absence of a host, and thus further limit inoculum potential.

88

Results obtained from. this study' showed. that the
isoflavonoid genistein does affect the potential of P.sojae to
infect the roots of soybean seedlings. Thus, genistein may
play Efll important role 1J1 the field tolerance (n? soybean
varieties to the pathogen. Factors responsible for
differential effects of genistein on different isolates of P.
sojae are not clear. Vedenyapina et al (1996) reported
variable effects of genistein on zoospores of different races
of P. sojae although genistein used in the study came from one
source. This observation tend to place the source of variation
in intrinsic qualities of the zoospores. In this study,
however, different isolates of P.sojae yielded variable field
tolerance values for each of the soybean varieties indicating
a possible second factor which resides in the host.

It is INN: known whether or run: differences 1J1 field
tolerance of soybean to P. sojae is due only to some intrinsic
qualities of zoospores. More information is needed on the role
of genistein in the variable field tolerance of soybean to P.
sojae. Understanding the Ixflfii of exuded genistein 1J1 this
variability will enhance the effort by breeders and growers to
identify more tolerant soybean varieties as the crop continues

to grow in importance to the nation’s agriculture and economy.

89

Literature cited

Banfalvi, Z., A. Nieuwkoop, M. Schell. L. Besl, and G.
8tacy.l988. Regulation of nod gene expression in

Bradyrhizobium japonicum. Mol. Gen. Genet. 214:420-
424.

Bauer, W.D., and G. Caetano-Anolles. 1990. Chemotaxis,
induced gene expression and competitiveness in
rhizosphere. Plant Soil 129:45-52.

Brett, M.. Tyler, Ming-hoi Wu, Jo-man Wang, Winnie Cheung,
and Paul F. Morris. 1996. Chemotactic Preferance and
Strain Variation in the Response of Phytophthora sojae
Zoospores toHost Isoflavones. Applied and
Environmental Microbiology,Vol.62, No. 8,p.281l-28l7.

Cameron, J.N., and M.J. Carlile. 1981. Binding of
isovaleraldehyde, an attractant of zoospores of the
fungus Phytophthora palmivora. Journal of Cellular
8cience.49:273-281.

Carlile, M.J. 1983. Motility, taxis, and tropism in
Phytophthora,p. 95-107. In D.C. Erwin, 8.Bartnicki-
Garsia, and P.H. Tsao(ed.), Phytophthora: its biology,
taxonomy,ecology and pathology. American
Phytopathological society, St. Paul, Minn.

Feng, Z. and D.L. Smith. 1996. Genisten accumulation in
soybean(Glycine max [L] Merr.) root systems under
suboptimal root zone temperatures. Experimental
Botany,Vol. 47, No.299,pp. 785-792.

Horio, T., Y. Kawabata, T. Takayama, Y. Fukushi, H.
Nishimura, and J. Mizutani. 1992. A potent attractant
of zoospores of Aphanomyces Cochlioides isolated from
its host Spinacia oleracea. Experimentia 48: 410-414.

Lerouge, P., P. Roche, C. Faucher, F. Maillet, G. Truchet,
J.C. Prome, and J. Denarie . 1990.8ymbiotic host-
specificity of Rhizobium meliloti is determined by
sulphated and acylated glucosamine oligosaccharide
signal. Nature (London) 344:781-784.

90

Morris, P.F., and E.W.B Ward. 1992. Chemoattraction of
zoospores of the soybean pathogen, P.sojae, by
isoflavones. Physiol. Mol. Plant Pathol. 40:17-22.

Nair, M.G., G. R. Safir, and J. O. Siquera. 1991. Isolation
and Identification of vesicular-arbuscul mycorrhiza
stimulatory compounds from clover (trifolium repens)
roots. Appl. Environ. Microbiol. 57:434-439.

Sekizaki, H., R. Yokosawa. 1988. Studies on zoospore-
attracting activity. I. Synthesis of isoflavones and
their attracting activity to Aphanomyces enteiches
zoospore. Chem.Pharm.Bull. 36:4876—4880.

Sekizaki, H., R. Yokosawa, C. Chinen, H. Adachi, and Y.
Yamane.1993. Studies on zoospore-attracting activity.
II.Synthesis of isoflavones and their attracting

activity to Aphanomyces enteiches zoospore. Biol.
Pharm. Bull. 16:698—701.

Vedenyapina, E.G., Gene R. Safir, Brendan A. Nieemira, and
Thomas E. Chase. 1996. Low concentrations of the
isoflavone Genistein influence in vitro Asexual
Reproduction and Growth of Phytophthora sojae.
Phytopathology 86:144-148.

Verma, D.P.S. 1992. Molecular signals in plant-microbe
communications. CRC Press, Boca Raton, Florida, USA.

Zambryski, P. 1988. Basic processes underlying
Agrobacterium-mediated DNA transfer to plant cells.
Ann.Rev. Genet. 22:1-30.

Chapter 4

Survey of P. sojae presence in soybeans infested with the
soybean cyst nematode.

Abstract

Phytophthora sojae (Kaufmann and Gerdemann) is a serious
but opportunistic pathogen of soybeans. P. sojae is known to
attack soybeans mostly when under stress from other
environmental factors such as cool and wet field conditions,
which put stress on plants but create favorable conditions for
the release enui dissemination cm? zoospores. Thus, it: is
possible that nematode feeding may augment the infection of
soybeans tn! P. sojae when both organisms are pmesent and
conditions are favorable. To examine the possible impact of
nematode activity on the infection of soybeans by P.sojae,
soybean plant samples were collected from a field trial study
of soybean varieties in a field naturally infested with H.
glycines. Soybean samples were scored for the presence of P.
sojae and mean values were compared to the average number of
nematode cysts in 100 (x: of soil from the rhizosphere of
soybean varieties. In the non—fumigated plots, significant
(Ps0.05) correlation between nematode cyst numbers and the
presence of Ehsojae were observed. In the fumigated plots,

lower cyst numbers and P.sojae scores were observed but there

91

92

was no correlation between the two organisms. These results
suggest. that H1 glycines may augment the infection of
soybeans by P.sojae: where the soybean crop is susceptible and

soil conditions support both organisms.

Introduction

Of the species of nematodes known to parasitize soybeans,
Soybean cyst nematode (SCN), Heterodera glucines Ichinoe, is
a major pest of soybean (glycine max) in the north central
United states. Illinois, Indiana, Iowa, Kansas, Michigan,
Minnesota, Missouri, Nebraska, Ohio, and Wisconsin have all
reported H. glycines infestation (Niblack, 1993). This
nematode is the major limiting factor in soybean production in
the north central region of the United States (W.G. Bird and
F.W. Warner, 1990). SCN probably had a long association with
soybean in Asia; it was first reported in Heilongjiang
province of China in 1938 (Nakata and Asuyana, 1938). The
presence of the nematode was documented in 1936 from Korea
(Yokoo, 1936), in 1958 from Taiwan (Hang, 1958), and in 1984
from the island of Java in Indonesia (Nishizawa, 1984).

In North America, If glycines occurs in tflma USA and
Canada. In the US the nematode was first reported in 1954 in
Hanover county in North Carolina and has since spread to most

soybean producing states ( Winstead , Skotland and Sasser,

93

1955; Brewer, 1981; and Mulrooney, 1988). In Canada, SCN was
first detected in Kent county in Ontario in 1957 (Anderson et
al., 1988). The nematode was first detected in Michigan in
1987 in Gratiot county (W.G. Bird and P.W. Warner, 1988), and
has since been reported in 25 counties in the state (G.W. Bird
and F. Warner; personal communication).

Temperature, soil water, and soil texture are the most
important physical factors that affect the ckwelopment of
nematodes. Juveniles do not develop beyond the second stage in
soybean roots grown at constant temperature of 10‘K2in water
baths in a greenhouse, and adult females do not develop at 35
3C (Ross, 1964). The calculated basal temperature threshold
is 5 0C and the thermal optimum for embriogenesis and hatch
with low mortality is 24 OC (Andeson et al., 1988).
Development within the egg stops at the first juvenile stage
at 15 to 30 OC. Hatch occurs at 20 to 30 0C but at 36 0C the
egg dies. However, soil temperature averages in excess of 34
0C during the month of July in Georgia did not lower juvenile
population as expected (Hussey and Boerma, 1983). Hatch of
juveniles declined in November (Ross, 1963) and this decline
could be attributed to induction of dormancy by decreasing
temperature (Hill and Schmitt, 1989).

For movement in the soil, nematodes require a film of

water in the pore space around soil particles (Wallace, 1964).

94

In 51 study by Heatherly et EH. (1982), the distribution of
cysts increased significantly at water potentials between -30
and - 40 kPa in sandy loam soil (49% sand, 42% silt, and 9%
clay). Baker et al (Baker and Koenning, 1989) reported that
at the middle of the growing season, soil water levels did not
affect the number of H. glycines, but late in the season low
soil water favored reproduction of the nematode. In the same
study, yield was suppressed by approximately the same
percentage in wet and dry treatments. These results suggest
that SCN damage cannot be overcome by irrigation, although
yield may be higher in infested fields with irrigation than in
non-infested. fields without irrigation“ Soil water and
nematode infestation effects on yield were approximately equal
in a study by Young and Heatherly (1988).

Soil particle size is a major determinant of pore size ,
which governs the ease of nematode movement through the soil.
SCN apparently does not maintain populations in fine—textured
soil such as sharkey clay (Heatherly and Young, 1991). SCN
number increased 60 days after planting in Dubbs containing
silt loam soil maintained at - 30 kPa but declined in Sharkey
clay soil kept at the same soil water levels (Heatherly and
Young, 1991). Soil texture may also influence damage potential
of SCN on soybean. Koennimg et al (Koenning et al.,1988),

reported that when sand content of soil is greater than 70% in

95
SCN-infested fields in Missouri, large differences in soybean
yield are associated with small changes in sand content of
soil. As sand content of soil decreased, differences in yield
between resistant and susceptible cultivars narrowed.

Effects of tillage on SCN have been inconsistent. However
population levels are lower and yields are higher in soils
that receive little disturbance (no-till) compared to soil
receiving conventional tillage practices (Tayler' et al.,
1987). Ihi a study tux Young (1987), more EKHJ (three-fold)
females were extracted (30 days from planting) from disturbed
soil. earlier tflmni front undisturbed smfld. cores (10 - cm-
diameter by 15cm- deep). Disturbance of soil appears to have
at least a short-term effect on SCN population dynamics and
yield , but the factors responsible are not known.

Most soybean disease complexes that involve SCN have a
soil-inhabiting fungus an; the other component. Significant
interactions which augment both fungal and nematode
populations in soybean roots occur between Calonectria
crotalaria Bell and Eflfli under' greenhouse conditions
(Overstreet and McGawley, 1988, 1990). Soybean plants growing
under‘ field conditions anmi parasitized in! SCN exhibited
augmented Fusarium wilt (Ross, 1965). Soybeans infested by SCN
in the presence of F. oxysporum or F. solani have exhibited

severe wilting (Roy et al., 1989). Race 3 of SCN and race 1 of

96

P. sojae increased seedling disease of soybean, although only
additively whereas interaction between Macrophomina phaseolina
and SCN has had variable results (Adeneji et al., 1975). In
one study, SCN augmented M2.,phaseolina activity (Todd et al.,
1987). Yet in another study (FranceI and wyllie, 1988), no
interaction between the two pathogens was evident. When
pathogenic and endomycorrhizal fungi associated with soybean
roots were surveyed, no consistent relationship was found
(Schenck and Kinloch , 1974).

Soybean genotype is an important factor controlling the
amount of disease caused by SCN. Because initial infection of
soybean plants seems ix) be more important than subsequent
infections, practices that reduce the initial SCN population
densities should 1x3 effective (Wrather enui Anand, 1988).
Resistant cultivars are effective in reducing SCN population
levels and increasing yield (Hartwig, 1981). However the value
of resistance is limited by selection of new races of the
nematode when the cultivars are planted frequently. Crop
rotation and application of nematicides have the same effects
except that ‘these pmactices do run; increase selection
pressure on nematode population for genotypes which can
reproduce on the resistant cultivars (Edward et al., 1988).

Nematicide application on soybean has been diminishing in

recent years because of toxicological, environmental, and cost

97

factors (Johnson and Feldmesser,1987; Kinloch, 1979; and Riggs
and Wrather, 1992).. Limitation of nematicide use in soybean
production has also been linked to the value of the crop. From
1973 until the early 19805, when soybean prices were
conducive, use of nematicides in the southern United States
was common. The combinatitmi of product removal and low prices
of soybean has resulted in the rapid decline of this
management tactic (Riggs and Wrather, 1992).

Biological control, the use of natural enemies to manage
the population levels and minimize damage by nematodes, has
been investigated (Carris and Glawe, 1989; Kerry, 1984;
Morgan-Jones and Rodriguez-Kabana, 1987; Tribe, 1977, 1980).
This management tactic has not achieved commercial application
in the control of SCN because most of the organism studied
have either exhibited limited success in trials or are
impractical (Cook, 1983). About 150 species of fungi have been
isolated from eight species of cyst nematodes; about 60% of
these are from SCN (Carris and Glawel989; Epps and Golden,
1967). However, most of the fungi have not been tested for
parasitism and efficacy as biocontrol agents on the nematode.
Also, some of the fungi are obligate parasites and cannot be
cultured on artificial media (Riggs and Wrather, 1992). Field
application of fungal biological control agents has further

been limited by time lack of suitable carrier material and

98
application, methods (Backman and Rodriguez Kabana, 1987;
Conway, 1986, Pereira and Roberts, 1990; and Walker and
Connick, 1983).

The bacterial parasite, Pasteuria penetrans (Sayre and
Starr), continues to receive attention because of its
effectiveness, resistancetxJadverseeenvironmental conditions,
and host specificity. 71 new strain cdf 1% penetrans ihas
recently been discovered in Korea, the U. S.A. (Riggs and
Wrather, l992)and Japan (Overstreet and Mcgawley, 1990), but
its obligatory status limits its usefulness. Application of
individual biological control agents has been further limited
by the interactive competition with other soil microorganisms.
However, combinationscflfmultiple agentscnithe improvement in
formulations, have considerable potential for biological
control of SCN (Tribe,1980).

Following the recent identification of highly virulent
isolates of P. sojae in lMichigan , plant samples were
collected from a field trial study of soybean varieties in a
field naturally infested with H. glycines. The objective of
the survey was to examine possible effects of SCN on P. sojae
infection of soybean varieties. P. sojae, the causal agent of
root and stem rot in soybeans, is an opportunistic pathogen as
it attacks soybeans mostly when under stress from other

environmental factors (Moots et al, 1988 and Kittle et al,

99
1979). Thus, it is possible that stress from nematode feeding
augments P. sojae infection of soybeans where both organisms
are present and conditions are favorable. It is hoped that
information from this survey will be useful in the development
and implementation of an inclusive IPM program for low input

production systems.

Materials and methods

Soybean plant samples were collected from a field trial
project of soybean varieties in a field naturally infested
win SCN in Saginaw county. The experimental set-up was a
random complete block design with 12 soybean varieties
(Anderson, NC250, Asgrow seed A2722, Callahan 6180, Ciba seeds
3311, Dekalb CD< 252, Great lake CH; 2415, ICI seeds E260,
Mycogen J 250, Pioneer 9171, Conrad, Corsoy and Jack), two
treatments (fumigated and non-fumigated) and seven
replications. The nematicide Telone II (1,3-Dichloropropene)
was applied at 30 gal./acre. Ten plant samples of each soybean
variety were randomly collected from each replication. This
gave a total of 70 samples per entry in each of the two
treatments. All samples were kept in polyethylene bags and
stored at 4”C to maintain fungal viability as samples were
being processed.

The Canaday—Schmittenner medium (Canaday and

100

Schmittenner, 1982), as described elsewhere, was used in the
detection and identification of Phytophthora from plants.
Roots and stems of plants were surface—sterilized with 10%
bleach for ten minutes and thoroughly rinsed 3 to 4 times with
sterile distilled water . Small sections of tissue were.taken
from the edges of advancing lesions, where visible, and placed
on the rmxhimn To Hdnimize bacterial contamination, plant
tissues were placed under the medium to limit oxygen
availability. Processed samples were incubated on benches at
room temperature and observed over a period of 4 days. Fungal
contamination was minimal and Phytophthora, when present, was
readily observable at 50X under the dissecting microscope.
Data was obtained as the number of samples with Phytophthora
per soybean variety (entry). Nematode cyst numbers were
determined for 100 cc of soil from the rhizosphere of each
plant sample.

Data were analyzed for correlation using the statistical

program Minitab.

Results

In the fumigated plots, and with the exceptions of the
varieties Jack and CX 252 (data excluded), which is believed
to have escaped infestation, significant (0.05) correlation

between nematode infestation and the presence of P. sojae in

101

Figure 4.1. Number of cysts of Heterodera glycines in the rhizosphere soil volumes of
soybean varieties from a field study of un-fumigated plots (in Saginaw county)
were compared to the presence of P. sojae in the soybeans. Soybean plant tissues
were surface-sterilized and incubated in low nutrient media. After two days, plants
were scored for presence or absence of P. sojae. Significant correlation between
SCN activity and P. sojae presence was observed.

102

250 ‘ _“i,,, ” .1- '7’; _# 77 ,1
f | +Number of cysts/1000csoil ‘
i g +Number of samples/1O .
'. (,1 _, M"ifli°fii-.- _ _
i

200 j-

150

i
. _l_.i_ .
l

/

Average number of cysts/100 cc soil

50 1

0 4 - -1” i - "+- -

‘6 ‘3 Q Q C ’L 0 ’\
3'30 qutbb Org?) sf.) 000‘? P33? $07,?) $3’\

Soybean varieties

.-___+-__ All i

Average number per ten plant samples yielding P.sojae

I
I
_._ ______.__ ___. _ A U .

103

Figure 4.2. Number of nematode cysts and the presence of P. sojae in fumigated plots.
Low scores of P. sojae and cyst nematode counts were obtained from the fumigated
plots and data did not support correlation between the two pathogens.

104

12

0
1

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7

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Soybean varieties

105

soybean varieties was observed (figure 4.1). Variety Jack
had lower nematode count but relatively’ high P. sojae
presence. It is possible that CX 252 wich is tolerant to SCN
but supports large populations of the nematode (Melakebern;
personal communication ) escaped infestation and was excluded
from the analysis. All other ten varieties showed positive
correlation between P. sojae occurrence and nematode
infestation. Soybean variety 61801uxithe highest incidence of
P. sojae (9 in ten samples). Corsoy and 9171 had the highest
nematode cyst populations, and also high P. sojae occurrence.

In the fumigated plots, lower nematode counts and P.
sojae occurrence were observed, and data did not show any
correlation between the two pathogens (figure 4.2). Varieties
with the highest nematode counts (Conrad and 3311) did not
have the highest incidence of P. sojae. The varieties with
high P. sojae occurrence (GL2415 and J251) had lower counts of
nematodes but the trend was not significant enough to indicate

an inverse relationship.

Discussion

P. sojae is essentially an opportunistic plant pathogen
that mainly attacks its host when under stress. It is known
that wet and cool soil conditions early in the growing season

favor tine development of INUR in soybeans (Kittle 6H; al.,

106

1979). These field conditions enable the release and
dispersal of zoospores but they put soybean seedlings under
stress by reducing metabolism and plant growth (Kittle et
al., 1979 ). Nematode feeding puts stress on soybean seedlings
by rendering roots inefficient i1] the uptake CHE water and
nutrients(Ross, 1965).. Feeding-furrows in the roots also make
it easier‘ for pathogen. propagules to enter and infect
soybeans(Ross, 1965).

It is noteworthy that the results reported here were
obtained from a field survey where P. sojae and SCN may not
have been evenly distributed; thus, it is possible that some
soybeans may have escaped infection by either organism. This
may explain why some soybean varieties had low P. sojae
occurrence despite high nematode cyst counts. Another possible
explanation is the absence of compatible races of P. sojae. If
compatible races are run: present, wounding Iby nematode
feeding alone may not ensure infection. Some inoculation
techniques in screening procedures also create wounds in
soybean seedlings but ck) not render' them. susceptible to
incompatible races cu? P. sojae. SCN also inns races that
selectively attack certain genotypes of soybeans, and the
presence of compatible races of the two organisms may be
necessary for enhanced disease condition. Adeneji (1975)

observed increased soybean seedling disease intflmainteraction

107

of race 3 of SCN and race 1 of P. sojae. Due to the race
specificity factor, information on the races of both SCN and
P. sojae occurring in a given field or growing region would
be important to growers. In Michigan, this would be
particularly important in areas where soil composition
(structure) and field topography are likely to support SCN and
P. sojae.

The results obtained in this survey agree in general with
reports from other workers. While positive interactions
between SCN and certain fungal plant pathogens have been
observed (Todd.et al.,l987; Rrancl and Wyllie,1988; Schenk and
Kinloch, 1974), lack of interaction and inconsistency have
also been reported. Interaction between SCN and Macrophomina
phaseoli had variable results with increased root colonization
(Todd et ,1987). In another study (Francl and Wyllie,1988), no
interaction was evident. When pathogenic fungi that are
associated with soybean roots were surveyed, no consistent
relationship between the occurrence of specific fungi and SCN
was evident (Schenk and Kinloch, 1974). Enhanced phosphorus
utilization and reduction of second—stage juveniles of SCN in
the presence of a vesicular arbuscular mycorrhizal (Glomus
fasiculatum) fungus has been reported (Tylka et al., 1988).
According to Roy (Roy et a1. 1989), F. oxysporum and F. solani

exhibited severe wilting in soybeans infested by SCN. Thus

108
Fusarium, which is less host-specific and survives under
various field conditions, may be the most significant
opportunistb: fungal plant pathogen ii] the infestation of
soybeans by SCN.

The lack of typical symptmms in most of the soybean
samples reported in this survey may be due to the possibility
that more aggressive races of P.sojae infect incompatible
soybean genotypes but do not cause disease in them. Stella
Avila (MS thesis) reported presence of Phsojae in non-host
crops such as wheat and dry beans in which the pathogen exist
without causing disease. It is possible that Phytophthora
behaves similarly in highly tolerant/resistant soybean
genotypes as it is not impossible to isolate P. sojae from
healthy-looking soybeans (P. sojae was isolated from healthy-

looking plants in our laboratory).

109

Literature cited

Adeniji, M.G., Edwards, D. I., Sinclair, J. B. and Malek, R.
B. 1975. Interrelationship of Heterodera glycines and
Phytophthora megasperma var. sojae in soybeans.
Phytopathology. 65:722-725.

Anderson, T. R., Welacky, T. W., Olechowski, T. H., Ablett, G.
an Ebsory, B.A. 1988. First Report of Heterodera
glycine in Ontario, Canada. Plant Disease Report.
72:453.

Bachman, P.A. and Rodriguez-Kabana, R. 1975. A system for the
growth and delivery of biological control agents to
soil. Phytopathology 65:819-821.

Baker, K.R. and Koenning, S.R. 1989. Influence of soil
moisture and texture on Heterodera/Glycine max
interaction(Abstr.) J. Nematology 21:550.

Bird, G.W. J.F. Davenport, and C. Chen. 1988. Potential role
of Heterodera glycines in dry bean production in
Michigan. J. Nematology 20:628—629.

Brewer, F.L. 1989. Special assessment of the soybean cyst
nematode Heterodera glycines problem.Joined problem
planning and evaluation staff paper. 08 Dept. Agric.
Washington DC.

Canaday, C.H. and A.F. Schmittenner. 1982. Isolating
Phytophthora megasperma f. sp. glycinea from soil with a
baiting method that minimizes Pythium contamination.
soil Biology and Biochemistry 14:67-68.

Carris, L.M. and Glawe, D.A. 1989. Fungi colonizing cysts of
Heterodera glycines. Illinois Agric. Exp. Stn. Bull.
786.93p.

Doupnik,B. 1993. Soybean production and disease loss estimates
for North Central United States from 1989-1991. Plant
Disease 77:1170-1171.

Conway, K.E. 1986. Use of fluid-drilling gels to deliver
biological control agents to soil. Plant Disease.
70:835-839.

110

Edward, J.H. Thurlow D.L. and Eason, J.T. 1988. Influence of
tillage and crop rotation on yields of corn , soybean
and wheat. Agronomy Journal 80:76-80.

Francl, L.J. and Wyllie, T.D. 1988. Influence of crop rotation
on population density of Macrophomina phaseolina in
soil infested with Heterodera glycines. Plant Disease
72:760-764.

Hartwig, E.E. 1985. Breeding productive soybeans with
resistance to soybean cyst nematode. Pages 394-399.in:
World Soybean Research Conference III, R.A. Shibles ed.
Westview Press, Boulder CO.

Heatherly, L.G., Young, L.D. 1991. Soybean and soybean cyst
nematode response to soil water content in loam and clay
soil. Crop Sci. 31:191-196.

Heatherly,L.G.,Young, L.D., Epps,¢Llf and Hartwig, E.E. 1982.
Effect of upper-profile soil water potential on numbers
of numbers of cysts of Heterodera glycines on
soybeans. Crop sci. 22:833-835.

Hill, N.S., and Schmitt, D.P. 1989. Influence of temperature
and soybean phenology on dormancy induction of
Heterodera glycines. J. Nematology 21:361-369.

Hung, Y. 1958. A preliminary report on the plant parasitic
nematode of soybean crop of the Pintung District,
Taiwan, China. Agric. Pest News 5:1-5.

Hussey, R.8. and Boerman, H.R. 1983. Influence of planting
date on damage to soybeans caused by Heterodera glycines.
J. Nematology15z253-258.

Johnson, A.W. and Feldmesser, J. 1987. Nematodes- A historical
review Pg. 448-454. in: Vista on Nematology, J.A. Veech
and D.W. Dickson eds. Society of Nematologists inc.
Hayattsville Md. USA.

Kerry, B. R. 1988. Fungal parasites of cyst nematodes. Agric.
Ecosysys. And Environ. 24:293-305.

Kerry, B.R. 1984. Nematophagous fungi and the regulation of
nematode populations in soil. Helminthol. Abstr. Ser.
B:53(1):1-l4.

111

Kinloch, R.A. 1979. Response of resistant cultivars to
fumigation at planting for the control of soybean cyst
and root knot nematodes. Nematropica 9:27-32.

Kittle, D.R. and Gray, L.E. 1979. The influence of
temperature, Moisture, porosity and bulk density on the
pathogenicity of Phytophthora Megasperma var. sojae.
Plant Disease 63: 231-234.

Koenning, S.R. Anand, S.C. and Wrather, J.A. 1988. Effect of
within field variation in soil texture on Heterodera
glycines and soybean yield.J. Nematol. 20:373-380.

Moots, C.K. , Nickell, C.D. and Gray, L.E. 1988. Effect of
soil compaction on the incidence of Phytophthora

megasperma f. sp. glycinea in soybean. Plant Disease.
72:896-900.

Morgan-Johns, G. and Rodriguez-Kabana, R. 1987. Fungal
biocontrol for the management of nematodes. Pages 94-99
in: Vistas on Nematology. J.A. Veech and D.W. Dickson,
eds. Society of Nematologists, Inc., Hayattsville, MD.
USA.

Mulrooney, R.P. 1988. Soybean disease loss estimates for
Southern United States in 1997. Plant Disease Report.
72:95.

Nakata, K. and Asuyana, H. 1938. Survey of the principle
diseases of crops in Manchuria. Br. Indus. Report 32:66.

Niblack, T.L. ed. 1983. Protect your soybean profits: Manage
soybean cyst nematode. Columbia, Mo: University of
Missouri Printing Service.

Nishizawa, T. 1984. Nematode survey on upland fields in JavaI
sland with special reference to soybean fields.Pg. 165-
185. in: Report on Japan-Indonesia Joint Agric. Res.
Proj.JR 84-74, Japan International Coop. Agency.

Overstreet, C. and McGowerley, E.C. 1990. Interaction between
Calonectria crotolaria and Heterodera glycines on
soybeans. J. Nematology 22:496-505.

Overstreet, C. and McGowerley, E.C. 1988. Influence of
Calonectria crotolaria on reproduction of Heterodera
glycines. J. Nematology 20:457-467.

112

Pereira, R.M. and Roberts, D.W. 1990. Dry mycelium preparation
of entomophagous fungi, Metarhizium anisopliae and
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Rodriguez-Kabana, R. 1992. Chemical Control in: Biology and
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Ross, J.P. 1965. Predisposition of soybean to Fusarium wilt by
Heterodera glycines and Meloidogyne incognita
Phytopathology 55:361-368.

Ross, J.P. 1964. Effect of soil temperature on the development
Heterodera glycine in soybean roots. Phytopathology
54:815-818.

Ross, J.P. 1963. Seasonal variation of larval emergence from
cysts of the soybean nematode Heterodera glycines.
Phytopathology 53:608-609.

Roy, K.W. Laurence, G.W. Hedges, H.H., McLean, K.S. and
Killebrew,J.F. 1969. Sudden death syndrom of soybeans:
Fusarium solani as incitant and relation of Heterodera to
disease severity. Phytopathology 79:191-197.

Schenk, N.C. and Kinloch, R.A. 1974. Pathogenic fungi,
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Todd, T.C. Peason, C.A.S., and Schwenk, F.W. 1887. Effect of
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40.

Tribe, H.T. 1980. Prospects for the biological control of
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Tyler,D.D., Chambers, A.Y. and Young, L.D. 1987. No-tillage
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Tylka, G.L. Hussey, R.S., and Roncadori, R.S. 1988.
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113

production and formulation of mycoherbicides. Weed Sci.
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Wallace, H.R. 1964. The biology of plant parasitic nematodes.
St. Martins Press, New York.

Winstead, N.N. Skotland, C.B., and Sasser, J.N. 1955. Soybean
cyst nematode in North Carolina. Plant Disease
Report.39:9-11.

Yokoo, T. 1936. Host plants of Heterodera Schactii Schmidt and
some instructions. Korea Agric. Expt. Stn. Bull. 8: 47-
174.

Chapter 5

Summary and Conclusions

Introduction

In this study, a survey was conducted on the occurrence
and virulence of P. sojae in Michigan soybean fields in the
1993-97 growing seasons, and genistein (4' EL '7—trihydroxy
isoflavone), a compound that is produced and exuded by soybean
roots was evaluated for potential as a molecular marker for
field tolerance in soybean to P. sojae. The effect of
genistein on the ability of P.sojae zoospores to infect and
cause disease in soybean seedlings was also studied. A soybean
field naturally infested with the soybean cyst nematode H}
glycines , was surveyed for possible correlation between
nematode activity and P. sojae infection, and the results are

also presented in the study.

Phytophthora sojae in Michigan soybean fields

Ninety of time P. sojae isolates obtained from soybean
fields were tested for virulence and race status. Fifty or 55%

of the isolates tested defeated more than seven Rps genes each

114

115
and were categorized as highly virulent. Ten isolates (13%)
showed intermediate virulence, defeating four to six Rps genes
each, while eighteen (20%) of isolates were of low virulence
defeating 1-4 Rps genes. Nine (11%) were avirulent as they
attacked neither of the Rps genes nor the susceptible variety
Williams (rps).

Rps genes Rps3b, Rps3a, Rpslb, Rpslk, and Rps6 were (in
that order) the most resistant to the field isolates. These
genes resisted 70-78% of the P. sojae isolates while Rpslaand
Rps7 had. the lowest rating resisting only 12-13% of 'the
isolates. Rpsla, Rpslc, Rpslk, Rps3a and Rps7 are incorporated
in varieties either being planted or developed in Michigan
(B.W. Diers and J.F. Boyse. Dept. of crop and soil sciences,
Michigan state university, East Lansing MI). In light of the
results obtained in this study, soybean lines with Rpsla and
Rps7 need to be monitored closely for their performance in
areas where P. sojae is known to occur or be replaced with
Rpsl,3 and Rpslw Incorporating' these genes singly (n: in
combinations inlflichigan soybean varieties in conjunction with
recommended cultural practices should provide improved
protection against most of the P. sojae races that occur in
the state. Due to race shift and the presence of rare but
compatible races of P. sojae, more enduring non-race specific

genetic protection in soybeans has become more attractive,

116
particularly when used as part cflfani IPM program. Genetic
defense of soybeans against P. sojae may be further enhanced
by a program where growers may have their popular soybean
lines evaluated for tolerance to races that are common to

their growing areas.

Effect of genistein on the infection of soybean roots
Exogenous application of 5 ppm of genistein solution to
pot cultures of two-day old soybean seedlings inoculated with
zoospores (3000/ml) <1f 1% sojae significantly (P $0.05)
reduced infection and increased dry weights of roots. It is
known that genistein hastens the encystment and germination of
zoospores (Morris and Ward, 1992) and may impact the initial
inoculum potential. This is particularly important since P.
sojae is a soil-born pathogen for which secondary inoculum has
little or no additive value to infection and disease progress.
Zoospores which come in contact with genistein germinate away
from soybean roots and the number of zoospores which come in
contact with roots is reduced. Further studies are needed in
order to determine the impact of genistein exuded by
individual soybean lines on disease. A study examining the
relationship between the concentration and, dispersion of
exuded genistein in the rhizosphere soil volume and disease

reduction may further elucidate the role of the isoflavone in

117

Phytophthora root and stem rot of soybeans. Determining the
effects of exuded genistein (from selected soybean varieties)
on races of P. sojae that interact variably with the beans,
will further elucidate the role of the isoflavone on field

tolerance.

Fluorescence of root exudates and field tolerance values of
soybeans

Fluorescence levels of root exudates and field tolerance
values (ME soybean ‘varieties gave ‘variable results. Three
highly tolerant varieties, Resnik, Williams 82, and PI 103 had
high fluorescence values while Harlon and Sloan with low
tolerance had low exudate fluorescence. Fluorescence and field
tolerance values for these five varieties tended to indicate
a correlation between field tolerance and fluorescence of root
exudates but values for the varieties Repley, Harosoy, Conrad
and Pella gave highly varied field tolerance values among the
isolates of P. sojae.

Field tolerance values above four are considered poor by
growers (A.F. Schmithenner. Personal communication) The
variety Repley with fluorescence of 700 nm had good tolerance
to Race 25, M80 32 and M80 23 but poor tolerance values of 4.5
and 6.0 to M80 25 and M80 10 respectively. Harosoy with

fluorescence at 680 nm had low tolerance to M80 10 and M80 32

118

but good tolerance to Race 25, M80 23 and M80 25. Due to these
observations and time highly ‘variable ‘tolerance ‘values to
isolates for Conrad and Pella, it was not possible to link
fluorescence of root exudates to field tolerance in soybeans.
Because of time variable response CH? soybeans ti) different
races of 1% sojae, finding a3 molecular marker timn: will
indicate tolerance levels to various races of the pathogen

may be difficult.

P. sojae in soybeans infested with H. glycines

With the exception of the varieties Jack and CX252, a
significant (P< 0.05) correlation between nematode infestation
of soybeans and the occurrence of P. sojae was observed in the
non-fumigated plots. In the fumigated plots, lower nematode
counts and p. sojae occurrence were observed and the data
obtained ch11 not support any cxmielation between time two
pathogens.

Studies on the interactions between H. glycines and fungal
pathogens have reported inconsistent results (Todd et al.,
1987 and Francl and Wyllie , 1988). Adeneji (1975) observed
increased soybean seedling disease in the interaction of race
3 of CSN and race 1.<df P. sojae. Due to the race specific
factor in both SCN and P.sojae, information on the races of

both pathogens in a given field or growing area would be

119

important to growers. Roy (Roy et al. 1989) reported severe
Fusarium wilting in soybeans infested by SCN. Thus non-
selective opportunistic soil pathogens such as F. solani and
F. oxysporum may be the most important organisms in the

infestation of soybeans by SCN.

120

Literature cited

Adeneji, M..O. , Edwards, D.I., Singlare, J.B. and Malek
R.B. 1975. Interrelationship of Heterodera glycines
and Phytophthora megasperma var. sojae in soybeans.

Francl, L.J. and Wyllie, T.D. 1988. Influence of crop
rotation on population density of Macrophomina

phaseolina in soil infested with Heterodera glycines.
Plant Disease 72:760-764.

Morris, P.F. and E.B.W.Ward. 1992. Chemoattraction of
zoospores of the soybean pathogen P.sojae by
isoflavones . Physiol. Mol. Plant Pathol. 40:17-22.

Roy, K.W., Laurence, G.W. Hedges, H.H. McLean, K.S. and
Killebrew. J.F.. 1969.8udden death syndrom of
soybeans: Fusarium solani as an incitant and relation
of Heterodera glycines to disease severity.
Phytopathology 79:191-197.

Todd, T.C., Peason, C.A.S., and Schwenk, F.W. 1987. Effect
of Heterodera glycines on charcoal rot severity in
soybean cultivars resistant and susceptible to the
soybean cyst nematode. Ann. App. Nematol.

(J. Nematology suppl.) 1:35— 40.