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thesis entitled
Analysis of the Omega-3 Fatty Acid Desaturase in Soybean
Genotypes A5 and A23; Resistance to Sclerotinia Stem Rot
in Soybean is Induced by 2,6-Dichloro-Isonicotinic Acid.

presented by
Joseph Byrum

has been accepted towards fulfillment
of the requirements for

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ANALYSIS OF THE OMEGA-3 FATTY ACID DESATURASE IN SOYBEAN
[Glycine max (L.) Merr.] GENOTYPES A5 AND A23; RESISTANCE TO
SCLEROTINIA STEM ROT IN SOYBEAN [Glycine max (L.) Merr.] IS INDUCED
BY 2,6DICHLORO-ISONICOTINIC ACID
By

Joseph Byrum

A THESIS

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

MASTER OF SCIENCE

Department of Crop and Soil Sciences
Plant Breeding and Genetics

1995

ABSTRACT

ANALYSIS OF THE OMEGA-3 FATTY ACID DESATURASE IN SOYBEAN
[Glycine max (L.) Merr.] GENOTYPES A5 AND A23; RESISTANCE TO
SCLEROTINIA STEM ROT IN SOYBEAN [Glycine max (L.) Merr.] IS INDUCED
BY 2,6—DICHLORO-ISONICOTINIC ACID

By
Joseph Byrum

Soybean genotypes A5 and A23 have reduced a-linolenate (18:3“3’6'9)
content in their seed oil when compared with wild type soybeans. A gene
controlling linolenic acid in A5 is designated fan(A5) and is independent of the
linolenic acid gene fan2(A23). DNA gel-blot hybridizations using the
microsomal w-3 linoleate desaturase cDNA indicate a missing DNA fragment in
the A5 when compared to A23 and lines wildtype for linolenic acid. It is
likely, therefore, that the phenotype of the A5 mutant is a result of a deletion of
a microsomal w-3 linoleate desaturase gene.

Soybean frequently suffer heavy losses from white mold under conditions
that are favorable for disease development. Systemic acquired resistance (SAR)
is a mechanism that is characterized by the development of a disease-resistant
state in plants. It was our goal to exploit this mechanism via the use of a novel
synthetic immunization compound 2,6-dichloro-isonicotinic acid INA. We
report here that the application of 2,6-dichloro—isonicotinic acid (INA) to

soybeans results in protection against white mold.

TABLE OF CONTENTS

List of Tables . . . . . . . . v

List of Figures . . . . . . . . viii

Chapter 1. Analysis of the Omega-3 Fatty Acid Desaturase in Soybean
Genotypes A5 and A23

Background . . . . . . . . . 2
Introduction . . . . . . . . . 5
Materials and Methods . . . . . . . 8
Results and Discussion . . . . . . . 11

Chapter 2. Resistance to Sclerotinia Stem Rot in Soybean is Induced by 2,6-
Dichloro-Isonicotinic Acid

Background . . . . . . . . . 17
Introduction . . . . . . . . . 24
Materials and Methods . . . . . . . 28
Results and Discussion . . . . . . . 33

iii

Conclusion

Literature Cited

iv

47

Table 1.

Table 2.

LIST OF TABLES

Cultivars utilized at the East Lansing and Zilwaukee sites.
Disease susceptibility ratings are based on grower
observations and can be influenced by any number of

cultural practices and environmental conditions. 27

Yield results from two blocks at the East Lansing site in
1993. "" Significantly different from control at LSD P s

0.01. . . . . . . . 35

Table 3.

Table 4.

D81 ratings of cultivars in 1993 at East Lansing and
Zilwaukee. Fifty plants were scored at random from the
center three rows of each plot. Reaction of cultivars to S.
sclerotiorum was rated on a 0-3 scale, where O = no
symptoms, 1 = lesion(s) only on lateral stems, 2 =
lesion(s) on main stems, and 3 = diseased plant dead
(Nelson et al. 1991). A disease severity index (DSI) was
calculated by the method of Sherwood and Hagedorn
(1958). * Significantly different from control at LSD P s

0.01. . . . . . . . 36

Yield results from the 1994 tests at East Lansing and
Zilwaukee sites. Rates (182 mM, 234 mM, and 339 mM)
are combined within each application of each cultivar since
there was not a significant rate effect. 11 = 8 for the
control (0) and n = 24 for applications 1, 3, and 4. "‘
Significantly different from control at LSD P s 0.01.

38

vi

Figure 1.

Figure 2.

Figure 3.

LIST OF FIGURES

DNA gel-blot analysis of the soybean genotypes using the cDNA
encoding the microsomal w-3 desaturase. Lanes 1 - 6 were
digested with Dra I. Lanes 7 - 12 were digested with Hind HI.
Lanes 1 - 6, 7 - 12 represent the double mutant parent, wildtype
parent, A5, A23, A16 and A17, respectively.

13
PCR analysis of the parental genotypes using 30-mer
oligonucleotides flanking the 5’ and 3’ end of the cDNA encoding
the microsomal w-3 desaturase. Lanes 1 - 6 represent A17, A16,
A23, A5, wildtype parent, and double mutant parent.

14
Colony diameters of S. sclerotiorum grown on INA-amended PDA

for 3 days, expressed as percentage of unamended control (8 cm).

32

viii

CHAPTER 1. ANALYSIS OF THE OMEGA-3 FATTY ACID

DESATURASE IN SOYBEAN GENOTYPES A5 AND A23

2

BACKGROUND

The fatty acid composition of an oil determines its physical and chemical
properties and thus it uses. Plants are attractive experimental objects for
genetic studies of lipid metabolism for several reasons. First, because plant oils
are of major economic importance and because the oil composition is less than
optimal in many cases, there is substantial interest in exploring the degree to
which genetic methods can be used to modify the oil composition of seeds.
Second, in many cases, relatively large alterations can be made in the fatty acid
composition of the storage triacylglycerol without exerting any obvious
deleterious effects on the growth and development of the organism. The major
edible oils have rather similar fatty acid compositions, in which 18 carbon fatty
acids with one to three double bonds predominate. Thus, for many edible uses
the various major oil sources are interchangeable and their consumption and
competitive status in the market-place is largely controlled by price. However,
the specific fatty acid composition of an edible plant oil may sometimes give it
special status. Concerns about the possible contributions of saturated fats to
heart disease have also put a slight premium on those vegetable oils with the
lowest levels of these constituents. For example, the entry of canola oil into

the edible oils market, although dependent on economic factors, has been

3

buoyed by publicity concerning its low saturated fatty acid content.

Soybean produces seed oil in which the fatty acid composition is not
ideally suited for the intended use. Linolenic acid has been identified as the
unstable component of soybean oil responsible for undesirable odors and
flavors. Therefore, there has been an ongoing interest in reducing linolenic
content below 3 %. One of the most striking examples of the utility of the
mutagenic approach is the isolation of two ethyl-methanesulfonate—induced
mutants of soybean. When the two mutations were combined to make the
double mutant, a line almost completely deficient in 18:3 in seed lipids was
obtained. There was apparently no deleterious effect on the viability, oil
content, or germination of the seeds. The simplest interpretation of these
results is that there are two genes encoding two desaturases in the seeds and the
products of these genes have been inactivated by the mutations. Since there
was little or no effect on leaf fatty acid composition, it seems likely that there
are additional desaturase genes which are leaf specific. Biochemical and
genetic studies of these mutants are important to our understanding of the 18:2
desaturation step because the enzyme is an integral membrane protein that has
been difficult to solubilize and, therefore, to investigate by traditional
enzymological methods. In the absence of a purified enzyme, genetic
techniques can be an alternative means to study the relevant genetic locus.

However, it seems likely that, because of the inherent limitations of mutation

4

breeding, many other desirable changes in seed oil fatty acid composition may
require the directed application of genetic engineering methods. Presently,
specific information about the biochemistry and regulation of lipid metabolism
has made it possible to predict the result of the introduction of one or a few
genes that might usefully alter seed lipid synthesis. This would allow scientists
to design new products that can provide expanded or new markets for excess

agricultural output.

INTRODUCTION

Soybean produces seed oil in which the fatty acid composition affects the
nutritional value as well as its physical and chemical characteristics (Neff et a1.
1992). The polyunsaturated fatty acid a-linolenate(18:3“’ 3"3"") is synthesized by
plants, but not by most other higher eukaryotes. This fatty acid is an essential
component of human nutrition because in mammals it acts as a precursor to
membrane lipids and families of signaling molecules (Smith and Borgeat,

1985). It is also commercially important because it has been identified as the
unstable component of soybean oil responsible for undesirable odors and flavors
(Liu et al., 1992). For these reasons, variation in the linolenic acid content has
been a target for genetic selection.

The application of conventional breeding methods, coupled in some cases
with mutagenesis, has resulted in the production of new lines with desirable
alterations in the fatty acid composition of the seed oil (Hammond and Fehr,
1983). Most of the genetic alterations in seed fatty acid composition appear be
the result of mutations disrupt normal fatty acid metabolism and leads to an
accumulation of intermediate fatty acid products in seed storage lipids

(Ohlrogge et al., 1991). It is unclear, however, what structural genes have

6

been mutated in existing public soybean germplasm.

The biosynthesis of linolenic acid is the result of a desaturation reaction
Catalyzed by a membrane associated omega-3 desaturase which introduces the
third double bond into the 18-carbon fatty acid. In leaf tissue, there are two
distinct pathways for polyunsaturated fatty acid biosynthesis, one located in the
microsomes and the other located in the plastid membranes. In Arabidopsis
thaliana, the plastid w-3 fatty acid desaturations are controlled by the FAD 7
locus (Browse and Somerville, 1991). The major enzyme responsible for the
synthesis of seed linolenic acid is the microsomal co-3 linoleate desaturase. Its
activity is controlled in Arabidopsis by the FAD 3 locus (Lemieux et al., 1990)
which may be equivalent to one or both of the FAN loci in soybean.

The lowest published content of a-linolenate(18:3“’3’6'9) in soybean was
identified at Iowa State University from the cross of two EMS induced mutant
lines, A5 and A23 (Fehr et al., 1992). A5 has a major gene controlling
reduced linolenic acid, at a locus independent of fan2(A23). The locus in A5 is
designated fan(A5). When the two mutants were crossed, segregates with
<25g kg'1 linolenic acid in seed lipids were obtained which were designated
A16 and A17. The simplest interpretation of these results is that there are at
least two genes encoding the microsomal w-3 linoleate desaturase in the seeds
and that the products of these genes have been inactivated by the mutations.

The objective of this project was to describe the molecular nature of the 03-3

7

fatty acid desaturase in soybean genotypes A5 and A23.

8

MATERIALS AND METHODS

Genetic Material. The genotypes utilized for the study included A5, A23, A16,
and A17. A23 has a major gene controlling reduced linolenic acid content, at a
locus independent of fan(A5). The locus in A23 is designated fan2(A23). A16
and A17 are considered to have the genotype:
fan(A5)fan(A5)fan2(A23)fan2(A23). A population of 70 FM derived
individuals segregating for fan(AS) and fan2(A23) was constructed from a cross
between Pioneer variety 9231 which has wildtype linolenate levels x double
mutant A89-269077. Seed and fatty acid composition of each line in the
population was kindly provided by Pioneer Hi-Bred International.

DNA Gel-Blot Analysis. Total genomic DNA from young soybean leaves was
isolated as previously described by Anderson et a1. (1992). Restriction digests,
electrophoresis on agarose gels, Southern blots, hybridizations, and
autoradiography was according to Berantzky and Tanksley (1986) with minor
modifications. Ten micrograms of parental DNA was digested with EcoRI, ,
Hind HI, Taq I, EcoRV and Dra 1. DNA from the parental genotypes was
analyzed by gel-blot hybridization to the cDNA coding for the soybean
microsomal w-3 linoleate desaturase (Yadav et al., 1993) to identify
polymorphisms. When polymorphisms were identified, DNA from each

segregant in the Ft, population was digested with EcoRI and Dra I. The

9

populations were then analyzed by gel-blot hybridization with the microsomal
w-3 linoleate desaturase cDNA. Analysis of variance was used to test for
significant association between the segregation of the cDNA and the linolenate
content of the lines. Data were analyzed using the general linear model
procedures of the statistical analysis system (SAS).

PCR Analysis. Eight random 30—mer oligonucleotides were designed so that
four primers were in the forward orientation and four primers were in the
reverse orientation based on the cDNA sequence of the microsomal w-3
linoleate desaturase. All possible primer combinations were amplified.
Amplification was carried out using standard reaction conditions in a Perkin
Elmer Cetus DNA Thermal Cycler 480 programmed for 94°C/4 min and then
30 cycles of 94°C/1 min, 60°C/l min, 72°C/3 min.

RNA Gel-Blot Analysis. Soybean embryo tissue was harvested and frozen in
liquid nitrogen from the following genotypes: A5, A23, A16, and A17 at 10
DAF and 30 DAF (DAF = Days After Flowering). Total RNA was isolated
as described by Ausubel et al. (1991). Yield was determined by
spectrophotomeric A260. After electrophoresis of 20pg of total RNA per lane in
a 1.2% formaldehyde agarose gel, RNA was transferred to Hybond—N+
membrane (Amersham) according to the manufacturers specifications. Nucleic
acid hybridizations were as described previously (Yadav et al., 1993). To

ensure that each lane of the RNA gel blots contained similar amounts of

10

undegraded RNA, filters that had been hybridized to the microsomal w-3
linoleate desaturase probe were subsequently stripped and rehybridized with a
32P labeled soybean actin gene (Psac 3). The amount of hybridization was
determined by measuring the amount of bound radiolabled probe using a

Molecular Dynamics (Sunnyvale, CA) Phoshorlmager.

11

RESULTS AND DISCUSION

A5 Mutant. Previous studies have shown the low linolenic acid phenotype of
fan(A5) to behave like a quantitative character (Fehr et al., 1992). The
complexity of the biochemical interactions and the genetics of this mutant
suggested that the mutation may not have directly affected a structural gene for
a desaturase, but the regulation of expression of desaturase activity (Ohlrogge et
al., 1991). Our study indicates a deletion of the structural gene encoding the
microsomal w-3 linoleate desaturase has occurred in this mutant. DNA gel-blot
hybridizations using the microsomal 02-3 linoleate desaturase cDNA indicates a
missing DNA fragment in the A5, A16, and A17 mutants when compared to
A23 and lines wildtype for linolenic acid (Figure 1). The 70 F45 individuals
segregating for the fan(A5) and fan2(A23) loci were scored for the presence or
absence of the fragment. The absence of the fragment was significantly (P ..<..
0.0001) associated with reduced linolenic acid and accounted for 67% of the
variation for linolenic acid in the population. The mean of the lines without the
DNA fragment was 43 g kg“ and the mean of the lines with the DNA fragment
was 77g kg". PCR was utilized to further investigate the deletion in fan(A5).
All possible pairwise oligonucleotide combinations generated one DNA
fragment in genotypes A5, A16, and A17 (Figure 2). In contrast, the wildtype

and A23 mutant generated two DNA fragments. The reduction of linolenic acid

12

of the fan(A5) mutant, is thus, the result of a single codominant nuclear
mutation at the microsomal w-3 linoleate desaturase that controls the level of
linolenic acid in seeds. However, the PI line from which A5 was derived via
EMS treatment was not available for this study. We cannot completely rule-out
the possibility that the deletion was present prior to the EMS treatment.

A microsomal w-3 linoleate desaturase gene mapped to linkage group B2
in the USDA-ARS G. max /G.soja population (Byrum et al., 1995). This is
consistent with the placement of fan(A5) at the same location in another
population (Brummer et al., 1995). This further suggests that the low linolenic
acid phenotype obtained with the fan(A5) locus is the result of a mutation in

the microsomal w-3 linoleate desaturase gene.

13

Figure 1. DNA gel-blot analysis of the soybean genotypes using the cDNA
encoding the microsomal w-3 desaturase. Lanes 1 - 6 were
digested with Dra I. Lanes 7 - 12 were digested with Hind III.
Lanes 1 — 6, 7 — 12 represent the double mutant parent, wildtype

parent, A5, A23, A16 and A17, respectively.

12 3456739101112

 

'

 

Figure 2.

14

PCR analysis of the parental genotypes using 30—mer
oligonucleotides flanking the 5’ and 3’ end of the cDNA encoding
the microsomal w—3 desaturase. Lanes 1 - 6 represent A17, A16,

A23, A5, wildtype parent, and double mutant parent.

123456

 

15

A23 Mutant. No polymorphisms were identified with the microsomal w-3
desaturase cDNA to map fan2(A23) in the F45 derived population segregating
for alleles of the fan(A5) and fan2(A23) loci. Therefore, the locus from which
fan2(A23) is derived was not definitively determined.

Northern analysis indicated no differential changes in steady state
contents of the RNA levels from the microsomal w-3 desaturase in A5, A16 and
A17 when compared with A23 and wildtype lines. Since A5, A16 and A17
have a deleted microsomal w-3 desaturase locus this would indicate the
existance of more than one gene. This observation is consistent with studies by
(Rennie and Tanner 1989) which reported the fan(A5) mutant phenotype to be
temperature sensitive. The temperature effect is independent of fan(A5) and,
thus, the result of another desaturase locus possibly but not necessarily

fan2(A23) .

CHAPTER 2. RESISTANCE TO SCLEROTINIA STEM ROT IN SOYBEAN

IS INDUCED BY 2,6—DICHLORO-ISONICOTINIC ACID

16

17

BACKGROUND

An important aspect of successful agriculture is the control of plant
diseases that reduce productivity, quality, and profitability. The use of
intensive farming and mass monoculture resulted in the extensive use of
chemical pesticides to control plant pests and diseases. The use of chemical
pesticides, however, will become increasingly restricted due to concerns for the
environment and health. The need for extensive toxicological and field testing
prior to release of new chemicals and development of pest or pathogen
resistance to some current pesticides may also contribute to a reduction in
pesticide utilization. Environmentalists campaign for reduction in the use of
pesticides and fertilizers that can find their way into air, water, and soil. A
segment of the public clamors for food that contains no pesticide residues, yet
wants an abundant supply of inexpensive, high-quality food. Extremists believe
that farmers should not use any synthetic chemicals. However, sincere these
proponents may be, such paradigms do not completely reflect contemporary
reality. This is not to say that agriculture cannot change. The changes of the
20th century have been revolutionary in scope. The changes of the 2lst century
will be even more profound. While it is clear that changes are needed, is also
is clear that we cannot abruptly shift into a lower gear that would cause

starvation and economic collapse. Change must be based on sound research

18

and economic considerations. We are faced with the challenge of finding more
effective, safer, and economical ways to protect plants against pests to help feed
an ever increasing world population. Utilization of plants own defense
mechanisms by application of technologies to induce resistance may contribute
toward a solution to this problem (Metraux et al. 1991). Plant immunization
can be a natural, safe, effective, persistent and durable alternative to pesticides
in controlling plant diseases. Chemical inducers may provide better means of
application possibilities for induction of resistance, providing that they are
easily accessible and not harmful. Plant immunization may find its niche in
sustainable agriculture as one of a variety of practices that promote plant health
and reduce plant disease.

It is a lesser known fact that plants can also be immunized against
disease-causing pathogens or feeding pests (Kuc, 1982). This phenomenon is
the result of the development of systemic acquired resistance (SAR) which is
characterized as an increase in resistance to subsequent pathogen attack in
inoculated and uninoculated parts of the plant. SAR in plants was reviewed
almost 60 years ago by (Chester, 1933). SAR to pathogens usually develops
after the appearance of a necrotic lesion around the inoculation site. This
localized cell suicide is called the hypersensitive response (HR). While the HR
effectively traps pathogens in and around lesions, it makes the whole plant more

resistant to a wide range of disease-causing microorganisms (Madamanchi and

19

Kuc, 1991; White and Antoniw, 1991). These local resistance responses
develop mere rapidly than SAR and involve cell wall and cuticle strengthening,
synthesis to toxins, antifeedants, and the production of defense-related proteins,
which include the pathogenesis-related (PR) proteins (Lamb and Dixon, 1990;
Madamanchi and Kuc, 1991; White and Antoniw, 1991; Ross 1966; Kuc 1982;
Ward et al., 1991; Paxton and Chamberlain, 1967; Svoboda and Paxton, 1972).
Local resistance may be partially mediated through relatively immobile
endogenous elicitor which include oligogalacturonide fragments of the plant cell
wall (Lamb and Dixon, 1990). The combined effects of local and systemic
resistance mechanisms protect the area around the HR lesion best against a
subsequent pathogen attack (Ross, 1961). For, example, SAR induced by
necrotizing viruses develops in uninfected tissues two to three days after
inoculation, lasts for several weeks, and may provide protection against
pathogenic bacteria, fungi, and other viruses.

Pathogen invasion triggers a complex pattern of responses in the host
plant that result in either the disease state or in the development of disease
resistance (Lamb et al., 1989). The choice between disease and resistance
represents the end-product of a complex dialog between host and pathogen
(Dixon and Lamb, 1990). A diverse array of pathogen virulence functions and
plant defense functions have been described. More recently, dissection of the

complex interplay between plant and pathogen has become possible through the

20

combined application of genetic and biochemical techniques (Daniels et al.,
1988). In many cases, disease resistance depends on early recognition of the
pathogen by the plant followed by elaboration of a rapid defense response
(Klement, 1982). It has been postulated that for certain plant/pathogen
interactions, specific recognition is mediated by pairs of "complementary"
genes: a pathogen-encoded avirulence (avr) gene and a host-encoded disease
resistance gene (Keen, 1990). Only interactions which involve both genes lead
to pathogen recognition by the host, induction of an active defense response,
and disease resistance. In several instances, race/cultivar specificities can now
be explained by interactions between defined avirulence gene/resistance gene
pairs.

Pathogen-induced HR and SAR are associated with the local and
systemic appearance of at least five families of proteins referred to as PR
proteins. Tissue localization, timing of appearance and known functions of
some PR proteins suggest their involvement in SAR. However, definitive proof
that the systemic induction of PR proteins causes SAR is still lacking. Plants
rely on a long-distance signal molecules that at low concentrations, can activate
resistance mechanisms‘in cells not directly invaded by the pathogen. The
existence of these signals was first hypothesized by Ross (1966) and
demonstrated in grafting experiments (Guedes et al., 1980). A signal in SAR

should be synthesized by the plant, increase systemically following attack by a

21

pathogen, move throughout the plant, induce defense-related proteins and

phytochemicals, and enhance resistance to pathogens.

22

INTRODUCTION

Sclerotinia stem rot or white mold caused by Sclerotinia sclerotiorum
(Lib.) de Bary is becoming an increasingly important soybean disease in
Michigan. S. sclerotiorum may cause extensive plant mortality during pod
development resulting in significant yield reduction. Soybean cultivars vary in
their reaction to S. sclerotiorum (Grau et al., 1984; Chun et al., 1987; Buzzell
et al., 1993). Cultivars range from moderately tolerant to extremely susceptible
within maturity groups 0 through II. However, less tolerance is reported
within maturity group III and later maturity groups (Nelson et al., 1991). It is
unclear whether tolerance is due to avoidance mechanisms such as plant
architecture, maturity, or lodging characteristics. A major unresolved issue is
whether or not physiological resistance accounts for observed differences in
cultivar reactions to S. sclerotiorum.

Disease control measures have traditionally exploited genetic resistance
to pathogenic organisms present in the environment. However, plants also
possess an inducible resistance mechanism called systemic acquired resistance
(SAR) which is a nonspecific "immunity" to infection. Once induced, the
resistance is effective against a broad’range of pathogens (Ryals et al., 1992)
and can last from a week to months after induction. SAR to disease has been

documented for more than 100 years (Chester, 1933) and has been extensively

23

studied in tobacco and cucumber (Ross 1966; Kuc 1982; Ward et al., 1991).

Evidence for induced resistance in leguminous plants was demonstrated
when common bean plants became resistant to virulent races of Colletotrichum
lindemuthianum after preinfection of hypocotyls with spores of avirulent races
of the same species (Elliston et al., 1971; Elliston et al., 1976; Elliston et al.,
1976; Elliston et al., 1977; Raje et al., 1969; Sutton, 1979). A bean cultivar
normally susceptible to all races of C. lindemthianum could also be made
anthracnose-resistance with a preinfection of the cucumber pathogen,
Colletotrichum lagenarium. This result shows that the presence of a strain-
specific resistance gene is apparently not required for SAR to develop. Elliston
et al., (1976a) tested” a number of different species and strains of Colletotrichum
in the same type of experiment. These researchers demonstrated that two
species failed to give localized protection; while, two others and a race of C.
lagenarium gave inconsistent protection. Conversely, C. trifolii and two other
races of C. lagenarium gave localized protection. If the process underlying
induction of systemic resistance is similiar in bean and curcurbits, then it is not
likely to be confined in its effectiveness to species of Colletorichum as
pathogens. More recently, Kutzner et. al., (1993) described SAR in bean
against TNV using bean rust as inducer.

Induced resistance in cultivars lacking resistance genes was examined

under microscopy of epidermal strips by Ellistion et al., (1976b). Protection

24

caused by C. lagenarium became evident 84 to 96 hours after inoculation with
C. lindemuthianum. When the primary mycelium of the C. lindemuthianum
ceased growing, the contents of the invaded plant cells became granular and
brown. Cloud and Deverall (1987) repeated the earlier experiments of Sutton
(1979) confirming the effectiveness of droplet inoculation on the first leaves in
greatly diminishing symptom development on the second leaves when these
were inoculated a week later.

Several lines of evidence indicate that SAR induces putative defense
compounds in soybean. Soybean seedlings that Were initially inoculated with
Phytophthora cactorum were protected from subsequent infections of P.
megasperma. This was associated with the accumulation of phytoalexins
(Paxton and Chamberlain, 1967; Svoboda and Paxton, 1972). More recent
work has shown that resistance induced against P. megasperma results in the
increased levels of pterocarpanoid phytoalexins, B-l,3-endoglucanase,
isoflavones, daidzein, and genistein (Hahn et al., 1985; Bonhoff and Grisebach,
1988; Yoshikawa et al., 1990; Morris et al., 1991; Graham and Graham,
1991). These defense-related proteins and phytochemicals induced during SAR
enhance resistance to pathogens.

In addition to biotic inducers, certain chemicals with no direct antibiotic
effect can also induce resistance in plants. These include natural products such

as salicylic acid (SA) (White, 1979) and synthetic immunomodulators such as

25

2,6-dichloro-isonicotinic acid (INA) (Metraux et al. 1991). Experiments with
INA in the common bean system showed that the application to the first leaves
not only protected the second leaves against C. lindemuthianum, but also
against Pseudomonas syringae pv. phaseolicola, the cause of halo blight, and
Uromyces appendiculatus, the cause of beans rust disease (Dann, 1991).
Additionally, INA treatments were equally or more effective as C.
lindemuthianum in inducing systemic resistance against the same foliar
pathogens, that is, the anthracnose and rust fungi (Dann and Deverall, 1995).
The discovery of new chemical compounds that stimulate natural disease
resistance mechanisms will provide innovative strategies for crop protection.
Pretreatments of crop plants to improve resistance against pathogens is an
attractive disease control practice, especially when protection covers resistance
against a wide range of pathogens (Kuc, 1982; Metraux et al., 1991; Ryals et
al., 1992). Such an approach would complement conventional chemical control
and assist crop management strategies. Progress toward implementing these
strategies has been limited. Considering the eventual breakdown of genetic
disease resistance coupled with the finite sources of genetic tolerance against S.
sclerotiorum, an alternative means of control would be desirable in soybean.
The objective of this study was to determine if the application of 2,6-dichloro-

isonicotinic acid (INA) to soybean would induce resistance to S. sclerotiorum.

26

MATERIALS AND METHODS

General Experimental Procedure. A two-year study was conducted at East
Lansing, M1 on a Capac loam (fine -loamy, mixed, mesic Aeric Ochraqualfs)
and at Zilwaukee, M1 on a Sloan silt loam (fine-loamy, mixed, mesic
Fluvaquentic Haplaquolicorganic). Four soybean cultivars were chosen for this
study that varied in disease reaction to S. sclerotiorum: NK 1990, Elgin 87,
Corsoy 79, and Williams 82 (Table 1). A combination secondary tillage tool
with cultivator shovels, cutting blades, spike tooth harrow, and rolling baskets
was used to prepare the seedbed at both locations. Soybean cultivars were
solid-seed planted at 385,000 seeds ha" on May 18, 1993 and May 15, 1994 at
both locations. The experiment was maintained weed-free with a postemergence
application of 0.78 kg ai ha" bentazon with 0.21 kg ai ha" acifluorfen plus crop

oil concentrate at 2.9 L ha“, followed by hand weeding twice.

27

Table 1. Cultivars utilized in tests. Disease susceptibility ratings are based
on grower observations and can be influenced by any number of

cultural practices and environmental conditions.

 

 

 

 

 

 

 

 

 

 

SOYBEAN CULTIVARS
CULTIVAR MATURITY DISEASE
SUSCEPTIBILITY
NK 1990 I Highly Tolerant
Corsoy 79 II Moderately Tolerant
Elgin 87 II Moderately Susceptible
Williams 82 III Highly Susceptible

 

28

Field Disease Pressure. Rainfall was supplemented with overhead irrigation to
total 4 cm of water per wk at East Lansing during flowering in 1993 and 1994.
The test at Zilwaukee was managed under rainfed conditions in 1993 and 1994
due to the relatively high water table at this location. Historically, the
Zilwaukee research site had a high level of natural S. sclerotiorum infestation
and was planted to susceptible crops in previous years. To ensure an adequate
level of inoculum in soil at East Lansing, sclerotia were collected from cull pile
screenings at a dry bean processing plant in the fall of 1990 - (present) and
lightly incorporated into soil with a cultivator.

Mycelial Growth on INA-Amended PDA. Wild—type isolates were used to
determine if INA had any direct antibiotic effect on S. sclerotiorum. Isolates
were maintained on potato-dextrose agar (PDA) at 4 C and cultured on PDA at
22 to 25 C. Four PDA (Difco) solutions were prepared at INA concentrations
of O, 130 mM, 260 mM, and 520 mM, autoclaved at 121 C for 20 min, then
poured into petri dishes and allowed to cool. Mycelium were grown by
transferring agar disks (3 mm diameter) cut from the actively growing colony
margins of individual isolates and placed in the center of each PDA plate with
the mycelium in contact with the surface of the medium. Four replicates of
each INA concentration were placed under fluorescent lights. Colony diameters
were determined after 3 days by taking the mean of two perpendicular

measurements .

29

General Chemical Procedure. INA (2,6-dichloro-isonicotinic acid) (CIBA-
GEIGY AG, Basel, Switzerland) was formulated as a wettable powder with
25% active ingredient. It was applied to 1.5 m by 6.1m plots with a hand-held,
COz-pressurized sprayer equipped with a four-nozzle boom with 8004 type flat
fan nozzles delivering a volume of 106 L ha’l at 207 kPa in 1993 and 1994.‘
The suspensions were prepared with sterile tap water. Crop injury was
assessed 14 and 28 d after treatment both years, using a 0 to 100% scale, with
0 = no injury and 100% = plant death.

1993 Field Study. In 1993, treatments of INA at the East Lansing and
Zilwaukee sites were arranged in a randomized complete block design with two
and five blocks, respectively. INA was applied at 78 mM, 130 mM, or 182
mM on July 5 and then the same rate applied again on July 15 and July 25.
The East Lansing site was harvested on November 13, 1993.

1994 Field Study. The experimental design in 1994 was a randomized complete
block design. This experiment included higher INA concentrations, an earlier
initial INA application, and a varying number of applications during the
infection period. Treatments at the East Lansing and Zilwaukee sites were
arranged in a randomized complete block design with four replications at each
location. INA was applied at 182 mM, 234 mM, and 339 mM either once on
June 25, three times on June 25, July 4, and July 15 or four times which

included a July 28 application. The experiment was harvested on November

30

11, 1994 at both locations.

Disease Ratings. Disease was evaluated on September 9, 1993 and September
30, 1994 at the East Lansing Site and on September 11, 1993 and September
28, 1994 at the Zilwaukee site. Individual plants were rated for S. sclerotiorum
on a 0-3 scale, where 0 = no symptoms, 1 = lesion(s) only on lateral stems, 2
= lesion(s) on main stems, and 3 = diseased plant dead (Sherwood and
Hagedorn, 1958). A score of 0 would be given to a plot where no plants rated
have disease symptoms and 100 to a plot where all plants rated were dead from
the disease. Fifty plants in 1993 and thirty plants in 1994 were scored at
random from the center three rows of each plot. A disease severity index (DSI)
was calculated by the method of Sherwood and Hagedorn (1958).

Statistical Protocol. Data were analyzed using the analysis of variance and
general linear model procedures of the statistical analysis system (SAS). Data
from each year was analyzed separately due to the different experimental

designs.

31

RESULTS and DISCUSSION

INA did not affect maturity, height or lodging of any cultivar in 1993
and 1994 (Data not presented). Treatments with INA were not accompanied by
any crop injury at the highest concentrations in 1993 and 1994. Evaluations of
fungal isolates indicate that the effect of INA is unlikely to result from a direct
antibiotic effect. There was no difference in colony diameters at any

concentration three days after plating (Figure 3).

32

Figure 3. Colony diameters of S. sclerotiorum grown on INA-amended PDA

for 3 days, expressed as percentage of unamended control (8 cm).

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A

_O.SCOO UFO

 

“so

0
4

LmHoEmfi >co_oO

_ .
O
2

 

 

.20
)

ation (In

INA C'onceriti"

M

34

Significant (P s 0.01) yield increases were seen with applications of
INA averaged over the four cultivars (Table 2). When cultivars were
individually evaluated for the effect of INA, only Corsoy 79 had a significant
(P S 0.01) yield increase. The nonsignificant effect of INA for individual
cultivars was at least partly because of the limited replication of the yield data.
Yield was taken only for the two replications of plots in Ingham County. Yield
response in INA treated soybeans were presumed to be the result of increased
protection against S. sclerotiorum. Ideally, a producer would want to "induce
resistance" before flowering. The ascospores are the primary inoculum for S.
sclerotiorum, first colonizing senescing flower parts and then progressing into
the stem tissues (Tourneau, 1979; Lumsden, 1979; Abawi and Grogan, 1979).
Significant disease reduction (P s 0.01) was observed with INA applications

averaged over the four cultivars (Table 3).

35

Table 2. Yield results from two blocks at the East Lansing site in 1993.

* Significantly different from control at LSD P S 0.01.

 

Cultivar INA Rates (mM)
0 78 130 182
(kg ha") -------------

NK 1990 4048 4442 4396 4434
Corsoy 79 3287 3954 4101* 4037
Elgin 87 3550 3839 4063 3521
Williams 82 2783 3237 3247 3121
Mean 3417 3868"F 3952* 3778

Table 3.

Cultivar

NK 1990
Corsoy 79
Elgin 87
Williams 82

Mean

36

D81 ratings of cultivars in 1993 at East Lansing and Zilwaukee.

Fifty plants were scored at random from the center three rows of

each plot. Reaction of cultivars to S. sclerotiorum was rated on a

0-3 scale, where 0 = no symptoms, 1 = lesion(s) only on lateral

stems, 2 = lesion(s) on main stems, and 3 = diseased plant dead

(Nelson et al. 1991). A disease severity index (DSI) was

calculated by the method of Sherwood and Hagedorn (1958). *

Significantly different from control at LSD P s 0.01.

0.3

1.8

14.5

37.9

13.6

INA Rates (mM)

78

 

0.3
0.9
8.7
20.9*

20.9*

130

(DSI) -------------

0.3
0.6
3.6*
16.6*

5.3*

182

0.5
1.1
2.7*
17.7*

5.5*

37

In 1994 soybean response to INA was influenced by application timing,
but not by application rate. The initial application on June 25 was applied
approximently six to ten days in advance of flowering. One application INA on
June 25 was not sufficient to provide significant protection against S.
sclerotiorum in any cultivar regardless of INA application rate.’ A significant
yield increases was seen with applications of INA for Williams 82 (Table 4).
Significant disease reduction (P s 0.01) was also observed for these INA

applications in Williams 82 (Table 5).

Table 4.

Cultivar

NK 1990
Corsoy 79
Elgin 87
Williams 82

Mean

38

Yield results from the 1994 tests at East Lansing and Zilwaukee
sites. Rates (182 mM, 234 mM, and 339 mM) are combined
within each application of each cultivar since there was not a
significant rate effect. it = 8 for the control (0) and n = 24 for

applications 1, 3, and 4. ’* Significantly different from control at

2932

LSD P S 0.01.
Number of INA Applications
0 l 3 4
------------- (kg ha“)-------------
3662 3650 3571 3486
2888 2876 2877 2757
2762 2900 2956 3062
1922 2057 2325 2493*
2809 2871 2950

Table 5.

Cultivar

NK 1990
Corsoy 79
Elgin 87
Williams 82

Mean

39

DSI ratings of cultivars in 1994 at East Lansing and Zilwaukee.
Thirty plants were scored at random from the center three rows of
each plot. Reaction of cultivars to S. sclerotiorum was rated on a
0-3 scale, where 0 = no symptoms, 1 = lesion(s) only on lateral
stems, 2 = lesion(s) on main stems, and 3 = diseased plant dead
(Nelson et al. 1991). A disease severity index (DSI) was
calculated by the method of Sherwood and Hagedorn (1958).
Rates (182 mM, 234 mM, and 339 mM) are combined within
each application of each cultivar since there was not a significant
rate effect. n = 8 for the control (0) and n = 24 for applications
1, 3, and 4. * Significantly different from control at LSD P s

0.01.

Number of INA Applications

 

0 1 3 4
(DSI) -------------

13.8 14.5 9.1 9.5

26.5 33.8 30.5 31.0

50.0 54.3 50.7 45.3

65.0 58.5 51.4 499*

38.8 40.3 35.4 33.9

40

The contrasting yield results between 1993 and 1994 could be the result
of: (i) the effect of INA was not sufficient enough to overcome the increase
disease pressure observed in 1994 versus 1993 or (ii) the environmental
conditions were not conducive for allowing the INA effective entry and
distribution into the plant. The INA derivative utilized in this study was in the
free acid form. One factor that would enhance the effectiveness in the field
would be the use of an appropriate surfactant for soybean.

S. sclerotiorum is an important fungal pathogen of many plant species
and is responsible for substantial losses in crop yields in North America each
year (Purdy, 1979). Disease symptoms on individual plants are often severe
under suitable environmental conditions. The destructive nature of these
symptoms is facilitated by its extensive host range and the production of
persistent sclerotia that produce apothecia. Cultural practices aimed at
minimizing S. sclerotiorum infection have had a marginal impact depending on
the environmental conditions. Additionally, the ability to identify and
manipulate heritable genetic resistance have been slow. The advent of synthetic
immunomodulators such as 2,6—dichloro—isonicotinic acid (INA) which activate
inducible defenses could result in a broad spectrum of protection not limited to
S. sclerotiorum in soybean. Evidence from a related signal molecule (SA)
indicates that the exogenous application of SA stimulates resistance to a variety

of lesion-inducing viral, bacterial, or fungal pathogens (Mills and Wood, 1984;

41

Penazio et al., 1987; Ye et al., 1989; Rasmussen et al., 1991). Confirmation
of broad spectrum control of disease in cucumber and arabidopsis with INA
was shown by Metraux et al. (1991) and Ward et al. (1991). In our tests, 2,6-
dichloro-isonicotinic acid (INA) protected the most susceptible soybean cultivars
from S. sclerotiorum under field conditions, suggesting that INA functions

indirectly by increasing the plants resistance.

42

CONCLUSION

Two lines of evidence suggest that the fungicidal response observed in
INA immunization results not from direct effects on the pathogen but from a
reaction of the plant to the chemical. First, neither INA nor its metabolites
have direct antibiotic activity (Metraux et al., 1991). Second, INA has been
shown to cause dramatic changes in plant gene expression that are similar to the
changes observed during pathogen-induced immunization (Ward et al., 1991).
Thus, INA may induce resistance by mimicking some aspects of pathogen
attack, possibly accelerating the normal responses of the plant to further
infection; therefore, the biological responses of soybean to chemical
immunization should be investigated. Plants should be treated with varying
concentrations of INA and, at increasing intervals after treatment, should be
inoculated with a pathogen such as Peronospora parasitica that causes downy
mildew. Trypan blue staining can be used to visualize the plant-fungus
interaction because it penetrates fungal cells easily and preferentially stains
plant cells that have suffered membrane damage (Keogh et al., 1980).
Additionally, in tobacco, INA and other immunizing agents induce the dramatic
accumulation of mRNA’s that encode a variety of extracellular proteins (Ward
et al., 1991). Using PAGE examination, proteins present in the intercellular

wash fluid after INA treatment should be compared with soybeans plants that

43

had been treated with a water control. This would lead up to experiments
defining the onset of SAR at the molecular level which can be achieved by the
isolation and characterization of proteins followed by cDNA identification with
oligonucleotides, cross-hybridization of known cDNA clones to previously
unknown related genes and differential screening of cDNA’s expressed in tissue
rendered systemically resistant by INA treatment. The identification of cDNA’s
would allow one to implement time-course studies of resistance development.

A significant issue is arising from observations of hyperinducibility of
defense genes and whether the hyperinducing signal combinations can induce
resistance to pathogens. Many genes are induced by several apparently
unrelated signals, such as osmotic stress and pathogen invasion, bringing into
question the relationship between inductive signals and gene function (Mason et
al., 1992; Xu et al., 1994). There is evidence that there are hyperinduced
states of plant defense genes that result from particular signal combinations.
Signal combinations may synergistically hyperinduce other plant defense genes
and that such synergistic signals may be more specifically related to gene
function than any single inductive signal, for example, using combined
treatments of SA and INA.

Another aspect of the induced resistance state is understanding the nature
and transmission of the systemic signal or signals. Elicitors of diverse chemical

nature, including carbohydrates, lipids, oligosaccharides and proteins and have

44

been shown to trigger plant defense responses at nanomolar concentrations
(Ebel and Cosio, 1994). The effects of elicitors on ion fluxes across the plasma
membrane have been considered to be part of elicitor-specific signal
transduction leading to the induction of plant defense responses in several plant-
pathogen interactions (Farmer et al., 1989; Dietrich et al., 1990; Felix et al.,
1991; Mathieu et al., 1991; Renelt et al., 1993). Thus, nonchemical signals
can not be excluded at this time and would be of great interest for further

investigation.

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45

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