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This is to certify that the

dissertation entitled

Isozyme Genetics, Cultivar Relationships, and Disease

Reaction of Creeping Bentgrass (Agrostis palustris Huds.)

presented by

Scott Eric Warnke

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Plant Breeding and Genetics

 

Date 7/2, 9S
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MS U i: an Affirmative Action/Equal Opportunity Institution

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DATE DUE DATE DUE

 

ISOZYME GENETICS, CULTIVAR RELATIONSHIPS, AND DISEASE
REACTION OF CREEPING BENTGRASS (Agrostis palusm's Huds.)
By

Scott Eric Wamke

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

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Sciences

1995

ABSTRACT

ISOZYME GENETICS, CULTIVAR RELATIONSHIPS, AND DISEASE
REACTION OF CREEPING BENTGRASS (AGROSHS PALUSTRIS HUDS.)

By
Scott Eric Warnke

Creeping bentgrass is a cool-season turfgrass species primarily used for the
establishment of golf course greens, tees, and fairways. Efficient breeding efforts with
this species will require that the level of genetic understanding of creeping bentgrass be
improved. Molecular markers such as isozymes can be an effective tool for studying
the genetic behavior of an organism and can lead to more efficient breeding stratagies
through marker assisted selection. The objectives of this research were to utilize
isozymes to improve the level of genetic understanding of creeping bentgrass to assess
the level of genetic diversity in the cultivated germplasm, and to determine the disease
reaction of bentgrasses to Sclerotinia homoeocarp Bennett. the causal agent of dollar
spot.

Inheritance data from tetraploid Agrostis palustris Huds. populations were
collected. Segregation data at the Ipi-I, 112112, Got-2, Pgi-2 and Pgm-Z loci were used
to identify allozymes and distinguish between allo- or autotetraploid segregation. Fixed
heterozygosity at the Ibi-Z and Got-2 loci and disomic segregation for a tetra-allelic
individual at the Pgi—Z locus provided strong genetic evidence to support allotetraploid
inheritance in cultivated creeping bentgrass.

The unweighted pair group method with arithmetic averages (U PGMA) cluster

analysis generated from the between cultivar genetic distance matrix divided the

cultivars into two clusters. One cluster contains ten cultivars all potentially related to
the cultivar Seaside. The second group contains the tightly clusterd cultivars
Pennlinks, Southshore, Pro/Cup and Lopez as well as a group of cultivars with some
unique allozyme characteristics.

Thirty-one cultivars of bentgrass representing 2 species (Agrostis palusm's
Huds. and Agrostis tennis Sibth.) were screened for their reaction to a single isolate of
Sclerotinia homoeocarpa Bennett. Plants were rated on a scale of 1 (dead plant) to 9
(no disease damage). The overall disease means of cultivars ranged from 1.0 to 2.6.
Sixty-three percent of the bentgrass population was killed by the pathogen and 96%

received a score of 1 or 2 indicating that resistance to this pathogen is very low.

To Mary Ann, Erik, and Samantha
' for all of your love, friendship, encouragement, and understanding

iv

ACKNOWLEDGMENTS

I would like to express my gratitude to my advisors, Dr. Bruce Branham, and Dr. Dave
Douche: for their guidance and friendship during my studies. They both offered me
tremendous support in the development of my academic and research skills. I would
also like to thank the members of my committee, Dr. James Hancock, Dr. Brian Diets,
and Dr. Joe Vargas. I learned much from each of them during the course of my
studies. I would also like to thank all the members of the turf group and potato group
for their support and encouragement. Thanks also to the Michigan Turfgrass
Foundation for their financial support of this research. And last but not least I would
like to thank my parents Ron, and Marilyn and my brother Kevin for their love

encouragement, and financial support throughout my studies.

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
INTRODUCTION
LIST OF REFERENCES
CHAPTER I: ISOZYME ANALYSIS SUPPORTS ALLOTETRAPLOID
INHERITANCE IN TETRAPLOID CREEPING BENTGRASS
(Agrostis palustris Huds.)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
LIST OF REFERENCES
CHAPTER II: RELATIONSHIPS AMONG CREEPING BENTGRASS
(Agrostis palusm's Huds.) CULTIVARS BASED ON ISOZYME
POLYMORPHISMS
ABSTRACT
INTRODUCTION .
MATERIALS AND METHODS

RESULTS AND DISCUSSION

viii

10

12

16

22

26

28

28

30

32

35

LIST OF REFERENCES
CHAPTER III: SCREENING CREEPING BENTGRASS
(Agrostis palustris Huds.) AND COLONIAL BENTGRASS
(Agrostis tennis Sibth.) FOR RESISTANCE TO DOLLAR SPOT
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION

LIST OF REFERENCES

vii

46

46

47

49

53

57

Table 1.1.

Table 1.2.

Table 1.3.

Table 2.1.

Table 2.2.

Table 2.3.

Table 3.1.

Table 3.2.

LIST OF TABLES

Clones included in creeping bentgrass segregation study, 13
the clone source variety, the crosses made, and the mating
system.

Expected gamete and progeny class frequencies for disomic 15
and tetrasomic inheritance in creeping bentgrass.

Segregation and chi-square values at 5 polymorphic loci in 19
creeping bentgrass.
Creeping bentgrass cultivars studied, the number of plants 33

scored in each, and the average within cultivar genetic distance
(AWGD) of each.

Cultivar allele frequencies for loci that were polymorphic within 37
A. palusm's.

Matrix of Nei’s coefficients of genetic distance for 19 creeping 38
bentgrass cultivars.

Results of artificial inoculation of 31 bentgrass populations with 50
S. homoeocarpa.

Visual rating criteria for bentgrass plants inoculated with 52
Scleorotinia homoeocarpa.

viii

Figure 1.1.

Figure 2.1.

Figure 2.2.

Figure 3.1

LIST OF FIGURES

.Allozyme phenotypes of creeping bentgrass. (A) Pgm-I and
Pgm-Z, (B) Pgi-I and Pgi-Z, (C) Got-2 showing fixed
heterozygosity for the Got-2’ and Got-22 alleles and 1:1
segregation for the Got-2’ allele, ) Tpi-I and Ipi-Z showing
fixed heterozygosity for the 1pi- and 1pi-22 alleles and 1:1
segregation for the Ipi-Z’ allele.

Dendrogram of 18 creeping bentgrass cultivars generated by
UPGMA cluster analysis.

Effects of sample size on the mean genetic distances among
creeping bentgrass cultivars. The differences between

population sizes of 25 and 70 were not significant (or = 0.05).

Scleortinia homoeocarpa mycelia development on creeping
bentgrass plants 12 hours after inoculation.

ix

18

39

43

54

INTRODUCTION

The bentgrasses are native to W. Europe (Harlan, 1992) with the genus Agrostis
consisting of approximately 200 species (Hitchcock, 1951). There are four species
which are commonly accepted as turfgrass types. These are Agrostis palnstris -
creeping Bentgrass (2n=4x=28), Agrostis canina — velvet bentgrass (2n=2x= 14),
Agrostis tennis - colonial bentgrass (2n=4x=28), and Agrostis gigantea - redtop
bentgrass (2n =6x =42). Considerable variation exists in the literature concerning the
naming of these species. For example, creeping bentgrass is referred to as Agrostis
palnstris Huds, Agrostis stolontfera L. and Agrostis stolontfera L. var. palnstris
(Huds.) Farw. Hitchcock (1951) considers A. stolonifera and A. palnstris to be two
distinct species with the primary difference being more prolific stolon production by A.
palnstris. Hitchcock lists the cultivated creeping bentgrass in the United States as
examples of A. palnstris.

According to Duich (1985), the first bentgrasses to be used for putting greens in
the United States were known as South German Bentgrass. South German bentgrass
was harvested from pastures in present day Austria and Hungary and later from other
areas of Europe. South German bentgrass consisted of a mixture of the four
bentgrasses mentioned above and over time would segregate into patches of
predominatly creeping bentgrasses and to a lesser extent velvet bentgrass in favorable
temperate climates. Many of the better appearing patches were selected and maintained
vegetatively by the United States Golf Association Green Section at the Arlington Turf

Gardens in Arlington, VA. Many hundreds of clones were eventually selected, some 0

f whichbecame known as the C-series creeping bentgrasses. The best of the C-series
bentgrasses were used for vegetative establishment of putting greens. The most widely
used C-series bentgrasses were Toronto (015), Cohansey (07), Washington (060),
Arlington (C-l), Congressinal (C-19), and Old Orchard (C-52) each of which was well
adapted to specific regions of the United States.

The only available seeded creeping bentgrass from the late 1920’s until the
1950’s was the variety Seaside. Seaside was discovered growing in tidal flatlands near
Coos Bay, Oregon. However, Seaside was much inferior in quality to the available
vegetatively propagated material. Therefore, putting greens in the United States were
primarily established vegetatively until the 1950’s when the desire to utilize creeping
bentgrass for fairways led breeders to begin developing an acceptable seeded creeping
bentgrass. Holt and Payne (1951) studied the growth rate, texture, density, type of
growth and drought tolerance of seedling progenies from 49 C-series clones of creeping
bentgrass to determine the extent of variability within and among Strains for these
characteristics. The results indicated that seed propagation of most of the available C-
series creeping bentgrass clones was not promising due to the high level of variability
in the seeded progeny.

In 1955, the variety Penncross was released from Pennsylvania State University
by Dr. H.B. Musser and established a new level of excellence for seeded creeping
bentgrasses. Penncross is often referred to as the landmark variety (Rogers, 1991) of
creeping bentgrasses. Penncross has much higher overall quality and is less likely to

segregate into distinct patches than Seaside. Penncross creeping bentgrass is the first

generation (Syn-0) seed only, produced by random crossing of 3 vegetatively
propagated clonal strains (Hein, 195 8).

Creeping bentgrass has the strongest stoloniferous growth habit of any cool-
season turfgrass species and it is also able to tolerate cutting heights of 1.25 cm or less
making it the species of choice under highly managed golf course conditions. The
increased popularity of golf and the resultant increase in golf course construction has
led to the use of creeping bentgrass on a much wider scale. Creeping bentgrass is now
commonly planted on golf course tees, greens, and fairways. The increased utilization
of creeping bentgrass has resulted in an increase in breeding efforts, with the 1993
National Turfgrass Evaluation Programs bentgrass variety trial containing 27 varieties.
However, the increase in creeping bentgrass varieties has not been accompanied by a
similar increase in the level of scientific knowledge about the genetics of this species.

Jones (1956a), in a set of three papers, examined the cytological relationships
in the key members of the Agrostis genus. The first paper examined A. canina L.
subsp. canina (2n=2x=14) and A. canina L. subsp. montana Hartm. (2n=4x=28).
Meiotic analysis of these two species indicated that the diploid regularly forms seven
bivalents and the tetraploid behaves as an autopolyploid.

The second paper (Jones, 1956b) examined the significance of chromosome
pairing in the tetraploid hybrids of A. canina subsp. montana. with A. tennis. and A.
stolonifera. A. tennis and A. stolonijera formed only bivalents at meiosis with A.
tennis possibly being a segmental allotetraploid and A. stolonzfera behaving as a strict

allotetraploid with well differentiated genomes. Chromosome pairing evidence in

crosses between A. tennis and A. stolonifera indicated that these two species might have
one ancestral diploid type in common.

The third paper (Jones, 1956c) dealt with Agrostis gigantea Roth. and its
hybrids with A. tennis and A. stolonifera. A. gigantea regularly formed twenty-one
bivalents at meiosis and the pentaploid hybrids of A. gigantea with A. tennis and A.
stolonifera all showed typical metaphase pairing of 14 bivalents and seven univalents.
The genome constitutions assigned to these three species were A. tennis A1A1A2A2, A.
stolonzfera A2A2A3A3 and A. gigantea A1A1A2A2A3A3. Meiotic pairing studies ean
provide useful information about genomic relationships, however, they are not a
definitive indicator of polyploid type because random bivalent pairing during meiosis
would result in allopolyploid chromosome pairing behavior with autopolyploid
segregation ratios. The most definitive method of determining genomic relationships is
through the use of genetic markers (Krebs & Hancock 1989).

Molecular markers are a fast, reliable, and readily available source of
information that can be utilized to provide an increased genetic understanding of a
species. Protein markers code for proteins that can be separated by electrophoresis to
determine the presence or absence of specific alleles (Tanksley, 1983). The most
widely used protein markers in plant breeding and genetics are isozymes. Isozymes are
multiple molecular forms of an enzyme having the same catalytic activity. Isozyme
analysis is fairly simple and inexpensive and has been used in a number of applications.
Outcrossing rates have been estimated by Shaw et al.(1981) and Shaw and Brown

(1982). Isozymes have been used to facilitate the introgression of genes from wild

species ('I‘anksley et a1, 1980) as well as for measuring genetic variability (Brown and
Weir, 1983), and for varietal patenting and protection (Bailey, 1983). Isozymes have
also been utilized for gene-centromere mapping (Douches and Quiros, 1987) and
quantitative trait locus mapping (Stuber, 1982). Additionally, isozymes have been very
useful for the determination of the mode of inheritance (disomic or tetrasomic) in
polyploid species (Quiros, 1982).

Creeping bentgrass is susceptible to a wide range of diseases including dollar
spot, brown patch, Fnsarinm blight, Pithinm blight, red thread, stripe smut and
Typhnla blight (Beard 1973), therefore, preventative fungicide programs are often
required. On golf courses in the temperate and hot humid regions of the United States
more money is spent to manage dollar spot than on any other turfgrass diseases
(Vargas, 1994). Sclerotinia homoeocarpa Bennett. the causal agent of dollar spot has
developed resistance to cadmium-containing fungicides (Cole et a1. 1968), benzimidizol
compounds (Warren et a1. 1974) and recently the dimetheylase inhibitor fungicides
(Vargas 1994). Therefore, the development of some level of genetic resistance to this
important pathogen of creeping bentgrass is important from both an economic and
fungicide management perspective.

The objectives of this research were to 1) improve the level of genetic
understanding of the cultivated species of creeping bentgrass Agrostis palnstris Hud. , 2)
assess the level of genetic diversity in the cultivated germplasm and determine the

location of germplasm that may be useful for broadening the genetic base of the

cultivated species, and 3) determine the disease reaction of bentgrasses to Sclerotinia
homoeocarpa Bennett. the causal agent of dollar spot. The information gathered from
this research will provide the ground work for future breeding work aimed at

improving this important turfgrass species.

LIST OF REFERENCES
Bailey, DC. 1983. Isozymic variation and plant breeders’ rights. In: Isozymes in Plant
Genetics and Breeding, Part A. Eds. S.D. Tanksley and T.J. Orton. Elsevier Science
Publishers, Amsterdam 425-440.

Beard, J .B. 1973. Turfgrass: Science and Culture. Prentice Hall. USA New Jersey pp.
73.

Brown, A.H.D., and BS. Weir 1983. Measuring gentic variability in plant
populations. In: S.D. Tanksley and T.J. Orton (eds.) Isozymes in Plant Genetics and
Breeding. Elsevier Co., Amsterdam.

Cole, H., B. Taylor, and J. Duich. 1968. Evidence of differing tolerance to fungicides
among isolates of Sclerotinia homoeocarpa. Phytopath. 58:683-686.

Douches, BS, and CF. Quiros. 1987. Use of 4x-2x crosses to determine gene-
centromere map distances of isozyme loci in Solannm species. Genome, 29: 519-527.

Duich, J .M. 1985. The bent grasses. Weeds trees and turf. 72-78.

Harlan, LR. 1992. Crops and Man. American Society of Agronomy, Madison, Wisc.
Hein, M.A. 1958. Registration of varieties and strains of grasses. Agron. Jour. 50:399.
Hitchcock, 1951. Manual of the grasses of the United States 2nd ed. Washington DC.

Holt, EC, and KT. Payne. 1952. Variation in spreading rate and growth
characteristics of creeping bentgrass seedlings. Agronomy Journal. 44:88-90.

Jones, K. 1956a. Species differentiation in Agrostis I. Cytological relationships in
Agrostis canina L. J. Genet. 54:370-376.

Jones, K. 1956b. Species differentiation in Agrostis II. The significance of chromosome
pairing in the tetraploid hybrids of Agrostis canina subsp. Montana Hartm., A. tennis
Sibth. and A. stolonifera L. J. Genet. 54:377-393.

Jones, K. 1956c. Species differentiation in Agrostis III. Agrostis gigantea Roth. and its
hybrids with A. tennis Sibth. and A. stolonifera L. J. Genet. 54:394-399.

Krebs, S., and J. Hancock. 1989. Tetrasomic inheritance of isozyme markers in the
highbush blueberry, Vaccininm corymbosnm L. Heredity 63: 11-18.

Quiros, CF. 1982. Tetrasomic inheritance for multiple alleles in alfalfa. Genetics
101:117-127.

Rogers M. 1991. Cool-season turfgrass varieties. Grounds Maintence. Sept pp. 22.

Shaw, D.V., A.L. Kahler and R.W. Allard. 1981. A multilocus estimator of mating
parameters in plant plant populations. Proc. Nat. Acad. Sci. 78:1298-1302.

Shaw, D.V., and A.H.D. Brown 1982. Optimum number of marker loci for estimating
outcrossing in plant populations. Theor. Appl. Genet. 61:321-325.

Stuber, C.W., M.M. Goodman and RH. Moll. 1982. Improvement of yield and ear
number resulting from selection at allozyme loci in a maize population. Crop Sci.
22:737-740.

Tanksley, S.D., and CM. Rick 1980. Isozymic gene linkage map of the tomato:
applieations in genetics and breeding. Theor. Appl. Genet. 57: 161-170.

Tanksley, S. D. 1983. Molecular Markers in Plant Breeding. Plant Molecular Biology
Reporter. 1:3-8.

Vargas, J .M. 1994. Management of turfgrass diseases. p. 23-26 2nd ed. CRC Press,
Inc., Boca Raton Fl.

Warren, C.G. , P. Sanders, and H. Cole. 1974. Sclerotinia homoeocarpa tolerance to
benzimidazole configuration fungicides. Phytopathology 64: 1139-1 142.

 

CHAPTER I
ISOZYME ANALYSIS SUPPORTS ALLOTETRAPLOID INHERITANCE IN
TETRAPLOID CREEPING BENTGRASS (Agrostis palnstris Huds.)

ABSTRACT
Inheritance data from tetraploid Agrostis palnstris Huds. populations produced by
crossing selected plants from cultivated creeping bentgrass varieties is repOrted. Twelve
enzyme systems were evaluated using the Histidine-Citrate pH 5.7 and Tris-
citrate/Lithium-borate pH 8.3 buffer systems. Four enzyme systems exhibiting
polymorphism in the Tris-citrate/Lithium-borate buffer system were selected for further
genetics studies. Segregation data at the 7pi-1, 7pi-2, Got-2, Pgi-2 and Pgm-2 loci
were used to identify allozymes and distinguish between allo- or autotetraploid
segregation patterns. Two allozymes were identified at the Pgm-Z and Ipi-I , loci while
three allozymes were identified at the Got-2 and 7pi-2 loci. The Pgi-Z locus was
highly polymorphic with six different allozymes identified. Fixed heterozygosity at the
Ipi-Z and Got-2 loci and disomic segregation for a tetra-allelic individual at the Pgi-Z
locus provided strong genetic evidence to support allotetraploid inheritance in creeping

bentgrass.

INTRODUCTION

The bentgrasses are native to Western Europe (Harlan 1992) with the genus
Agrostis consisting of approximately 200 species (Hitchcock 1951). The four species
commonly accepted as turfgrass types are Agrostis palnstris Huds. - creeping bentgrass
(2n=4x=28), Agrostis canina - velvet bentgrass (2n=2x=14), Agrostis tennis —
colonial bentgrass (2n=4x=28), and Agrostis gigantea - redtop bentgrass
(2n=6x=42). Creeping bentgrass is the most widely utilized of the turf-type
bentgrasses because of its excellent tolerance of low mowing heights and a strong
stoloniferous growth habit that makes it well suited for the establishment of golf course
greens and fairways.

Limited genetic research has been conducted on the genus Agrostis, however,
Jones (1955) examined the cytological relationships in the key members of the Agrostis
genus. He examined the significance of chromosome pairing in the tetraploid species
hybrids of A. canina subsp. montana Hartm. with A. tennis Sibth. and A. stolonifera L.
Jones concluded that A. tennis and A. stolonifera form only bivalents at meiosis with A.
tennis possibly being a segmental allotetraploid and A. stolonifera behaving as a strict
allotetraploid with well-differentiated genomes.

A. stolomfera and A. palnstris are two separate species according to Hitchcock
(1951) with the primary difference being more prolific stolon production by A.
palnstris. Hitchcock cites some early vegetatively-propagated creeping bentgrass
varieties as well as naturalized populations in the pacific northwest as examples of A.
palnstris. The creeping bentgrass variety Seaside originates from a naturalized

population in Coos Bay, Oregon (Duich 1985) and is likely to have provided much of

10

11

the initial germplasm for many of the current creeping bentgrass varieties (Wamke et
a1. 1995).

Previous isozyme analysis in creeping bentgrass have focused on varietal
identification. Wilkinson and Beard (1972) used acrylamide gel disc electrophoresis to
distinguish one md-propagated and five vegetatively-propagated creeping bentgrass
varieties. Yamomoto and Duich (1994) utilized bulk plant leaf samples to identify
cross-pollinated bentgrass species and cultivars with starch gel electrophoresis.
However, isozymes have not previously been used for genetic analysis or to determine
polyploid type in creeping bentgrass. The objectives of this research were to study
isozyme segragation patterns in controlled crosses of creeping bentgrass and used this

segregation data to infer the type of polyploidy occurring in creeping bentgrass.

MATERIALS AND METHODS

Approximately 650 clones randomly selected from creeping bentgrass varieties
included in the National Turfgrass Evaluation Program’s 1989 bentgrass variety trial
were screened for isozyme polymorphism. Progeny for isozyme analysis were
developed from. controlled crosses among highly fertile bentgrass clones selected for
their differences in isozyme banding patterns. Crosses were made by placing
reproductive tillers from field-grown plants in 30ml test tubes held upright in clay pots.
Clear plastic bags supported by wooden stakes were placed over the tillers to maintain
pollination isolation. The pots were placed in the greenhouse and the test tubes filled
with water as needed. Seed harvested from the two parents of each cross was kept
separate to examine whether any self-fertilization had occurred (Table 1.1).

Isozyme analysis was performed on individual creeping bentgrass plants that
were at least 2 months old. Plants were analyzed 7 to 10 days after fertilization with
20-20-20 soluble fertilizer. A crude protein extract was obtained by macerating 4 to 5
newly-expanded leaves in 80ul of chilled extraction buffer (Weeden, 1989) composed
of 75mM Tris-HCL buffer, pH 7.5, 5% PVP-40 (w/v), 14mM mercaptoethanol (0.2%
v/v). The extraction was done in chilled, 12 sample, porcelain plates with a plexiglas
rod rounded on one end. The crude extracts were absorbed onto 3 x 8 mm Whatman
3MM wicks and stored over night at
-20°C.

Phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), glutamate
oxaloacetate transaminase (GOT), triosphosphate isomerase (TPI), anodal peroxidase
(PRX), esterase (EST) and alcohol dehydrogenase (ADH) were resolved in Tris-
citrate/Lithium borate pH 8.3 gels (Weeden,l989), after 4.5h of electric current at 50

to 60 mA. Acid phosphatase (APS), 6-phosphogluconate dehydrogenase (6-PGDH),

12

13

Table 1.1. Clones included in creeping bentgrass segregation study, the clone source
variety, the crosses made, and the mating system.

 

 

l n r Matias
3mm
PE 91-25 Penneagle @2115 X NA9l-22)* Cross
PE 91-24 Penneagle 9121421 X PE9l-24) Cross
PV 91-21 Providence (EV21-21 X NA9l-22) Cross
NA 91-22 National (25%;: X PV91-21) Cross
Syn 91-3 Syn 89-1 (311121;: x EM91-6) Selt‘
EM 91-6 Emerald Mk2 X SR91-30) Cross
Nor 91-7 ' Normarc 101 (Regent)
SR 91-30 SR 1020

 

*Underlined clone in each cross is the female parent
‘Was scored as a self-fertilization of Syn91-3

14

aconitase (ACO), malate dehydrogenase (MDH), and shikimic acid dehydrogenase
(SDI-I) were resolved in a Histidine—Citrate pH 5.7 buffer system after 4.5h of electric
current at 35 mA (Weeden, 1989). Gel slabs consisted of 10% potato starch (Starch
Art, Smithville, TX). Gel trays which provided for a direct contact between the gel and
tray buffers were used. Enzyme activity stains were prepared according Vallejos
(1983). PGM activity was improved by the inclusion of 1.5 ml of 0.5% (w/v in H20)
glucose 1-6 diphosphate to the staining solution.

In the anodal section of the gel, the fastest migrating zone of activity in each
enzyme system was designated the first locus and the next zone the second locus. The
fastest migrating allele at each locus was designated the first allele, the next allele was
the second allele and, so on until all alleles were assigned a number. Loci designation
in allopolyploids is complicated by the fact that each locus contains homoeoalleles that
form heterodimers but the chromosomes they are located on do not pair during meiosis,
therefore, two loci are present at each designated locus in this study. Allopolyploid
wheat isozyme loci have been assigned genomic designations based on studies with
monosomic lines (Hart, 1983). However, genomic designation is not currently possible
in creeping bentgrass, therefore, all homoeoloci have been grouped together with no
genomic designations made. Chi-square tests were used to test hypothesized genetic
ratios. Whenever possible, plants homozygous for alternate alleles at a locus were
selected for crossing to determine if self-fertility occurs. Plants appearing to exhibit a
duplex allelic state at a putative locus were also selected since inheritance data from
these plants provides the clearest differentiation between disomic and tetrasomic

inheritance in self and testcross progenies (Table 1.2).

15

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RESULTS

A total of 12 enzyme systems were examined, however, only five showed
excellent resolution and exhibited polymorphism in the anodal region of the gel using
the his-citrate, lithium borate pH 8.3 buffer system, therefore, Pgi-2, 7pi—1, 7pi-2,
Got-2, and Pgm-Z were selected for further study. WH, SDH, EST, and ACO
exhibited complex banding patterns; 6PGDH and APS were poorly resolved with the
buffer systems used; and PRX was monomorphic, so these enzyme systems were not
studied further.

Phosphoglucomutase (PGM) In A. palnstris, PGM is a monomeric enzyme,
which encodes for two putative loci designated Pgm-I and Pgm-2 (Figurela). In this
study, segregation data were not obtained for the Pgm—I locus. Pgm-I often exhibited
fixed heterozygosity, therefore, segregating populations were difficult to find.
Segregation data were available from one cross at the Pgm-Z locus and this cross
followed a test cross segregation pattern for the Pgm-Z2 and Pgm-Z’ alleles (Table 1.3).

Phosphoglucoisomerase (PGI). Two zones of activity were present in the four
crosses scored for PC]. Zone 1 was poorly resolved and difficult to score reliably
(Figure lb). Zone 2 designated Pgi-Z has a complex banding pattern which has been
previously reported by Yamamoto and Duich (1994). Six alleles were scored in the
four crosses we evaluated (Table 1.3). Data from an extensive evaluation of creeping
bentgrass varieties indicates that one or two additional putative alleles may exist in the
cultivated creeping bentgrass population (Wamke et al. 1995).

Pgi-Z is a dimeric enzyme and in many cases the alleles present at this locus
have very similar mobilities, making allele designation difficult. However, the clone

NA91-22 has one copy of the slow migrating Pgi-27 allele and the clone PV91-21 has

16

17

one copy of the slow migrating Pgi-26 allele (Figure 1b.) making it possible to score
allele presence or absence, in the segregating progeny, based on heterodimer location.
The cross between these two clones produced four progeny classes indicating that
PV91-21

18

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20

has one copy of the Pgi-Z‘ allele and three copies of the Pgi-Z‘ allele while the NA91-22 clone
has one copy of the Pgi-z’ allele and three copies of the Pgi-z‘ allele. PV91-21 and NA91-22
were both crossed to PE91-25 and eight progeny classes were detected from each cross
indicating that the P139 1-25 clone is tetra-allelic having the Pgi-Zz, Pgi-23, Pgi—Z“ and Pgi-Z’
alleles. The fourth cross at Pgi-Z involves N0191-7 X SR91-30 and exhibits test cross
segregation for the Pgi-Z“ and Pgi—Z‘ alleles indicating that Nor91-7 is homozygous for the
Pgi-Z‘ allele and SR91-30 has three copies of the Pgi-Z“ allele and one copy of the Pgi-Z‘
allele.

Glutamate oxaloacetate transaminase (GOT) In the three crosses studied, two zones
of activity were detected in the GOT zymograms. The most anodal zone corresponds to Go:-
1, however, this locus was monomorphic in the crosses studied. The slower migrating zone
corresponds to Got-2. Fixed heterozygosity for the Got-2’ and Got-22 alleles was exhibited in
the cross PE91-25 X PV91-21. Segregation at the Got-2 locus was very similar to the Ipi-Z
locus in that three alleles were present in one of the parental clones and fixed heterozygosity
was observed for the Got-2’ and Got-22 alleles. The cross PE91-25 x NA91-22 was another
cross between di-allelic and tli-allelic genotypes similar to the Ipi-Z locus and again only two
progeny classes were observed. One class had the Got-21 and Got-22 alleles and the other had
the Got-2’ , Got-22, and Got-2’ alleles, indicating that the di-allelic clone exhibted fixed
heterozygosity (Fig lc). The final cross to examine the Got-2 locus was the self of Syn 91-3
and a 3:1 segregation ratio for the Got-2' and Got-23 alleles was exhibited. (Table 3).

Triosphosphate isomerase (TPI). The TPI zymogram revealed two very closely
migrating zones of activity, which are coded by two unlinked loci. The separation of these
two loci can be seen more clearly in diploid A. canina populations (data not shown). At the
1PM locus we observed segregation data for two allozymes. The faster allozyme was
assigned Ipi-IZ and the slower band was assigned 7))i-13. Two additional allozymes, one with

21

a faster migration than Ipi-IZ and the other having a slower migration than 1pi-13 have been
observed in other populations (Wamke et al. 1995).

The cross PV 91-21 X PE 91-24 is homozygous for alternate alleles at DH and all
205 progeny examined exhibited a three-banded heterozygous pattern indicating all progeny
were of hybrid origin. The second cross segregating at the Tpi-l locus, Syn 91-3 x Em 91-6,
exhibited a high level of self-fertility based on homozygous alternate alleles at the Tpi-2 locus.
The selfed progeny of Syn91-3 fit a 3:1 ratio of heterozygotes to homozygous individuals
supporting a hypothesis that this clone has one copy of the Ipi-II allele and three copies of the
not-1’ allele.

The Tpi-2 locus has alleles which are quite easily scored. Three crosses were scored for
Tpi-2 segregation and in all cases fixed heterozygosity was observed which again supports
disomic inheritance in this species. The crosses PE9l-25 X PV91-21 and PV9l-21 X PE91-24
both involve a tri-allelic clone having the Tpi-Zl , Tpi-Zz and Ibi-Z’ alleles crossed to a diallelic
clones having the 7pi-2’ and Tpi-Z2 alleles. Only two progeny classes were observed, one
having the 1221121 and 1121122 alleles and the other having the Tpi-Z' , 7pi-22, and 1721323 alleles

indicating that the diallelic clones are exhibiting allotetraploid segregation (Fig 1d).

DISCUSSION

Genetic data from co—dominant molecular markers can clearly distingish between allo-
and auto—polyploidy due to a difference in the progeny classes observed from crosses. A
diploid organism with two different alleles at a locus (A and 0) produces one heterozygote
class (Aa). Selfing of this individual will produce a progeny array of 1AA:2Aa: 100.

However, with tetrasomic inheritance, in an autotetraploid, three different classes of
heterozygotes can be produced (AAaa), (Aaaa) and (AAAa). Selfing of an individual having
the (Mac) genotype will result in a progeny array of 1AAAA:8AAAa:18AAaa:8Aaaa: laaaa
(Muller, 1914; Haldane, 1930). In contrast, preferential pairing of genomes in an
allotetraploid having AA on a chromosome pair in one genome and aa on a chromosome pair
from the other genome would produce only AAaa progeny. Therefore, allopolyploidy results
in “fixed ” heterozygosity which would not be expected in a autopolyploid.

Tetrasomic inheritance has been documented using codominant isozyme loci in a
number of plant species including Medicago sativa (Quiros, 1982), Solanum tuberosum L.
(Martinez- Zapater and Oliver, 1984; Quiros and McHale, 1985), Haplopappus spinulosus
(Hauber, 1986), Tolmiea menziesii (Soltis and Soltis, 1988), Heuchera micrantha (Soltis and
Soltis, 1989) and Vaccinium corymbosum L. (Krebs and Hancock, 1988). Disomic inheritance
in allopolyploids has been documented in tetraploid Tragopogon mirus and T. miscellus (Roose
and Gottleib, 1976) tetraploid Prunus cerasus L. (Beaver and Iezzoni, 1993) and for triplicated
loci in hexaploid Tn'ticum aestivum (Hart, 1983) and quadruplicated loci in octaploid Fragaria
X ananassa (Arulsekar et. al., 1981)

A. palnstris, which is the primary cultivated species of bentgrass in the United States,
is not well-understood genetically. If genetic advancement is to be made, an understanding of
the creeping bentgrass genome must be developed. Co—dominant molecular markers provide a

tool for use in understanding polyploid inheritance

22

23

patterns. The segregation data from crosses that exhibit fixed heterozygosity as well as
the tetra-allelic segregation, observed for the cross of PE91-25 with NA 91-22 and
PV91-21, provides strong genetic evidence for disomic rather than tetrasomic
inheritance in creeping bentgrass (Table 3). Tetrasomic inheritance, either by the
formation of quadravalents or random bivalent pairing, would result in 12 different
progeny classes rather than the 8 which are observed in these crosses and expected with
disomic inheritance (Table 1.2).

Mating systems in allopolyploids and autopolyploids generally compliment the
type of polyploidy exhibited. For example, all autopolyploids maintain heterozygosity
via cross fertilization and exhibit low fertility in response to self-fertilization.
However, most allopolyploids are self-compatible with several being highly self-
polliriated (Mackey, 1970). Creeping bentgrass would appear to be an exception to this
system in that it is a predominately outcrossin g allotetraploid. The high level of
outcrossing in creeping bentgrass results in some characteristics which are normally
attributed to autopolyploids such as the many tri and tetra-allelic genotypes observed in
creeping bentgrass.

In performing crosses with creeping bentgrass, it is not possible to emasculate
flowers due to small floret size, and attempts at self fertilization were ineffective.
Therefore when possible, crosses that had loci fixed for alternate alleles were made to
allow for the establishment of the level of self-fertilization. The clones PV 91-21 and
PE 91-24 are homozygous for alternate alleles at the Tpi-I locus and all 205 progeny
exhibited a three-banded heterozygous pattern indicating that no self fertilization
occurred. However, based on homozygous alternate alleles at the Tpi-Z locus in the
cross of Syn91-3 X Em91-6, only 17 out of 146 progeny were crosses. Interestingly,
the clone Syn91-3 is much less polymorphic than other clones used in this study.
Moreover, scrne of the selfed progeny of this clone were homozygous at all loci

examined. Homozygous alternate alleles were not available for all crosses studied,

24

however, in all cases seed harvested from the two parents was kept separate and alleles
from the proposed male parent segregated in expected ratios inferring little or no self-
fertilization had occured. With all crosses except Syn9l-3 X EM9l-6, seed was
present on the parent tillers of only one of the two clones placed in isolation,
suggesting that a timing mechanism may be involved in reducing self-fertility. The
data from this study indicates that both cross— and self-fertilization can occur in
creeping bentgrass'with outcrossing being the predominant mating system.

Dudeck and Duich (1967) studied the utility of a systematic program of
inbreeding and selection for breeding colonial bentgrass, Agrostis tenuis Sibth. Some
loss of vigor, increased plant mortality and an increase in self-sterility were reported as
the degree of inbreeding increased. However, only two generations of selfing were
reported, therefore, no conclusions can be drawn as to the long-term effects of selection
for self-fertility in A. tennis. Current bentgrass varieties are synthetics, therefore,
segregation for important characteristics can occur during the seed production process.
However, inbreeding might provide a method of reducing the segregation of desirable
characteristics which would create more uniform varieties. The utilization of
inbreeding in creeping bentgrass would require a more thorough understanding of its
effects on fertility and plant vigor as well as the mechanisms of self incompatibility in
this species.

The Pgi-2 locus is very highly polymorphic in creeping bentgrass, with 7 or 8
scorable alleles present in the cultivated population, additionally, Pgi-2 has the highest
staining activity of the enzyme systems tested. The combination of these two factors
indicates that this locus may have potential for the fingerprinting of creeping bentgrass

varieties. PGI allozymes have been useful in the fingerprinting of ryegrass varieties

(Gilliland et al. 1982) and Yamamoto and Duich (1994) were able to distinguish 12

25

creeping bentgrass varieties based on banding patterns at the Pgi-Z locus. However, in
an analysis of additional bentgrass varieties, Wamke et al. (1995) were not able to

distinguish all creeping bentgrass varieties based on the Pgi-Z locus.

Isozymes provide a relatively inexpensive, reliable, and informative source of
genetic markers for creeping bentgrass. Isozymes may also have utility in establishing
varietal and species relationships and estimating the genetic diversity of the cultivated
and non-cultivated species (Wamke et al. 1995). Isozymes were chosen for the initial
studies in creeping bentgrass because they are polymorphic and technically less
complicated and less expensive than DNA markers such as RAPDs and RFLPs. The
information provided by isozyme markers can be utilized to develop highly informative
mapping populations for future studies with DNA-based markers. Genetic maps
developed with RFLP and RAPD markers could aid in the dissection of quantitative
traits through QTL analysis and provide a means of rapid and reliable selection for

simply inherited traits in breeding populations.

LIST OF REFERENCES

Aruldekar, S'., R.S. Bringhurst, and V. Voth. 1981. Inheritance of PGI and LAP
isozymes in octaploid cultivated strawberries. J. Amer. Soc. Hort. Sci. 106:679-683.

Beaver, J .A., and A.F. Iezzoni. 1993. Allozyme inheritance in tetraploid sour cherry
(Prams cerasus L.) J. Amer. Soc. Hort. Sci. 118(6):873-877.

Dudeck, A.E. , and J .M. Duich. 1967. Preliminary investigations on the reproductive
and morphological behavior of several selections of colonial bentgrass, Agrostis tenuis
Sibth.

Duich, J .M. 1985. The bent grasses. Weeds trees and turf. 72-78.

Gilliland, T.J., M.S. Camlin, and CE. Wright. 1982. Evaluation of
phosphoglucoisomerase allozyme electrophoresis for the identification and registration
of cultivars of perennial ryegrass (Lolium perenne) Seed Sci. & Technol. , 10:415-430.
Haldane, J BS 1930. Theoretical genetics of autopolyploids. J. Genetics 22:359-372.
Harlan, J .R. 1992. Crops and Man. American Society of Agronomy, Madison, Wisc-

Hart, 6.13. 1983. Genetics and evolution of multilocus isozymes in hexaploid wheat. In
Isozymes: Current Topics in Biological and Medical Research 10:365-380.

Hauber, D.P. 1986. Autotetraploidy in Haplopappus spinulosus hybrids: evidence from
natural and synthetic tetraploids. Amer. J. Bot. 73:1595-1606.

Hitchcock, 1951. Manual of the grasses of the United States.

Jones, K. 1956. Species differentiation in Agrostis I. Cytological relationships in
Agrostis canina L. J. Genet. 54:370-376.

Jones, K. 1956. Species differentiation in Agrostis II. The significance of chromosome
pairing in the tetraploid hybrids of Agrostis canina subsp. Montana Hartm., A. tenuis
Sibth. and A. stolonifera L. J. Genet. 54:377-393.

Jones, K. 1956. Species differentiation in Agrostis IH. Agrostis gigantea Roth. and its
hybrids with A. tennis Sibth. and A. stolonifera L. J. Genet. 54:394-399.

Krebs, S., and J. Hancock. 1989. Tetrasomic inheritance of isozyme markers in the
highbush blueberry, Vaccinium corymbosum L. Heredity 63:11-18.

MacKey, J. 1970. Significance of mating systems for chromosomes and gametes in
polyploids. Hereditas 66: 165-176.

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27

Martinez-Zapater, J.M. and J .L. Olivier. 1984. Genetic analysis of isozyme loci in
tetraploid potatoes (Solanum tuberosum L.). Genetics 108:669-679.

Muller, H.J. 1914. A new mode of segregation in Gregory’s tetraploid Primulas.
Amer. Nat. XLVIII, 508-512.

Quiros, C.F. 1982. Tetrasomic inheritance for multiple alleles in alfalfa. Genetics
101:117-127.

Quiros, C.F., and N. McHale. 1985. Genetic analysis of isozyme variation in diploid
and tetraploid potatoes. Genetics 111: 131-145.

Roose, M.L., and L.D. Gottlieb. 1976. Genetic and biochemical consequences of
polyploidy in Tragopogon. Evolution 30: 818-830.

Soltis, D.E. , and PS. Soltis. 1988. Electrophoretic evidence for tetrasomic segregation
in Tolmiea menziesii (Saxifragacea). Heredity 60: 375-382.

Soltis, DE, and PS Soltis. 1989. Tetrasomic inheritance in Heuchera micrantha
(Saxifragaceae). J. Hered. 80:123-126.

Vallejos, E. 1983. Enzyme activity staining. In S.D. Tanksley and T.J. Orton (eds.)
Isozymes in plant genetics and breeding, Part A, 469-516, Elsevier, Amsterdam.

Wwden, N.F., and J .F. Wendel 1989. Visualization and interpretation of plant
isozymes. In D.E. Soltis and PS. Soltis (eds.) Isozymes in Plant Biology 5-45.

Wilkinson, J .F. , and LB. Beard. 1972. Electrophoretic identification of Agrostis
palustris and Poa pratensis cultivars. Crop Sci. 12:833-834.

Yamamoto, I. , and J .M. Duich. 1994. Electrophoretic identification of cross-pollinated
bentgrass species and cultivars. Crop Sci. 34:792-798.

CHAPTERII

RELATIONSHIPS AMONG CREEPING BENTGRASS (Agrostis palnstris Huds.)
CULTIVARS BASED ON ISOZYME POLYMORPHISMS
ABSTRACT
An understanding of the genetic variability within a crop species is essential to

its improvement. The objectives of this research were to study the utility of isozyme
patterns for creeping bentgrass cultivar discrimination and to estimate the relationships
between creeping bentgrass cultivars based on isozyme patterns. Seventy plants from
each of 18 creeping bentgrass cutivars and 25 plants from one plant introduction were
scored for 24 isozyme polymorphisms representing six loci. All cultivars except a
small group containing the cultivars Pennlinks, Pro/ Cup, Southshore and Lopez could
be uniquely characterized based on a 20% or greater band frequency in one cultivar
verses absence of the band in the most closely clustered cultivar. The isozyme patterns
from each plant were used to generate a genetic distance matrix for the plants within a
cultivar, and the average band frequency within a cultivar was utilized to create a
genetic distance matrix between cultivars. The cultivars Pennlinks, Pro/ Cup,
Southshore and Lopez had the highest average within-cultivar genetic distances
indicating that additional marker loci will be needed to distinguish these cultivars. The
UPGMA cluster analysis generated from the between cultivar genetic distance matrix
divided the cultivars into two clusters. One cluster contains the variety Seaside as its
base suggesting that this variety may have provided some initial germplasm for this

group. The second group contains the cultivars Pennlinks, Southshore, Pro/Cup and

28

29

Lopez as well as a group of cultivars with some unique allozyme characteristics. The
plant introduction P1251945 was distantly related to the cultivated germplasm indicating
that European material may be a source of genetic diversity to broaden U.S bentgrass

germplasm.

INTRODUCTION

The bentgrasses are native to Western Europe (Harlan 1992) with the genus
Agrostis consisting of approximately 200 species (Hitchcock 1951). The four species
commonly accepted as turfgrasses are Agrostis palnstris Huds - creeping bentgrass
(2n=4x=28), Agrostis canina - velvet bentgrass (2n=2x=14), Agrostis tennis -
colonial bentgrass (2n=4x=28), and Agrostis gigantea - redtop bentgrass
(2n=6x=42). Creeping bentgrass is the most widely utilized of the turf-type
bentgrasses because it has excellent tolerance of low mowing heights and a strong
stoloniferous growth habit making it ideally suited for the establishment of golf course
greens and fairways in areas where cool-season turfgrasses are adapted.

An understanding of the genetic diversity present in the cultivated germplasm of
a species as well as the location of new sources of genetic variability is important for
the optimal utilization of genetic resources. Plant brwders must have an understanding
of the genetic variability of elite germplasm because continued reselection within this
germplasm can narrow the genetic base of elite material and ultimately increase the
potential vulnerability to pests and abiotic stresses. Information about the location of
new sources of genetic variability can help broaden the genetic base of elite material
and maintain long-term improvement.

Information about the relatedness of creeping bentgrass cultivars is limited
because it is an allogamous species and in many cases the parental clones used in
synthetic cultivar development are of unknown origin making accurate estimates of
relatedness based on coefficients of parentage impossible. However, in a number of

species, estimates of genetic similarity between cultivars have been determined based

30

31

on molecular markers such as isozymes in soybeans (Cox et al. , 1985) and potatoes
(Douches and Ludlam, 1991), RFLP’s in barley (Melchinger et al., 1994), maize
(Messmer et al., 1993) and tall fescue (Xu et a1. 1994) and RAPD markers in lima
bean (Nienhuis et al., 1995), and olive (Fabbri etal., 1995). Isozyme markers are not
as numerous as RFLP or RAPD markers, however, they are polymorphic in creeping
bentgrass populations (Wamke et al. , 1995) and technically simpler than RFLP or
RAPD markers with large population sizes. The objectives of this research were to (1)
assess isozyme patterns for creeping bentgrass cultivar identification, (2) determine the
optimum sample size for estimating allozyme frequencies from creeping bentgrass
populations, and (3) estimate the genetic relationship between creeping bentgrass

cultivars based on these allozyme frequencies.

MATERIALS AND METHODS

Eighteen Agrostis palnstris Huds. cultivars representing most of the named
creeping bentgrass cultivars commercially available in the United States and one plant
introduction were assayed for isozyme polymorphisms. Seed was obtained from the
1993 National Turfgrass Evaluation Programs (NTEP) bentgrass cultivar trial or
directly from the seed company that produced the cultivar. In a few cases seed from
the 1989 NTEP bentgrass cultivar trial was used (Table2.1).

The condition of plants for analysis is vital for successful isozyme resolution. It
was determined that etiolated tissue from plants maintained in the laboratory for 1 week
prior toianalysis produced more consistant enzyme activity. Individual plants at least
two months old were used for isozyme analysis. Plants were maintained in the
greenhouse and fertilized every two weeks with a 20-20-20 water soluble fertilizer
solution and trimmed regularly to promote tiller production. A 4 - 5 leaf sample of
newly expanded leaves was collected from each plant and crushed in 80ul of an
extraction buffer composed of 75mM Tris-HCL buffer, pH 7.5, 5 % PVP-40 (w/v),
and 14mM Mercaptoethanol (0.2% v/v). The extraction was done in chilled 12-sample
porcelain color plates and macerated with a plexiglass rod rounded on one end. The
crude extracts were absorbed onto 3 x 8 mm Whatman 3MM wicks and stored over
night at -20°C, or at -80°C when longer term storage was needed.

Phosphoglucose isomerase (PGI, E.C.5.3. 1.9), phosphoglucomutase (PGM,
E.C.5.4.2.2), glutamate oxaloacetate transaminase (GOT, E.C.2.6.l.l), and

triosphosphate isomerase (TPI, E.C.5.3. 1.1) were resolved in tris-citrate/lithium borate

32

33

Table 2.1. Creeping bentgrass cultivars studied, the number of plants scored in each,
and the average within cultivar genetic distance (AWGD) of each.

 

 

Cultivar , Year' Source Number Evaluated AWGD°
1 Penneagle 1978 1993 NTEP bentgrass trial 72 0.233
2 Penncross. 1955 1993 NTEP bentgrass trial 73 0.210
3 Trueline 1995 1993 NTEP bentgrass trial 73 0.267
4 Crenshaw 1993 1993 NTEP bentgrass trial 73 0.203
5 Southshore 1992 1993 NTEP bentgrass trial 73 0.326
6 Providence 1988 1993 NTEP bentgrass trial 70 0.230
7 National 1988 1989 NTEP bentgrass trial 70 0.312
8 Viper 1995 International Swds 72 0.173
9 18th Green 1995 1993 NTEP bentgrass trial 73 0.257
10 Cobra 1988 1989 NTEP bentgrass trial 73 0.180
11 Emerald 1973 1989 NTEP bentgrass trial 73 ‘ 0.138
12 Pennlinks 1987 1993 NTEP bentgrass trial 73 0.387
13 SR1020 1987 Seed Research 73 0.265
14 Putter . 1988 Jacklin Seed 73 0.161
15 Seaside 1924 1993 NTEP bentgrass trial 73 0.258
16 Lopez 1994 1993 NTEP bentgrass trial 72 0.325
17 Pro/Cup 1994 1993 NTEP bentgrass trial 73 0.340
18 Cato 1993 1993 NTEP bentgrass trial 70 0.152
19 P1251945 1958" Plant Intro. Station Pullman,WA 25 0.104
a year of release .

b year collected

c Average within-cultivar genetic distance calculated using Nei’s 1972 distance formula

34

pH 8.3 buffer system (Weeden, 1989), after 4h of electric current at 50 to 60 mA. Gel
slabs consisted of 10% potato starch obtained from Starch Art, (Smithville, TX). Gel
trays that provided for a direct contact between the gel tray and buffer were used.
Enzyme activity stains were prepared according to the method of Vallejos, (1983).

Each gel contained 25 plants from a cultivar and 2 check plants, of known
allozyme composition, to aid in band scoring. Allelic bands (Wamke et. al. 1995)
were scored as 1 present or 0 absent for each plant. A total of 75 plants from each
cultivar were analyzed, however, due to enzyme degradation in some samples, fewer
plants were used to establish band frequencies (Table 2.1). The band frequency in the
population was obtained by dividing the number of plants containing the band by the
total plants examined. Genetic distances between and within each cultivar were
established using Nei’s 1972 distance formula. A dendrograrn based on the distance
matrix was constructed by applying the unweighted pair group method with arithmetic
averages (UPGMA) cluster analysis. The distance matrix and dendrograrn were both
constructed using' NTS YS-pc version 1.7 (Rohlf, 1992). Between-variety genetic
distances were calculated using Sample sizes of 4, 8, 12, 16, 20, 25 and 70 to establish
the optimum population sample size for estimating the genetic distance between

varieties.

RESULTS AND DISCUSSION

A total of 24 alleles were scored for 70 plants in each of the 18 cultivars.
Segregation data is available for 14 of the 24 bands scored (Wamke et al. 1995). Six
of the bands for which no segregation data are available come from the Pgm-I and
Pgm-Z loci. Genetic analysis (Wamke et al. 1995) has shown this to be a monomeric
enzyme so assignment of additional alleles at these loci were based on band mobility
differences compared to the check plants run with each gel. The Pgi-Z locus in creeping
bentgrass is highly variable with seven scorable alleles present, however, the dimeric
structure of this enzyme and close mobilities of many alleles does not allow for the
accurate classification of all alleles present in some genotypes without progeny testing.
Therefore, only the fastest and slowest migrating alleles for each plant, i.e. those that
were easily detected, were scored. Average band frequencies ranged from 0.01 for the
Pgm-I‘ and Pgm-Z‘ bands and 1.00 for the Pgm-I’ and Got-2l bands (Table 2.2).

Distance values (Table 2.3) ranged from 0.007 for Southshore and Pennlinks

to 0.277 for Cato and P1251945. The dendrogram resulting from the UPGMA cluster
analysis is shown in Figure 2.1. The UPGMA cluster analysis separates the cultivars
into two main groups. The first group includes 10 cultivars (Penneagle, Putter,
Penncross, Trueline, Viper, Emerald, 18th Green, Cobra, Crenshaw and Seaside).
With the exception of Crenshaw these are strongly creeping cultivars having a prostrate

to semi-erect growth habit. The cultivar Seaside is at the base of this group and may

35

36

have provided much of the initial germplasm for creeping bentgrass cultivar
development over the last 40 years. Seaside originated as a naturalized population

growing in tidal flats near Coos Bay, Oregon and was the only widely availiable seeded

bentgrass in

37

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38

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40

theU.S. from the 1920’s until 1955 when Penncross was released (Duich, 1985). The
second cluster contains nine cultivars that can be divided into two groups. Four of
these cultivars (Southshore, Pennlinks, Pro/ Cup and Lopez) cluster quite closely and
are difficult to distinguish from one another with the isozyme polymorphisms studied.
One reason for the tight clustering of these four cultivars is that they possess most of
the isozyme bands observed in this study. This isozyme diversity is evident in the fact
that these four cultivars have the highest average within-cultivar genetic distances
(AWGD) of any of the cultivars tested (Table 2.1). The cultivar Southshore is a very
broad-based cultivar derived from the progenies of 203 selected clones (Hurley et al. ,
1994) which would explain its high AWGD The last four cultivars in the second group
(Providence, SR1020, National and Cato) do not group closely with any of the other
cultivars and, in fact, these cultivars all possess some unique isozyme characteristics.
The plant introduction P1251945 was included in the study to gain some insight into the
European bentgrass germplasm. P1251945 was collected in 1958 from Austria and,
based upon these results, it is quite distantly related to the United States cultivars
examined, suggesting that European germplasm may be a means to broaden the genetic
diversity of U.S. germplasm. The cultivars SR1020 (5 clone synthetic) and Crenshaw
(six clone synthetic) share three parental clones in common (Engelke et al. , 1995).
Despite this common genetic base they have very different isozyme profiles and do not
cluster closely in the dendrogram. These cultivars are synthetic populations and
differences in the initial parent clones that make up these cultivars can result in large

genetic distances between the cultivars due to numerous isozyme band differences. For

41

example, the exclusion of two SR1020 parental clones from Crenshaw could result in
the exclusion of isozyme bands present in SR1020 from Crenshaw. Furthermore, the
addition of three new parental clones to the cultivar Crenshaw could result in bands
being present in Crenshaw that are not in SR1020 leading to a large number of band
differences between the two cultivars and a large genetic distance estimate.

Creeping bentgrass cultivar identification via isozymes is complicated by the
fact that cultivars are synthetics and considerable within-cultivar variation exists. The
establishment of isozymes as a reliable characteristic for fingerprinting requires that
band frequencies in a given cultivar be stable in different environments and
generations. In this study all cultivars except Southshore, Pennlinks, Pro/Cup and
Lopez could be distinguished based on a band being present in one cultivar at a
frequency greater than 20% and absent from the most closely-related cultivar. The
cultivar Lopez is close to meeting this requirement at the Got-2’ allele. However,
further research needs to be done on swd lot variability and post-establishment effects
on isozyme band frequencies to determine the overall utility of isozymes for creeping
bentgrass cultivar discrimination. Additionally, it appears that more enzyme systems
will need to be evaluated to reliably distinguish Pennlinks, Pro/Cup and Southshore.

Yamamoto and Duich (1994) examined the utility of isozyme analysis with bulk
plant leaf sampling and were able to distinguish twelve creeping bentgrass cultivars
using only the Pgi-Z locus. Estergaard and Nielson (1981) investigated the utility of
using the Pgi-Z locus for cultivar identification in tetraploid ryegrass and found that
different seed lots did not differ with respect to frequencies of allele classes.

Additionally, seed size differences and low germination frequencies did not lead to

42

significant within-cultivar allozyme frequency differences. Hayward et al. (1978)
demonstrated Pgi-2 isozyme profiles in perennial ryegrass did not differ significantly
between seed stocks and samples from three year old swards. Similar studies must be
conducted for creeping bentgrass to establish the utility of isozyme analysis for
creeping bentgrass variety discrimination in the turf industry.

Sample size is an important consideration in designing experiments for
outcrossing species. One method for determining optimum sample sizes was discribed
by Xu et al. (1994) in an RFLP analysis of tall fescue cultivars. Average genetic
distances are calculated for different population sizes and the point at which there is no
longer a significant difference between the average genetic distance of two population
sizes is considered the most efficient sample size. In our study average genetic
distances between cultivars were calculated using data from 4, 8, 12, 16, 20, 25, and
70 plants. The mean distance between cultivars decreased and became consistent as
population size increased (Figure 2.2). The difference between means (a = 0.05) was
not significant for population sizes of 16 and 20 or 16 and 25, however, there was a
significant difference between population sizes of 16 and 70 as well as 20 and 70 but
not between 25 and 70 indicating that a random sample of 25 plants is the minimum
number of plants nwded to accurately estimate the isozyme variation within these
creeping bentgrass cultivars. In the study by Xu et al. (1994) the largest population
size examined was 20 and there was no significant difference between population sizes
of 16and 20 plants. These results indicate that 16 to 25 plants is an adequate

population size for sampling the genetic variation within these two outcrossing species.

43

 

0.18
0.16
0.14
0.12

0.10
E one
on;
5
E

0.04
0.02
om

 

 

 

 

Figure 2.2. Effects of sample size on the mean genetic distances among
creeping bentgrass cultivars. The difference between population sizes

of 25 and 70 were not significant ((1 = 0.05).

LIST OF REFERENCES

Bailey, D.C. 1983. Isozymic variation and plant breeders’ rights. In: Isozymes in Plant
Genetics and Breeding, Part A. Eds. S.D. Tanksley and T.J. Orton. Elsevier Science
Publishers, Amsterdam 425-440.

Cox, T.S., J.P., G.L. Lookhart, D.E. Walker, L.G. Harrell, L.D. Albers, and D.M.
Rodgers. 1985 . Genetic relationships among hard red winter wheat cultivars as
evaluated by pedigree analysis and gliadin polyacrylamide gel electrophoretic patterns.
Crop Sci. 25:1058-1063.

Douches, D.S. , and K. Ludlam. 1991 . Electrophoretic characterization of North
American potato cultivars. American Potato Journal. 68:767-780.

Duich, J.M. 1985. The bent grasses. Weeds trees and turf. 72-78.

Engelke, M.C., V.G. Lehman, W.R. Kneebone, P.F. Colbaugh, J.A. Reinert, and
WE. Knoop. 1995. Registration of ‘Crenshaw’ creeping bentgrass. Crop Sci. 35:589.

Fabbri, A., J .I. Hormaza, and VS. Polito. 1995. Random amplified polymorphic
DNA analysis of olive (Olea europaea L.) cultivars. J. Amer. Soc. Hort. Sci.
120(3):538-542.

Harlan, J .R. 1992. Crops and Man. American Society of Agronomy, Madison, Wisc.

Hayward, M.D., NJ. McAdam, T. Balls, and M. Zaruk. 1978. The use of isoenzymes
as genetic markes. Report. Welsh Plant Breeding Station. 47.

Hurley, R.H., V.G. Lehman, J.A. Murphy and CR. Funk. 1994. Registration of
‘Southshore’ creeping bentgrass. Crop Sci. 34:1124-1125.

Melchinger, A.E., A. Graner, M. Singh, and M.M. Messmer. 1994. Relationships
among European. barley germplasm: 1. genetic diversity among winter and spring
cultivars revealed by RFLPs. Crop Sci. 34: 1 191-1 199.

Messmer, M.M., A.E. Melchinger, R.G. Herrmann, and J. Boppenmaier. 1993.
Relationships among early European maize inbreds: II. Comparison of pedigree and
RFLP data. Crop Sci. 33:944-950.

Nei, M. 1972. Genetic distance between populations. Amer. Natur. 106:283-292.
Nienhuis, J ., J. Tivang, P. Skroch, and J .B. dos Santos. 1995. Genetic relationships

among cultivars and landraces of lima bean (Phaseolus lunatus L.) as measured by
RAPD markers. J. Amer. Soc. Hort. Sci. 120(2):300—306.

44

45

Ostergaard, H. and G. Nielsen. 1981. Cultivar identifieation by means of isoenzymes
I. Genotypic survey of the Pgi—2 locus in tetraploid ryegrass. Z. Pflanzenziichtg.
87: 121-132

Rohlf, F.J. 1992. NTSYS-pc. Numerical taxonomy and multivariate analysis system,
version 1.70. Exeter Software, New York.

Xu, W.W., D.A. Sleper, and GP. Krause. 1994. Genetic diversity of tall fescue
germplasm based on RFLPs. Crop Sci. 34:246-252.

Yamamoto, I. , and J .M. Duich. 1994. Electrophoretic identification of cross-pollinated
bentgrass species and cultivars. Crop Sci. 34:792-798.

CHAPTERIII

SCREENING CREEPING BENTGRASS (Agrostis palnstris Huds.) AND COLONIAL
BENTGRASS (Agrostis tenuis Sibth.) FOR RESISTANCE TO DOLLAR SPOT

ABSTRACT

Thirty-one cultivars of bentgrass representing 2 species (Agrostis palnstris
Huds. and Agrostis tenuis Sibth.) were screened for their reaction to a single isolate of
Sclerotinia homoeocarpa Bennett. Two inoculations were conducted and plants were
rated on a scale of 1 (dead plant) to 9 (no disease damage). The overall disease means
of cultivars ranged from 1.0 to 2.6. Sixty-three percent of the bentgrass population
was killed by the pathogen and 96% received a score of 1 or 2 indicating that resistance
to this pathogen is very low. Those plants showing enhanced tolerance to S.
homoeocarpa were selected and will be evaluated further in an effort to understand the

mechanisms involved in there survival.

46

INTRODUCTION
Creeping bentgrass (Agrostis palnstris Huds.) is a cool season turfgrass species

primarily used for golf course putting greens and fairways. Dollar spot, caused by
Sclerotinia homoeocarpa Bennett, is a major disease of creeping bentgrass in areas
where high humidity, prolonged leaf wetness and temperatures greater than 20 C occur.
S. homoeocarpa causes straw-colored, sunken spots approximately 5 cm in diameter on
creeping bentgrass greens and fairways. Foliar lesions are typically straw colored with
brown margins.

On golf courses in the temperate and hot humid regions of the United States
more money is spent to manage dollar spot than on any other turfgrass disease (Vargas,
1994). In areas where frequent fungicide applications are practiced S. homoeocarpa
has shown an ability to develop resistance to Cadmium-containing fungicides (Cole et
al. 1968), Benzimidizol compounds (Warren et al. 1974) and recently the Dimethylase
inhibitor fungicides (Vargas, 1994).

Endo et al. (1964) and Endo and Malca (1965) found a toxin associated with
stunting and necrosis of bentgrass roots. The lack of pathogenicity at 10 and 32 C was
associated with a lack of toxin production (Endo, 1963). The toxic substance was
found to have chemical similarities to D-galactose (Malca and Endo 1965), however,
plant response differences between the toxin and D-galactose lead them to conclude that
D-galctose is not the toxic substance produced in viva (Endo and Malca 1965).

A practical means of slowing resistance to these fungicides and reducing
fungicide application costs is to develop host plant resistance to this disease. Research

on the host pathogen interaction of A. palnstris and S. homoeocarpa is limited. Cole et

47

48

al. (1969) in a study of the influence of fungal isolate and bentgrass variety on dollar
spot development screened 18 different creeping bentgrass populations with 4 different
dollar spot isolates and found a significant fungal isolate variety interaction. Reports
from regional variety trials (Colbaugh & Engelke, 1993) (Hsiang & Cook, 1993)
(Doney & Vincelli, 1993) provide somewhat conflicting information, however, the
varieties SR1020 and Emerald are consistently highly susceptable, while the colonial
bentgrasses have shown the highest levels of resistance.

The improvement of creeping bentgrass resistance to dollar spot will require a
more thorough understanding of the methods of pathogenesis utilized by S.
homoeocarpa as well as the plant defense mechanisms available in creeping bentgrass.
Determinining plant resistance mechanisms under field conditions can be misleading,
because it is difficult to determine if a plant without disease symptoms is resistant or
was not exposed to disease pressure. This problem can be avoided by using artificial
inoculations under controlled environmental conditions. The objectives of this research
were to 1) establish a means of inoculating individual bentgrass plants under controled
environment conditions, 2) establish the range of reaction among and within bentgrass
populations to S. homoeocarpa and, 3) select the most resistant plant for further

genetics studies. ’

MATERIALS AND METHODS

Seeds from 31 different bentgrass cultivars and plant introductions (Table3. 1)
were planted in flats containing 128 cells of 3.5 cm2 volume filled with Bacto soil-less
media. The flats were misted twice a day until plants reached the three-leaf stage. The
healthiest 100 plants from each cultivar and plant introduction were selected and
watered as needed, fertilized monthly with a 20-20-20 soluble fertilizer, and clipped
twice monthly.

A S. homoeocarpa isolate obtained from dollar spot infected turf at the Hancock
Turfgrass Research Center East Lansing, MI was used to prepare infested topdressing
according to the method of Goodman and Burpee (1991). A mixture of sand and
cornmeal (2:1) was placed in 30 x 40-cm aluminum baking pans lined with aluminum
foil. The sand cornmeal mixture was smoothed to a depth of l-cm and autoclaved at
121 C twice for 60 minutes then moistened to 6% (v/v) with 1% lactic acid in sterile
deionized water. Three fungal colonies on 20ml of potato dextrose agar in 9-cm petri
dishes were cut into 100-200 pieces and placed on the 1200cm3 of sterile sand-
commeal. After incubation for 2 weeks at room temperature the media was sliced and
forced through a 2.5-mm mesh screen.

A ‘. Five-month old creeping bentgrass plants from the 31 different bentgrass
varieties and plant introductions were inoculated by top-dressing with 0.75g of S.
homoeocarpa infested sand cornmeal and then placed in a growth chamber.
Populations to be screened were split into two groups due to growth chamber space
limitations. Controls of both uninoculated and inoculated plants with noninfested sand—

commeal were included with each screening. The flats and growth chamber walls

49

50

Table 3.1. Results of artifical inoculation of 31 bentgrass populations with S. homoeocarpa.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

try Speciw Origin Mean Nunber of Plants in Clam Number
. ' Rati ‘ l 2 3 4 5 6 7 Scored
DF-l Creepipg Tee-Z-Green Com. 2.6 13 60 3 6 12 4 2 100
A-l Creeping Tee-2-Green Corp. 2 27 61 3 4 2 3 0 100
Lopez Creeping Finelawn Research 1.9 24 71 1 3 1 0 0 100
ICobra Creeping International Seeds 1.7 42 52 2 4 0 0 0 100
Syn 92-1-93 Creeping Texas A&M Univ. 1.7 52 40 0 6 2 0 0 100
Bar Ws 4210 Creeping BarenbrugHolland 1.6 48 45 3 3 l 0 0 100
Pennlinks Creeping Tee-2-Green Corp. 1.6 58 33 1 7 1 0 0 100
88CBE Creeping International Seeds 1.5 42 32 5 0 0 0 0 79
18th Green Creepin Johnson Seeds 1.5 54 46 0 0 0 0 0 100
Southshore Creeping Loft's Seed, Inc 1.5 52 46 0 2 0 0 0 100
P1 235541 Creeping Pullman WA. 1.5 65 20 2 6 1 0 0 94
Providence Creepin Seed Research 1.5 49 48 0 l 0 0 0 98
|G-2 Creepifl Tee-2-Green Cog. 1.5 44 55 0 0 0 0 0 99
lG-s Cming Tee-2-Green Corp. 1.5 49 50 0 1 o 0 0 100
Penneagl: Creeping Tee-2-Green Cgp. 1.5 57 40 0 3 0 0 0 100
SR1020 Creepipg Seed Research 1.4 61 36 3 0 0 0 0 100
Penncross Creeping Tee-2-Green C03 1.4 63 36 0 1 0 0 0 100
PI 235440 Creepin Pullman WA. 1.3 70 20 0 2 0 0 0 92
P1251945 ' Creeping Pullman WA. 1.3 72 25 1 2 0 0 0 100
A-4 Creeping Tee-2-Green Corp. 1.3 78 20 0 1 1 0 0 100
Syn 92-5-93 Creeping Texas A&M Univ. 1.3 73 23 2 2 0 0 0 100
Syn 92-2-93 Creeping Texas A&M Univ. 1.3 76 22 0 1 l 0 0 100
Pro/Cup Creeping Forbes Seed 1.2 81 19 0 0 0 0 0 100
Emerald Creeping International Seeds 1.2 81 18 0 1 0 0 0 100
|Crenshaw Creeping Texas A&M Univ. 1.2 75 25 0 0 0 0 0 100
[Cato Creeping Texas A&M Univ. 1.2 79 21 0 0 0 0 0 100
Trueline Creeping Turf Merchants 1.2 72 20 0 0 0 0 0 92
SR7100 Colonial Seed Research 1.1 90 9 0 0 0 0 0 99
Seaside Cregiing Standard 1.1 89 11 0 0 0 0 0 100
Tendez Colonial Finelawn Research 1 94 1 0 0 0 0 0 95
Syn 1-88 Creeping Texas A&M Univ. 1 86 14 0 0 0 0 0 100
Total or mean 1.4 1916 1019 26 56 22 7 2 3048
% of total plants 63 33 0.8 1.8 0.7 0.2 0.1
% of plants 3.7
selected
* Rating from 1—9 l=dead plant 9=no
damage

 

 

 

 

 

 

 

 

51

were misted daily at 1700 h to increase relative humidity. The growth chamber was
shut down every night during the inoculation phase to eliminate air movement. The
flats were maintained at 75 to 85% relative humidity and 25 to 30 C from 1700 h to
0900 h. Temperatures were 25 to 30 c and relative humidity 30-60% from 0900 to
1700. Disease pressure was maintined for seven days and each group of varieties
received two inoculations to control for the possibility of escapes. A 1-9 rating scale
was established (Table 3.1) to assess the level of damage to each plant. Disease ratings
were taken two weeks after inoculation because this allowed for a more accurate

assessment of plant damage.

Table 3.2. Visual rating criteria for

 

 

 

 

 

 

 

 

 

 

 

bentgrass plants inoculated with

Sclerotinia homoeocarpa.

Score Visual criteria

1 dead plant

2 one or two surviving
tillers at edge of plant

3 three or four surviving
tillers

4 five or six surviving
tillers

5 seven or eight surviving
tillers

6 nine or ten surviving
tillers

7 greater than 10 surviving
tiller with leaf necrosis
very visable

8 greater than 10 surviving
tillers with some leaf
necrosis visable

9 .no plant damage visable

 

 

 

52

53

RESULTS AND DISCUSSION

The range of disease scores went from 1 to 7 with 96% of the plants receiving
a. score of 1 (dead plant) or 2 (1 or 2 surviving tillers) (Table 3.1). The average
disease rating was a 1.4, indicating that overall disease pressure was very high. The
inoculation procedure resulted in very uniform disease pressure based on visual
observation of the level of mycelia growth on individual plants (Figure 3.1). One
hundred thirteen plants (3.7% of the plants screened) received a score from 3 to 7 and
these plants were selected for further study. The variety DF-l had the highest level of
resistance with a mean score of 2.6 and 6 plants with a score of 6 or better. However,
this variety was still heavily damaged by the disease with 73 out 100 plants having a
score of 1 or 2. The two colonial bentgrass varities SR7100 and Tendez showed high
levels of susceptability under these inoculation conditions having mean scores of 1.1
and 1.0, respectively.

Plants employ a wide range of defense mechanisms to restrict damage from
potential pathogens. Defense mechanisms can be classified as avoidance, resistance
and tolerance (Parlevliet, 1981). Avoidance mechanisms are those that reduce the
contact of potential pathogens with the host species. Resistance and tolerance
mechanisms operate after pathogen contact has been established. Resistance
mechanisms are mostly of a chemical nature and result in a reduction in the growth and
development of the pathogen. Tolerance mechanisms generally do not reduce pathogen
growth, however, the level of damage to the host is reduced.

Lumsden (1978) in a review of pathogenesis in plant diseases caused by
Sclerotinia species states that the success of Sclerotina spp. as a pathogen appears to be

54

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55

dependent on a complex combination of factors that can overwhelm the host plant by
rapidly acting before the host can respond. This type of pathogenesis would appear to
occur with bentgrass as well because extensive fungal development can occur within 12
hours of inoculation. This type of pathogenesis is likely to circumvent plant resistance
mechanisms such as appositional cell wall formation, which has been shown to be
important for fungal resistance in the Garnineae (Sherwood and Vance, 1980).

Avoidance-type mechanisms such as open plant canopies that reduce plant
moisture levels have been shown to reduce disease incidence in other plant species
affected by Sclerotina spp. (Schwartz et al. 1978). Bentgrass plant canopy
characteristics are not well understood so the importance of this factor in dollar spot
resistance cannot be estimated. However, high levels of turf moisture are essential for
dollar spot development and management factors which reduce turf moisture levels,
such as early morning dew removal, have been shown to reduce the incidence of dollar
spot under field conditions (Williams et al. 1993). Therefore, open canopy types that
reduce turf moisture levels and plants with reduced levels of guttation fluid excretion
might be expected to exhibit an avoidance type of defense mechanism under field
conditons. Colonial bentgrass is a rhizomatous species with a more erect leaf
orientation than creeping bentgrass which could result in a microclimate less condusive
to high moisture levels and explain why this species shows lower levels of dollar spot
than creeping bentgrass under field conditons. However, this type of plant defense
mechanism would be eliminated from our growth chamber experiments because plants
were misted daily to maintain leaf wetness which could explain why the colonial

bentgrasses preformed so poorly in this study.

S6

The fact that no completely resistant plants were observed in this experiment
might indicate that a tolerance type of defense mechanism is being exhibited by the
higher rated surviving plants. This type of mechanism would be important for bentgrass
recovery from dollar spot injury, however, its importance in the reduction of disease
incidence under field conditions needs further evaluation. A potential tolerance
mechanism could be reduced susceptibility to the pathogen produced toxin or an ability
to dispose of the toxin. The experimental variety DF-l was developed by selecting salt
tolerant clones from the variety Seaside, therefore, it is possible that this variety has an
efficient mechanism of dealing with toxic substances.

Further research needs to be conducted to determine the importance of turf
canopy characteristics and turf wetness levels on dollar spot development.

Additionally, an understanding of the importance of disease tolerance under field
conditions nwds to be determined. If disease avoidance and tolerance mechanisms are
shown to be important in disease reduction under field conditions then genetic
information needs to be developed for these characteristics so that effective breeding
strategies can be established. A more thorough understanding of the mechanism of S.
homoeocarpa pathogenesis and elucidation of mechanisms by which bentgrass might
resists infection should provide the scientific base from which cultivars with improved

resistance to this important disease can be developed.

LIST OF REFERENCES

Colbaugh, P.F. and M.C. Engelke. 1993. Sclerotinia dollar spot on bentgrasses.
Biological and cultural tests. 8:111.

Cole, H., J .M. Duich, L.B. Massie, and W.D. Barber. 1969. Influence of fungus
isolate and grass variety on Sclerotinia dollarspot development. Crop Sci. 9:567-570.

Doney, J.C. Jr., and RC. Vincelli. 1993. Reactions of bentgrasses to dollar spot and
brown patch. Biological and Cultural Tests. 8:118.

Endo, R.M. 1963. Influence of temperature on rate of growth of five fungus
pathogens of turfgrass and on rate of disease spread. Phytopathology 53:857-861.

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