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This is to certify that the

thesis entitled
DEVELOPMENT OF AN ASSAY FOR RESISTANCE TO SCLEROTINIA

SCLEROTIORUM INFECTION OF SOYBEAN AND INVESTIGATIONS INTO
THE CAUSE OF THE VARIABILITY WITHIN A GENOTYPE
FOR TOLERANCE TO THE FUNGUS

 

 

presented by

Clay Hurd Sneller

has been accepted towards fulfillment
of the requirements for

Master . Crop and Soil Sciences
degree in

 

 

   

Major professor

Date February 27, 1987

 

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DEVELOPMENT OF AN ASSAY FOR RESISTANCE T0 SCLERQIIHIA
SCLERQILQRQM INFECTION OF SOYBEAN AND INVESTIGATIONS INTO
THE CAUSE OF THE VARIABILITY'VITHIN A GENOTYPE FOR TOLERANCE
TO THE FUNGUS

BY

Clay Hurd Sneller

A THESIS

Submitted to chhtqan State Unlverslty In partial

fulflllment of the requlrements for the degree of

MASTER OF SCIENCE

Department of Crops and Soil:

1987

ABSTRACT

DEVELOPMENT OF AN ASSAY FOR RESISTANCE T0 W
W INFECTION OF SOYBEAN AND INVESTIGATIONS INTO
THE CAUSE OF THE VARIATION WITHIN A GENOTYPE FOR RESISTANCE
TO THE FUNGUS

By

Clay Hurd Sneller

Soybeans [W (L.) Merr.) exhibit variability
within a cultivar for resistance to WM
0:.) De Bary. The cause of the variability was
investigated. Stem sections were cut from greenhouse plants
and Inoculated In the laboratory with mycelium plugs. The
resulting lesions were measured. Inoculations on the intact
cuticle resulted in escapes and delayed Infections which
contributed to residual variability. These problems were
minimized by inoculating on wounded tissue. The wound
Inoculation technique detected significant differences In
lesion lengths among cultivars. The results matched field
resistance and the occurrence of restricted lesions as
detected by Inoculations made on sites where cuticular waxes
were removed. Attempts to increase the expression of
resistance and the sensitivity of the method by lowering the

duration of inoculation or the nutritional content of the

Inoculum medium failed. Investigations into the cause of
the residual variability of the wound Inoculation technique

implicated the growth environment of the plants.

This thesis and my all my efforts to complete my Masters
degree are dedicated to my wife Sharon for without her help
and support I probably would have never returned to school

to pursue these endeavors.

ACKNOWLEDGEMENTS

This thesis was produced with the help of many

Individuals. I would particularily like to acknowledge the

assistance of my thesis committee, Dr. Thomas G. Isleib, Dr.

John L. Lockwood and Dr. .Jhn Kelly who all provided

Invaluble advice and guidance to my research efforts.

TABLE OF CONTENTS

List of Tables viii
List of Figures x
Literature Review ‘ 1
Introduction 1
Epidemiology 2
Histology 12
Avoidance and Resistance 25
Summary 38
Proposed Research 41
List of References 43

Development of an an Assay for Resistance to

5g1g2931n1§_§g1gngtignum Infection of Soybean 55
Introduction 55
Materials and Methods 56
Results 61

Inoculation on Intact Stem Tissue 61

Inoculation on Sites Where the Either the Cuticle

or Cuticular Waxes Were Removed 66
Inoculation on Wounded Stem Tissue 68
Limited Duration of Inoculation 74

Lower Concentration of Nutrients in the Inoculum
Medium 76

v1

v11

Inoculation on Stem Tissue Where the Cuticular
Waxes Have Been Removed

Inoculation on Stem Tissue Varing in Age
Discussion

List of References

Sources of Variability Within a Genotype
for Resistance to Wm
Infection of Soybean.

Introduction

Materials and Methods

Results

Discussion

List of references

Implications of Thesis Findings to a Breeding Program
for Resistance to W.

77
83
88
96

99
99
101
104
110

114

116

LIST OF TABLES

Table 1. The experimental design used In Experiments 1
through 22.

Table 2. The Incidence of Infection from Experiments
1 through 5.

Table 3. Disease Incidence in four cultivars over four
experiments.

Table 4. Comparison of the final average lesion lengths
from Experiment 3 (10/2/85) between stems infected by
the second day and stems which developed lesions after
the second day.

Table 5. Summary of lesion length data 48 hours after
inoculation from Experiment 22 (10/28/86) and
Experiment 6 (11/14/86).

Table 6. Summary of average lesion length data from
experiments utilizing the wound inoculation (WI)
technique.

Table 7. Averages of the statistics in Table 6
pooled across all experiments.

Table 8. Summary of the effect of duration of
Inoculation on lesion development, Experiment 12
(2/23/86).

Table 9. Summary of the effect of inoculum media
nutrient concentration on the Incidence of infection
and on lesion length, Experiment 21 (4/30/86).

Table 10. Summary of wax removal experiments.

Table 11. Results of the wound inoculation (WI) and
wax removal (WR) methods of Inoculation pooled across
all experiments.

Table 12. Summary of the Incidence of restricted and

non-restricted lesions in the wax removal (WR)
experiments

viii

ix

Table 13. Summary of the incidence of restricted and
non-restricted lesions of a subset of the data in
Table 11. 82

Table 14. Summary of the Incidence of restricted
lesions by position from data combined from
Experiments 18 - 20. 87

Table 1. Analysis of variance for lesion length for SPFs
of five cultivars, Experiment 1 (1/24/86. 1/30/86,
2/23/86). 104

Table 2. Analysis of variance by runs for lesion length
for the SPFs tested In Experiment 1. 105

Table 3. Mean squares for the sources of variation
in lesion length within a cultivar. 106

Table 4. Analysis of variance for lesion length for
SPFs of five cultivars, Experiment 2 (5/22/86). 107

Table 5. Correlation of the average lesion lengths of
the SPFs tested in Experiment 1 with the average lesion
lengths obtained In Experiment 2. 108

Table 6. Summary of height data for the plants used
in Experiments 1 (run 2) and 2. 109

Table 7. r values and probabilities for the
correlation of plant height and lesion length from
Experiments 1 (run 2) and 2. 110

List of Figures

Figure 1. Cumulative percentage of lesion initiation and

average lesion length of

lesions initiated on a

particular day of Experiment 4.

Figure 2. Average lesion

Figure 3. Average lesion
lesions omitted from the

Figure 4. Average lesion
18, 19 and 20.

length by position.

by position with restricted
data set.

by cultivar from Experiments

6'?
e4

85

86

L I TERATURE REV I EW

I NTRODUCT I ON

The fungus WW (Lib.) De Bary can
Infect a multitude of plant hosts prompting Purdy (103) to
describe It as "among the most nonspecific, omnivorous. and
successful of plant pathogens“. Adams (10) reported a host
range of 64 families, 225 genera, and 361 species. The

W. We and W: each have over 17
genera and 30 species which can host the pathogen.

In soybeans. [W (L.) Merr.l, WIN
causes Sclerotinia stem rot. In 1946, Weiss (134) reported
the presence of the disease in New York, Maryland, Iowa. and
Virginia. It has since been reported in Illinois (26. 27),
Minnesota (45), New Jersey (53). Maryland (11), Wisconsin
(47), Virginia (98), Arizona (56), Michigan (67). Ontario
(23, 55). South Africa (124). France (112) and Brazil (136).
It Is primarily a localized problem, often occuring in
fields previously planted with a more susceptible host (45,
56. 98). Two recent reports indicate that the disease
Incidence is increasing as soybean production expands into
areas where more susceptible hosts have been grown (27, 47).

Field observations of the disease’s effect on yield
vary from no effect (98) to severe (56). Grau and Radke
(47) found a negative correlation between disease severity
ratings and yield where soybean cultivars were planted in
either narrow or wide rows in a disease nursery. Based on
previous studies, narrow rows were expected to yield 21%

1

2

more than wide rows. Instead the narrow row soybeans
developed higher disease severity indexes. yield was
decreased 42 ’6 as compared to the yeild of less diseased
wide rows. Lockwood and Isleib (69) also reported that
yield and disease Incidence were negatively correlated ( r -
-.94).

mm Is also the cause of white mold in
W L. This disease is very'slmilar to
Sclerotinia stem rot of soybeans. White mold is by far the
more studied of the two diseases. In all cases where
sufficient research on Sclerotinia stem rot exists. it
appears to parallel white mold research. This review will
include white mold research to emphasize and fill in voids

in Sclerotinia stem rot research.
EPIDEMIOLOGY

While the disease is often found In soybeans rotated
with more susceptible hosts, it can also spread to
previously uninfected fields by means of windblown
.ascospores (6). Ascospores can occasionally travel up to 10
kilometers from the point of release (25). Another method
of long distance transmission is through contaminated seed.
From 1969 to 1970, Nicholson “x931 found S_._
W Internally seedborne In 28 of 37 lots of ’Lee
68’ soybeans. In 1971. 1% of ’Cutler’ lots and 295 of the
’Amsoy’ and ’Beeson’ lots were similarly contaminated. The

fungus could still be recovered from the infected lots 18

months later. The plantings from these seed lots developed
Sclerotinia stem rot though it was not shown that the
infections originated from the infected seeds. In L
11.1mm. Steadnan (118) found that 489‘ of the infected
fields produced seed Infected with the fungus. The Infected
seed produced healthy plants so it was concluded that the
seedborne fungus was only important In disease
dissemination. Soybean seed can also be contaminated with
sclerotia which can spread the disease (98, 124). 5‘
W has also been found In the seeds of certain
crucifers (90) and W has been found contaminating
peanut (AnaghIs_nypggga L.) seed (130).

Other methods of dissemination include movement of soil
contaminated with mycelium or sclerotia (6). contaminated
manure or straw (22. 115) and irrigation water (119).

Sclerotia give rise to the infectious forms of the
disease, mycella and ascospores. Increased scierotlum
numbers in a field Increases the amount of inoculum that can
be produced. With leaf drop disease In lettuce, caused by
$‘_mIng:. disease Incidence Is correlated to the number of
sclerotia per unit area of soil (39). Imolehin and Brogan
report similar results (63). Sclerotia numbers In a field
are primarily increased In a susceptible host, though
sclerotia have been shown to increase in the soil (30. 135).
Adams (9) counted up to 1000 sclerotia of mg: formed on
a single lettuce plant. This would translated into an.

increase of 50 sclerotia per kg of soil. This Is a dramatic

4

Illustration of the potential buildup of Inoculum In a field
planted to a susceptible host. Sclerotium levels were
lowered when all Infected lettuce plants were removed over
three years (97). The number of sclerotia of S_._
W In a bean field appears to range from 0-7
sclerotia per kilogram of soil (1, 108, 134). Abawi and
Grogan (I) obtained surface counts of 16.1 and 5.4 sclerotia
/ 30 cm2- In bean fields that had been severely infected.
After plowing only 0.2 and 0 sclerotia / 30 cml were
found. A concentration of only 0.2 scierotlum per kg of
soil was enough to infect 46 t of a dry bean canopy (110).

Inoculum can also be produced outside of the field.
Apothecla were found In fence rows and wooded areas (I) and
this was considered to be an Important source of Inoculum.
Wm can also Infect many weedy hosts including
MW Gandooer-Hl). Amnesia
MW L440). WEI: (1..) Media-
<85>. was R. Br. (85). W51: L.
(86) and W L. (24, 86). All these are weeds
comnonly found In soybean fields.

S. sclerotiorum survives from year to year primarily as
sclerotiaf Cook gt, 1L.(30) reported that 7596 of sclerotia
buried for 3 years at depths from 5 to 20 cm germinated.
They also found that the fungus could overwinter as mycelia
in bean seeds but not in crop residues. Adams (9) found
that sclerotia could survive for 16 months when buried up to

30.4 cm but that survival at 61 cm was poor. Merriman gt

5

51,482) reported that more sclerotia survived at the soil
surface than when buried. Plowing reduced disease by
burying the sclerotia.

Soil moisture level Is an important factor in
scierotlum survival with several reports Indicating that
survival Is greater in drier conditions (7. 63. 83).
Sclerotia also survive better in soils that have never been
Infected before (7, 63). This is apparently due to the
absence of sclerotia parasites. WM (126,
125) has been shown to lower scierotlum survival as has
WWII! <87) and Wanna app. (57, 63. 64.
78).

While rotations with non—host crops have been reported
to be an effective method of controlling the disease (115,
137), It must be a long rotation. A three year rotation to
a non-host crop in Nebraska following susceptible dry beans
produced no significant reduction In scierotlum numbers
(110). Surprisingly the growing of susceptible dry beans
for three years did not increase sclerotia levels. Adams
and Ayers (10) concluded that the sclerotia of S_._
Wm may remain viable in the soil for 4-5 years.

Wm can infect a host either as mycelium or
as ascospores. Sclerotia can produce mycelia and apothecia.
The apothecla then produce and release ascospores. Much of
the research determining which form of the pathogen Is the
primary form of Inoculum has been done in WILL! and

points to ascospores being the primary Inoculum of

6

Infections. Similar research with soybeans Is lacking,
though parallels can be drawn which indicate ascospores are
also the primary inoculum In soybeans.

The growth of mycelium through the soil appears to be
very restricted. Infections of beans by soil mycelium were
only successful when an additional energy source was
provided (1). Even then Infections were sporadic if the
inoculum was placed further than 1 cm from the plant.
Mycelium growing from a food base failed to Infect lettuce
(W L.) seedlings which were further than 2 cm
from the food base (92). The scierotlum does not appear to
supply the mycelium with the nutrition necessary for
extended growth (1, 102). Purdy (102) showed that mycelium
cannot Infect a host unless It Is supplied with an exogenous
energy source and that the energy source acts as a bridge to
the host. However, Abawi and Grogan (1) reported no
Infections accomplished in this way. Cook gt gl.(30)
concluded that less than 10% of the infections in their
study could be attributed to this process. It was also
noted that mycelium did not to grow from the ground up on
the outside of the stem to Initiate above-ground lesions.
Sclerotium produced mycelium can Infect the host but it
appears to be of minor Importance except In certain
situations where there is abundant senescent tissue and the
tissue is In contact with both the soil and the host.

Infections and epidemics of W in soybeans
and W predominately originate in the plant canopy

(1. 26. 30. 45, 46. 56, 89. 115. 124). Cook gt gL.(30)
stated that above ground lesions can only be initiated by an
airbourne propagule. This indicates that ascospores are the
primary inoculum. The presence of ascospores has been
positively correlated with disease development (1). When
bean tissue was collected from various fields, disease
synptoms only developed on the tissue from fields where
ascospores were also found. Mycellum can Infect plant
debris which could become windborne and cause Infections
(89), but the nature of epidemics and the proven
pathogeniclty of ascospores (27. 30. 92. 102, 110. 134)
Indicate that ascospores are indeed the primary inoculum of
Wain.

An exogenous energy source Is required for any
substantial infection to occur by either ascospores or
mycelium. While Infections can occur without the benefit of
an energy source (92, 102, 121), they are generally
restricted to a spotting or flecking of the surface.
Ascospores failed to infect tomato leaves (Lyggggggtggn
gsgylgntnm Mill.) without energy being supplied from either
flower tissue or a nutrient solution (101). Grogan and
Abawi (48) found that spore survival was closely correlated
with the nutrient content of the medium upon which the
spores were germinated. Spore survival was related to the
amount of growth and formation of appressoria. The presence
of the energy source appears to promote appressoria

production (1, 2, 46, 102. 123). This allows the pathogen

to penetrate the host through the cuticle (2, 73, 102,
121). Purdy (102) placed ascospores In distilled water and
noted that no appressoria formed. Infections of host tissue
did not occur unless a carbon source was added. In the same
study. mycelium formed appressoria without an energy source
being supplied but were still unable to infect. Sutton and
Deverall (121) noted Infections of young soybean tissue by
ascospores lacking an appressorium or an energy source.
These Infections were restricted to a few cells under the
ascospore. While penetration was achieved without an energy
source. colonization of the host tissue did not occur. The
ascospores formed appressoria If supplied with pollen
grains, but spreading infections only occurred when flower
tissue. a more substantial energy source. was provided.
This evidence along with the work of Purdy (102) Indicates
that while an exogenous energy source promotes the formation
of appressoria from ascospores, It must also have a further
role In promoting Infection.

In Eg_yu1ggnt§ the apparent energy source used to fuel
infections is senescent tissue, primarily flower blossoms
(2). While wounded tissue can be infected at any time (1.
3. 92). epidemics only occur after flowering. In a New York
study. Natti (89) found that white mold epidemics In beans
occurred 8 to 14 days after flowering. regardless of the
planting date. The Infections appeared to originate in leaf
and branch axils where blossoms were lodged. Other authors

have also reported that lesions originated in branch axils

9

where spent bean blossoms were found (1. 30). Studies in
other species also Indicate that the presence of flower
tissue is Important for Infection (66, 81. 101). In
greenhouse studies of soybeans. Cline and Jacobson (27)
found that plants were infected by ascospores only after the
plants had flowered. Sutton and Deverall (121) reported
that soybean blossoms were a suitable energy source for
ascospore infections of the host.

The blossom tissue of several WI; and 2.,
ggggtngug (111) lines was screened for resistance to
colonization by ascospores or mycelium, but no differences
were found.

Environmental factors influence the inoculum
concentration and the Infection process of Sg_gglgngttgnmm.
One of the key factors is moisture. Grau and Radke (47)
showed that Increasedlmoisture due to Irrigation resulted In
greater disease severity in soybeans. Similar effects of
moisture have been noted by other researchers in soybeans
(56, 112) and In 2t_1ulggntg (34. 64. 84. 89. 110, 111).

For sclerotia to produce apothecia they must be in
nearly saturated soil for approximately 10 days (6).
Apothecla and ascospores may be produced throughout the
growing season If the conditions are favorable (1, 6).
Others have also observed Increased apothecia production In
wetter conditions (29. 34. 110). In lettuce fields.
apothecia could only be found In Isolated wet areas under

the plant canOpy (92). Laboratory studies have also shown

10

the Importance of sufficient moisture for carpogenic
germination (48. 62). The Importance of sufficient moisture
for apotheclum germination In relation to white mold
epidemology was shown by Nattl (89). This research showed a
positive correlation between disease severity and high July
rainfalls before flowering, but no correlation between
disease severity and rainfall during blossoming. The July
rainfall favored carpogenic germination of sclerotia and
ascospore production thus creating the primary inoculwm for
infections.

Apothecla formation has also been shown to be
Influenced by herbicides (105) and depth of burial. Singh
and Singh (113) reported maximum carpogenic germination of
sclerotia on the soil surface and that germination decreased
with depth of burial. However, in another study germination
was greatest when the sclerotia were buried (82). This
would no doubt be Influenced by the precipitation pattern
and drainage of a particular site.

Ascospore survival is Influenced by relative humidity.
In an laboratory experiment. spore survival was highest In a
low relative humidity (48). The longest survival time was
21 days at 7% relative humidity. In field studies In mhich
ascospores were exposed to fluctuating relative humidity and
temperature, survival ranged from 9 to 12 days. Thus
ascospores may land on the host and remain viable for a
period of time during which they may germinate when

conditions are favorable.

11

Moisture Is also critical for ascospore germination and
host infection. In a field study on the effect of rainfall,
temperature. relative humidity and leaf wetness on disease
severity, only leaf wetness was positively correlated with
disease (1). Weiss gt gl.(133) also found the duration of
leaf wetness to be the most important factor determining
disease incidence. Attempts to infect bean tissue with
mycelium grown on agar 'were only successful when free
moisture was present at the Infection interface for 16 to 24
hours (1). Successful Infections with colonized, dry
blossom tissue or ascospores needed free moisture at the
Infection interface for 48 to 72 hours before host
penetration was observed. Shorter durations of moisture may
also support Infections (19).

Lesion development Is also Influenced by moisture.
Abawi and Grogan (1) found that lesion development ceased If
the lesion and stem surface were dry. Even a relative
humidity of 99 % was Insufficient to maintain lesion growth.
The arrested lesions required a 48 to 72 hour exposure to
free moisture before they would resume growth. Growth of
mycelia] colonies on agar medium declined as the water
potential of the medium drops below -20 bars (48), again
illustrating slower growth with drier conditions.

Temperature also plays an Important role in Inoculum
production and host infection. Apothecium production
appeared to be optimum at 10°- 20° C (I, 110) though

production has been noted at 25° C (1), Indicating a broad

12

range of temperatures suitable for the production of
ascospores. A broad range of temperatures (10 - 30° C) was
also reported as favorable for ascospore germination with
25° C appearing optimal (I). Germ tubes were reported to
grow fastest at 25° C. Lesion and mycelium growth In beans
and soybeans appeared optimum at 20° C (1. 98) though 250 C
also appeared adequate (133). Lesions did not appear to
expand at 10° or 30° C (1, 133). The optimum temperatures
are In the ranges expected in soybean and bean fields at the
critical flowering period. Weiss gt ‘gl.(133) reported
favorable temperatures 82 % of the time In a regularily
Irrigated bean field In Nebraska. As such It Is doubtful
that temperature is a limiting factor in the disease

epidemology In the areas where Wm is

traditionally a problem.
HISTOLOGY

Upon germinating on the host, the pathogen can Infect
through the stomata (65), although penetration through the
cuticle appears the most conmon form of Infection (2, 75.
76, 102, 123). Generally ascospores will colonize a bean
blossom and produce mycelium. The mycelium continues the
Infection process (2, 121).

Generally an appressorium-like structure (infection
cushion) develops from a hypha, and penetration occurs by
purely mechanical means (73, 75, 76, 123). ‘This also'
appears to be true for other smlgngttnta species (76, 100,

13

114). The Infection cushions vary in complexity by the
number of hyphae but basically consist of several hundred
parallel hyphal tips which are perpendicular to the host
surface and some longer peripheral hyphae (76). The
Infection cushions form upon physical contact of the hyphae
with the host tissue (2, 36. 41. 102). The hyphae then
develop the dome-shaped Infection cushions (2, 41. 73. 102).
This is followed by the actual penetration, which may be
accomplished by a penetration peg (2, 102). While
penetration appears to be physical. there Is evidence that
some Sglgmtinn species (100, 114) produce enzymes that
precede penetration. Cell death was observed beneath the
point of penetration prior to the actual penetration (76.
77).

Abawi gt gl.(2) found the complexity of the infection
cushion to be associated with the nutrient level of the
medium. Tariq and Jefferies report (123) that nutrition was
Important for formation of Infection cushions but did not
Influence the complexity. They found that the physical
resistance of the surface Influenced the complexity of the
resulting Infection cushion with more complex Infection
cushions formed on the surface of Eg_ggggtngug leaves than
on the softer Eg_1ulggntg leaves.

Upon penetration the pathogen forms a vesicle (73, 102)
from which Infection hyphae form which continue the
Infection (73, 74. 102). In beans the infection hyphae grow

radially from the vesicle and grow Intercellularly (2, 73,

14

76, 102) between the cuticle and epidermal cell layer and In
the cortex (73). The subcutlcular hyphae branch. and the
branches run parallel to each other. forming an Infection
front (73. 76). The subcutlcular hyphae grow faster than
the cortex hyphae though It Is the later that girdle the
stem (73). Up to this point all hyphal growth has been
interceilular.

Ramifying hyphae form from the Infection hyphae 12-24
hours after infection (73, 76). These hyphae form to the
rear of the infection front and branch extensively (73.).
The ramifying hyphae grow both Inter- and Intracellularly in
healthy and dead cells and Invade the vascular tissue (73,
100). It Is the ramifying hyphae which emerge from the stem
through stomata or breaks in the cuticle (2, 73, 76).

The Infection hyphae appear to be responsible for
changes In the host’s pectic material and for the death of
host cells that occur In advance of any hyphae (73). These
functions are aided by production of diffusible toxins and
enzymes.

To colonize host tissue and move Intercellularly the
middle lamella must be degraded. Wm produces
polygalacturonases which have pectolytic activity and
accomplish this task (18. 42, 50, 74. 86). Lumsden (74)
found that the enzymes can be produced as early as 12 hours
after infection and the amount of pectic material decreases
as the polygalacturonases Increase (42, 74)..

Endo—polygalacuronase (Endo-pg) is produced first and has

15

been found both In yttng (18, 42, 50) and in 2119 (50, 74).
Endo—pg Is localized In the advancing margins of lesions In
the early stages of infection (24 hours) after which It
.qulckly declines (74). Early reports claimed that no
correlation existed between pathogenicity and enzyme acivity
(54, 86). Held (54) did report that a degenerate strain of
Sg__t:tfigltgzuml lacked. the ability to produce the wilt
symptoms. Lumsden (74) later found a positive relationship
between Endo-pg production and virulence and postulated that
the relationship was missed in the earlier reports due to
the early Inactivation (48 hrs.) of the enzyme.

Exo-polygalacturonase (Exp-pg) Is also produced and Is
believed to break down the substrate left from the Endo—pg
digestion (74). It Is found In mature lesions (74) and Is
correlated to the rapid growth phase of an Infection. Both
Exo- and Endo-pg have a peak activity at a pH between 4.5
and 5.5 (42, 50, 74).

Another key enzyme In pathogenesis Is pectin
methylesterase (PME). This enzyme occurs naturally In
uninfected bean tissue but PME from the host and the
pathogen can differentiated (74). PME Is also produced early
In the infection process (42. 50, 74) and is associated with
the advancing margins of lesions. PME content Increases
with time (74). Hancock showed that the PME levels are two
times higher In Infected than In uninfected tissue and that
as PME Increases. methoxy group content decreases (50). PME

functions by demethylating pectin to pectate (50, 74) which

16

is more rapidly degraded by the polygalacturonases (50).
PME has not been correlated with pathogenicity (74, 86)
which may be expected as the Endo—pg can degrade methylated
pectin substances.

Both Endo-pg and PME can be found in the middle lamella
approximately two cells in advance of the Infection hyphae
(74) suggesting that these enzymes are produced by the
infection hyphae. The middle lamella was already altered
one to two cells in advance of the §g_§g1gngtigngm hyphae In
apple (Malgs spp.) infections Indicating the role of these
two enzymes in facilitating infections. In later stages of
Infection the PME probably works In concert with Exo-pg.

Cellulases and hemicellulases are also produced by St
sglgggttgngm (18, 70, 74). Cellulase Is abundant In
diseased tissue and appears to provide the pathogen with
nutrition via cellulose breakdown as well as to facilitate
cell Invasion (70). The disease severity of a tissue has
been associated with the. cellulase levels. Cellulose
content of diseased tissue declines as the tissue is
degraded (70).

Phosphatidase is another enzyme that is produced early
in the Infection process (71). It is capable of hydrolyzing
phosphorus-containing components of the cell wall. The
enzyme Is induced by calcium and the optimum pH for activity
is 4.0.

Oxalic acid has long been associated with St

W Infections (35, 80, 96) and Is believed to

17

cause many of the changes that occur in the host tissue
during pathenogenesis. The pH of healthy bean tissue is
approximately 6 to 7 while the margins of advancing lesions
have a pH close to 4.0 (50. 72, 74, 80. 86). The drop in pH
has been correlated with the presence of oxalic acid (80).
The lower pH favors the activity of the polygalacturonases,
cellulases, hemlcellulases and phosphatidase (50. 51, 70,
71. 74).

A synergistic relationship between oxalic acid and
polygalacturonases has also been found with Wm
MI, (15). Oxalic acid chelates calcium ions which
otherwise would also Inhibit the activity of both Endo- and
Exo-pg (15. 50. 51) and tissue maceration. The removal of
calcium from the calcium-pectate complexes renders them more
susceptible to polygalacturonase degradation (15, 80). The
lowering of the pH and the chelation of calcium Ions may
account for the cell death In advance of the pathogen (75).
Calcium oxalate crystals have been found In xylem vessels
(73) and may be associated with wilt symptoms (75). Oxalic
acid also stimulates the activity of PME (75).

Maxwell and Lumsden (80) showed a positive correlation
between the disease severity In bean tissue and the level of
oxalic acid. An Isolate of mm which produced
slow disease development produced less oxalic acid than an
isolate which rapidly Infected bean hypocotyls. The

isolates were from different hosts.

18

Oxalic acid treatment of sunflower (flgttantngg_gnnggg

L.) shoots produced wilt symptoms Identical to those
produced by St_sglgngttgzum.infections (94). Wilted leaves
had 15 times more oxalic acid than healthy leaves. Oxalic
acid moved through the xylem and the xylem sap pH was one
unit lower than normal, 2 cm ahead of lesions. The fact
that oxalic acid accumulates In the leaves Indicates the
mobility of this compound within the plant.

Physiological resistance to W has been
noted In soybeans (23. 27, 45) and 205519.13: species (41.
109. 111) and Is characterized by the development of
restricted, reddish brown lesions. The Interaction of
pathogen produced enzymes and the structural components of
the tissue has not been studied. Dow and Lumsden (41) found
that penetration of the cuticle of resistant Ehaggglus
ggggtngug occurred less often and was more difficult than in
more susceptible genotypes. Upon penetration the infection
hyphae were smaller than normal and rarely developed beneath
the cuticle where rapid growth normally occurs. They
concluded that the resistant tissue slowed the infection
process and thus allowed the plant to actively respond to
the pathogen.

As polygalacturonases and oxalic acid have been
correlated with virulence (74. 121), it Is of Interest to
review evidence on whether tissue can be resistant to enzyme
degradation and If such resistance could account for

differences In host-pathogen compatibility.

19

W Kuhn produces an enzyme complex
similar to Sg_ggtgnttgumm (14). In bean hypocotys Infected
with W. calcium was found to accumulate within
lesions and In the tissue surrounding a lesion (14). This
Indicates an active tissue alteration In response to the
pathogen. The calcium Inhibited polygalaturoanase activity
and the oxalic acid chelated the calcium. Bateman (14)
hypothesized that the calcium accumulation liberates the
host PME from the cell walls. The PME then demethylates
pectic substances exposing carboxyl groups which have a high
affinity for calcium Ions. The calcium Ions become strongly
complexed with the pectic materials and thus Inhibit
polygalacturonase activity and limit lesion growth.

The tissue of bean hypocotyls becomes resistant to Rt
ggtgnt with Increasing age (16, 17). The odder tissue Is
characterized by changes in cell wall polysaccharide
composition (91). changes In the pectic materials (16) and
higher calcium content (16). The older tissue was more
resistant to maceration than younger tissue (16) and the
older cell walls were more resistant to enzmme degradation
than younger cell walls (17). This evidence shows that
tissue can vary in resistance to the enzyme complex. The
age-related changes have not been conclusively linked to the
resistance of the older tissue though the changes would seem
to play an important role In slowing the Infection process.
Bateman et al.(17) reported that young hypocotyls can also

rapidly limit lesion growth which indicates a response

20

beyond the age-related changes. Phytoalexin accumulation
was also found In response to the infections. The authors
concluded that lesion limitation was probably the result of
a combination of Induced cell wall alterations and
phytoalexin accumulation.

Resistance to Rg_gglgnt is associated with cell wall
maturity in bean hypocotyls. Within a species the sequence
of events leading to maturity does not vary though the time
needed to reach maturity can (91). Among three Eg_1ulgantg
cultivars the time required to attain maturity varied by
four days. The slower maturing cultivars were more
susceptible to Rg_§glgnt. This was not a very large test
but It does suggest that cultivars that mature faster may be
more resistant. This resistance could be due to the
age-related changes already covered.

Stockwell and Hanchey (120) found Increased calcium
levels In lesions caused by Rg_gglgnt on Eg_yulggntg. More
calcium accumulated In the older. more resistant tissue.
The authors failed to find increased calcification of the
cell walls at the lesion border. They concluded that the
calcium accumulation may have been cytoplasmic and that an
Increase In phenolics In the lesion tissue may have been
responsible for lesion limitation. Cultivar responses were
not Investigated.

Cell suspension cultures from sunflower cultivars
resistant or susceptible to St_gglg£gttgnum have been tested

for reaction to oxalic acid (94). The cultures derived from

21

resistant cultivars were also more resistant to cell lysis
by oxalic acid. As the middle lamella had already been
dissolved this Indicates that the resistance was In the cell
wall or the cytoplasm.

Calcium accumulations have been noted In other
host-pathogen Interactions. Hamond and Lewis (49) found
calcium accumulations and ultrastructural changes In the-
leslons produced by W on W
stems. The increase in calcium was associated with an
accumulation of a lignIn-like material. The changes often
occurred well ahead of the hyphal front. The calcium was
mainly deposited on the cell walls. In the expanding phase
of the Infection the hyphae grew through these
calcium—lignln barriers and even when lesion growth ceased
the hyphae were still several cells away from the completely
lignified cell walls. The authors concluded that while the
changes may slow hyphal growth, they could not account for
the cessation of growth. Since little calcium accumulated
In the middle lamella. It Is doubtful that It became more
resistant to polygalacturonase degradation. They proposed
that the changes were an attempt by the host to
compartmentalize the Infection and that the effect of the
calcium could be the result of other metabolic functions.

In W f. sp. mm Infections of
squash (W spp.), calcium accumulated In the lesions

and was deposited on the cell wall and the middle lamella

22

(52). Despite this, the calcium did not appear to slow the
rate of tissue maceration.

LignIn accumulation In response to fungal Infections Is
well documented (106. 129). Vance gt_a,]_._( 129) reported that
heavy lignificatlon Is an Induced host response to fungal
pathogens In resistant reactions In W L.,

W L. var. am 0.0.. mm L. and
Cucumis species. In general lignificatlon Is thought to

Impede hyphal growth. It makes tissue more resistant to
compressive forces and enzyme degradation. Ride (106)
hypothesized that the lignificatlon may prevent the
diffusion of pathogen produced toxins from the inlnediate
Infection site and that Its phenolic precursors may be toxic
to the pathogen. It is unclear whether lignificatlon Is the
primary cause of resistance or a result of resistance.

The role of calcium and lignin In lhniting .5;
gglgngttgzum infections has not been studied. The fact that
W: tissue can show a physical resistance to S_._
aglgngtjmm Infections (41) Indicates that the tissue Is
somehow resistant to the pathogen. In this system Is not
known whether the resistance Is due to a passive or Induced
tissue difference or due to some other host response. The
evidence from other host-pathogen systems which Involve
similar enzymes suggests that tissue alterations occur in
response to a pathogen and that these changes are more
pronounced In incompatible reactions. The role of the

alterations are unknown.

23

Soybeans and E. yulgacis produce phytoalexins In
response to Wm Infections (122). The type of
phytoalexin produced varied with the Inoculated tissue
(leaves or hypocotyls) and with the Inoculum type
(ascospores or mycelia). Ascospore Inoculation of bean and
soybean leaves resulted In hypersensitive reactions. High
concentrations of phaseollin or phaseollldin accumulated In
the bean leaves while no phytoalexins were detected in the
soybean leaves. In addition no inhibitors of fungal growth
were detected In the soybean leaves using bioassays. There
remains a possibility that the phytoalexins accumulated in
only the few hypersensitive soybean cells whereas the assay
was run using the entire leaf thus possibly diluting the
phytoalexin to a concentration too low to detect.

Bean leaves Infected with mycelium did not contained
detectable phytoalexins and had spreading lesions. Bean
hypocotyls contained only kievitone 24 hours after
Inoculation and the lesions maintained a steady growth rate
over a 100 hour period at 18° C . The rate of growth was
much slower at 28° C and it ceased after 42 hours.
Phaseollin was detected and kievitone levels were
significantly higher at 280 C. The concentrations of these
two compounds further increased from 42 to 100 hours.
Soybean leaves accumulated low levels of glyceollin but
soybean hypocotyls were not tested. The soybean leaf
lesions were similar to the bean leaf lesions. The various.

phytoalexins produced were tested for their effects on the

24

growth of Wm ascopores and mycelia In Him.
Phaseollin was the most toxic while glyceollin was the least
toxic.

The levels of phaseollin and kievitone In the bean
hypocotyls at 28°C would be expected to suppress fungal
growth based on the In yitng assays. The glyceollin levels
In the soybean tissues were never very high though the
concentrations may have been higher In the cells adJacent to
the Infections. These higher concentrations could be toxic
to the fungus. The role of the phytoalexin In resistance Is
uncertain. The concentration of phytoalexin at the hyphal
tip must be shown to be high enough to cause the cessation
of fungal growth.

Red clover (Intjgltum__gngtgngg L.) produced
phytoalexins in response to Sg_t;11gltg;um.Infections (37).
Pterocarpans were found to accumulate in the lesions. The
lesions on all the cultivars tested continued to spread but
the rate was slower on the two cultivars which consistently
accumulated higher pterocarpan levels.

Phytoalexin production has been associated with
resistance to several fungal diseases (79). Resistance to
W var. aging In soybeans has been
associated with glyceollin accumulation. This association
was found whether resistance was due to a gene- for-gene
response, a light-induced response or an age- related

response .

25

Sutton and Deverall (122) showed that soybeans and
beans produce phytoalexins In response to Wm
Infections that are fungitoxic and that In beans. these
phytoalexins accumulate to toxic concentrations. Both crops
produce phytoalexins In response to other pathogens (17,
79). The evidence indicates that the phytoalexins play an
important role in limiting infections. Differences In
phytoalexin accumulation among cultivars of a species may be
a determining factor In differences In compatability. It is
probable that tissue alterations and phytoalexins work In
concert in response to pathogen Invasions. The nature of the

Interaction In unknown.
AVOIDANCE AND RESISTANCE

To minimize the losses from Wm researchers
have attempted to Identify resistant genotypes which could
be employed In a breeding program. There have been two
different approaches to this problem. One approach has been
to develop genotypes that avoid becoming Infected by 3_._
gglgmttgmm. The other approach is the development of
genotypes that are physiologically resistant or tolerant to
Infections.

There have been many reports of disease avoidance in
W species. The principle of avoidance is that
certain plant architectures create a microclimate which Is
not conducive to the Infection processes. White mold

severity was found to be lower In wide rows versus narrow

26

rows and in determinate versus indeterminate Great Northern
isolines (116). This indicated that the microclimate could
be controlled to reduce disease severity. Coyne gt
31,431) noted that mechanical or chemical alteration of a
cultivar’s architecture influenced the disease severity.
They found that dense, compact canopies resulted in a higher
disease severity than upright. open plant canopies. Plants
with dense canopies developed more humid microclimates which
favored the disease. An ideotype was proposed for
avoidance. The ideal type would be upright, determinate or
short Indeterminate, fewer main stems. long internodes,
fewer and shorter branches and snail trifoliate leaves.
These traits enhance air movement through the foliage and
lead to conditions that are unfavorable for disease
development.

Their observations of the effects of the more open
canopy on the nflcroclimate were confirmed in later studies
that showed that temperatures were cooler and that leaves
remained wet for longer periods of time when plant canopies
are dense and compact (19, 132). Plant architecture can
also Influence the production of primary inoculum. Steadman
gt al.(IIO) found fewer apothecia beneath open canopied bush
beans than under viney, dense canopied beans.

Later studies have changed the ideotype somewhat.
Determinacy In Itself does not appear to be very Important
as a determinate pinto bean. ’Ouray’, remained susceptible

(13, 32). Further tests on nearly Isogenic determinate and

27

indeterminate lines showed no difference in apparent
susceptibility (32). Schwartz gt al.(III) suggested that the
height of the canopy from the soil was a key component to
avoidance. Other researchers have found that early maturing
lines avoided the disease better than late maturing lines
(32, 43). This suggests that a plant can avoid infection by
either plant architecture, maturity or a combination of
both.

It is difficult to study and determine the extent of
disease avoidance In the field because of the confounding
factors such as avoidance through plant maturity or
architecture and physiological resistance. For example
’Black Turtle Soup’ bean cultivar has a dense, viney
indeterminate growth habit and as such should be quite
susceptible to white mold. But ’Black Turtle Soup’
apparently possesses a physiological trait that confers
resistance (13, 32, 33). To further complicate field tests
of avoidance, it has been found that the plant architecture
of the adJoinIng rows influences the disease severity in any
given row (43). These problems as well as an apparently low
heritability for disease avoidance (narrow sense -.12) (12)
indicate that selecting for this trait In a breeding program
will not be very efficient.

In soybeans there have been no studies on disease
avoidance. Results of row width studies (47, 136) on disease
development suggests that an avoidance Ideotype could be

effective. But with the trend to plant soybeans in narrow

26

rows it is doubtful whether this strategy will be accepted
In soybean production.

The second approach is to find sources of physiological
resistance and Incorporate them through a breeding program.
This resistance would be expressd as an ability to prevent
or limit infections to non-lethal proportions. This could
be accomplished by preventing any of the steps in the
Infection process after the Inoculum arrives at a suitable
site.

In both soybeans and Ehasgglu: species there have been
mentions of physiological resistance from field studies. In
soybeans, Grau and BIssonette (45) noted differences In
susceptibility among seven cultivars. In later studies
cultivar differences were again found significant (46, 47).
Among the cultivars that were tested over more than one
year, ’Gnome’ appeared susceptible while ’Corsoy’ and
’Hodgson’ and ’Hodgson 78’ appeared tolerant to the disease.
Lockwood and Isleib (68, 69) have also found significant
differences among cultivars. Of the cultivars tested In over
years, Gnome and ’Weber 84’ were susceptible while ’Corsoy
79’, ’Hardln’, Hodgson 78, ’Pella’ and ’Evans’ were
tolerant. Studies from Virginia (98) and Brazil (136) have
also noted differences In resistance among soybean
cultivars.

The field studies suggest differences In physiological
resistance, but other factors may be confounded In the

results such as maturity and plant architecture avoidance,

29

Interrow effects (43) and inoculum distribution. Lockwood
and Isleib (68) noted that the disease was variable in
locations within a test. The distribution of inoculum of at
m was shown to have a significant effect on the severity
of leaf drop In lettuce (39, 63). Grau and Bissonette (45)
noted that Corsoy, and to a lesser extent ’Chippewa 64’,
could restrict Infections to localized, reddish brown
lesions which indicated a physiological reaction to an
Infection. The localized lesions did not result in the
death of the portions of the plant above them.

Laboratory and greenhouse research can remove many of
the confounding factors of field studies. In an early
greenhouse study, 14-day-old soybean seedlings were
Inoculated on wounded cotyledons with mycelia grown on
potato dextrose agar (45). Steps were taken to keep the
inoculations moist. All cotyledons were destroyed but In
some cultivars the infections did not spread beyond the
cotyledon In all plants. In general the results matched
their field results and Corsoy even developed red,
restricted lesions as In the field.

A different technique was employed by Lockwood and
Isleib (67, 68). Stem sections were cut from greenhouse
plants and inoculated In the laboratory by placing mycelial
plugs on unwounded tissue at the apiacl end of the stem.
The length of the resulting lesion was then measured. There.
were escapes and variability within a cultivar, but despite

this, significant differences were found among genotypes.

30

Gnome and Weber 84 developed longer average lesion lengths
than Corsoy. This techique and a modification of it has
been used to screen plant Introductions and several have
been Identified as being more resistant than Corsoy (68,
69).

Cline and Jacobson (27) compared several greenhouse
inoculation techniques. Ascospores were used as Inoculum
and were either sprayed on flowering plants or on flower
blossoms that were then placed in leaf axils. In other
techniques, mycelium was grown on carrot root discs or
celery petioles. The carrot discs were placed on the leaves
while the celery pieces were placed on the stem of the
soybean plants between the cotyledon and first trifoliate
node. The celery pieces were then removed after 24 hours.
In all experiments the environment was controlled to
maintain conditions optimal for Infection. The celery
petiole technique, called limited term Inoculation (LTI) and
originally developed by Hunter gt gl.(60), produced the most
uniform results. The ascospore Inoculations were quite
variable as infected blossoms often fell off. Placing the
flower blossoms In the leaf axils reduced the number of
escapes and increased disease severity. The LTI method
produced significant differences among the cultivars tested.
’Elf’, ’Evans’ and ’Wells II’ developed more severe symptoms
than Corsoy, ’Wllliams’ and ’UnIon’. Gnome also developed
more severe symptoms than the more resistant cultivars

though the difference was not significant. All three of the

31

resistant cultivars developed restricted, reddish brown
lesions though not on all of the tested plants of a given
cultivar. The cause of the variation within a cultivar was
not determined. The disease severity was Influenced by the
age of the plants and the light Intensity of the growth
enviroment.

The LTI technique was used by Boland and Hall (23) to
evaluate soybean cultivars. They obtained variable results
and concluded that several trials were needed to make solid
conclusions. They noted variability within a cultivar and
the appearance of the reddish brown, restricted lesions.
The restricted lesions were more common In the more
resistant cultivars.

In all the above-mentioned assays there remains
variation In the ratings of resistance Including variation
within a cultivar. There Is also a possibility of escapes.
Nevertheless the results matched, to some extent the field
observations of resistance, indicating that these techniques
may be useful Indicators of physiological resistance.

Physiological resistance has also been noted In
Ehggggtug species In field studies. Black Turtle Soup (13,
32, 33), ’Charlevoix’ (33, 109), a red kidney bean and
’Valentine’ (109), a snap bean, have shown physiological
resistance In the field. All appear to restrict Infections
to reddish brown lesions though this ability was diminished
In Valentine (109). The restricted lesions were rarely

found in the most susceptible genotypes. Black Turtle Soup,

32

Valentine and Charlevoix all produced the resistant reaction
In growth room conditions (111). Several Eg_ggggtngu§ lines
have been noted to exhibit physiological resistance In the
field (33, 115, 117). .

An early greenhouse study was conducted by Adams gt
gt.(8). Cultivars were tested by Inoculating 10 day old
bean plants with mycelium Infected oat seeds. Of the 180
genotypes tested nine appeared to exhibit resistance. All
Eg_ggggtngg§ lines were resistant.

Abawi ,gt yal.(5) sprayed flowering plants with
ascospores and placed the plants In a mist chamber.
Resistance was noted and the Inheritance investigated
utilizing ascospore inoculation. The F1, F2, 8C1, 8C2 and
parents from a cross of W cultivar ’Bush Blue
Lake’ x W; were evaluated for resistance. The
data pointed to resistance being controlled by a single
dominant gene. The authors stated that the resistance was
associated with the blossom tissue though no reasons were
given. Eg_ggggtngg§ is also resistant to Infections from
mycelial Inoculations (4, 8) which Implicates some factor
Involving stem tissue.

In a study comparing various Inoculation techniques the
method of Abawi gt 31,45) produced Inconsistent results
(59). This was attributed to differences in the ages of
flowers and to flowers falling off the plant. The technique
was modified by removing one-day-old blossoms, spraying them

with ascospores and placing them In leaf axils. The results

33

of this method were still variable with escapes occurring.
The results were more consistent than before and several
lines previously considered resistant by Abawi gt_g]_.(5)
appeared .susceptible. This clouds the conclusion that
resistance may be controlled by a single dominant gene (5).
Further modifications led to the development of the
limited term Inoculation (LTI) technique (60). Mycelium
infected celery pieces were attached to the stems of four
week old soybeans between the cotyledonary and first
trifoliate node. The best results were achieved when the
Inoculum was removed after 24 hours. During Inoculation the
bean plants were kept In a growth chamber under conditions
favorable for Infection. The plants were then placed on a
greenhouse bench and rated for disease severity four days
later. This technique was less variable than using
ascospores and significant variation among cultivars for
disease severity was detected. Some variability within
cultivars remained and escapes were possible. The results
were similar to field observations with Bush Blue Lake being
susceptible and Wing”: lines being resistant. Plant
age and the light Intensity of the growth environment had an
effect on disease severity. A similar comparison of
techniques In soybeans produced the same results (27).
The LTI technique was used to evaluate plant
introductions for resistance to white mold In mm,

(60). A line was considered resistant if 50% of the plants

34

survived. This Illustrates the variability of the assay.
Despite this 13 of 310 lines exhibited resistance.

Dickson gt__gl.(38) screened three segregating
populations for resistance to white mold. Population 1
consisted Of 19 F2 families generated from 4-way crosses
among susceptible E, 2919;“: lines. Population 2 was
produced from eight crosses between Intermediate resistant x
Intermediate resistant and Intermediate resistant x
resistant Eg_1ulggntg and Eg_ggggtngug lines. Population 3
was generated from 21 combinations of 10 E;_ggggtngu§ lines
resistant to white mold. In all populations F2 families
were tested in the F3 generation. In Populations 1 and 2
the survival rates when subJected to LTI treatment were only
0.8% and 2.0% respectively. Population 3 had a 3.8%
survival rate. The survivors of Populations 1 and 2 were
advanced to the F4 and retested. The combined survival
rate of the two populations was 17%. Population 3 was not
tested In the F4. The authors concluded that repeated
selection Increased the resistance of the population. This
probably occurred through selection for and accumulation of
additive resistance genes from the various susceptible
lines. While not tested they felt that the heritability of
this trait was high enough for efficient selection based on
the Increase In the percentage of survivors In one
generation.

The Inheritance of the physiologlal resistance has been

Investigated In several field trials. Tests of the

35

segregating progeny from a cross between a resistant, late
maturing, viney Black Turtle Soup bean and a susceptible,

early, viney Great Northern bean Indicated the the
Inheritance is quantitative with a low heritability (33).
Early maturing, resistant recombinants were recoverd.

Limited populations derived from selfed plants in 8C1 and

8C2 of a cross between W and Em

segregated In a pattern Indicative of a single dominant gene
controlling resistance (33). Escapes could have affected
these results.

In another study, a half diallel was constructed
between three resistant and three susceptible mm
W lines (44). Fifteen F2 populations and the six
parent lines were tested In the greenhouse, where Inoculum
was evenly distributed, and In the field where the effect of
the adJacent row (43) was eliminated with control rows. The
results showed no cross x environment effect or parent line
x environment effect. There was less disease development In
the field which was attributed to possible avoidance. This
has also been noted elsewhere (109). Avoidance was not. a
factor In the greenhouse. The results were the same in both
environments with GCA effects being highly significant
Indicating additive gene action. Heterosis only accounted
for 5% of the genetic variation. There was a suggestion of
partial dominance in one of the resistant lines. While the
results were the same In both environments the authors noted

that this does not Indicate that the same genes are Involved

9

36

in both environments as the field resistance could be due to
avoidance. It Is Interesting that the three resistant lines
may have different genes controlling resistance.

Resistance In 2g_1g1gantg appears to be a quantitative
trait (33, 38, 44) while resistance In Eg_ggggtngu§ appears
to be controlled by a single dominant gene (5, 33). While
some of the results have been questioned (59), it remains
possible for the two species to have different genetics of
resistance. Walker (131) reported that In some cabbage
(W L.) lines, resistance to Win
W Is controlled by a single dominant gene while in
other lines resistance is quantitatively Inherited.

There have been several attempts to use crude filtrates
of Wm cultures or oxalic acid In
potential screening methods. ’Ex Rico 23’ a cultivar of
white beans that had been reported to be resistant to white
mold In the field (127) had a lower disease Incidence and
smaller lesions Including a smaller water soaked area than
susceptible cultivars. Oxalic acid has been. noted to
produce the typical wilt symptoms of a Wm
Infection (35, 39, 94, 96). By Imerslng the petioles of
excised leaves In [MCI-labeled oxalic acid, the rate of
oxalic acid diffusion through the leaf was followed (128).
Oxalic acid diffused slower In the resistant Ex Rico 23
than In the susceptible cultivars. Furthermore the labeled

oxalic acid was restricted to the main veins In Ex Rico 23

37

whereas a uniform distribution was found in the suceptlble
cultivars. No other resistant cultivars were checked.

A crude enzyme filtrate of a Sg_§glgngttgnmm culture
was used to test sunflowers for resistance to Infection
(58). The filtrate mimicked field symptoms and cultivar
differences were detected for reaction to the filtrate.
There was variability within a cultivar for the reaction.
The results were not compared with any field study so the
method’s ability to predict field reactions is unknown.

Sunflower cell suspension cultures have been tested for
tolerance to oxalic acid (94). Cultures were generated from
cultivars that had been rated as tolerant or susceptible
based on field and greenhouse studies. The cultures from
the resistant cultivars showed a higher tolerance to oxalic
acid than the cultures from the susceptible cultivars as
measured by cell lysis. The tissue had already been
macerated to release the cells so the tolerance must be due
to the properties of the cell wall or the cytoplasm, not the
middle lamella.

Blanchette and Auld (21) used a heat stable element
from a crude culture filtrate and oxalic acid to Induce
field symptoms of mm. Non-host crops did not
developed symptoms. The ability of the technique to
distinguish cultivar differences was not reported.
Resistance of alfalfa (mm L.) to Wm
has been tested by spraying seedlings with oxalic acid

(107). While the spray induced symptoms of the disease It

38

was concluded that reaction to oxalic acid was not
correlated to field reactions to the pathogen. The other
oxalic acid assay mentioned in this review were performed by
Immersing stem or petiole ends in an oxalic acid solution.
It Is possible that spraying oxalic acid on the leaf surface
did not simulate the ‘way the plant normally encounters
oxalic acid (I.e. via subcutlcular production from hyphae)
and therefore did not test for resistance to the normal
Infection process.

This author has tried to cover primarily St
sglgpgttgngm in soybeans and Engsggtgs species. But it Is
still Interesting to note that sources of resistance have
been found in other crops such as alfalfa (107), peanuts
(28, 99), lettuce (77), safflower (anthgmg§__ttngtgntg§

L.)(88), peas (Wm L.)(20) and sunflowers (94,
95, 104).

SUMMARY

Sg_§glg;gttg;um causes Sclerotinia stem rot in soybeans
and white mold In MIM- Both diseases can cause
considerable crop loss. Sclerotinia stem rot usually
appears in soybeans when they follow a more susceptible crop
In a rotation. The disease spreads from field to field
mainly by windblown ascospores and infected seed. The main
reason for the recent reports of an increase in the-
incidence of Sclerotinia stem rot appears to be the spread

of soybean cultivation Into lareas *where the environment

39

favors the disease and where susceptible crops are
traditionally grown.

The disease overwinters as sclerotia that survive In
the soil and In the stalks of diseased plants. Survival is'
Influenced by the depth of burial', soil moisture, and the
presence of microparasltes. Sclerotium numbers In a field
Increase primarily by means of their production Inside an
Infected host.

Sclerotia undergo carpogenic germination In the spring
and early summer when the temperature and moisture
conditions are favorable. The sclerotia produce apothecia
which In turn produce ascospores. The ascospores are
ejected Into the air and may land on the plant surfaces.
Ascospores are the primary form of Inoculum and can survive
on the plant surfaces for approximately two weeks.

Mycelium from germinating ascospores colonize senescent
tissue, primarily blossom tissue. The pathogen needs to
colonize this Intermediary tissue as an energy source so It
can infect stem or leaf tissue. The energy is necessary for
the formation of Infection cushions and successful cuticle
penetration which apparently occurs by mechanical means.
Ascospore germination and host penetration require free
moisture for extended periods of time. Cooler temperatures
(200 C) also favor Infections.

Upon entering the host, the pathogen produces Infection
hyphae which grow Intercellularly between the cuticle and

the epidermis to form an Infection front. Behind the front

40

the Infection hyphae give rise to smaller ramifying hyphae
which grow Inter- and Intra-cellularly. The pathogen
produces enzymes and oxalic acid which precede the Infection
hyphae by one or two cells. This enzyme complex alters and
degrades the pectic materials of the middle lamella and cell
walls and facilitates fungal growth. Other enzymes are
later produced which digest cellulose and cell- contents.
Oxalic acid reproduces disease symptoms when applied alone
to host tissue, and tolerance to oxalic acid has been
correlated with Wm field resistance. The
quantitative production of polygalacturonase and oxalic acid
has been correlated with the virulence of an Isolate.

Plants escape lethal Sg_gglgngttgnum,Infections through
avoidance of the disease and/or by physiological means. In
Eg_yulgant§ avoidance has been attributed to differences In
maturity and through modified plant architecture which
alters the microclimate so It Is less favorable for the
Infection process. Physiological resistance has been found
in Eg_1ylggntg, Eg_§g&glngu§ and soybeans and Is expressed
by restricted, reddish brown lesions. This trait has been
studied in the field and with greenhouse and laboratory
techniques. Field studies of resistance can produce results
which are confounded with avoidance, escapes, uneven
inoculum distribution and Interrow effects.

The Inheritance of physiological resistance has been.

studied in was and W. In mum-.11 the

41

resistance appears to be polygenic while the resistance in
ngggingug appears controlled by a single dominant gene.
Physiological resistance In Eg_ggggtngu§ Is expressed by
a resistance to Infection and the ability of the host to
restrict hyphal growth. Phytoalexins are produced In
soybeans and 2g_yu1gantg In response to Sg_gglgggttgnum, At
the present there have not been any specific studies
performed on the actual causes of resistance to St

W.

PROPOSED RESEARCH

For a resistance assay to be of use to a breeder it must
produce repeatable results, correlate with field resistance
and be sensitive to difference among cultivars. Many of the
reports of laboratory and greenhouse studies on resistance
covered in this review report variation within a cultivar
for resistance to Sg_gglgngttgnum (20, 26, 41, 45, 58, 60,
61, 67, 94). This variation contributes to the error term
and lowers the sensitivity of the test to genotypic
differences. The cause of the variability has not been
determined. Except for the filtrate and oxalic acid tests
the variation could arise from escapes. Other possible
causes could reside In the techniques themselves, in the
growth environment of the materials to be tested and

residual genetic heterogeneity within a cultivar for

resistance to W.

42

The studies reported hereafter were conducted to
develop an Improved screening technique which lowers the
variability In response to the disease within a cultivar.
An Improved assay would Increase the selection efficiency of
a resistance breeding program. The cause of the residual
variability was Investigated so that It could be eliminated
or reduced. A

The starting point for modification of the assay was
the technique of Lockwood and Isleib (69). It was familiar
to cooperative researchers and had the advantage over other
assays of ease, speed, controlled environment and a

quantitative assessment of disease development.

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Schwartz, H.F., J.R. Steadman and D.P. Coyne. 1977.
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smuerotInIa_scIerotIocwm. Rep. Bean Improv. Comm.
20:71-71.

Schwartz, H.F. and J.R. Steadman. 1978. Factors
affecting scierotlum populations of, and apotheclum

production by. SclerotInIa_scIerotIorum. Phytopath-
68:383-388.

Schwartz, H.F, J.R. Steadman and D.P. Coyne. 1978.
Influence of Enasggtg§_yglgants blossoming

characteristcs and canopy structure upon reaction to

SclerotInIa_scIerotIorume Phytopath 68:465-470-

Slgnoret, P.A., P.C. Bernaux and B. Polnso. 1975.
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Singh, U.P. and R.B. Singh. 1983. The effect of soil
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on carpogenic germination of Sglgngttnta
gglgngtlgngm. ZPP 90:662-669.

Smith, V.L., PunJa, Z.K. and S.F. Jenkins. 1986. A
histological study of Infection of host tissue by

SclerotIum_noIstI. Phytopath 76:755-759.

Starr, C.H., H.J. Walters and C.H. Bridgemon. 1953.
White mold (Sglgngttnta) of beans. Wyo. Agric. Exp.
Sta. Bull. 322.

Steadman, J.R, D.P. Coyne and G.E. Cook. 1973.
Reduction of severity of white mold disease on Great
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Steadman, J.R., D.P. Coyne and H.F. Schwartz. 1974.
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Steadman, J.R. 1975. Nature and epidemiological
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Steadman, J.R., C.R. Maier, H.F. Schwartz and E.D.
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120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

53

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132.

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137.

54

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DEVELOPMENT OF AN ASSAY FOR RESISTANCE TO
W INFECTION 0F SOYBEAN

I NTRODUCT I ON

WW (Lib.) De Bary is the cause of
Sclerotinia stem rot In soybean [film (L.) Merr.l.
This disease has been reported in many soybean producing
states (5, 6, 11; 13, 17, 31). The disease Is not
widespread and is more prevalent In cool, wet areas (13, 14,
32) and when soybeans follow a more susceptible host In a
crop rotation (11, 14, 24). The disease can be quite
devastating and causes considerable yield losses (13, 14,
19). Cultivars that are resistant to Sclerotinia stem rot
could minimize the losses from this disease.

Resistance has been reported among soybean cultivars
In the field (4, 11, 18, 24, 32) and in greenhouse and
laboratory experiments (4, 6, 11, 17, 18). In field studies
the expression of physiological resistance or susceptibility
can be confounded with disease avoidance (27), inoculum
distribution (8, 16), and interrow effects (10).. Greenhouse
and laboratory studies minimize these problems but may
require considerable space, growth and/or mist chambers, may
be labor intensive and escapes are still possible. The
laboratory method of Lockwood and Isleib (17) offers the
advantages of requiring little space, having a quick and
easy Inoculation procedure, a constant Inoculation and
Incubation environment between experiments, and offers a
quantitative assessment of disease development. At present
the method lacks the sensitivity to distinguish the
difference between the closely related lines that would need

55

56

to be screened in the advanced stages of a resistance
breeding program.

The following experiments were undertaken to improve
the technique of Lockwood and Isleib (17) by Identifying the
causes of the residual variability and either eliminating

them or accounting for them In the experimental procedures.

MATERIALS AND METHODS

All the plant material was grown In the greenhouse
under natural and supplemental light (16 hrs. day, 8 hrs.
night). The plant age varied by experiment as some
experiments required longer stems. The ages of the plants
used In the experiments are summarized in Table 1 along with
the experimental design used In the experiments. The plants
were grown In 20 cm diameter clay pots containing a 5:3:2
mixture of sandy loam soil, sphagnum peat and torpedo sand.
In the experiments where the plants were grown for more than
45 days, each pot received a fertilizer treatment consisting
of 4 gm Rapid-Gro Plus fertilizer (23-19-17) in 1000 ml of
water. Four plants were grown In each pot. Each pot was
randomly assigned one genotype. All pots were randomly
placed 130 cm below the light source on the greenhouse bench
such that the pot rims touched each other.

The assay was performed on stem sections Inoculated in
controlled laboratory conditions. In Experiments 1, 2, 3, 6
and 22 the stem sections were prepared from the top 15 cm of

the main stem. The leaves and petioles were then cut from

57'

 

Table 1. The experimental design used In Experiments 1
through 22.
Stems per
EXP. 1.80. of trtmnt/rep Inoculations Plant
nmme—mwm
-- days --
I RCBO 3 4 1 30 16 genotypes
2 RCBO 3 4 I 30 9 genotypes
3 R080 3 4 I 30 2 x 9 factorial
(2 Inoculation site preparations by 9 genotypes)
4 RCOO SP 3 6 3 74 4 genotypes
5 RCBO SP 2 2 7 79 5 genotypes
6 RCBO 3 4 1 39 9 genotypes
7 CRO unequal I 1 50 3 genotypes
8 CRO 21 1 1 40 5 genotypes
9 RCBO 3 12 I 38 5 genotypes
10 RCOO 3 12 1 38 5 genotypes
II RCBO 3 12 1 38 5 genotypes
12 R080 3 5 1 37 5 x 3 factorial
(5 durations of inoculation by 3 genotypes)
13 RCOO 2 4 1 58 5 genotypes
14 CRO unequal 1 1 60 4 genotypes
15 RCBO 3 unequal 1 47 5 genotypes
16 R080 SP 2 3 5 60 2 genotypes
17 CRO 4 1 4 42 2 genotypes
18 RCBO SP 2 4 4 58 5 genotypes
19 CRO unequal 1 4 36 4 genotypes
20 CRO 5 I 4 72 4 genotypes
21 RCBO 2 5 1 60 2 x 2 factorial
(2 media concentrations by 2 genotypes)
Wanna! 1 1 W

 

‘1' R080 - randomized couplete block design, 0120 I coupletly randomized
design, RCBO SP = randanized camiete block design with a split plot.

58

this 15 cm stem section. The sections were then placed In
plastic bags and transported to the laboratory. In
Experiments 6 and 22 the top 0.5 cm was removed to create a
wound. In Experiments 4, 5 and 17 the uppermost 24 cm, 51
cm and 38 cm respectively were cut and trimmed for study.
In Experiments 7 through 15 and Experiment 21, the stem
sections were prepared as follows: one cut was made 1.5 cm
above the node bearing the uppermost fully expanded
trifoliate leaf. The second cut was made 15 cm below this
node, thus producing a 16.5 cm section. In the laboratory,
Immediately before Inoculation, a final cut was made
perpendicular to the length of the stem through the node
which bore the uppermost trifoliate leaf. The final cut
produced a 15 cm stem section for testing. ‘The stem
sections were Inoculated on this freshly cut surface. The
stem sections for Experiment 16 and Experiments 18 through
20 were prepared as follows: The top 24 cm of the main stem
was removed and discarded. A second cut was made below this
cut to produce stem sections of 51 cm for Experiments 16,
19 and 20, and stem sections of 38 cm for Experiment 18.
All the stem sections were Inoculated and Incubated In
clear plexiglass boxes (56 x 53 x 8 cm), to maintain a humid
environment for disease development. A 4 cm layer of
‘medium grade vermiculite was placed In each box. The
vermiculite was then moistened with 1500 ml of distilled
water. The stem sections were then randomly assigned to

locations within the box.

59

All experiments were performed In a room cooled to
approximately 20° C. The temperature did flucuate upwards
to 24°’ C during the day.

The inoculum for all experiments was grown on 2% millet
seed medium. The millet seed was ground In a wiley mill
with a 20 mesh screen. The Inoculum for an experiment was
started by placing a .5 by .5 cm plug of medium and mycelium
from another culture In the center of a 15 cm diameter petri
dish of medium. The dish was then sealed with Parafllm and
Incubated for 5 days In darkness at ambient room
temperatures (approximately 24" C). Plugs of Inoculum were
cut with a 0.4 cm cork borer at a distance of 4 to 6 cm from
the center of the dish. Approximatly 60 plugs were prepared
per dish. Before removalof the plugs, the agar surface was
covered with a 0.3% water agar solution. The plugs of
inoculum were removed from the dish with a dissecting needle
and were placed mycelium side down on a particular location
on a stem section. In Experiment 21, 0.3% millet seed media
was tested.

In Experiments 1 through 4 and on half the stems in
Experiment 5, the Inoculations were made on Intact tissue.
In Experiments 1, 2 and 3 the Inoculum was placed on the
area of the stem Just below the uppermost node. In
Experiments 4 and 5 multiple Inoculations were made per stem
section. In Experiment 4, three Inoculations were made
approximately 6.2, 12.0 and 18.0 cm from the apical end of

the stem section. In Experiment 5 seven inoculations were

60

made approximately 6, 12, 19, 25, 32, 38 and 45 cm from the
apical end of the 51 cm stem section. In Experiments 6
through 15 and Experiments 21 and 22 the Inoculum.was placed
on the freshly cut apical end of the 15 cm stem section.

In Experiments 16 through 20 and on half of Experiment
5, all Inoculation sites were first coated with clear nail
polish. The nail polish was allowed to dry and then
carefully peeled from the site with a dissecting needle and
fingers. This was termed the wax removal (WR) technique.
In Experiments 16 through 20 there were multiple
Inoculations per stem section. In Experiments 17 and 18
four Inoculations were made approximately 7, 16, 24 and 32
cm from the apical end of the 38 cm stem section. In
Experiments 19 and 20 four Inoculations were made
approximately 10, 22, 32 and 44 cm from the apical end of
the 51 cm stem section. In Experiment 16 there were 5
Inoculations approximately 8, 18, 25, 34 and 44 cm from the
apical end of the 51 cm stem section. No inoculations were
made on nodal tissue In any of the multiple Inoculation
experiments.

Lesion lengths was measured from the point of
Inoculation to the point of the lesion where the epidermal
tissue was no loner easy to peel off with the edge of a
plastic ruler. In the multiple Inoculation experiments the
lesions grew in both directions on the stem. In these
experiments lesion length was measured as the distance

between the two leading edges of the lesion.

61

Two different methods of inoculation were used In
Experiment 3. Half of the stems were Inoculated on Intact
tissue. The Inoculation sites of the other half of the
stems were first treated with chloroform. The Inoculation
site (same site as described learlier for experiment 3)
received two quick wipes with a cotton swab soaked In
chloroform. The site was then wiped with distilled water
and allowed to dry. The Inoculum was then placed on the

site as before.
RESULTS
Inoculation on Intact Stem Tissue

Inoculations on Intact tissue (Experiments 1 - 5) often
failed to produce infections. The escapes were given a
value of zero, resulting In a high error term. In one
experiment the lesion lengths ranged from 0 to 11.5 cm for
’Weber 84’ and from 0 to 7 cm for ’Gnome’. The percentage
of Inoculations that resulted In Infections varied among
experiments and among cultivars within an experiment (Table
2).

The difference among experiments In the percentage of
Inoculations producing infections was highly significant
<P< .001. x2 = 107.9, 4 df). The experiment totals could be
Influenced by the cultivars Involved in a‘ particular
experiment. Only Experiments 4 and 5 Involved the same set

of cultivars and as such were directly comparable.

62:

Table 2. The Incidence of infections from Experiments 1
through 5.

 

 

 

Experiment 1 2 3 4 5

Date 6/10/85 8/21/85 10/2/85 4/5/86 4/10/86 % of
Plant age 30 days 30 days 30 days 74 days 79 days total

41+

Gnane 4 8 11 I 7 5 19 35 1 27 35.6
PI 297514 0 12 5 7 3 9 I9 35 0 28 22.9
Corsoy 1 II 7 5 6 6 12 42 0 28 22.0
Weber 84 II 1 8 4 5 7 8 46 0 28 27.1
Wells II 2 10 4 8 9 3 - - - - 41.7
century 4 8 9 3 6 6 - - - - 52.8
Corsoy 79 4 8 4 8 9 3 - - - - 47.2
PI 358314 3 8 7 5 3 9 - - - - 36.1
Evans 2 10 - - 8 4 - - - - 41.7
Ozzie - - 9 3 - - - - - -
t of total

F3?

MIME—203 59-1—51‘L—J2—Je9

I I - Number of Inoculations resulting In Infections. NI - Number of
inoculations not resulting in Infections.

63

Experiment 5 was conducted with plants left over from
Experiment 4 and which were five days older. The space
between the Individual plants was Increased In Experiment 5
as compared to Experiment 4 which Increased the light
Intensity on the stems and may have resulted In a thicker
cuticle (23). Experiment 5 had fewer Infections than
Experiment 4 which could have been due to the thicker
cuticle being a more effective barrier to infection.

As not all cultivars appeared In all experiments, the
cultivar totals In Table 2 could be Influenced by the varing
conditions of the experiments In which a cultivar appeared.
By creating a subset of the data In Table 2, unequal effects
were removed from the margin totals which allowed a more
extensive analysis of experiment and cultivar effects on the
incidence of Infection (Table 3). The results of Experiment
5 were so different from the other four experiments that It
was excluded from the data subset.

There was a highly significant effect of experiments
(P< .001.3Xz = 26.4, 3 df) on the incidence of Infection.
Experiments 1, 2 and 3 varied only by the time of year when
the plants were grown, suggesting that the growth
environment Influenced the Incidence of Infection. In
Experiment 4 there were multiple inoculations per stem with
some Inoculations made on older tissue than In the other
three experiments. The older tissue may have had a thicker

cuticle than the younger tissue which could account for the

64

Table 3. Disease Incidence in four cultivars over four
experiments.

 

 

 

Experiment X2 for differences
among experiments
1 2 3 4 Total minim
-------------- % Infected------------
Corsoy 8.3 58.3 58.3 22.2 30.0 11.1 *
Gnome 33.3 91.7 58.3 35.2 45.5 14.2 **
PI297514 0.0 41.7 25.0 35.2 30.0 6.6
ygpgr84 91.7 6&7 41.7 1448 35.5 31.5 11*
Total % 33.3 64.6 45.8 26.8 35.3 %
27.7** 6-8 3-0 8-3* Xz for differences among

cultivars within an_gxpgtlmgnt
* and ** denote significance at the .05 and .01 alpha levels

respectively

low* Infection rate of Experiment ‘4. Experiment. 4 used
74-day-old plants, while 30-day-old plants were tested In
the other three experiments. Despite this age difference
the incidence of infection was not significantly different
(P) .05, X2 = .79, 1 df) between Experiments 1 and 4.

In Experiments 1 and 4 there was a significant cultivar
effect on the Incidence of infection. Houiever, the two
experiments were different with regard to the ranking of the
cultivars. Weber 84 had the highest Incidence of Infection
In Experiment 1 and the lowest Incidence of infection In
Experiment 4. The differences In the ranking of cultivars
between experiments suggested a cultivar by experiment
Interaction. This Interaction seemed to prevent a
repeatable cultivar influence on the Incidense of infection.

A Chi-square test on cultivar totals, pooled over

65

experiments, showed that the differences in the Infection
rates among the cultivars was no greater than expected by
chance alone (X2 = 6.9, 3 df)

The second problem with the technique of inoculating on
the intact cuticle was that the resulting Infections were
not all initiated at the same time. On the second day of
Experiment 3, 6.5% of the stems had become Infected. By the
fifth day, 51.9 % of the stems were Infected. At the end of
the experiment the stems that were Infected on the second
day had longer lesions than the stems which became Infected
afterwards (Table 4). This contributed to variability
within a cultivar. Only four of the nine cultivars In
Experiment 3 had Infected stems on day 2 and are Included in

Table 4.

Table 4. Comparison of the final average lesion lengths
from Experiment 3 (10/2/85) between stems infected by the
second day and stems which developed lesions after the
second day.

 

Average lesion length for Average lesion length for

 

 

 

stems with lesions on stems developing lesions
m We Difference
-------------------------- cm ———----——-------——--—-—_--
Weber 84 7.2 3.9 3.3
PI 297514 7.0 1.3 5.7
Corsoy 79 8.0 3.8 4.2
PI 358314 8.0 3.0 5.vi

 

In Experiment 4, data were collected on lesion

development for ten days after inoculation. The data for a

66

particular day was pooled over all cultivars and the average
lesion length of a particular day was calculated only from
the lesions that. were initiated on that day» .Not all
cultivars were equally represented In the average lesion of
each day as not all cultivars developed a new lesion on each
day. New Infections were Initiated on each of ten days.
While the average lesion length for each day was Influenced
by the cultivars developing infections on that day, there
was still a strong trend of longer lesions with earlier

Infection (Fig. I).

Inoculation on Sites Where Either the Cuticle or the

Cuticular Waxes Were Removed

The role of the cuticle In preventing and delaying
infections was Investigated In Experiments 3 and 5. In
Experiment 3, a total of 108 stems from nine cultivars were
Inoculated on the Intact cuticle. Another 108 stems from
the same cultivars were treated with chloroform to remove
the cuticle (M. Bukovac, personal communication). The
chloroform treatment also wounded the underlying tissue as
shown by scarring of unlnoculated control stems treated with
chloroform.

Only 51.9% of the 108 untreated stems became infected
five days after Inoculation while 100% of the treated stems
developed lesions. After two days, 93.5% of the stems

treated with chloroform were already Infected.

average lesion length (cm)

67

 

 

 

 

25
average leelon length H

S of total lesions initiatul H I.”

zo-I so
'0
+70 %
2
‘5‘ ‘0 e-
n
c
o
POO '5
.9.
104 40 :3
*0
+00 "6
R

5. i-20

I40

0 I T fl
1' 2 i I 5 I 7 I o 10

day of experiment

Figure 1: Cumulative percent of lesion initiation
and average lesion length of lesions Initiated on
a particular day of Expenrnent 4.

68

In Experiment 5, half of the Inoculations were made on
the Intact cuticle and the other half were made on sites
where only the cuticular waxes had been removed with clear
nail polish (M. Bukovac, personal communication). This
treatment also caused a slight wounding of the underlying
tissue by removing the trichomes. This wounding was
considerably less than caused by the chloroform treatment.
Of the 140 Inoculations on the Intact cuticle only 1 caused
an infection while 129 of 140 (92.1 %) Inoculations of

treated tissue caused an infection.
Inoculation on Wounded Stem Tissue

The chloroform treatment of the stems created a wound
and resulted in a more uniform Incidence of Infection. An
Inoculation technique concurrently under study by Dr. J. L.
Lockwood produced wounds and was adapted as It was a faster
and easier method of circumventing the cuticle barrier.
Experiments 5 and 22 were performed using this technique.
The apical bud was removed 'from the stem section and the
inoculations were made on this site. Infection rates of
100% were achieved utilizing this technique.

Lesions were measured every 24 hours (Table 5). All
stem sections were Infected after 48 hours. Variation In
average lesion lengths at 48 hours was not large either
among genotypes or among the stems within a genotype with
the exception of Weber 84 In the Experiment 22. This

Indicated that uniform infection occurred.

69

Table 5. Summary of lesion length data 48 hours after
Inoculation from Experiment 22 (10/28/85) and Experiment 6
(11/15/85).

 

 

 

 

Experiment 6 Experiment 22
Average Standard Average Standard
WW
..... cm -_--- ----- cm ----
Gnome .5 -.9 .7 .12 .6 -1.0 .8 .13
P1297514 .2 -.8 .5 .18 .3 - .8 .6 .13
P1358314 .3 -.8 .6 .13 .2 -1.1 .7 .25
Weber 84 .6 -.9 .7 .13 7 -4.4 1.7 1.05
Corsoy .3 -.9 .5 .17
Corsoy 79 .4 -.7 .5 .08
Wells II .5 -.8 .7 .09
Century .5 -.7 .6 .09
sztgL. .5 -.9 17, .111

 

Further disease development was variable. Some of the
lesions on the young tissue ceased growing and dried out,
often at the first node encountered. This led to
variability within a cultivar. The Inoculation technique
was altered by making the final cut through the middle of
the node which bore the uppermost fully expanded trifoliate
leaf and then Immediately Inculating non this cut. This
avoided the young meristem tissue and provided a large
surface on which to place the Inoculum. This technique was
termed wound Inoculation (WI).

Lesion initiation with the WI technique was uniform as
in Experiments 5 and 22. Further growth continued in a
fairly uniform fashion within a cultivar but significant
differences In average lesion lengths developed among

cultivars. Several experiments were conducted to determine

70

If there were repeatable differences .mmong cultivars In
their ability to limit or slow lesion growth (Table 6).
Only data from stems Inoculated via the WI technique and
with inoculum grown on the standard medium Is Included In
Table 6. The experiments varied by size, date (and thus the
environment In which the plants were grown), plant age, and
the length of the experiment as shown by the duration of
Incubation.

The cultivars Gnome and Weber 84 were tested because
they were reported to be susceptible to $g_§glgngttgnmm In
field trials (12, 13, 18, 19) and In previous greenhouse and
laboratory tests (6, 18). Corsoy was selected because It
had expressed resistance in field trials (12, 13, 19) and In
greenhouse and laboratory tests (6, 11, 18). The plant
introductions were selected because both had exhibited
greater resistance than Corsoy In laboratory trials (18).

Several trends were apparent from these experiments.
The most obvious was that Gnome developed the longest
average lesion in eight of the nine tests. PI 297514
developed the shortest average lesion In six of the nine
tests.

A more general trend was a grouping of the cultivars as
"susceptible" and "resistant”. The more susceptible
cultivars were Gnome and Weber 84. The more resistant
cultivars were Corsoy, PI 297514 and PI 358314 which always
developed a shorter average lesion than Gnome or Weber 84.

The difference was significant (P< .05) except for the

71

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a N.m a c.« so o.m 1 a n.m A A.m 0A c.» 1 1 mouuoo
a s.@ 1 A 5.3 a.c.c~ a <.- a A.o~ As a.» a c.- a h.m so uerz
A o.o u ~.~ a A.s A N.“ u m.o~ p ~.h 0 A.~ A n.» A n.A «Anson no
a n.o a A.AA a N.A~ a ~.on a A.- a n.c~ a u.m 0 ~.- a m.m 08060
as
mass u asap 5 saws 5 shop A mass A shop A when n shop a shop A coauaaauocw
. mo cowueuso
asap he uses cc asap hm uses an mass mm shop on mass on shop as uses on one ocean
owxwuxm omxmuxo Am\N\A oo\n~\~ AA\MN\~ Amxcn\~ AA\<N\~ nmxnnxuu mo\n~\- open
AA «A n— NA AA o~ o m A ucoawuooxu

 

. 335.03 35

dowuaasuopw p::o:.oAu mduuqauus aucaflauoouo Baum nuns Aumcou defined ounuo>a up huuladm .A OHAaH

72

comparison between Gnome and Corsoy In Experiment 15. In
Experiment 9, the difference between PI 358314, Corsoy and
Weber 84 was not significant. In Experiment 15 the
difference between Corsoy and Weber 84 was not significant.
PI 297514 always developed a significantly shorter average
lesion than Gnome or Weber 84.

The rankings within the resistant group varied by
experiment. PI 297514 had the lowest average lesion length
when data from all the tests were pooled and always
developed a significantly shorter average lesion than the
two susceptible cultivars. This Indicated that PI 297514
was probably the most resistant cultivar tested. Table 7
contains the average values of the statistics In Table 6
pooled across all experiments. All five cultivars appeared
In the same experiment five times (Euperiments 9, 10, 11,
13, 15). The average lesion lengths from these five

experiments are also Included in Table 7.

Table 7. Averages of the statistics In Table 6 pooled
across all experiments.

 

 

__Imm_l.:aian__
all experiments

MW
nnnnnnnn a -o------
Range 3.1 cm Gnome 10 4 10.5
L.S.O.o. 5 .82 Weber 84 9 9 9.9
Range / £.S.O. 3.73 Corsoy 8 6 8.9
Pi 297514 / Gnome 75.6 t PI 358314 8 1 8.0
2.50 on P1 297514 7 9 8.0

Gnome-P1297514
W

A

73

An assay’s ability to detect significant differences
among cultivars Is determined by an assay’s ability to
discriminate among cultivars (i.e. range of values) and Its
sensitivity to cultivar differences (L.S.D. or C.V.). The
value In Tables 6 and 7, calculated by dividing the
range by the L.S.D. expresses the range In units of
significant difference. This value Incorporates the
discrimination and sensitivity of an experiment In one
descriptive term. The value would be Influenced by the
the technique used In an experiment, the conditions of the
experiment and the material tested. The range/L.S.D. showed
that Experiment 12 was the most sensitive test of cultivar
differences even though it did not produce the lowest
coefficient of variability or the widest range of values.
PI 297514 and Gnome appeared In all tests so the range
between these cultivars divided by the L.S.D. was presented
as a comon check for all experiments. This value again
showed Experiment 12 to be the most sensitive test.

The WI nwthod only measured a stem’s ability to slow
lesion growth. Resistance was not expressed In any stem of
any cultivar as no lesions stopped growing and all the
lesions would have been lethal to a plant.

The WI method was modified In an attempt to increase
the sensitivity of the W1 method and allow for an Increased

expression of resistance.

74
Limited Duration of Inoculation

One change was the duration of Inoculation. An adapted
form of limited term Inoculation (6, 15) was used In
Experiment 12 where plugs of inoculum were removed after a
specific time and replaced with plugs of 2% water agar. The
control treatment was inoculum left on for the duration of
the experiment (146 hours). In a later study It was found
that the 2% water agar plugs .had. no effect on lesion
development.

Durations of inoculations of 32, 46 and 146 hours
essentially behaved the same as reflected In the number of
escapes, the average lesion lengths and the sensitivity of
the tests (Table 8). Durations of Inoculations of 10 and
23 hours were characterized by stems escaping Infection and
higher error rates. The Incidence of escapes among the
cultivars at 10 and 23 hours was not significantly different
(Xz’= .48, 2 df). When escapes were omitted from the data
set, the average lesion length for 10 and 23 hours were
similar to those of the longer durations. While there was a
significant difference between the average lesion lengths
omitting escapes among durations of Inoculations, this
significance was not be repeated In another experiment.

Shorter durations of Inoculations resulted In more
escapes but did not increase the range between the
susceptible and resistant cultivars. Shorter durations of

inoculations resulted in a reduced sensitivity to cultivar

75

'rabnie 8.

Summary of the effect of duration of Inoculation

on lesion development, Experiment 12 (2/23/86).

 

 

 

 

Duration of Inoculation (hours) Overall
10 23 32 46 146 average
ELI” t A.L. A.L. A.L. A.L. Igstgn_
Gnome 4.9 a 10.2 a 10.1 a 0.5 a 9.7 a 11.7
Weber 84 4.9 a 10.0 a 10.3 a 0.3 a 9.3 a 10.0
PI 297514 4 3 a 5.2 b 7.5 b 7.8 b 6.3 b 7.0
Overall average 4.7 8.5 9.3 9.5 8.4
lesion
CV (%) 94.5 23.3 11.4 1.1 20.9
I"Si-0.05 4.6 2.8 .8 1.0 1.5
Range (cm) .6 5.0 2.8 2.7 3.4
Range / L.S.D. .1 1.8 3.4 2.8 2.3
We 19 3 0 J L
W
----- cm ----—
Gnome 8.1 b 10.2 a - - -
Weber 84 10.5 a 10.0 a - - -
PI 297514 6.4 c 6.4 b - - -
Overall average
lesion omitting 8.1 b t 9.1 a 9.3 a 9.5 a 8.4 b

___£§£§2£S

Means within a column with the same letter are not significantly

different at alpha = .05.

‘1 A.L. = average lesion length

$-Means within this row with the same letter are not significantly

different at alpha = .05.

76

differences. The incidence of escapes was not Influenced by
cultivar and therefore did not appear to be an increased
expression of resistance between cultivars. There was no
significant interaction of cultivar by duration of

Inoculation on average lesion length.
Lower Concentration of Nutrients In the Inoculum Medium

The concentration of the Inoculum media was lowered In
an attempt to weaken the pathogen. In Experiment 21
inoculum was grown on the standard 2% millet seed medium and
on a medium containing only .3% millet seed powder (Table

9).

Table 9. Summary of the effects of Inoculum media nutrient
concentration on the incidence of infection and on lesion
length, Experiment 21 (4/30/86).

 

 

 

 

Average lesion Average lesion
per medium Cultivar with escapes Cultivar
concentration average omitted average
2.% .3 % lesion .¥_2 t .3 % Igstgn_
-------------------------- cm —----—---—----—-—---—-
PI 297514 4.9 2 9 3.9 5 4 5.4 5.6
Gnome 7.6. .4.0 5.8 7.6 6.4 7.1
average lesion 6.2 a 3 5 b 6 5 a 5.9 a

PI 297514 1

Gnqm: 0

Means within a row with the same row with the same letter are not
significantly different at alpha = .05.

A01

The lower nutrient concentration of the medium produced

a significantly lower incidence of Infection than the 2%

77

standard medium (X.L == 8.5, 1 df) Indicating that the more
dilute medium may have weakened the pathogen and allowed the
host to better resist Infection. PI 297514 had six escapes
while Gnome had four though this difference was not
significant (XL = .53, 1 df). When the escapes were
omitted from the data set, the average lesion lengths
resulting from the two concentrations were not significantly

different. There was no significant interaction of cultivar

by concentration on average lesion length.

Inoculation on Stem Tissue Where the Cuticular Waxes Have

Been Removed

In Experiment 5, the wax portion of the cuticle was
removed from the inoculation sites. Inoculations on these
sites often resulted In restricted lesions. The restricted
lesions were initiated at approximately the same time as
normal spreading lesions but ceased growing at a length of
approximately 1 cm or less. Often a restricted lesion would
not spread beyond the 0.5 cm diameter scar caused by the wax
removal. The restricted lesions ranged In color from a
lIght buff to a rusty red, became dry by the end of an
experiment, did not girdle the stem and would not have been
lethal to the upper portions of an intact plant.

Several experiments were performed to Investigate the
potential of the: wax removal (WR) technique as an

Improvement over the WI method (Table 10).

78

Table 10. Sumary of the wax removal experiments.

 

Experiment 5 16 17 18 19 20

Date 4/10/86 4/30/86 5/12/86 6/2/86 6/20/86 6/11/86
Plant age 74 days 60 days 42 days 58 days 72 days 60 days
Duration of

 

 

 

WWW
Cll
Gnane 7.7 a 5.3 a 8.2 a 5.0 a 5.7 a 10.5 a
PI 297514 4.2 bc .6 b 6.0 b 3.6 b .6 b 4.1 b
Corsoy 3.2 cd 2.6 be 4.8 a 2.0 b
PI 358314 2.0 d 1.9 c .4 b 3.1 b
M84 4.7 b 3.7 b
cv (a) 58.4 82.3 21.3 47.9 93.4 47.9
L'S'D'OJIS 1.36 1.26 1.09 1.24 1.70 3.18 ¢
Range (cm) 5.7 4.8 2.1 3.1 5.3 8.5

W.A.—2.8L

Gum-'P1297514 305 ‘08 201 104 501 604
P1297514/Gnome 54.5% 10.55 73.7% 72.0% 10.53 39.1%
WEI—.25 3-9 43.0 1-1 EL—ZJL.‘

 

 

Means within the same column with the same letter are not significantly
different at alpha - .05.

‘l Gnm . Gnane, 297 - PI 297514,

* Experiment 20 had unequal replications.1his L.S.D. Is calculated
using the rep X genotype line from the ANOVA.

£3 The L.S.O. Is the appropriate one for cmparing Gnane to P1 297514.

79

The ranking of the cultivars by average lesion length
under WR conditions was similar to that produced by the WI
method. Gnome and Weber 84 always developed longer average
lesions than the three "resistant“ cultivars (Corsoy, PI
297514 and PI 358314). Differences between of average
lesions of the two plant introductions and Gnome were always
significant. The difference between the average lesion
lengths of Corsoy and Gnome was not significant In
Experiment 19.

Weber 84 appeared In only two of the six WR tests and
the difference between the average lesion length of Weber 84
and any of the three resistant cultivars was only
significant when comparing Weber 84 with Corsoy and P1
358314 In Experiment 5 and with P1 358314 In Experiment 13.
Weber 84 had a significantly shorter average lesion than
Gnome in both tests. The results of the WR method would
place Weber 84 In the resistant group though Weber 84 was
not extensively tested.

Despite the high coefficients of variability of the WR
experiments, the results were repeatable with the trends
established in the WI method, with the exception of Weber
84, being repeated (Table 11).

A direct comparison of the two methods Is not valid due
to unequal representation of cultivars between methods and
the different environmental conditions In which the plants
were grown for each experiment. The following observations

are made under the assumption that the confounded factors

80

Table 11. Results of the wound Inoculation (WI) and wax
removal (WR) methods of Inoculation pooled across all
experiments.

 

Wax removal,

 

 

Wound Wax lethal lesions
WI only
CV 14.40 % 59.17 %
Range (cm) 3.1 5.0
L.S.D. 0.05 .82 1.63
Range / L.S.D. 3.73 3.09
PI 297514/Gnome X 100 75.60 % 43.26 %
Gnome - PI 297514 2.50 3.74
Gngmg;£l_292514 / L.S.D. .3 04 .2.30
W
............... cm -----—-—--—
Gnome 10.4 6.7 7 4 (i38)'f
Weber 84 9.9 4.2 5.2 (48)
Corsoy 8.6 3.1 4.2 (67)
PI 358314 8.1 1.9 3.8 (55)
PI 297514 7.9 3.0 5 3 (86)
Susceptible =t-(Gnm, W84) 10.2 6.0
Resistant (Cor, 297, 358) _§t2 2.2
Difference 2.0 3.3

 

'? The number in parentheses Is the number of lethal lesions that
developed across all experiments.

Gnome,
PI 358314.

1* Susceptible or resistant based on WI results. Gnm
W84 = Weber 84, Cor = Corsoy, 297 = P1297514, 358

81

did not greatly influence the statistics in Table 11. Due
to the occurrence of restricted lesions, the WR method
produced a larger coefficient of variability than the WI
method and was less sensitive to differences among cultivars
despite producing a wider range of values between the
average lesions of the susceptible and resistant groups.

The frequency and distribution of restricted lesions
from the WR experiments are summarized In Table 12.
Experiment 17 was omitted as It did not produce restricted
lesions. The absence of restricted lesions may have been
due to the young, succulent plant material. Four of the
five experiments had a significant cultivar effect on the
distribution of restricted lesions at alpha 8 .05 and all
were significant at alpha = .08. The cultivar totals In
Table 12 contain unequal experiment contributions and are
not comparable. An analysis of cultivar and experiment
effects was performed on a subset of data from Table 12
which omitted data from Experiment 16 and Weber 84 (Table
13).

The effect of cultivars on the incidence of restricted
lesions was significant In Experiments 5, 19 and 20. A Chi
square test of the distribution of restricted and
non-restricted lesions among cultivar totals pooled over all
experiments was highly significant (X2' = 40.2, 3 df ) while
a Chi-square test among the three resistant genotypes was-
not, indicating that it was the difference between the three

resistant genotypes and Gnome that was responsible for the

82!

Table 12. Summary of the incidence of restricted and
non-restricted lesions In the wax removal (WR)
experiments.

 

Experiment 5 16 18 20 19
Date 4/10/86 4/30/86 6/2/86 6/20/86 6/11 86
Plant age 74 days 60 days 58 days 72 days 60 days
Duration of
Inoculation 4 days 6 days 4 days 5 days 7 days Total

 

 

Gnome 0 28 6 24 5 27 3 17 0 '16 11.1
Weber 84 1 27 - - 11 21 - - - - 25.0
Corsoy 7 21 - - 7 25 8 12 11 9 33.3
PI 297514 3 25 25 5 10 22 18 2 8 16 47.8
__£1_3§8314 8 .20 - - 14 18 17 a. .14 14 .jatfi
Total % R 13.5 51.7 29.4 57.5 37.5 33.4
X2 for cultivar
effects within 15.5 as 24.1 as 7.2 32.1 as 14.3 as

W03-

! and is denote significance at the .05 and .01 alpha levels
respectively.

7 R - the number of restricted lesions NR - the number of
non-restricted lesions

Table 13. summary of the Incidence of restricted and
non-restricted lesions of a subset of the data in Table 11.

 

 

 

X2 for
Experiment 5 18 20 19 Total experiment
+
Gnome 0 28 5 27 3 I7 0 16 8.3 7.2
Corsoy 7 21 7 25 8 12 11 9 33.3 7.5
P1297514 3 25 10 22 18 2 8 6 37.5 32.8 as
21358311__fl__20 14 18 17 a 14 14 49.1 15.4 gg____
% R 15.9 28.1 57.5 37.5 32.6
X2 for
genotype 10.9 I 7.1 32.1 as 14.8 as
effects

 

a and Ni denote significance at the .05 and .01 alpha levels
respectively.

t R s The number of restricted lesions, NR . The number of
non-restricted lesions

83

significance in the overall Chi-square test. This Indicated
that there was a relationship between the WI average lesion
lengths and the ability to limit an Infection to non-lethal

dimensions.
Inoculation on Stem Tissue Varing in Age

Through the use of multiple Inoculations per stem, the
effect of tissue age on the nature of an Infection was
studied. The position of an Inoculation on the stem had a
significant effect on the Incidence of restricted lesions
and on lesion length In all WR experiments. Older tissue,
as designated by higher position numbers, developed shorter
average lesion lengths (Fig. 2) even when the data from
restricted lesions was removed from the data set (Fig. 3).
Experiments 18, I9 and 20 were comparable when the data for
Weber 84 were removed. In these experiments Gnome developed
a longer average lesion than the three resistant genotypes
at all four positions on the stems (Fig. 4).

The position of the inoculation had a significant
effect on the appearance of restricted lesions within the
resistant cultivars but not with Gnome (Table 14). The
sample size for Gnome may have been too small to detect
significance. Of the Gnome stems, 77.7% developed a lethal
lesion at the lower positions while only 57.0 % of the

resistant stems did so.

average lesion length (cm)

84

 

HExp.18
HExp.17
HExp.18
H519.18
HExp.20
HExp.5

 

 

 

 

 

1 W f r l
2

3 4 5
position on stem

Figure 2: Average lesion length by position.

 

85

 

11111
551%:

  

average lesion length (cm)

‘1!

 

 

i 5 i 6

position on stem

Figure 3: Average lesion length with restricted
lesions omitted from the data set.

 

average lesion length (cm)

86

 

  
  
 

e—e Gnome
H Pl 297514
H Pl 358314

8- H Corsoy

 
 
 
 
 

\l
1

a:
l

 

 

 

   

 

l I

 

ll 2 3
position on stem
Figure 4: Average lesion length"? 2cultivar from
Experlgrnents 18 1

87

Table 14. Summary of the Incidence of restricted lesions by position
from data combined from Experiments 18 - 20.

 

Resistant
Percentage of inoculations
resulting in restricted lesions 11.8 46.9
Percentage of Inoculations Position 1 5.9 36.8
resulting In restricted Position 2 5.9 36.8
lesions analyzed by Position 3 11.8 43.9
position Position 4 23.5 70.1
X2 for position effects on the 3.4 13.2 **
distribution of restricted lesions
Percentage of stems with lethal 77.7 57.0

_J_eai_an.LaLeJ_therp081tions 3 or 4
1' The resistant genotypes are Corsoy, PI 297514 and PI 358314.

4* denotes significance at alpha = .01 level

88
D I SCUSS I ON

The results Indicated two reasons for the low
sensitivity of the assay when Inoculating on intact tissue.
The first problem was that not all Inoculations resulted In
Infections. In two experiments, genotype had a significant
effect on the Incidence of infection but the pattern could
not be repeated. The cause of the Interaction between
cultivars and experiments is unknown. The interaction
prevented a repeatable cultivar effect the Incidence of
infection and contributed to error both within and between
experiments.

The second problem was that Infections were not
uniformly initiated when Inoculations were made on the
Intact cuticle. The later an infection was initiated, the
shorter the resulting lesion was. Delayed Infections and
escapes led to variability within a genotype thus raising
the error term of the experiment and lowering the
sensitivity of the assay to differences among cultivars.

The cuticle was a barrier to Infections, though not
necessarily the cause of resistance. Clrcumventing the
cuticle allowed for 100 % Infection rates and a highly
uniform Initiation of lesions among all stems. Removal of
only the cuticular waxes resulted In a significant Increase
In infection rates, suggesting that the wax components of
the cuticle may play a key role In preventing Infections.

These conclusions and particularily the role of the wax

89

components must be tempered by the fact that the removal of
the whole cuticle or Just the wax layers was always
confounded with some degree of wounding of the underlying
epidermal tissue which In Itself would facilitate infection.
However, histological studies (20, 21) Indicate that
unwounded epidermal tissue would not be likely to resist an
infection.

The development of a cuticle is Influenced
quantitatively by the greenhouse environment with high light
intensity, high temperatures and low humidity favoring the
formation of a thicker cuticle and wax portion of the
cuticle (23). Variation In these factors probably account
for the experiment-by—cultivar Interaction observed ‘when
Inoculating on the cuticle. Whether cuticle and/or wax
differences could account for variability for resistance to
Infection within a species Is debated (22). Variation In
cuticle and wax properties exist between species (3, 20) but
the variation within a species has not been extensively
studied.

There appears to be differences among Ehgggglus species
for resistance to penetration by mm with P_,_
W showing greater resistance than W (9,
29) though not necessarily a lower Incidence of Infection.
The greater resistance was thought to be the result of a
physical characteristic of the tissue. Even the ability to
delay an Infection could be Important. As environmental

factors must be favorable to Initiate the infection process

90

(2), delays In the completion of an infection could allow
for environmental changes to occur which may be less
conducive to Infection or for the depletion of the nutrients
supporting Infection. Evidence from this study Indicated
that the ability to resist Infection was Influenced by the
environment. It may be worth further study to develop more
sensitive methods for evaluating the effect of the cuticle
and wax components on Infections to see If there exists
useful genetic variation among soybean cultivars for the
ability to resist or delay Infections.

The WI technique corrected the problems associated with
inoculating on intact tissue. Inoculating on the node which
bore the uppermost fully expanded trifoliate leaf and
trimming the stem Immediately before inoculation nunimized
the problems of inoculation site deslcation and Inoculum
plugs falling off the stem sections that occurred with
Inoculations on the apical bud wound. Also most Infections
in the field occur low In the plant canopy (5, 12, 26), so
Inoculations on a lower node were more representative of the
tissue likely to become Infected than inoculations on the
apical end of the stem. The WI method resulted In uniform
Initiation of Infections and Increased variation among
lesion lengths over time would be due to variation In the
growth of the pathogen through the stem tissue.

The grouping of cultivars by average lesion length was-
repeated in nine WI experiments conducted over a six month

period on plants grown In various environments Indicating

91

that the WI technique produced reliable results. The WI
technique grouped Gnome and Weber 84 as “susceptible" and
Corsoy, PI 297514 and PI 358314 as “resistant“. This
corresponds to field observations of the susceptiblity of
Gnome (4, 12, 13, 18) and Weber 84 (18, 19) and the
resistance of Corsoy (12, 13, 19). The results also match
those of other greenhouse and laboratory ,experiments for
Gnome (6, 18) and Corsoy (6, 11, 18). The behavior of Weber
84 was variable In a previous laboratory test (18). The
plant Introductions have not been tested in field conditions
but had been found to be more resistant than Corsoy In
previous laboratory experiments (18). Thus the WI technique
appeared to be an accurate predictor of field resistance.
More genotypes should be tested In the field and by the WI
method to confirm this relationship. It should be noted
that field ratings of disease severity may not always be
accurate indicators of physiological resistance due to the
confounding factors of avoidance and inoculum distribution.
Few soybean cultivars have been thouroughly tested for
resistance In trials conducted over years and with
consideration to the confounding factors that may be
confused with the expression with physiological resistance.

The WI method only measured a cultivar’s ability to
slow lesion growth. Check cultivars must be used with this
method as the significance of the results were relative to a
susceptible and resistant cultivar Included In each test.

Complete resistance was not expressed as all the lesions

92

would have been lethal to a plant. The conditions of the WI
test favored the pathogen In the host-pathogen interaction
and may have precluded the expression of complete
resistance. Under the WI conditions It Is possible that a
moderate or strong resistance Is overcome leaving the slower
lesion growth as a manifestation of Its expression.

Through the WR technique, restricted lesions developed.
Similar lesions have been noted In soybean (6, 4, 11), L
ygtgantg (26, 27) and Eg__ggggtngg§ (9, 26) genotypes
resistant of Sg_§g1gngttgzgm Infections. The Incidence of
restricted lesions was Influenced by genotype. The
cultivars rated resistant by the WI method developed
significantly fewer lethal lesions than Gnome. Thus the WI
results appeared to relate not only to field results but
also to an ability to restrict an Infection to non-lethal
porportlons. Despite the Increased frequency of restricted
lesions In the resistant cultivars, all cultivars developed
some lethal lesions. The increased levels of resistance in
some cultivars appeared to only lead to a lower probability
of a lethal lesion. Resistance was Influenced by the
environment as shown by differences among experiments in the
Incidence of restricted lesions.

Inoculations made on older tissue resulted In fewer
restricted lesions than inoculations made on younger tissue
and the spreading lesions that did occur were shorter than
the spreading lesions on younger tissue. The reduction in

the Incidence of restricted lesions on older tissue was more

93

pronounced in the resistant cultivars. This Indicated that
the resistant cultivars were not only less apt to develop
lethal lesions but also that the lethal lesions were less
likely to occur low on the stem where they would result In
more damage. More extensive testing Is needed to see If
there Is genetic variability for this ability to minimize
damage from a lethal infection.

Attempts to further Improve the sensitivity of the WI
test failed. Shorter durations of Inoculation or Inoculum
grown on weaker medium both resulted In a higher Incidence
of escapes. The lesions that did occur were no different
than those achieved via the standard W1 procedures. There
was no cultivar effect on the Incidence of escapes and
therefore the modifications did not Increase the ability of
the assay to discriminate among cultivars. The
modifications influenced the Infection process, apparently
favored the host In general, but they did not appear to
increase the expression of resistance in the resistant
cultivars and did not influence further lesion growth. The
result of the modifications were a higher error term and a
lower sensitivity to cultivar differences.

The lower concentration of nutrients In the medium
resulted In significantly fewer infections probably by
weakening the pathogen. Nutrition has been linked to the
ability of Sg_sglg;gttgngm to successfully Infect a host (1,
15, 28, 29). In the field the pathogen uses flower tissue

to supply the nutrition .for infection (2). This suggests

94

that If there existed genetic variation for the nutrient
content of flower tissue, then this variation could be used
to breed cultivars with flower tissue that could lower the
Incidence of Infections. Schwartz gt_a_]_.(27) found no
difference among nine lines of WALLS and W
for blossom resistance to ascospore colonization. The
ability of the blossoms to support Infections was not
tested.

The WI method still developed a degree of unexplained
variability between the stems of a cultivar as shown by the
average coefficient of varibllity of 14.4 %. This
Indicated that the test would not be accurate on single
plants and will require the testing of several progeny to
evaluate a line. In a breeding program this will require
testing in later generations when a line produces more
homogeneous progeny.

The WI method only measured a cultivar’s ability to
slow lesion growth which appeared to correspond to field
resistance and the ability to restrict lesions to non-lethal
dimensions. This Is Just one trait that could lead to the
failure of an Inoculation to result In a lethal lesion. The
probability of a lethal lesion can be thought of as a
function of the probabilities of the occurrence of the
required steps. Any break In the chain of events that leads
to a lethal lesion will result In resistance. Some factors

other than slow lesion growth which may lead to a lower

probability of a lethal Infection by Wm are

95

suggested by this and other studies. A lower probability of
an Infection can result from disease avoidance. ,Modlfied
plant architecture has been employed In Eg_1glggnts to avoid
white mold (7). Flower tissue may be nutritionally
unsuitable to support an Infection therefore lowering the
probability of an Infection. Also the probability of an
Infection may be lowered. if the cuticle layer is able to
resist or delay penetration. Variation for the last two
traits has yet to found. All the possible resistance
mechanisms mentioned appear to be Incomplete and be

expressed as a lower probability of a lethal lesion.

 

LIST OF REFERENCES

LIST OF REFERENCES

. Abawi, G.S., F.J. Polach and W.T. Molin. 1975.

Infection of been by ascospores of Whgtzgltnta
gglgngttgngm. Phytopath 65:673-678.

Abawi, G.S. and R.G. Grogan. 1979. Epidemiology of

diseases caused by Sgtgzgttnta species. Phytopath.
69:899-903.

Baker, E.A and J.T. Martin. 1967. Studies on plant
cuticle. X. The cutlcles of plants of related families.
Ann. Appl. Biol. 60:313-319.

. Boland, C.J. and R. Hall. 1968. Growthroom evaluation

of soybean cultivars for resistance to Sglgngttntg
gglgngttgngm. Can. J. Plant Sci. 66:559-564.

Chamberlain, D.W. 1951. Sclerotinia stem rot of
soybeans. Plant Dis. Rep. 35:490-491.

Cline, M.N. and B.J. Jacobson. 1983. Methods of
evaluating soybean cultivars for resistance to
SclerotInIa_scIeratIarum. Plant Dis. 67:784-786-

Coyne, D.P. J.R. Steadman and F.N. Anderson. 1974.
Effect of modified plant architecture of Great Northern

dry bean varieties (Enaggglg§_xgtggnt§) on white mold
severity, and components of yield. Plant Dis. Rep.

58:379-382.

. Dillard, H.R and R.G. Grogan. 1985. Relationship

between sclerotial spatial pattern and density of

Sgtgngttn1a_mtngn and Incidence of leaf drop.
Phytopath. 75:90-94.

Dow, R.L. and R.D. Lumsden. 1975. Histology of
resistance of Bhasgglu§_ggggtngu§ (Scarlet Runner

bean) to SclerotInIa_scIeratIQLum- Proc. Am.
Phytopath. Soc. (Abstr.) 2:122.

96

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97

Fuller, P.A., D.P. Coyne, J.R. Steadman and R.F Mumm.
1984. Inter- and intra-row inter-genotypic competition
influences selection for avoidance of white mold
disease In dry edible beans. J. Amer. Hort. Sci.
109:567-572.

Grau, C.R. and H.L. Bissonette. 1974. Whetzelinia stem
rot of soybeans In Minnesota. Plant Dis. Rep.
58:693-695.

Grau, C.R., V.L. Radke and F.L. Gillespie. 1982.
Resistance of soybean cultivars to Sglgngttntg

sglgzgttgngm. Plant Dis. 66:506-508.

Grau, C.R. and V.L. Radke. 1984. Effects of cultivars
and cultural practices on Sclerotinia stem rot of
soybean. Plant Dis. 68:56-58.

Hine, R.B. and J.E. Wheeler. 1970. The occurrence of
some previously unreported diseases In Arizona. Plant
Dis. Rep. 54:179-180.

Hunter, J.E., M.H. Dickson amd A. Cigna. 1981. Limited
-term Inoculation: A method to screen bean plants
for partial resistance to white mold. Plant Dis.
65:414-417.

Imohelln, E.D. and R.G. Grogan. 1980. Factors affecting
survival of sclerotia, and effects of Inoculum density,
relative position and distance from the host on

Infection of lettuce by Sglgggttntg_mtngg.
Phytopath. 70:1162-1167.

Lockwood, J.L. and T.G. Isleib. 1983. Resistance of
soybeans to white mold. Annual Report to the Soybean
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Lockwood, J.L. and T.G. Isleib. 1984. Resistance of
soybeans to white mold. Annual Report to the Soybean
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Lockwood, J.L. and T.G. Isleib. 1985. Resistance of
soybeans to white mold. Annual Report to the Soybean
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Lumsden, R.D. 1079. Histology and physiology of
pathogenesis In plant diseases caused by
Sglgggttnta species. Phytopath. 69:890-896.

Lumsden. R.D. and W.P. Wergin. 1980. Scanning-electron
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plants. 347 pages. Edward Arnold Ltd. Edinburgh

Phipps, P.M. and D.M. Porter. 1982. Sclerotinia blight
of soybeans caused by Sglgggt1n1g_mtngn and

ScIeLatInIa_scIeratIarum- Plant Dis. 66:163-165-

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and infection by SciarotInIa_scIeratIarum» Phytopath
48:605-609.

Schwartz, H.F., J.R. Steadman and D.P. Coyne. 1977.
Resistance of Charlevoix and Valentine to infection by

SclerotInIa_scleLatIorum- Rep. Bean Improv. Comm.
20:71-71.

Schwartz, H.F, J.R. Steadman and D.P. Coyne. 1978.
Influence of Engsgglg§_tglgant§ blossoming

characteristcs and canopy structure upon reaction to

SalsratInIa_scIeratIarumm Phytopath 68:465-470.

Sutton, D.C and B.J. Deverall. 1983. Studies on
Infection of bean (Enastgg1g§_1glgant§) and soybean
(fiflxcine_max> by ascospores of Sclerotinia
sglgngtigngm. Plant Path. 32:251-261.

Tariq, V.N. and P. Jefferies. 1984. Appressorlum
formation by SclerotInIa_scIeratIarum: Scanning
electron microscopy. Trans. Brit. Mycol. Soc.
82:645-651.

Thompson, A.H. and G.C.A. Van Der Westhuizen. 1979.
ScleratInIa_scIeratIorum <Lib.) De Bary on soybean in
South Africa. Phytophylactica 11:145-148.

Weiss, F. 1946. Check list revision. Plant Dis.
30:130-137.

Yorinori, J.T. and M. Homechin. 1985. Sclerotinia stem
rot of soybeans, Its Importance and research In
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proceedings. ed. Richard Shibles. Westview press.
Boulder.

SOURCES OF VARIABILITY WITHIN A GENOTYPE FOR

RESISTANCE T0 W INFECTION 01"
SOYBEAN

INTRODUCTION

Sclerotinia stem rot in soybean (W (L.)
Merr.) Is caused by the fungus Wm
(LIb.) De Bary. Though reported In the maJor soybean
producing areas of the midwest (3, 4, 8, 10, 15, 21), the
disease has not been a widespread problem. It is more
likely to be a problem when soybeans are planted In
rotations with more susceptible hosts (8, 11, 19) and In
cool wet environments that favor disease development (10,
22). In these situations the disease can be devastating to
whole fields or sections of them causing considerable yield
reductions (10, 17). It would be desirable to have soybean
genotypes that exhibit resistance Sclerotinia stem rot for
those regions where the disease has been a problem.

To breed for resistance there must be a method to
screen genotypes for their reaction to the pathogen. A
method should be quick, easy, repeatable, sensitive to
differences among cultivars and correlated with field
resistance. The wound inoculation (WI) method developed by
Lockwood and Isleib (17) and adapted by Sneller gt
al.(unpublished) meets the above mentioned criteria plus It
has the advantage of a quantitative rating of disease
development. The WI technique still produced unexplained
variability among the stems of cultivar for reaction to the
pathogen. This variation contributed to the error term and
reduced the sensitivity of the test. In one WI experiment,

99

100

lesions on stems of ’Weber 84’ ranged from 5.9 to 10.8 cm
and P1297514 lesions ranged from 2.3 to 10.1 cm.

Variability within a cultivar for reaction to S...
gglgngttgngm is common In other assays and In other species
and is reported In soybeans (2, 4, 8, 15), Ebgsgglug
yglggLL§_Lt (13, 14), Hgltgntngg_annuu§ L. (12, 18, 20) and
Elsum gatiygm L, (1).

If the cause of the variability was known, steps could
be taken to minimize It which would result in an Increased
sensitivity of a test at differentiating between cultivars.
Because the WI assay has many desirable features the
following study was undertaken to see If the source of the
variation could be ascertained.

There are several possible explainations for the
variation in lesion lengths among the stems of a cultivar.
There could be residual genetic heterogeneity within a
cultivar for reaction to $g_§glg:gtigngm, Three preliminary
studies by the author were inconclusive with two studies
supporting this hypothesis and one rebutting It. Another
source of variability could originate In the growth
environment of the plants used for testing. Other sources
of variability could arise from the Inoculum, the
Inoculation technique and from variation In the microclimate

of the actual testing environment.

101

MATERIALS AND METHODS

Twenty seven single plants were harvested from field
plots of five cultivars In October of 1985. Each plant was
threshed separately and the seed kept separate. In this
fashion five sets of twenty seven single plant families
(SPF) were developed, one set for each cultivar to be
tested. The cultivars had appeared to be the most variable
In previous tests (Sneller gt al. unpublished) and had been
reported to be resistance or susceptibility to $_,_
gglgzgttgzgm. The susceptible genotypes were Weber 84 (16,
17) and ’Gnome’ (4, 9, 15, 16, 17). The resistant cultivars
were ’Corsoy’ (4, 10, 15, 17), P1297514 (16), and P1358314
(16).

The plants were grown in the greenhouse under
artificial lIght (16 hour day, 8 hour night) In 20 cm
diameter clay pots, four plants per pot. The soil was a
5:3:2 mix of sandy loam, sphagnum peat and torpedo sand.
Each pot was randomly assigned a cultivar and then each
quarter section of the pot randomly assigned a seed from one
of the SPFs to be tested. Pats were randomly assigned to
locations on the greenhouse bench and were spaced 130 cm
below the lights such that the rims of the pots touched one
another. The plants In Experiment 1 were 38 days-old while
Experiment 2 was performed on 47 day-old-plants.

The WI method was employed to test the disease reaction

of the plants. This Involves the Inoculation of 15 cm stem

102

sections In the laboratory. The stem sections were prepared
by cutting the main stem 1.5 cm above the node bearing the
upper most trifoliate leaf and making a second cut 15 cm
below this node resulting In a 16.5 cm stem section. The
leaves and petioles were then cut from the stem section
The stem sections were then transported to the laboratory In
plastic bags. Immediately prior to inoculation, a final cut
was made through the middle of the described node
perpendicular to the long axis of the stem, resulting in a
15 cm stem section. The Inoculum was placed on the surface
of the final cut.

The stems were inoculated and Incubated 'in clear
plexiglass boxes (56 x 53 x 8 cm). The boxes were filled
with a 4 cm thick layer of medium grade vermiculite. The
vermiculite was moistened with 1500 ml of distilled water.
After Inoculation the boxes were covered with plastic wrap
and a clear plastic lid was put In place. All stems were
randomly assigned to locations within the boxes.

Inoculum was grown on 2% Bacto agar medium supplemented
with 2% millet seed powder and was cultured from mycelium
grown on the same medium. The Inoculum was grown In 15 cm
diameter petri dishs in the dark for five days at ambient
room temperature (approximately 24°C). A 0.3% percent water
agar solution was poured in the dish of Inoculum to cover
the surface of the medium. Plugs of Inoculum were cut with
a 4 nun cork borer from a distance of 4.0 to 6.0 cm from the-

center of the dish, then removed with a dissecting needle

103

and placed mycelium side down on the final cut at the apical
end of the stem section. One dish of Inoculum was used per
replication.

Lesion measurements ‘were taken five days after
Inoculation. A lesion was measured from the point of
Inoculation to the area of the lesion where the epidermis
could no longer be easily peeled off with the edge of a
plastic ruler.

The experiments were conducted In a room cooled to
approximately 20° C though this temperature fluctuated
upwards to 24"C during the day.

In Experiment 1, 27 SPFs from each cultivar were
tested. The experiment was conducted in three runs due to
the space constraints of the boxes. Each run was a
randomized complete block design with three replications
(boxes) with each cultivar was tested In each run and In
each box. Nine different SPFs from each cultivar were
tested In each run. Within each run, each box contained
three different SPFs from each cultivar. Thus the SPF’s
were nested within boxes, runs and cultivars. In each box,
each SPF was represented by four stems.

Experiment 2 was conducted In one run. The number of
SPFs tested was not equal among the five cultivars. This
was due the failure of enough plants of some SPF’s to grow
to the proper length. Nine SPFs were tested from Gnome,
Corsoy and PI 358314. Eight SPFs of Weber 84 and seven SPFs

of PI 297514 were tested. The overall design was a

104

randomized complete block with three replications (boxes).
One stem from each SPF of each cultivar was represented In
each replication. Thus the SPFs are not nested in boxes as

in Experiment 1.

RESULTS

The analysis of variance from Experiment one showed
that the variation among the SPFs of a cultivar was not
significantly greater than the variation among the stems

within a SPF (Table 1).

Table 1. Analysis of variance for lesion length for SPFs
of five cultivers, Experiment 1 (1/24/86, 1/30/86,
2/23/86).

 

 

m df MS F test
Run 2 197.52 xx
Cultivar 4 142.30 xx
Run x Cultivar 8 20.22 xxx
Box (Run) 6 5.96 x
Cultivar x Box (Run) 24 2.23

SPF (Cultivar Box Run) 90 2.14

Stem (SPF Cultivar Box Run) 405 1.91

will“ = 14.66%
x, xx, and xxx denote significance at the .05, .01 and .001 alpha levels

respectively.

Due to the design of the experiment the Interaction of
runs or boxes with SPFs could not be calculated. The effect
of runs was significant as was the run by cultivar
Interaction. It was then assumed that the run In which a

particular SPF was tested probably Influenced the resulting

105

average lesion length of a SPF. To eliminate the possible
run effect on SPF lesion lengths, three IANOVA’s were

performed, one for each run (Table 2).

A Table 2. Analysis of variance by runs for lesion length for
the SPFs tested in Experiment 1.

 

 

Date 1/24/86 1/30/86 2/23/86
Run 1 Run 2 Run 3

df MS 2 tgst MS E tgst as E tgst
Box 2 7.5 .8 9.6
Cultivar 4 21.8 x 81.2 xxx 79.8 xxx
Box x Cultivar 8 3.6 .9 2.1
SPF (Box Cultivar) 30 1 8 .9 x .4
Stem (SPF Box Cultivar) 135 I 7 .5 3.5

% .7.9 % 17.7 %

 

O

x, xx and xxx denote significance at the .05, .
respectively.

1 and .001 alpha levels

Box effects and the box by cultivar interactions were
not significant In any of the runs. The assumption was then
made that box effects would not Influence the comparisons of
SPFs among the boxes within a run. Only In Run 2 was the
variation among the SPFs significantly greater than the
variation among the stems within a SPF which may be
reflective of the better sensitivity or to the existance of
a larger difference among the 45 SPFs tested In Run 2 than
among the 45 SPFs tested In each of the other runs.

The SPF (Box Cultivar) line from the ANOVA of each run

was partitioned into five lines, each measuring the

106

contribution of an Individual cultivar’s SPFs to the overall

effect (Table 3).

Table 3. Mean squares by runs for the source of variation In lesion
length within a cultivar.

 

 

 

Run 1 Run 2 Run 3
SPF<Box2 Error t W
Corsoy 2.28 2.00 1.08 .76 4.64 8.96
Gnome 2.28 x .89 .36 .33 .94 1.00
Weber 84 1.21 3.05 1.63 xxx .30 3.59 2.00
PI 358314 1.88 1.60 .62 .72 2.62 1.55
21.297514 1.10 .90 .96 .54 6.86 4.06
x and xxx denote significance at alpha = .05 and alpha =.001 levels

respectively. Significance was tested with the reported error and not
with the overall experimental error of each run.

1' Error = Stem (Box, Cultivar)

The mean square of box effects was significant only for
Weber 84 in Run 1. Therefore It was assumed that box
effects do not significantly contribute to the variation
among the SPFs. The effect of SPFs on lesion lengths was
only significant for Gnome in Run 1 (P < .05) and for Weber
84 In Run 2 (P < .001). Significance was only found In the
two combinations of cultivar and run with the lowest error
term. The fact that significance was only found In two of
the fifteen cases may be due to significant differences
among the SPFs randomly assigned to these two tests or to
chance alone as two of fifteen populations deviating beyond

95 9: of normal Is not very different from the one out of

107

twenty expected by chance alone. This makes the results of
Experiment 1 Inconclusive. .

The SPFs from each cultivar with the longest and
shortest average lesion length in each run were retested In
Experiment 2 to see If the results could be repeated.

The overall effect of the SPFs on lesion development
was not significant (Table 4). Upon partitioning, only the
effect of the PI 297514 .SPFs was significant. Separate
analyses of variance were performed for each cultivar and
the effect of the SPFs were tested with the resulting
errors. Analyzed In this way the effect of the SPFs of

Weber 84 was found significant at P < .058 .

Table 4. Analysis of variance for lesion length for SPFs
of five cultivars, Experiment two (5/22/86).

 

 

Souroe df MS F test
Box 2 14.05 xx
Cultivar 4 22.02 xx
Box x Cultivar 8 2.64
SPF (Cultivar) 37 1.40

Corsoy 8 .76

Gnome 8 .41

Weber 84 7 .78

PI 297514 6 4.44 x

PI 358314 8 1.29
Stem (SPF Cultivar Box) 74 1.91
Wombiiitv 17.79 ’1

 

x, xx and xxx denote significance at the .05, .01 and .001
alpha levels respectively.

108

Table 5 compares the average lesion lengths obtained
for the SPF’s of the cultivars tested In Experiment 1 and

Experiment 2.

Table 5. Correlation of the average lesion lengths of the
SPFs tested In Experiment 1 with the average lesion lengths
obtained in Experiment 2.

 

 

r Significance
All SPFs .158
Corsoy SPFs -.513
Gnome SPFs .695 x
Weber 84 SPFs -.434
P1 297514 SPFs -.735
21 358314 SEE: -.045

 

x denotes significance the .05 alpha level.

While the effect of the SPFs of PI 297514 and Weber 84
were significant In Experiment 2, the average lesion lengths
of these SPFs *were not significantly correlated to the
average lesion lengths from Experiment 1 (Table 5). Only
the average lesion lengths of the SPFs of Gnome were
significantly correlated between experiments. The effect of
the SPFs of Gnome were not significant In Experiment 2.

Height data was collected on all the plants tested In
Experiment 2 and on the plants tested In Run 2 of Experiment
1 (Table 6) to see If there was a correlation of plant

heigth with lesion length.

109

Table 6. Summary of height data for the plants used In
Experiments 1 (run 2) and Experiment 2.

 

 

 

1/30/86 5/22/86
Experimmz—

Average Standard Average Standard
heiqht Wagon—Range.
-- cm -- -- cm -- -- cm -- -- cm --
Corsoy 52.2 13.7 33 - 83 64.8 7.6 51 - 79
Gnome 65.8 12.4 38 - 91 61.8 5.3 48 - 68
P1 297514 40.0 6.1 28 - 56 55.2 8.6 38 - 68
PI 359314 53.9 12.1 36 - 81 63.1 6.8 51 - 76
nggr 84 45:3. 8.6 30 - 83 62.6 5.8 53 - 73

 

There was considerable variation In plant height which
was probably due to variation within the growth environment
of the plants.

From Experiment I, run 2, data was collected on plant
height and on the pot In which the plant was grown. An
analysis of variance showed that the pot in which a plant
was grown had a very significant ( P < .001) effect on plant
height Indicating that conditions among pots varied enough
to cause significant variation In plant height. The effect
was significant for all cultivars except Weber 84.

In Experiment 1, Run 2, the correlation of height and
lesion length was significant for the stems of Corsoy, PI
297514, and Weber 84 (Table 7). A significant negative
correlation was observed for the stems of PI 297514 In
Experiment 2. The range of heights was less In Experiment 2
than in Experiment 1 and there may not have been a

sufficient range of heights to develop a good correlation.

110

Table 7. r values and probabilities for the correlation of

plant heigth and lesion length from Experiments 1 (run 2)
and 2.

 

 

Experiment 1 (Run 2 only) Experiment 2
r r

All genotypes .533 xxx - .152
Corsoy .596 xxx - .040
Gnome .328 .345
P1 297514 .875 xx - .458 x
PI 358314 .415 - .001
Weber 84 .461 x - .151

 

x, xx, xxx denote significance at the .05, l01 and .001
alpha levels respectively.

DISCUSSION

A soybean cultivar Is selected from a single plant at
some point In a breeding program and the progeny are bulked
thereafter. The earlier the generation In which the single
plant is selected, the greater the heterozygosity of the
selected plant. This will increase the heterogeneity of the
future bulked generations. A population (future cultivar)
would tend to have more heterogeneity among the individuals
for a trait that Is quantitative In nature and for which
there has not been selection pressure. In dry beans there
Is evidence that resistance to Sg__sg1gngttongm Is a
quantitative trait (5, 6, 79. At present there have not
been any soybean cultivars developed using reaction to
Sclerotinia stem rot as a major criterion of selection.
Thus heterogeneity within a cultivar for reaction to

Sclerotinia stem rot seemed a probable cause of the

 

111

unexplained variability In lesion lengths among the stems of
a cultivar as determined by the WI assay.

The results Indicated that genetic heterogeneity was
not a maJor cause of the residual variation In the WI assay.
The significant differences among the average lesion lengths
of the SPFs In Experiment 1 were not repeated In Experiment
2 and the overall correlation of the average lesion lengths
of the SPF’s In the two experiments was not significant. It
is possible that the WI assay was not sensitive enough to
detect such heterogeneity. Only the most sensitive analyses
performed found significant differences among SPFs. It Is
also possible that no variation existed among the 27 SPFs
per cultivar that were tested and that a larger sample would
uncover some residual heterogeneity. 1f heterogeneity does
exists, It did not contribute significantly to the overall
error term. When the SPF sum of square was returned to the
error term It only accounted for 19.9 % and 26.8 % of the
residual variability In Experiments 1 and 2 respectively.
These contributions were In line with the contribution of
the degrees of freedom associated with SPFs.

In some ancillary studies, all the stems of a single
cultivar were derived from. a single SPF. Considerable
residual variability remained despite the genetic
homogeneity of this material. In one of these tests the
lesions on the stems of Corsoy ranged from 3 to 6.6 cm.

P1297514 developed lesions ranging from 5.4 to 8.2 cm.

112

This suggests that environmental effects and
methodological inconsistencies were the source of the
residual variation. Variation In the microclimate of the
Inoculation sites within a box seemed unlikely due to
relatively small box size and the fact that the boxes were
well sealed. There also should be little variation in the
Inoculum. All the plugs of Inoculum were drawn from
approximately the same distance from the starting point of
the culture assuring that the age of the: mycelium was
similar for all plugs. One plate of inoculum was used per
replication so that variation among the plates was
confounded with replications. Variation due to the
inoculation technique was also minimized. All stems were
Inoculated immediately after they were cut to the final
length and plugs of Inoculum rarely fell off. The
experiments were checked about five hours after Initiation
and any plugs which fell off were repositioned or replaced
with fresh plugs. With the WI technique, all lesions
appeared to Initiate at approximately the same time and
overall variation increased with time (Sneller gt g1.
unpublished). This Indicated that the Inoculation process
Is uniform and that the factors causing the residual
variability affect lesion growth, not lesion initiation.

By elimination of the other possible sources of
variation, the growth environment of the plants appeared to_
be the most likely source of the residual variation. While

efforts were made to maintain a uniform growing environment

 

113

among the plants, It became more variable as more plants
were needed for an experiment. This variability was
reflected In the height data taken on the plants used in
this study. The height variation was not correlated with
lesion length. Variation In the microclimate in the
greenhouse may influence reaction to W
Independently of plant height.

Residual variation in the WI method was not reduced in
this study. Progeny testing remains necessary to assure an
accurate rating of a genotype. This will require testing a
soybean line in a late generation in a breeding program to
assure a high degree of genetic homogeneity among the
progeny.

Studies are presently under way to Investigate the
possible causes of variation In the growth environment
affecting resistance to $g_§glgngt1gggm. By controlling the
source of the variation It Is hoped that the sensitivity of
the test can be further Improved. This would allow testing
with fewer plants per line and Increase the number of lines

that can be screened at one time.

1.

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Blanchette, B.L., and D.L. Auld. 1978. Screening field
peas for resistance to white mold. Crop Sci.
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. Chamberlain, D.W. 1951. Sclerotinia stem rot of

soybeans. Plant Dis. Rep. 35:490-491.

. Cline, M.N. and B.J. Jacobson. 1983. Methods of

evaluating soybean cultivars for resistance to
SoIoLotInIa_soIerotIorum- Plant Dis. 67:784-786.

Coyne, D.P., J.R. Steadman and H.F. Schwartz. 1977.
Genetic variation, inheritance and breeding strategy
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(Engggglgg_1glggnt§ L.). Rep. Bean Improv. Comm.
20:19-21.

. Dickson, M.H., J.E. Hunter, M.A. Boettger and J.A.

Cigna. 1982. Selection for resistance In Ehgggglgg
yglggnig L. to white mold caused by Sglgggttntg
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Inheritance of resistance to white mold disease In a
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Grau, C.R. and H.L. Bissonette. 1974. Whetzelinia stem
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. Grau, C.R. and V.L. Radke. 1984. Effects of cultivars

and cultural practices on Sclerotinia stem rot of
soybean. Plant Dis. 68:56-58.

Hine, R.B. and J.E. Wheeler. 1970. The occureance of

some previously unreported diseases in Arizona. Plant
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IMPLICATIONS OF THESIS FINDINGS TO A BREEDING
PROGRAM FOR RESISTANCE T0 SCLERQILNLA_SCLERQILQRQM

The WI method appeared to be a reliable indicator of
field resistance to $g_gglgggttgggm. Several genotypes were
Identified which exhibited resistance to the fungus. This
Indicates that It would be possible to breed for resistance
to the disease by transferring the resistance genes to
adapted genotypes. Fortunately one of the most resistant
cultivars was ’Corsoy’ which Is already adapted to the
primary soybean producing regions of Michigan. Two plant

Introductions were also Identified as being resistant. It

would be Interesting to Investigate the genetic control of
resistance in these sources to see If they could be used to
further Increase the resistance of Corsoy.

All the lesions resulting from the WI technique would
be lethal to a plant. Complete resistance was not expressed
and the ratings were relative. A resistant and susceptible
check must be Included in each experiment. The resistance
as measured by the WI method, appeared to relate to the
ability to limit Infections to non-lethal dimensions. All
three resistant cultivars still developed some lethal
lesions, though significantly fewer than the more
susceptible cultivar, Gnome. Thus resistance appeared to be
incomplete and was ultimately expressed as a lower
probability of a lethal lesion. The expression of
resistance was also Influenced by the environment as shown
by variation between experiments.

116

117

There remained variation within a genotype for reaction
to Sg_gglgngttgnum, Genetic heterogeneity within a cultivar
for resistance to the fungus was ruled out as a cause of the
variability as were possible sources associated with the
inoculation procedures. The variation appeared to be
associated with some factor Influencing lesion growth after
lesion initiation. The most likely source of the variation
appeared to be the growth environment of the plants.
Environmental variation within an experiment lowers the
heritability of the trait regardless of the genetic control
of resistance. Selection efficiency will therefore be low
unless the cause of this variation can be managed.

The variation necessitates the testing of several stems
to obtain a reliable rating. The testing of progeny in a
breeding program will require testing lines in later
generations (P., or later) when a line produces relatively

homogeneous progeny.

 

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