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thesis entitled

BIOLOGY AND PATHOGENICITY FACTORS OF RUTSTROEMIA
FLOCCOSUM AND THE EFFECTS OF LIGHTWEIGHT ROLLING
ON DOLLAR SPOT DISEASE INCIDENCE IN CREEPING
BENTGRASS PUTTING GREENS.

presented by

Paul Ryan Giordano

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Plant Pathology

 

 

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BIOLOGY AND PATHOGENICITY FACTORS OF RUTSTROEMIA FLOCCOSUM
AND THE EFFECTS OF LIGHTWEIGHT ROLLING ON DOLLAR SPOT DISEASE
INCIDENCE IN CREEPING BENTGRASS PUTTING GREENS.

By

Paul Ryan Giordano

A THESIS

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

MASTER OF SCIENCE
Plant Pathology

2010

- llll

ABSTRACT
BIOLOGY AND PATHOGENICITY FACTORS OF RUTSTROEMIA FLOCCOSUM
AND THE EFFECTS OF LIGHTWEIGHT ROLLING ON DOLLAR SPOT DISEASE
INCIDENCE IN CREEPING BENTGRASS PUTTING GREENS.
By

Paul Ryan Giordano

A study to investigate the effects of light-weight rolling on the reduction of dollar spot
was conducted between 2008 and 2009. Treatments rolled in the afternoon exhibited
similar disease reduction as treatments rolled in the morning when compared to the
control. A twice day'l rolling treatment exhibited signiﬁcantly less disease than all other
treatments and resulted in greater rootzone soil volumetric water content (V WC) in both
2008 and 2009, when compared to the non-rolled control. Rolled treatments exhibited
increases in fatty acid abundances associated with common bacteria when rootzone soil
phospholipid fatty acids (PLF A) were analyzed. Furthermore, a general trend towards
higher total bacterial PLFA abundances was present in rolled treatments. These results
suggest that daily, season-long, light-weight rolling on putting greens may contribute to
dollar spot reductions through the alteration of microbial populations in the upper
rootzone. Additionally, aﬁemoon rolling treatments suggest mechanisms other than dew
and guttation removal to be responsible for disease reductions. R. ﬂoccosum isolates were
examined for oxalic acid (0A) and cell wall degrading enzymes (CWDE) production.
Non-virulent isolate Sh12B displayed high levels of CWDES and 0A production to a
lesser extent when compare to virulent isolates. This suggests that OA and CWDES may

not be sole pathogenicity factors necessary for R. ﬂoccosum infection on turfgrass.

 

 

 

To my loving parents, Patricia and Paul for all that you have done to help shape the

person that I am today; I can never thank you enough.

iii

ACKNOWLEDGMENTS

It would be impossible to thank every individual who has made a positive
impact on my life in the past two years, but there are many people I owe a sincere
thanks to for their aide and support during this time of my academic career. First and
foremost, to my major professor, Dr. Joe Vargas, thank you for your inspiring wisdom
and guidance; and for giving me the opportunity to learn and grow as an independent
researcher. I owe tremendous thanks to my committee members Dr. Ray
Hammerschmidt and Dr. Thom Nikolai for their keen advice and ideas, without which,
none of this would be possible. I would like to thank my parents Patricia and Paul
Giordano for your unconditional love and support, both personal and ﬁnancial. My two
sisters Lisa and Lori, thank you for all that you do. Much love to my wonderful ﬁancee
Courtney Rose for your unyielding support and encouragement throughout this
endeavor that keeps me grounded and driven. To my co-workers and friends, Nancy
and Ron, thank you for always lending a helping hand and being there with listening
ears and engaging ideas. To Liewei Yan, thank you for the insightful conversations and
camaraderie. To Kevin Budsberg and Dr. Teri Balser who aided in the PLFA analysis.
To Dr. Chris Vandervoort for her assistance with HPLC. To the whole MSU turf team,
including everyone at the Hancock Turfgrass Research Center for being so helpful and
setting a standard of excellence unmatched in this great industry. To my fellow
graduate students and everyone in the department of Plant Pathology I extend sincere

thanks and appreciation.

iv

TABLE OF CONTENTS

Page

LIST OF TABLES ................................................................................ vi

LIST OF FIGURES ............................................................................... vii

LITERATURE REVIEW .......................................................................... 1
Dollar Spot .......................................................................................... 1
Extracellular Enzyme and Acid Production by R. F loccosum ............................... 6
Lightweight Rolling .............................................................................. 10
Microbial Populations in Turfgrass Rootzones .............................................. 13
Literature Cited ................................................................................... 18

CHAPTER ONE

DOLLAR SPOT REDUCTION THROUGH LIGHTWEIGHT ROLLING ON

CREEPING BENTGRASS PUTTING GREENS
Abstract ............................................................................................ 26
Introduction ....................................................................................... 27
Materials and Methods .......................................................................... 29
Results and Discussion .......................................................................... 32
Conclusions ....................................................................................... 48
Literature Cited ................................................................................... 53

CHAPTER TWO

TURFGRASS ROOTZONE MICROBIAL RESPONSES TO ROLLING
Abstract ............................................................................................ 55
Introduction ....................................................................................... 56
Materials and Methods .......................................................................... 59
Results and Discussion .......................................................................... 63
Conclusions ....................................................................................... 69
Literature Cited .................................................................................. 75

CHAPTER THREE

ACID AND CELL WALL DEGRADIN G ENZYME PRODUCTION BY

RUT STROEMI F LOCCOSUM
Abstract ............................................................................................ 82
Introduction ....................................................................................... 83
Materials and Methods .......................................................................... 85
Results and Discussion .......................................................................... 90
Conclusions ....................................................................................... 97
Literature Cited ................................................................................. 102
Appendix .......................................................................................... 105

Table 1.01

Table 1.02

Table 2.01

Table 2.02

Table 2.03

Table 3.01

Table 3.02

Table 3.03

LIST OF TABLES
Means and LSD comparisons for treatment effects of rolling on dollar
spot disease incidence on different dates in 2008 ........................... 34

Means and LSD comparisons for treatment effects of rolling on dollar
spot disease incidence at different dates in 2009 ............................ 35

Relative abundance (mol %) of PLFA indicators in turfgrass root
zones .............................................................................. 65

PLFA markers used for taxonomic microbial groupings .................. 67
Bacterial organisms with antagonistic effects against common turfgrass
pathogens ......................................................................... 72
Rutstroemiaﬂoccosum isolates studied and their source of

obtainment ........................................................................ 85

Disease ratings of R. ﬂoccosum isolates on swards of creeping bentgrass
(Agrostis palustris Huds. cv. Crenshaw) ...................................... 91

Mean culture pH measurements of four R. ﬂoccosum isolates growing in
potato dextrose broth for 15 days ............................................. 93

vi

Figure 1.01

Figure 1.02

Figure 1.03

Figure 1.04

Figure 1.05

Figure 1.06

Figure 1.07

Figure 1.08

Figure 1.09

Figure 1.10

Figure 2.01

Figure 3.01

LIST OF FIGURES

Seasonal dollar spot means among different rolling treatments on
creeping bentgrass (Agrostis palustris Huds.) putting green plots in
2008 ............................................................................... 36

Seasonal dollar spot means among different rolling treatments on
creeping bentgrass (Agrostis palustris Huds.) putting green plots in
2009 ............................................................................... 37

Quality ratings on creeping bentgrass (Agrostis palustris Huds) turf plots
for rolling treatments on different dates in 2008 ............................ 40

Seasonal turfgrass quality ratings for rolled treatments on creeping
bentgrass (Agrostis palustris Huds.) putting green plots in 2008. . . . .....41

Quality ratings on creeping bentgrass (Agrostis palustris Huds) turf plots
for rolling treatments on different dates in 2009 ............................ 42

Seasonal turfgrass quality means for rolled treatments on creeping
bentgrass (Agrostis palustris Huds.) putting green plots in 2009.. . ......43

Percent volumetric water content means for rolling treatments on
creeping bentgrass (A grostis palustris Huds.) plots in 2008 ............... 44

Seasonal percent volumetric water content means for rolling treatments
on creeping bentgrass (Agrostis palustris Huds.) plots in
2008 ............................................................................... 45

Percent volumetric water content means for rolling treatments on
creeping bentgrass (A grostis palustris Huds.) plots in 2009 ............... 46

Seasonal percent volumetric water content means for rolling treatments
on creeping bentgrass (A grostis palustris Huds.) plots in 2009 ........... 47

PLFA group abundance (mol %) in turfgrass root zones subjected to
different rolling treatments ..................................................... 68

Mean media color change ratings from acid production of four R.
ﬂoccosum isolates grown on bromophenol blue-amended potato dextrose
agar for 15 days .................................................................. 92

vii

Figure 3.02

Figure 3.03

Figure 3.04

Figure 3.05

Figure A.1

Figure A.2

Oxalic acid production of R. ﬂoccosum isolates grown in potato dextrose

broth for 15 days ................................................................. 94
Oxalic acid production related to mycelial dry weight of R. ﬂoccosum
isolates grown in potato dextrose broth for 15 days. ....................... 95

Pectin methylesterase activity of four R. ﬂoccosum isolates grown in
liquid culture with creeping bentgrass (A grostis palustris Huds.) cell

walls ............................................................................... 96
Cellulase activity of four R. ﬂoccosum isolates grown in liquid culture
with creeping bentgrass (Agrostis palustris Huds.) cell walls ............ 97

Oxalate oxidase speciﬁc activity in creeping bentgrass (Agrostis
palustris Huds.) tissue from different rolling treatments ................. 105

Peroxidase speciﬁc activity in creeping bentgrass (Agrostis palustris
Huds.) tissue from different rolling treatments ............................ 106

viii

LITERATURE REVIEW
DOLLAR SPOT

Causal Agent. Dollar spot is a disease of turfgrass found throughout the world
(Smiley et al., 1992). It is caused by the fungal pathogen Rutstroemia ﬂoccosum syn.
Sclerotinia homoeocarpa F .T. Bennet (Smiley et al., 1992) and is considered the most
economically important disease on golf courses nationwide. More money is spent to
manage dollar spot than any other pathogen on golf courses in the United States (Vargas,
2005). When grown in vitro, the fungus exhibits a characteristic white mat of fast-
growing, ﬂuffy mycelium, which turns to a gray or brown as the culture ages. After 15-
30 days of growth in culture, the white mycelium can be accompanied by planes of dark
stroma (Smiley et al., 1992).

Symptoms. The name dollar spot was given to the disease due to the symptoms it
produces on the host turfgrass tissue, about the size of a silver dollar (Vargas, 2005). The
disease appears on putting greens and low mown turf as small, straw colored patches, less
than 6 cm, and commonly 1-3 cm, in diameter. In taller grass, like home lawns and
athletic ﬁelds, the patches may reach 15 cm in diameter. As the disease progresses and
conditions favorable for development persist, individual spots may often coalesce into
irregularly shaped patches, destroying large areas of turf. Lesions on individual leaf
blades are distinguished by pale, bleached, water soaked bands, bound by a tan or
reddish-brown margin at the outer edge of the lesion in bentgrass, Ketucky bluegrass,
ﬁne-leaf fescue, zoysiagrass, and bermudagrass. During periods of leaf wetness, such as

early morning hours when heavy dew and guttation ﬂuid are present, the active mycelium

is oﬁen visible as white, cottony or cobwebby growth (Smiley et al., 1992; Vargas, 2005;
Beard and Tani, 1997). Mycelia disappear as the leaves dry.

Disease Cycle. Rutstroemia ﬂoccosum can survive as dormant mycelia in
previously infected tissue. The disease is spread primarily by mowers, maintenance
equipment, and people carrying mycelia and infected plant tissue on shoes or other
equipment. The mycelium rapidly invades healthy leaf tissue and extends into moisture-
saturated air spaces surrounding infected leaves when the turfgrass environment favors
activity of the fungus. Mycelia] infection is thought to occur through out ends of leaves
or through stomata on leaf blades (Smiley et al., 1992). Although infertile aborted
apothecial primordial have been produced by the fungus in culture and fertile apothecia
have been observed from Festuca turf in ﬁeld conditions (Baldwin and Newell, 1992),
neither sexual nor asexual spores are considered important in the epidemiology of the
disease (Fenstermacher, 1979; Jackson, 1973)

Epidemiology. Dollar spot is known to infect many species of turfgrass (Smiley
et al., 1992). In the northern US, it is most damaging to creeping bentgrass (Agrostis
palustris Huds.), annual bluegrass (Poa annua), colonial bentgrass (Agrostis tenuis), and
ﬁne-leaf fescues (F estuca spp.) (Vargas, 2005). The disease can be prevalent on
turfgrass throughout the US. from the latter part of the spring through the end of the fall.
Most epidemics occur in July and again in late August through September (Smiley et al.,
1992). Conditions that favor the development and infection of dollar spot are
temperatures of 15-30 C. Many different vegetative compatibility groups exist that cause
the disease at various temperature and humidity regimes (Vargas, 2005). Guttation ﬂuid

is thought to be an ideal food source for the ﬁrngus. Guttation water is a ﬂuid rich in

carbohydrates and amino acids that is exuded from the tip of the grass blade through
hydathodes when turgor pressure builds up in the plant (Vargas, 2005). Dollar spot is
more severe under conditions of low nitrogen fertility, dry soils, and/or water stress. The
fungus can survive as mycelia and stroma on leaf tissue when conditions for infection are

unfavorable.

DOLLAR SPOT MANAGEMENT

gm Dollar spot management is often an integrated plan implemented by
golf course superintendents, and turf managers alike. Cultural, chemical, and biological
control techniques are all used in the management of this disease. Methods of cultural
control such as fertilization, irrigation, and cultivation are often effective in reducing the
amount of diseased turfgrass, and can aide in the reduction of chemical inputs.
Maintaining high nitrogen fertility, especially when applied at times of high disease
pressure, reduces disease (Vargas, 2005). Maintaining proper soil moisture levels, thus
avoiding plant drought stress, is thought to also contribute to disease control. Couch and
Bloom (1960) found low soil moisture to be important in the development of dollar spot,
and Howard and Smith reported more dollar spot in seasons with less rainfall (cited in
Vargas, 2005).
One of the most frequently used techniques in the cultural management of dollar spot is
the removal of dew or plant exudates known as guttation ﬂuid from the leaf blades in the
early morning hours after mowing. Prolonged leaf wetness duration (LWD) increases the
severity of dollar spot and other fungal diseases by providing a favorable environment for

fungal penetration of leaf tissues (Huber and Gillespie, 1992; Williams et al., 1996; Gross

et al., 1998; Walsh, 2000; Uddin et al., 2003). Walsh (2000) found that the minimum
LWD for the development of dollar spot for one isolate of S. homoeocarpa on creeping
bentgrass (Agrostis palustris Huds.) was 12 h at 175°C. A longer LWD was required for
infection when the temperature was lower. Dew or guttation removal is widely
implemented in the turfgrass industry, particularly on golf course putting greens, and is
thought to inhibit fungal mycelium growth by reducing LWD or the amount of food
source available for the fungus. Rolling, whipping, dragging, or syringing are among
some of the techniques used by turf managers to accomplish adequate removal or
dispersion of the moisture. Many studies have elucidated the beneﬁts of early morning
mowing, syringing, and other dew removal techniques in order to decrease LWD
(Williams and Powell, 1995; Ellram et al, 2007) ultimately reducing dollar spot
incidence. Williams and Powell (1995) noted that guttation droplets secrete from wound
exudates and these droplets are rich in nutrients that pathogens may use during hyphal
growth. Release of these exudates may be exacerbated in the early dawn hours due to a
combination of a fresh wound being produced by mowing and that turgor pressure may
be high at this time. Nikolai (2005) proposed the process of dew/guttation removal as an
underlying mechanism behind dollar spot reduction among other hypotheses such as
altered soil microbial populations, and plant phytoalexin defense responses (Nikolai,
2002)

Chemical. While cultural management of dollar spot is relatively effective, the
most efﬁcient dollar spot management can be obtained when good cultural practices are
combined with a sound fungicide program. Contact fungicides, like chlorothalonil, are

effective at controlling dollar spot for short (7-14 days) periods of time. Resistance to

contact fungicides has not been reported and is unlikely to develop due to the multi-site
mode of action these fungicides exhibit (Golembiewski et al., 1995). Effective systemic
fungicides include propiconazole, fenarimol, iprodione and vinclozolin, among others.
Systemic fungicides are able to give longer periods of control than contact fungicides, but
have a much greater likelihood of becoming ineffective due to pathogen resistance
developing. Resistance can be deﬁned as reduced efﬁcacy and shortened control intervals
of a previously sensitive fungal population. Since 1972, resistance has been reported to
many major systemic fungicide classes, which include the benzimidizoles (Warren,
1974), dicarboximides (Detweiler et al., 1983), and the demethylation inhibitors (DMI’s)
(Golembiewski et al., 1995). R. ﬂoccosum resistance to demethylation inhibitors was
conﬁrmed only 11 years after this family of ﬁmgicides was introduced to manage this
disease (Golembiewski et al., 1995). Today, demethylation inhibitors are ineffective
against dollar spot on many golf courses where this fungicide family has been used
extensively (Vargas, 2005).

Biological. The introduction of the biological control agent Pseudomonas
aureofaciens strain Tx-l sold as Spot-less (Turf Science Laboratories Inc., National City,
CA) for use in managing dollar spot has been a successful addition to the cultural and
chemical means available to manage dollar spot on golf courses (Dwyer, 1999). To date,
P. aureofaciens has been only mildly successful as a biological agent used in ﬁeld
situations for the control of dollar spot. Satisfactory disease suppression during years of
light to moderate dollar spot infection has been shown with composted materials

containing Enterobacter cloaca (Nelson et al., 1991, Nelson et al., 1992) as well.

EXTRACELLULAR ENZYME AND ACID PRODUCTION BY RUTSTROEMIA
FLOCCOSUM

While many products are available to control the dollar spot disease, much
remains unknown about the true life cycle and epidemiology of the fungus. One key to
disease control is having a keen understanding of the infection process of the pathogen of
interest. While a great deal of effort and research has been dedicated to dollar spot
control and temporal variations (i.e. vegetative compatibility groups), infection
mechanisms are yet to be elucidated. Knowledge of these mechanisms and pathogenicity
factors are important, and can play critical roles in the development of novel, effective
methods of control.

Infection Meﬂanisms. Plant pathogens must overcome many obstacles in order
to successfully infect their host. Some bacteria, for example, enter their hosts through
wounds or natural openings such as hydathodes in the plant. Other microorganisms may
be able to infect by means of mechanical pressure created by specialized structures that
aide in the facilitation of microbial entry (Agrios, 1988). Plant cells are surrounded by
rigid walls in which a multitude of polysaccharides with speciﬁc structures are
interconnected (Baur et al., 1973; Burke et al., 1974; Wilder and Albersheim, 1973).
These interconnected polysaccharides form initial barriers which serve as primary
defense mechanisms against invading pathogens. The evolutionary struggle between
pathogen and host most likely gave rise to cell wall degrading enzymes being secreted by

pathogens in order to aide in infection and overall virulence.

Cell Wall Degrgdation. Many pathogens achieve host infection by disrupting the
plant cell wall (Albersheim et al., 1969). Plant cell wall degradation results from the
action of pathogen-produced enzymes that are capable of cleaving speciﬁc linkages in the
wall matrix of their host (Albersheim et al., 1969). Extracellular proteins secreted by
fungi and bacteria are often able to macerate plant tissues and degrade plant cell wall
components. Pathogens must thus contain all of the enzymes corresponding to the types
of glycosidic linkages present in the cell wall polysaccharides of their target host.
Multiple enzymes can be sequentially secreted by a single fungal pathogen when grown
on isolated host cell walls (English et al., 1970; Jones et al., 1972). This observation is
logical when considering the array of polysaccharides associated with different plant cell
walls. Some pathogens can control the concentration and variety of mono- and
polysaccharide degrading enzymes they produce depending on the environment they are
growing in and the amount and type of substrate available (Albersheim et al., 1969).
Extensive research involving the elucidation of enzymes capable of degrading major
constituents of creeping bentgrass and determining whether speciﬁcity exists within the
dollar spot pathogen has not been conducted.

Plant Cell Wall Comp_osition. The composition of plant cell walls varies
signiﬁcantly from one cell type to another, one species to another, and between
accessions within species (Hazen et al., 2003). For instance, a typical dicotyledonous
plant cell wall contains around 30% cellulose, 30% hemicellulose, 35% pectin and 1—5%
structural protein. By contrast, a typical grass species contains around 25% cellulose,
55% hemicellulose and only 10% pectin (Cosgrove, 1997). The extent of cell wall

disruption often varies among plant pathogens and relies upon the secretion of

extracellular enzymes. Soft rot pathogens, for instance, are able to separate individual
cells causing severe tissue maceration (Mount et al., 1970; Stephens and Wood, 1975).
Other pathogens are limited to mycelia penetration through the cell wall, which aides in
the infection process (Weinhold and Motta, 1973).

Oxalic Acid. Many Sclerotim‘a species are capable of producing compounds and
enzymes which may contribute to the virulence on their particular host. Oxalic acid (0A)
is thought to play multiple roles in the virulence of Sclerotinia species and has been
shown to be required for the pathogenicity of a multitude of species which infect a wide
range of hosts (Maxwell and Lumsden, 1970; Noyes and Hancock, 1981; Marciano et al.,
1983; Godoy et al., 1990; Button and Evans, 1996; Zhou and Boland, 1999). Since many
of the pathogens in this genus produce cell wall degrading enzymes, which are optimally
active at a low pH, 0A is thought to play a major role in lowering the pH of the apoplast
during the infection process (Bateman and Beer, 1965). Direct toxicity of 0A in the
plant is also thought to be a factor in weakening the host, facilitating greater invasion of

the pathogen (Noyes and Hancock, 1981). Other theories as to what role 0A plays in

pathogen virulence include the chelation of Ca2+ by the oxalate anion, which is thought to

compromise the integrity of the host cell wall as well as inhibit Cay-dependent defense
responses (Bateman and Beer, 1965). In the case of Sclerotinia sclerotiorum, the
oxidative burst of the host plant is suppressed by 0A through the inhibition of H202
production, most likely via the blocking of a signaling step in the oxidase
assembly/activation stream, in tobacco and soybean cells (Cessna et al., 2000).

Beaulieu (2008) showed that the dollar spot pathogen R. ﬂoccosum (Sclerotinia

Homoeocarpa) indeed produces oxalic acid by analysis of secretions via gas

chromatography. However, concentrations of secreted 0A were substantially lower than
other Sclerotinia species such as S. Sclerotiorum. While the dollar spot pathogen may no
longer be considered a member of the Sclerotinia genus (Powell, 1998) the production of
oxalic acid is a commonality that cannot be ignored with regard to the elucidation of
infection mechanisms and virulence.

Hypovirulence. Sclerotinia species infect a wide range of hosts and are
responsible for extensive economic loss due to their widespread distribution.
Hypovirulence has been reported to occur in species such as S. sclerotiorum, S. minor,
and S. homoeocarpa (R. ﬂoccosum) (Boland, 2004). Hypovirulence refers to the reduced
ability of selected isolates within a population of a fungal plant pathogen to infect,
colonize, kill and (or) reproduce on susceptible host tissues (Elliston, I982). Often times
these isolates may be associated with various phenotypic characteristics such as reduced
growth rate or sporulation, and altered colony morphology or color. Hypovirulence in
Sclerotinia spp. is often due to the infection by fungal viruses and associated double—
stranded RNA elements (Boland, 2004). In the case of S. homoeocarpa (R. ﬂoccusom),
the causal agent of dollar spot, hypovirulence has been associated with the presence of
double-stranded ribonucleic acid (dsRNA) (Zhou and Boland, 1997). However, some
isolates detected as hypovirulent have been variable in expression of the phenotype, or
were not associated with detectable concentrations of dsRNA.

Presence of the hypovirulence associated virus Ophiostoma mitovirus 3a
(OMV3a) in numerous strains of hypovirulent S. homoeocarpa has been reported,
however many isolates testing positive for the presence of the virus do not display the

hypovirulent phenotype, leading to the presumption of possible latent infection by the

OMV3a virus (Melzer et al., 2003). Conclusive evidence has been difﬁcult to ascertain
due to inconsistencies in the determination of the true mechanism contributing to
hypovinrlence in the dollar spot fungus.

Hypovirulent isolates of various plant pathogens have been exploited to better
understand pathogenicity factors and virulence. Pathogenesis by fungi in the genus
Sclerotinia has been associated with the production of aforementioned cell wall
degrading enzymes (Hancock, 1967; Lumsden 1976, 1979; Lumsden, 1969) as well as
oxalic acid (Maxwell and Lumsden 1970). Oxalic acid has been conﬁrmed as a
pathogenicin determinant in S. sclerotiorum by using oxalic-acid-deﬁcient mutants
(Godoy et a1. 1990). Double stranded RNA-associated hypovirulence in isolates of S.
scerotiorum was shown to be associated with reduced and delayed production of oxalic
acid, in comparison to virulent isolates (Zhou and Boland, 1999). Mechanisms
associated with dsRNA hypovirulent strains of the dollar spot fungus in relation to
pathogenicity factors have yet to be elucidated, particularly with regard to oxalic acid and

extracellular enzyme production.

LIGHTWEIGHT ROLLING

Turfgrass as a commodity is subject to unique agricultural management practices
compared to most cash crops. Many cultural techniques implemented in the maintenance
of high quality turfgrass are not applicable with other families of managed plants. The
ability of some turfgrasses to withstand not only frequent (daily) mowing, but low (< 1
inch) mowing heights, as well as heavy trafﬁc volumes are some of the deﬁning

characteristics of this classiﬁcation of plant. On a typical golf course, the putting greens

10

are the most intensely managed areas. Golf course superintendents and turf managers
alike have been experimenting with different techniques to improve the quality of their
greens for centuries. One commonly used cultural practice that has been found to be
beneﬁcial in numerous aspects of management is rolling. Lightweight rolling is most
often used to increase ball roll speed and distance on highly maintained putting greens.
The rollers, weighing from 200 to 1000 lbs (91 to 454 kg), are manufactured in many
different shapes and sizes and can have a signiﬁcant affect on the playability of a putting
green.

Green Speeds. The Stirnpmeter is a device used to measure the speed of a golf
course putting green by applying a known force to a golf ball and measuring the distance
travels in feet. Designed by golfer Edward Stimpson, Sr. in 1935, the Stimpmeter, is an
angled track that releases a golf ball at a known velocity so that the distance it rolls on a
putting green's surface can be measured. The further a golf ball rolls, the “faster” the
green is considered. It was ﬁrst used by the USGA during the 1976 US. Open at Atlanta
and was made available to golf course superintendents in 1978 (Radko, 1977). Since the
inception of the Stimpmeter, golf course superintendents have used a variety of cultural
and chemical practices in an attempt to obtain ﬁrmer, faster greens; rolling being one of
them (Throssell, 1986). The implementation of these practices is often thought to be
detrimental to the health and quality of the turfgrass on the putting green (Kussow, 1998;
Stier, 2006), especially during times of heat stress.

Rolling Histog. In the 1920's, numerous publications addressed roller weight,
frequency, compaction and soil texture (Harban, 1922; Piper and Oakley, 1921;

Anonymous, 1926) without coming to any valid conclusions. Rolling had been a

ll

common practice on most golf courses at that time, mainly for the purpose of improving
surface uniformity. Shortly thereafter, the practice of frequent rolling ceased, as turfgrass
research showed a link between high levels of soil compaction and turf root grth
(DiPaola and Hartwiger, 1994). The negative connotations around rolling persisted
within the turfgrass industry until the 1990’s (Hartwiger, 1996). Some turfgrass scientists
believed that rolling may be causing unwanted damage to a healthy stand of turf, in
addition to the near century old concerns regarding soil compaction. These same
scientists felt the need to investigate the potential for above ground turfgrass problems
associated with continual season-long turf rolling and the possibility that pathogens may
invade crushed tissues, leading to diseased turf (Beard, 1994).

Disease Suppression. With demand for faster putting green speeds increasing,
with continued emphasis on turfgrass quality, rolling has become a major component of
putting green maintenance. During a rolling experiment designed to compare green
speed, Nikolai et a1. (2001) also noticed that morning rolling (3 times per week) not only
increased green speeds but decreased the incidence of dollar spot signiﬁcantly on
creeping bentgrass maintained at putting green height. After conducting the study for
several years, with all results yielding signiﬁcant dollar spot reduction, many theories
began to arise as to why and how rolling was inhibiting the development of this proliﬁc
turfgrass disease. Many of the theories revolved around the belief that rolling, which was
conducted immediately after morning mowing, was removing excess dew or guttation
water exuded by the plant and serving as a nutrient source for R. ﬂoccosum.

The beneﬁts of rolling have proven to be numerous and because experiments

involving rolling have shown to reduce dollar spot disease on creeping bentgrass greens

12

(Nikolai et al., 2001), many hypotheses to explain this phenomenon have been proposed.
Dispersion of dew and guttation ﬂuid (Nikolai et al., 2001), enhancement of phytoalexin
production, increased surface water holding capacity, and microbial changes in the upper
rootzone have all been proposed as possible factors responsible for reduced dollar spot
incidence, although the actual mechanism(s) remains unknown (Nikolai, 2005). Routine
rolling can produce a more prostrate turf canopy and limit the gradual elevation of plant
crowns at the thatch—soil interface during the growing season (Beard, 2002). These
effects could reduce the amount of leaf blade and leaf sheath tissue removed or damaged
at low mowing heights. This could also enhance photosynthetic capacity because the
youngest leaf blades, which would be most often removed by mowing, are the most
photosynthetically active (Youngner, 1969). Additionally, maintaining the position of
crowns lower in the mat layer may reduce plant expOsure to high temperature stress
because temperatures are often greatest just below the surface of dense, short-mowed turf

(Beard, 1973).

MICROBIAL POPULATIONS IN TURF GRASS ROOTZONES

The rhizosphere (as well as the phyllosphere) is an infection epicenter where soil
borne plant pathogens encounter their host and establish a parasitic relationship. The
rhizosphere is also where a complex community of both soil microﬂora and microfauna
can interact with pathogens, as well as the host, and inﬂuence the outcome of infection of
the plant. The rhizosphere can be considered a battleﬁeld, where complex communities

of microbial players interact with one another, an environment where pathogenic and

13

beneﬁcial microorganisms constitute groups that have a major inﬂuence on plant grth
and development (Lynch, 1990).

Agricultural management practices can have signiﬁcant impacts on the size and
activity of soil microbial communities (Bolton et al., 1985; Fraser et al., 1988; Kirchner
et al., 1993; Powlson et al., 1987). Microorganisms in soil are critical to the maintenance
of soil function in both natural and managed agricultural soils because of their
involvement in key processes such as soil structure formation, decomposition of organic
matter, toxin removal, and nutrient cycling. In addition, many microorganisms have been
found to play key roles in suppressing soilbome plant diseases, promoting plant growth,
and changing natural vegetation (Doran et al., 1996).

Suppressive Soils. A suppressive soil can be deﬁned as one with a broad array of

 

antibiosis-related ﬁrnctions, some of which associated with the suppression of plant
pathogens (van Elsas et al., 2008) The mechanisms by which soils are suppressive to
different pathogens, although not well understood, can often involve biotic and/or abiotic
factors. These factors may often vary with the pathogen as well as host plant in the
environment. The main agents responsible for soil suppressiveness are thought to be
microbial in nature, due to the observation that sterilization by autoclaving, stem
pasteurization, and irradiation has rendered soils more conducive to pathogens
(Malajczuk, 1983). The mechanisms by which these microorganisms make soil
suppressive can be divided into several categories: nutrient competition, microbial
antagonism, parasitism, and systemic induced resistance (Raaijamkers et al., 2009).
Commtition. Competitive colonization of the rhizosphere and successful

establishment in the root zone is a prerequisite for effective biocontrol, regardless of the

14

mechanism(s) involved (Weller, 1988; Raaijmakers et al 1995). Competition for
nutrients can often times be an effective biocontrol mechanism, particularly competition
for organic compounds which are necessary for reactivation of propagules and
subsequent proliferation of rhizosphere-dwelling pathogens (Paulitz et a1 1992; Van Dijk
and Nelson 2000; Fravel et a1. 2003). Competition for micronutrients, essential for
grth and activity of the pathogen, can take place as well.

Ant_agonism. Representatives of a range of bacterial (Pseudomonas,
Burkholderia, Bacillus, Serratia, many Actinomycetes) and fungal (T richoderma,
Penicillium, Gliocladium, Sporidesmium, nonpathogenic F usarium spp.) genera have
been identiﬁed as antagonists of soilbome plant pathogens. One of the most important
groups containing antagonistic microorganisms is the group containing ﬂuorescent
Pseudomonads. Several antibiotic-producing Pseudomonas spp. were isolated from soils
suppressive to diseases such as take-all of wheat, black rot of tobacco and F usarium wilt
(Keel et al. 1996; Tamietti et a1. 1993; Weller et a1. 1988). Naturally occurring root-
associated ﬂuorescent Pseudomonas spp. producing the antibiotic 2,4-DAPG were
numerously populated in take-all-suppressive soil and are key components of speciﬁc
suppression of Gaeumanonmyces graminis var. tritici (Raaijmakers and Weller, 1998;
Raaijmakers et al., 1997). This suppressive activity was lost when 2,4-DAPG-producing
Pseudomonas spp. were eliminated and, conversely, conducive soil gained
suppressiveness to take-all when 2,4-DAPG-producing Pseudomonas strains were
introduced. Biocontrol microorganisms may adversely affect the population density,

dynamics, and metabolic activities of pathogens.

15

Ingrced Systemic Resistance. Along with biocontrol activity of microorganisms
in the rhizosphere, several microbial groups can have a direct positive effect on plant
growth and health. Phytostimulatory and biofertilizing microbes can promote plant
health by making the plant “stronger”. Many rhizosphere microorganisms can induce a
systemic response in the plant as well, resulting in the activation of plant defense
mechanisms (Pieterse et al., 2003). Induced systemic resistance (ISR) does not
necessarily confer complete resistance, but rather protects the plant from various types of
pathogen infection. The capacity to convey this type of ISR has been identiﬁed in a wide
range of bacteria (Van loon et al., 1998; Haas and Defago, 2005), not only in greenhouse
experiments, but under ﬁeld conditions as well (Zehnder et al., 2001; Pieterse et al.,
2003).

Microbial Populations in Soil. Several studies have identiﬁed trends within the
microbial activity of soil related to and responsible for the suppression of pathogens. For
example, van Os & van Ginkel (2001) showed a clear relationship between the
suppression of Pythium root rot in bulbous Iris and soil microbial biomass and activity.
Their ﬁndings showed that high microbial biomass and activity induced the suppression
of Pythium growth and development in the soil. Relationships have been found between
microbial diversity and root disease suppression as well (Nitta, 1991; Workneh and van
Bruggen, 1994). Rovira & Wildennuth (1981) indicated that the microbiota in a “rich”
soil tends to reduce the severity of attack by many soilbome plant pathogens, or, in other
words, soils higher in microbiota content and diversity tend to Show trends towards
general disease suppression. Tippett (1978) provided an example of the importance of

the soil microbiota on the level of suppressiveness. By adding soil containing large

16

quantities of microorganisms to microbially deﬁcient soil, they were able to eliminate P.
cinnamoni. Many antagonistic microorganisms are naturally present in soil and exert a
certain degree of biological control over plant pathogens, regardless of human activities.
However, this level of natural control is often insufﬁcient for consistent, reliable disease-
free cropping. Researchers are, therefore, attempting to enhance the effectiveness of
antagonists, thus increasing suppressiveness (Hoitink et al., 2003).

Management of the biotic and abiotic properties of a soil is an important approach
in promoting the activity and diversity of beneﬁcial microorganisms. Cultural practices
have been proposed as means to decrease soil inoculum levels, or increase
suppressiveness, thus limiting the densities and activities of rhizosphere pathogens.
Disease suppression has been obtained through crop rotation (Cook et al., 2002), as well
as other practices such as intercropping (Schneider et al., 2003), residue destruction
(Baird et al., 2003), organic amendments (Tilston et al., 2002), and tillage management
practices (Sturz et al., 1997; Pankhurst et al., 2002).

With public perception and environmental stewardship at the forefront of
turfgrass management concerns, alternative options for management of diseases are
becoming highly desired in the turfgrass industry. Dollar spot is considered to be the
most important disease on turfgrass, particularly in the northeastern regions of the United
States (Vargas, 2005). If used as a tool in the management of dollar spot, rolling could
potentially provide major economical beneﬁts to golf course superintendents as well as
boast signiﬁcant conservational beneﬁts. However, literature regarding R. ﬂoccosum

suppression relating to rolling is sparse, particularly regarding mechanisms involved in

17

disease reductions. A multi-faceted approach was taken in an attempt to elucidate
particular hypotheses regarding the rolling effect on dollar spot.

R. ﬂoccosum infection mechanisms, including enzyme, and toxin production were
studied. Understanding speciﬁc plant-pathogen interactions and mechanisms related to
pathogen virulence is important in elucidating novel methods of disease control. These
have gone relatively uninvestigated with regard to the dollar spot pathogen on turfgrass.
Rolling hypotheses and R. ﬂoccosum infection and virulence are discussed in the chapters

that follow.

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Zehnder, G. W., Murphy, J. F., Sikora, E. J., and Kloepper, J. W. 2001. Application of
rhizobacteria for induced resistance. Euro. J. of Plant Pathol. 107: 39—50.

25

CHAPTER ONE
DOLLAR SPOT REDUCTION THROUGH LIGHTWEIGHT ROLLING ON
CREEPING BENTGRASS PUTTING GREENS

ABSTRACT

A study to investigate the effects of light-weight rolling on the reduction of dollar
spot was conducted at the Hancock Turfgrass Research Center on the campus of
Michigan State University in 2008 and 2009. Creeping bentgrass (Agrostis palustris)
plots subjected to daily (5 days week") rolling treatments resulted in signiﬁcantly less
disease when compared to a non-rolled control. Morning rolling treatments and afternoon
rolling treatments were implemented to investigate the effects of dew and guttation ﬂuid
removal and dispersal. Treatments rolled in the p.m. (after dew and guttation naturally
dissipated) exhibited similar disease reduction as treatments rolled in the am. (while dew
and guttation ﬂuid were present) when compared to the control. Cumulative effects of
rolling were investigated with treatments rolled twice (2x) day]. The 2x rolling
treatment exhibited signiﬁcantly less disease than all other treatments and resulted in
Signiﬁcantly higher upper rootzone soil volumetric water content means in both 2008 and
2009, when compared to the control. These results suggest that mechanisms other than
dew and guttation dispersal may be responsible for disease reduction due to the observed
dollar spot inhibition from afternoon rolling. Additionally, data suggests that season-long
lightweight rolling twice day'1 may have an effect on upper rootzone volumetric water

content in sand based creeping bentgrass putting greens.

26

INTRODUCTION

Turfgrass is subject to unique agricultural management practices compared to
those of other agronomically important crop systems. Many cultural techniques
implemented in the maintenance of high quality turfgrass are not applicable with other
species of managed plants, even within the family Gramineae. One commonly used
cultural practice on golf course turf is lightweight rolling. Lightweight rolling is most
often implemented to increase ball roll speed and distance during putting as well as to
enhance surface uniformity on intensively maintained putting greens. The rollers,
typically weighing from 200 to 1000 lbs (91 to 454 kg), are manufactured in many
different shapes and sizes, and can have a signiﬁcant affect on the playability of a putting
green thus having dramatic impacts on the game of golf.

The rolling of putting greens has been common on most golf courses for decades,
and prior research has led to even more questions regarding its effects on turfgrass and
underlying soil. Many negative associations with rolling have persisted within the
turfgrass industry (Hartwiger, 1996). Theories that rolling may be causing unwanted
damage to a healthy stand of turf, as well as concerns regarding soil compaction issues,
have existed since the 1920’s. Turf scientists have debated over the need to investigate
the potential for above ground turfgrass problems associated with continual, season-long
turf rolling and the possibility that pathogens may invade crushed tissues, leading to
diseased turf (Beard, 1994).

Nikolai et al. (2001) conducted a multiple year rolling study at Michigan State
University, aimed at the effects of rolling and fertility on putting green speed, disease

incidence, and soil properties in different root zone mixes. This study gave rise to

27

surprisingly signiﬁcant data regarding numerous aspects of plant management. Morning
rolling (3 times per week) was found to dramatically decrease the incidence of the fungal
disease dollar, spot caused by Rutstroemia ﬂoccosum, on creeping bentgrass maintained
at putting green height, thus improving overall turfgrass quality. These results were
observed in several years of the study. Since that time, others have observed similar
results with lightweight rolling. Inguagiato et. al. (2009) observed slight reductions in
turfgrass anthracnose, caused by Colletotrichum cereale, on plots rolled every other day.
These results were contrary to the previous suggestions that rolling enhances disease
severity (Demoeden, 2000; Smiley et. al., 2005).

The ﬁndings of decreased disease occurrence through rolling spurred many
hypotheses and theories with regard to possible suppression mechanisms. Many of the
theories stem from the belief that rolling, which typically is conducted immediately after
morning mowing, removes excess dew or guttation ﬂuid exuded by the plant. This
moisture is thought to serve as a nutrient rich food source for foliar feeding pathogens
like R. ﬂoccosum (Williams and Powell, 1995). Dew removal and reduced leaf wetness
duration (LWD) are widely accepted techniques used to reduce dollar spot incidence on
turfgrass. Many studies have revealed the beneﬁts of early morning mowing, syringing,
and other dew removal techniques in order to decrease LWD (Williams and Powell,
1995; Ellram et al, 2007), ultimately reducing dollar spot incidence.

If used as a tool in the management of dollar spot, rolling could have substantial
economic and environmental beneﬁts. However, the literature is sparse on rolling

studies, particularly on the topic of disease suppression mechanisms.

28

The objective of this study was to test speciﬁc hypotheses regarding rolling and
its effect on dollar spot incidence on creeping bentgrass putting greens. Different rolling
treatments were implemented for the duration of this study in order to generate data used
to draw conclusions related to each hypothesis. The ﬁrst objective was to investigate the
hypothesis that morning rolling contributed to the removal or dispersal of secondary
dew/guttation ﬂuid after mowing, and that the reduction of disease is directly related to
this practice. Other hypotheses related to soil microbial populations and plant defense
responses induced by rolling are addressed in forthcoming chapters. Phenomena related
to lightweight turfgrass rolling, dew/guttation removal, and dollar spot reductions were
examined. Investigations into possible cumulative effects of repeated daily rolling were

also undertaken in order to elucidate the potential for expedited disease reduction.

MATERIALS AND METHODS

Field research was conducted from June through October in 2008 and 2009 at the
Hancock Turfgrass Research Center on the campus of Michigan State University, East
Lansing, Michigan (42°43'48" N, 84°28'35" W). A research putting green, constructed in
2005, consisting of a mixed sward of creeping bentgrass (Agrostis stolonifera cv
‘Independence ’) and annual bluegrass (Poa annua L.) was used.

A 60 x 60 ft (18.3 x 18.3 m) research area was divided into twelve 7 x 12 ft (2.13
x 3.65 m) evenly spaced plots. Prior to initiating the study, each plot was randomly
assigned a rolling treatment. Alleys of three feet separated each plot so that the roller had
adequate space to stop without impeding into neighboring plots. All plots were mowed

in the morning between 6 and 7 am. before morning rolling treatments were

29

implemented. Rolling treatments were carried out 5 days week'1 (Monday-Friday) and

were as follows: 1) control (no rolling), 2) rolled once (1x) in the am. immediately after
mowing, 3) rolled once (1x) in the pm. when turf was dry or dew and guttation water had
dissipated, and 4) rolled twice (2x) in the am. immediately after mowing. The morning
rolling treatments were implemented between 7:00 and 8:30 am. and the afternoon
rolling between 1:00 and 2:00 pm. Rolling of plots was conducted with a Tru-Turf ride-
on greens roller, model RS48-11B (Tm-Turf Pty. Ltd, Molendinar, Queensland, AUS),
with a 39 inch (99 cm) roll swath weighing 562 pounds (255 kg) without an operator. A
single rolling treatment consisted of rolling across the plot using multiple passes in
opposite directions to ensure complete coverage of the plot with minimal overlap of
rolled areas. Once a single rolling pass was made, the process was repeated immediately
on plots rolled twice per day. To ensure study uniformity, the rolling treatment was the
only practice that differed among plots. All other cultural and chemical practices
remained constant among treatments for the duration of the study.

The root zone soil consisted of a sand based 80:20 (sandzpeat v/v) mix
constructed to USGA recommendations (U SGA, 1993). Sand topdressing was applied to
the entire research area on a light, frequent (bi/tri-weekly) basis throughout the growing
season in order to simulate typical golf course putting green maintenance practices. No
vertical mowing or core cultivation occurred on the research plots during the course of
the study in order to minimize turfgrass and soil disruption. Fungicides were not applied
to any of the research plots during the duration of the study in order to encourage dollar
spot disease infection. Insecticides and herbicides were applied as needed, and were

applied uniformly over the entire study area.

30

Nitrogen fertility was applied at a rate of 24.4 kg N ha_1 mo 4 (0.5 lbs N 1000 ft.

sq.‘l mo'l) during the growing season from April to September of each year. Irrigation

was applied via four Rain Bird Eagle irrigation heads, model 750 (Rain Bird Distribution
Co., Azusa, CA), which were located at the comers of the research area. Irrigation was

applied as needed in order to keep the turf stand healthy and free of wilting symptoms.
Plots were mowed at a height of 0.156 inches (3.96 mm) six days week'1 with a walk-

behind Toro Greensmaster 1000 greens mower (Toro Co., Bloomington, MN). and
clippings were collected throughout the study.

Dollar spot disease counts were taken when disease was active and symptoms
occurred (generally once per week or bi-weekly) by counting the number of individual
infection centers per plot. Spots greater than 3 cm in diameter were counted as one
infection center. Larger, coalescing spots were broken down into smaller spots when
rating and considered to be multiple infection centers. All ratings were taken in the
morning before mowing, when conditions were conducive to counting infection centers.

Quality ratings were taken on a regular basis throughout the growing seasons
(generally once per month from May — October). Quality was rated on a scale of l- 9
where l=dead/necrotic, 6=acceptable, and 9=excellent. Quality ratings were based on a
combination of characteristics such as color, density, unifOrmity, and playability. Ratings
of 6 and above were regarded as acceptable turf for a creeping bentgrass putting green.

Percent volumetric water content (%VWC) was measured using a F ieldScout
TDR 300 Soil Moisture Meter (Spectrum Technologies Inc., Plainﬁeld, IL) with probe
rods at a depth of 1.5 inches (3.81 cm). Twenty measurements were taken at random

locations in each plot and averaged in order to obtain a representative %VWC for each

31

plot on each measurement date. All volumetric water content measurements were taken at
least one full day (24 hours) after a rain event, with-holding irrigation, in order to ensure
consistent VWC ratings.

The experimental design was a randomized block design with three replications.
Analysis of variance was performed on observational measurements in order to determine
signiﬁcant effects, followed by Fisher’s protected Least Signiﬁcant Difference (LSD) if
differences were found at the probability level (P) < 0.05. Treatment differences were
analyzed using the Proc GLM procedure of the Statistical Analysis System (SAS
Institute, 2009), and when appropriate, means were separated using Fisher’s LSD

procedure at the 0.05 level of probability unless otherwise noted.

RESULTS AND DISCUSSION
Dollar spot disease incidence data were collected on a total of 15 dates between
2008 and 2009 (Tables 1.01 and 1.02, respectively). Three dates (18-Sep, 7-Oct, 20-Oct)

in 2008 resulted in signiﬁcant treatment effects on dollar spot, with the twice rolled (2x)
a.m. treatment having less dollar Spot than the control. Treatments rolled 1x day], either

in the morning or afternoon, were not signiﬁcantly different from one another on any of
the dates recorded (P > 0.05). In 2008, no signiﬁcant treatment effect for either 1x
rolling treatments were found compared to the control; and neither of the 1x treatments
were signiﬁcantly different from the 2x treatment according to Fischer’s LSD (P > 0.05).
While differences were not signiﬁcant on individual measuring dates, combined
seasonal dollar spot means from 2008 indicated a signiﬁcant rolling effect on dollar spot

incidence in both the 1x pm. and 2x a.m. treatments (Figure 1.01). The 1x am.

32

treatment was not signiﬁcantly different from any other treatment (P > 0.05). This lack
of statistical separation is likely due to one replication within the 1x a.m. treatment
having uncharacteristically higher dollar spot incidence than the other two replications.
The high dollar spot counts throughout the season on that particular plot resulted in a
much higher seasonal mean, thus rendering the 1x a.m. treatment statistically similar to
the control.

In 2009, dollar spot ratings were taken on nine separate occasions (Table 1.02).
Disease pressure was much higher in 2009 than in 2008, resulting in an average increase
in disease incidence of 2.72 times between the two yearly means. Dollar spot incidence
occurred earlier in the season and at much higher levels throughout the season as
indicated by the number of infection centers recorded. The 2x a.m. treatment had
signiﬁcantly less dollar spot incidence than the control on all but the ﬁrst two rating dates
in 2009 (Table 1.02). When dollar spot incidence was most severe (September and
October), all rolling treatments resulted in signiﬁcantly lower disease counts compared to
the control. July 31, was the only rating date where signiﬁcant differences between 2x
am. and either of the 1x rolling treatments were observed (Table 1.02). However, the 2x
a.m. rolling treatment showed a trend towards lower dollar spot incidence. The 1x am.
and 1x p.m. treatments were not signiﬁcantly different from one another on any
observation date, but both had a signiﬁcant effect on dollar spot when compared to the

control on four of the nine rating dates (25-Aug, 3-Sep, 22-Sep, 17-Oct) (Table 1.02).

33

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These data support earlier observations reported by Nikolai et al., (2001) who
found that dollar spot severity was reduced on rolled plots with every dollar spot outbreak
that was recorded over a six-year period.

Combined 2009 seasonal dollar spot means indicate a signiﬁcant treatment effect
on dollar spot in all rolling treatments (Figure 1.02). Additionally, the 2x a.m. treatment
resulted in signiﬁcantly less dollar spot compared to all other treatments (P < 0.05). Both
the 1x am. and 1x p.m. treatment resulted in signiﬁcantly lower dollar spot incidence
compared to the control (P < 0.05), and no signiﬁcant differences were observed among
the two 1x rolling treatments ((1 = 0.05).

In 2008, seven quality ratings were taken and are reported in Figure 1.03.
Turfgrass quality was rated on a scale of 1-9, with 9 signifying excellent quality, 6 and
above acceptable, and 1 indicating necrotic or dead turf. All rating dates resulted in a
signiﬁcant effect on quality for the 2x a.m. rolling treatment in 2008 (P < 0.05) compared
to the control. The 1x rolling treatments (am. and p.m.) had signiﬁcant effects on
turfgrass quality when compared to the control at three observation dates (S-Jul, 26-Jul,
lO-Oct). No differences in turfgrass quality were observed among the two 1x rolling
treatments at any date in 2008 (P > 0.05). The 2x a.m. treatment had signiﬁcantly better
turfgrass quality than the 1x a.m. treatment on three dates (5-Jul, 26-Jul, lO-Oct) in 2008
(P < 0.05). Additionally the 2x a.m. treatment resulted in signiﬁcantly better turfgrass
quality compared to the 1x p.m. treatment on two dates (5-Jul, 26-Jul) in 2008 (P < 0.05).

When a seasonal mean was calculated for 2008 turfgrass quality, both 1x rolling

treatments were signiﬁcantly better than the control while the 2x a.m. treatment, which

38

averaged a rating of 7.7, resulted in signiﬁcantly better turfgrass quality than all other
treatments (Figure 1.04).

In 2009, quality ratings were taken on 7 different dates and are reported in Figure
1.05. The 2x a.m. treatment resulted in signiﬁcantly better turfgrass quality compared to
the control on six of the seven rating dates. The 1x a.m. treatment was signiﬁcantly
better than the 1x p.m. treatment on only one occasion early in the grong season (19-
Jun); the rest of the rating dates resulted in statistically similar ratings for both 1x rolling
treatments. Both 1x rolling treatments resulted in signiﬁcant treatment effects on the
ﬁnal four rating dates in 2009 (Figure 1.05).

Quality ratings from all rating dates in 2009 were averaged in order to get
seasonal turfgrass quality means for treatments, which are reported in Figure 1.06. Both
1x treatments resulted in signiﬁcantly better turfgrass quality compared to the control (P
< 0.05), but were not signiﬁcantly different from one another (P > 0.05). The 2x a.m.
treatment resulted in signiﬁcantly better turfgrass quality compared to all other treatments
(P < 0.05).

Percent volumetric water content (%VWC) of the top 1.5 inches (3.81 cm) of soil
was measured in 2008 and 2009 on six and four occasions, respectively. In 2008,
signiﬁcant treatment effects were observed on three reading dates, data is reported in
Figure 1.07. The 2x a.m. treatment had signiﬁcantly higher %VWC compared to the
control on three different occasions in 2008 (13-Jul, lO-Sep, and l7-Oct). The 1x rolling
treatments (1x am. and 1x pm.) were not signiﬁcantly different from one another on any

rating date, and only resulted in a signiﬁcant treatment effect on one measurement date.

39

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47

The ﬁnal measurement date (17-Oct) resulted in a signiﬁcant treatment effect on
%VWC for all rolled treatments. Treatment means for individual dates were combined to
obtain a seasonal %VWC mean for treatments in 2008 and are reported in Figure 1.08.
The 2x a.m. rolling treatment resulted in a higher seasonal %VWC compared to the
control and was the only treatment with a signiﬁcant effect on %VWC (P < 0.05).

Two of the four dates in 2009 in which VWC measurements were taken resulted
in a signiﬁcant treatment effect on %VWC in the 2x a.m. treatment, (90-Aug and13-Oct);
results are reported in Figure 1.09. No signiﬁcant treatment effects on VWC were
observed with either of the 1x rolling treatments at any measurement date in 2009.
Treatment means from each measurement date were averaged in order to obtain a
seasonal %VWC mean for treatments; results are reported in Figure 1.10. Similar to
2008, only the 2x a.m. rolling treatment was signiﬁcantly different from the control
(P<0.05), resulting in a 3.35% increase in %VWC. Neither of the 1x rolling treatments

had a signiﬁcant effect on %VWC (P. 0.05).

CONCLUSIONS

It is widely accepted that the removal and dispersal of dew from turfgrass by early
morning mowing plays a signiﬁcant role in reducing dollar spot disease (Williams and
Powell, 1995; Ellram et al., 2007). However, results from this study indicate alternative
mechanisms in disease control. Many questions remain regarding lightweight rolling,
particularly concerning the mechanisms involved in dollar spot reductions. A common
theory revolves around dollar spot incidence being reduced on rolled plots due to the

removal or dispersal of excess moisture on the turfgrass canopy after initial mowing. The

48

moist, humid environment is thought to not only serve as a microclimate conducive for
fungal growth and infection, but guttation exuded from freshly mown turfgrass is thought
to supply a nutrient-rich food source to the dollar spot pathogen, Rutstroemia ﬂoccosum
(Williams and Powell, 1995).

In an attempt to challenge the notion that dollar spot reduction is solely due to the
removal of dew and guttation ﬂuid by morning rolling, one rolling treatment was
implemented in the morning hours, while dew was still present on the plant canopy, and
one in the aftemoon hours, after surface moisture had naturally evaporated. Both
morning and afternoon rolling treatments resulted in signiﬁcant reductions in disease. No
signiﬁcant differences were found between the 1x a.m. treatment and the 1x p.m.
treatment in either year of the study. These results conﬁrm the observation that daily
rolling of turfgrass plays a role in disease suppression but challenge the hypothesis that
dew and guttation removal from early morning rolling is the underlying mechanism
contributing to dollar spot reduction on creeping bentgrass putting greens. Afternoon
rolling treatments proved to have a statistically equal treatment affect on dollar spot
reduction as morning rolling treatments. Additionally, no statistical differences existed
between the aftemoon and morning treatments. This suggests that, aside from the
physical removal/dispersal of surface moisture, possible biological or physiological
mechanisms could be involved in the reduction of dollar spot on creeping bentgrass
putting greens when daily lightweight rolling is taking place.

Rolling ﬁve days week'l, regardless of the time of day, consistently resulted in
lower disease incidence, as well as superior turfgrass quality ratings, in 2008 and 2009.

In order to investigate whether or not rolling has cumulative effects on dollar spot

49

reduction, the 2x a.m. treatment was implemented. This 2x day'l rolling treatment
resulted in consistently lower dollar spot observations compared to both the control and
the 1x rolled treatments, as well as higher turfgrass quality ratings in both years of the
study. Greater reductions in dollar spot counts, signiﬁcantly better turfgrass quality, and
high probabilities of treatment effects on plots rolled 2x day'l, particularly at the
conclusion of the second year, are indications of a possible cumulative effect rolling may
be having on disease suppression and turfgrass health. These results are consistent with
Nikolai et al. (2001) in the respect that greater differences with regard to disease
occurrence, existed between rolled and non-rolled plots as the study progressed for
multiple years. Contrary to previous rolling research, Horvath et al. (2009) observed no

differences in dollar spot severity on rolled and non-rolled plots of creeping bentgrass.
However, rolling treatments were implemented 2-3 times week'1 and results were based

on six weeks of data collection. Cumulative effects of rolling that have been observed
herein as well as in Nikolai et al. (2001) are based on multiple years of data collection,
signiﬁcant treatment effects were typically a result of season long rolling, not observed
until later observation dates in the season, or until seasonal averages were obtained.
Average volumetric water content was signiﬁcantly higher in the 2x a.m.
treatment in both years of the study, and both 1x rolling treatments trended towards
higher %VWC when compared to the control. These observations not only suggests that
rolling may be contributing to to greater water holding capacity in the upper root zone of
the turfgrass canopy, but support observations by Couch and Bloom (1960) and Liu et al.
(1995), where higher soil moisture resulted in reduced dollar spot development and

incidence. Reports by Danneberger (1989), Hamilton et al. (1994), and Hartwiger et al.

50

(2001) indicated rolling did not increase soil compaction of putting greens constructed
with a high sand content root zone. While this may be true, rolling may be contributing
to a decrease in pore size in the top 1.5 in. (3.81 cm) of the root zone. Smaller pores

equate to a greater attractive force by which water can be held. Additionally, volumetric

V
6 = l
water content can be deﬁned as VT, where V.,. is the volume of water and V T is the

total volume associated with the soil (i.e. soil volume + water volume + void space). If
pore size is decreased due to slight compaction in the upper (top 3.8] cm) root zone, a
reduction in void space takes place, thus lowering VT in the equation. This may be
responsible for increasing 9 or the total volumetric water content measurements in rolled
treatments.

The phenomenon by which rolling inhibits disease incidence was investigated.
Rolling had a signiﬁcant effect on dollar spot incidence in both years of this study.
Morning and aﬁemoon rolling treatments had similar effects on dollar spot reduction
compared to the control. These similarities between morning and aftemoon rolling
suggest mechanisms other than secondary dew/guttation removal may be responsible for
dollar spot reductions. Many theories still remain, and future rolling research should be
conducted in order to investigate the potential biological or physiological impacts
associated with turfgrass rolling. This study also revealed a trend towards cumulative
effects of rolling on turfgrass health and quality. Multiple rolling applications per day
resulted in substantially better turfgrass quality with less disease when compared to
treatments rolled once per day. Continued research investigating this effect, particularly
in aftemoon or dew/guttation free settings should be pursued. The mechanism by which

rolling is detrimental to dollar spot incidence is most likely multi-faceted, involving

51

aspects of soil physics, microbial ecology, plant physiology, and plant pathology or
epidemiology. In the forthcoming chapter, one particular mechanism regarding microbial

populations is discussed.

52

 

LITERATURE CITED

Beard, J. B. 1994. Turf rolling. Grounds Maintenance. 29: 44-52.

Couch, H. B. and Bloom, J. R. 1960. Inﬂuence of environment on diseases of turfgrasses.
II. Effect of nutrition, pH, and soil moisture on Sclerotinia dollar spot. Phytopathology.
50: 761-763.

Danneberger, K. 1989. No speed limit. Landscape Manage. 29: 66—70.

Demoeden, P. H. 2000. Creeping bentgrass management: Summer stresses, weeds, and
selected maladies. John Wiley & Sons, Hoboken, NJ. pp. 10-12, 47.

DiPaola, J. M. and Hartwiger, C. R. 1994. Green speed, rolling and soil compaction. Golf
Course Management. 62(9): 49-51, 78.

Ellram, A., Horgan, B., and Hulke, B. 2007. Mowing strategies and dew removal to
minimize dollar spot on creeping bentgrass. Crop Sci. 49: 2129-2137.

Hamilton, G. W. Jr., Livingston, D. W., and Grover, A. E. 1994. The effects of
lightweight rolling on putting greens. Science and Golf II. pp. 425-430.

Harban, W. S. 1922. The effect of trampling and rolling on turf. Bulletin of the Green
Section of the US. Golf Association. 2: 148-150.

Hartwiger, C. 1996. The ups and downs of rolling putting greens. USGA Green Section
Record. 34: 1-4.

Hartwiger, C. E., Peacock, C. H., and DiPaola, J. M. 2001. Impact of lightweight rolling
on putting green performance. Crop Sci. 41: 1179-1184.

Horvath, B. J., Nichols, A. 13., and Cutulle, M. A. 2009. The effects of mowing height
and rolling on ball speed, quality and disease severity of creeping bentgrass. USGA Turf.
and Env. Res. Online. 8: 1-5.

Inguagiato, J. C., Murphy, J. A., and Clarke, B. B. 2009. Anthracnose disease and
annual bluegrass putting green performance affected by mowing practices and
lightweight rolling. Crop Sci. 49: 1454-1462.

Kussow, W. R. 1998. Putting green management systems. Wisc Turf Res. Reports. XV:
85-93.

Liu, L. X., Hsian. T., Carey, K., and Eggens, J. L. 1995. Microbial populations and
suppression of dollar spot disease in creeping bentgrass with inorganic and organic
amendments. Plant Dis. 79: 144-147.

53

Nikolai, T. A., Rieke, P. E., Rogers, J. N. III, Vargas, J. M. Jr. 2001. Turfgrass and soil

responses to lightweight rolling on putting green root zone mixes. International Turfgrass
Society Research Journal. 9: pp. 604-609.

Piper, C. V. and Oakley, R. A. 1921. Rolling the turf. Bulletin of the Green Section of the
US. Golf Association. 1: 36.

Radko, A. M. 1977. How fast are your greens? USGA Green Section Record. 15: 10-11.
Richards, J., Karcher, D., Nikolai, T., Richardson M., Patton, A., and Landreth, J. 2008.
Mowing height, frequency, and rolling frequency affect putting green speed. Arkansas
Turfgrass Report 2007. Ark. Ag. Exp. Stn. Res. Ser. 557: 52-56.

Smiley, R. W., Demoeden, P. H., and Clarke, B. B. 2005. Compendium of turfgrass
diseases. 3rd ed. Am. Phyto. Soc., St. Paul, MN.

Stier, J. 2006. Shorter mowing heights are hazardous to summer health. The Grass Roots
35: 4-5,7,9.

Throssell, C. 1986. Management practices affecting putting green speed. The Bull Sheet
39: 4,7,9.

USGA Green Section Staff. 1993. USGA recommendations for a method of putting green
construction. USGA Green Section Record. 31: 1-3.

Vargas, J. M. Jr. 2005. Management of turfgrass diseases. 3rd ed. CRC Press, Inc., Boca
Raton, F L. pp. 19-22.

Williams, D. W. and Powell, A. J. Jr. 1995. Dew removal and dollar spot on creeping
bentgrass. Golf Course Management. 63: 49-52.

Youngner, VB. 1969. Physiology of growth and development. In Hanson, A. A. and
Juska, F.V. (ed.). Turfgrass science. ASA, Madison, WI. pp.187-216.

54

 

CHAPTER TWO

TURFGRASS ROOTZONE MICROBIAL RESPONSES TO ROLLING

ABSTRACT

Relative abundance of microorganism community populations is useful
information for estimating the effects of particular management strategies on rhizosphere
soil (REF). A two-year rolling study was conducted during the growing seasons of 2008
and 2009 and relative abundance of soil microorganisms under a creeping bentgrass
(Agrostis palustris) putting green was elucidated via phospholipid fatty acid (PLF A)
analysis (REF). Treatments rolled twice in the morning (2x a.m.) and once in the
morning (1x a.m.) both showed signiﬁcant increases in particular PLFA abundance when
compared to the control (P < 0.05) all of which are bacterial biomarkers. When combined
to form speciﬁc PLF A taxonomic biomarker groups, relative abundance was found to be
higher in all rolled treatments compared to the non-rolled control, while ﬁmgal and other
PLF A relative abundance remained relatively constant. A trend towards higher bacterial
abundance in rolled treatments suggest a potential biological effect of lightweight rolling
possibly attributed to the aforementioned increase in volumetric water content observed
in rolled treatments (Chapter 1). Increases in bacterial relative abundance may play a role
in dollar spot (Rutstroemia ﬂoccosum) reductions due to various mechanisms of fungal

disease suppression often observed in many soil dwelling bacterial organisms.

55

 

INTRODUCTION

The rhizosphere is a complex community of soil microﬂora and microfauna.
Along with facilitating plant infection and disease, many microorganisms play beneﬁcial
roles in the root zone of a turfgrass stand. Microbial communities have been implicated
in suppressing soilbome plant diseases, promoting plant growth, and in changing
vegetation (Doran et al., 1996). Several studies have elucidated the signiﬁcance of
microbial activity in soil related to and responsible for the suppression of plant
pathogens. For example, relationships have been found between microbial diversity and
root disease suppression (Nitta, 1991; Workneh and van Bruggen, 1994). Rovira &
Wildermuth (1981) indicated that the microbiota in a “rich” soil tends to reduce the
severity of attack by many plant pathogens or, in other words, soils higher in microbiota
content and diversity show tendencies towards general disease suppression.

Bacterial populations in the soil and rhizosphere have long been of interest to
agronomists and plant scientists, partially due to the observation that antagonism and
competition between bacteria and many important plant pathogens can be exploited as
possible control mechanisms. An active microbial population performs and plays a role
in many beneﬁcial activities and processes such as organic matter decomposition,
assisting in plant nutrient acquisition, availability and recycling, and plant pathogen
suppression (Sylvia et al., 1997). Thus, turfgrasses grown in rootzones containing lower
microbial populations may be less healthy and possibly more easily affected by turfgrass
pathogens, resulting in an overall lower quality turfgrass than those grown in the presence
of higher microbial populations (Hodges, 1990; Couch, 1995). In turfgrass systems,

successful suppression of fungal diseases such as dollar spot (Rutstroemiaﬂoccosum) and

56

anthracnose (Colletotrichum cereale) has been achieved both in vitro and with ﬁeld
applications of an antibiotic producing Pseudomonas spp. (Powell, 1993; Uddin and Viji,
2002). These bacterial metabolites are thought to contribute to the antagonistic effects on
plant pathogenic fungi.

Bolton et al., (1985), Fraser et al., (1988), Kirchner et al., (1993), and Powlson et
al., (1987) have documented associations between agricultural management practices and
microbial community make-up and activities. Particular management practices in
different systems may alter the composition and structure of soil microbial communities.
This has been documented in agricultural soils by examining either whole soil microbial
communities or speciﬁc functional groups (Ka et al., 1995; Lundquist et al., 1999;
Webster et al., 2002; Clegg et al., 2003). Microbial populations ﬂuctuate, and their
community structure and composition is highly sensitive to change with intense
management (Donnison et al., 2000). Disease suppressiveness has been achieved through
crop rotation (Cook et al., 2002), intercropping (Schneider et al., 2003), residue
destruction (Baird et al., 2003), organic amendments (Tilston et al., 2002), and tillage
management practices (Sturz et al., 1997; Pankhurst et al., 2002). All of these cultureal
practices contribute to altering the microbial community in a way that favors plant health
and hinders disease development.

A useful approach to estimating the fungal and bacterial abundance in soil is to
measure chemical components that are speciﬁc for microbial groups. One such approach
relies on bacteria and fungi having different fatty acid compositions in their
phospholipids (Hardwood and Russell, 1984; Tunlid and White, 1992). Phospholipid

fatty acids (PLFAs) are widely accepted as biomarkers that indicate viable microbial

57

biomass and provide a microbial community ‘ﬁngerprint’ (Vestal and White, 1989;
Zelles, 1999). Changes in soil microbial communities under many different experimental
applications have been successfully measured using PLF A analysis. Fatty acid patterns
have been used to distinguish microbial ﬂuxes between different types of cultivation
(Zelles et al. 1995), fertilizer treatments (Guo and Wang, 2009), pH gradients (Baath and
Anderson 2003), agricultural management systems, seasons, soil types (Bossio et al.
1998), and changes in the soil microbial community due to pollution (Baath et al., 1992).
A quantitative measure of microbial communities can be obtained using PLFA analysis
as opposed to most current DNA-based ﬁngerprinting methods (Muyzer et al., 1993;
Kowalchuk et al., 1997).

While many studies have shown the direct effect that high populations of
microbes have on plant pathogens in the rhizosphere, much remains unknown with regard
to their disease suppression mechanisms. Numerous species within several genera of
bacteria have been reported to be effective biological control agents for root and foliar
diseases on turfgrass. The mechanisms of biological control are thought to occur mainly
through the process of antagonism. Antagonism can be deﬁned as active opposition that
results from the production of substances by one organism that are toxic to other
organisms. Such substances can be classiﬁed as antibiotics that cause lysis or death of
microbial competitors (Rovira and Wildermuth, 1981). Additionally, competition for
food, oxygen, and space is often considered a form of antagonism or a distinct
mechanism of biological inhibition all together (Baker and Cook, 1974).

The purpose of this study was to determine the effects of lightweight rolling of

creeping bentgrass putting greens in relation to soil microbial populations. Results of

58

dramatic decreases in disease incidence caused by the fungal pathogen R. ﬂoccosum,
have been outlined in the previous chapter. Microbial community assessment was
explored in order to test the hypothesis that rolling is having an effect on the community
dynamics, possibly related to disease suppression. Microbial community composition
was estimated using PLFA analysis of root zone samples from treatment plots in order to

make comparisons among rolled and non-rolled turf stands.

MATERIALS AND METHODS

Field research was conducted at the Hancock Turfgrass Research Center on the
campus of Michigan State University, East Lansing, Michigan, on an experimental
putting green constructed in 2005 and seeded with creeping bentgrass (Agrostis
stolonifera cv ‘Independence ).

Research plots were 7 ft x 12 ft (2.13 m x 3.65 m) arranged in a randomized block
design with three replications for each rolling treatment. Prior to initiating the study,
each plot was randomly assigned a rolling treatment. Mowing took place between 6:00

and 7:00 a.m. and was implemented on all plots prior to any rolling. Each rolling

treatment was carried out 5 days week.l (Monday-Friday) and were as follows: 1)

Control (no rolling), 2) rolled once (1x) in the a.m. immediately after mowing, 3) rolled
once (1x) in the p.m. when dew and guttation water had dissipated, and 4) rolled twice
(2x) in the a.m. immediately after mowing. The morning rolling treatments were
implemented between 7:00 and 8:30 a.m., and the aﬁemoon rolling between 1:00 and
2:00 p.m. Rolling of plots was conducted with a Tru-Turf ride-on greens roller, model

RS48-11B, with a 39 inch (99cm) roll swath weighing 562 pounds (255kg) without an

59

operator. To ensure study uniformity, rolling was the only treatment that differed in each
plot.

The root zone mix consisted of a sand based 80:20 (sandzpeat v/v) mix
constructed to USGA recommendations (U SGA, 1993). Sand topdressing was applied to
the entire research area on a light, ﬁequent (bi/tri-weekly) basis throughout the growing
season in order to simulate typical golf course putting green maintenance practices. No
vertical mowing or core cultivation occurred on the research plots during the course of
the study in order to minimize turfgrass and soil disruption. Fungicides were not applied
to any of the research plots during the duration of the study. Insecticides and herbicides
were applied only on an as needed basis, and were applied uniformly over the entire
study area when necessary.

Soil samples from plots were taken at a depth of 1 in (2.54 cm) with a 1 in (2.54
cm) diameter soil probe. Ten cores were randomly taken from each plot on September 25,
2009. Top growth, including turfgrass leaves, stems, roots, and debris was removed from
each core by cutting with a sterile razor blade just below the thatch/soil interface. Cut
ends were gently shook so that loose soil that remained in thatch and roots would be
included in the sample. Unused top growth was discarded. The remaining upper rootzone
soil was homogenized by vigorous mixing in plastic bags. Thirty grams from each of the
representative, homogenized soil samples was measured and placed into separate 3 1/8 X
5 1/2 inch paper coin envelopes before being lyophilized. Freeze dried soil samples from
each plot were ground using a pestle and mortar until they passed through an ATM #40
(425 um) U.S. standard testing mesh sieve (Advantech Manufacturing Inc., New Berlin,

WI). Ten grams of the ground material from each sample were stored in 2 oz. Nasco

60

Whirl-Pak bags at -20° C prior to their shipment to the University of Wisconsin,
Madison, Department of Soil Science for PLFA analysis by Dr. Teri Balser’s laboratory
staff.

Lipids were extracted from 3 g (dry weight) subsamples using a modiﬁed Bligh
and Dyer (1959) technique as described in Balser and Firestone (2005). Samples were
analyzed using a Hewlett-Packard Agilent 6890A gas chromatograph (GC) (Agilent
Tech. Co., Santa Clara, CA) equipped with an Agilent Ultra-2 (5% phenyl)-
methylpolysiloxane capillary column (25m by 0.2m by 0.33pm) and ﬂame ionization
detector (F ID). Peaks were identiﬁed using a mix of known FAME standards and
comparing retention times or estimated chain lengths (ECL) from each sample output to a
naming table. These naming tables have ECL’s for hundreds of known lipids and a
f‘naming window”, which is the accepted amount that an unknown can vary from the
ECL and still be assigned a particular name. The fatty acid analyses were carried out by
an MIDI Sherlock microbial identiﬁcation system (Version 4.5, MIDI, Newark, NJ).

Lipid data were converted from raw peak area to nmol/g soil in order to facilitate
statistical analysis. Peak area was converted to ug carbon (C) using conversion factors
from internal standards run on the GC. Conversion factors are equal to pg C of the
standard divided by the area of the standard on the GC and are typically the averages of
two internal standard lipids, 9:0 and 19:0 in order to capture the response of both short-
and long-chain lipids. This number was then divided by the unit weight soil in each
sample (approximately 3g), and ﬁnally converted to umol C g soil’I using the molecular

weight of each lipid.

61

Processed lipid data are expressed as abundance (nmollipid gsoil-l), mole fraction

(nmollipidx nmoltotampid-IQ (O-l)), or mole percent (mole fraction*100, (0-100%)). Mole

fraction and mole percent are normalized by the total biomass in a sample and are thus
measures of the relative abundance of any given lipid. Mole fraction is appropriate for
use in ordination analyses (after transformation, e.g. arcsine, square-root), while mole
percent (mole fraction multiplied by 100) is an easy-to-interpret value. Lipid abundances
are the absolute amount of a given lipid extracted per gram of soil. Because the quantity
of lipid per cell is reasonably constant, and the lipid extraction is highly quantitative (i.e.
close to 100% extraction efﬁciency), abundance is, in effect, an estimate of microbial
biomass. Total abundance is total biomass, and the abundance of key indicators reﬂects
the biomass of the group it represents (T. Balser, pers. comm).

Fatty acid nomenclature used in this study is as follows: total number of carbon
atomsznumber of double bonds, followed by the position of the double bond ((0) from the

methyl end of the molecule. Cis and trans geometries are indicated by the sufﬁxes c and
t, respectively. The preﬁxes a and 1' refer to anteiso- and iso-branching, respectively.
Methyl groups on the tenth carbon atom from the carboxyl end of the molecule are
indicated by. The positions of the hydroxyl (OH) groups are noted when necessary, while
ey indicates cyclopropane fatty acids (Bossio et al., 2006).

Each PLFA value is represented by the mean of three soil extraction replications.
Particular individual fatty acids extracted from samples have been used as signature
indicators for various taxonomic groups of microorganisms.

Combinations of particular PLFAs were considered to be representative of groups

0f organisms of interest in the soil. The PLFAs 18:1(o9c, 18:2(o6c, and 18:3(o6c were

62

used to represent soil fungi (Myers et al., 2001 and Vestal and White, 1989). Gram-
positive (gm+) bacteria were represented by i15:0, 015:0, 15:0, i16:O, 17:0, i17:0 and
a17:0, while 16:1m7c, cyl7:0, cyl9:0 and 18:1co9t were used to indicate gram-negative
(gm-) bacteria (Ratledge and Wilkinson, 1988 and Zogg et al., 1997). Total bacteria
were represented by the combined gm- and gm+ bacterial indicators (Ratledge and
Wilkinson, 1988 and Zogg et al., 1997).

Treatment effects on PLFA relative abundance were analyzed using the PROC
Mixed procedure in the SAS software (SAS Institute Inc. 2009, Cary, NC, USA).
Analysis of variance (ANOVA) followed by Dunnett’s test, for the comparison of
treatment means to the control for individual fatty acid mole percentages, were carried
out. Combined fatty acids representing groups of microorganisms were also subjected to
ANOVA as well as mean comparisons based on Dunnett’s test. A principal component
analysis (PCA) of the arcsine transformed mole fraction PLFA data was carried out with

the correlation matrix.

RESULTS AND DISCUSSION

Mole percent is considered an indication of relative abundance in relation
to total PLF A abundance. As FID response from gas chromatography is proportional to
molecular mass, results were expressed as molar percentages. Thirty fatty acids were
identiﬁed for use in data analysis. Individual fatty acid relative abundances were
compared in order to investigate possible differences due to season long lightweight
rolling treatments. All PLFAs used in analyses were evaluated for normal distribution

before being subjected to signiﬁcance testing. Signiﬁcant differences in particular fatty

63

acid mole percentages existed between rolled and control treatments in rootzone soils of
the creeping bentgrass putting green. These PLFA indicators, mole percentages, and
references, are reported in Table 2.01.

Of the thirty microorganism indicator PLFAs extracted, eight were signiﬁcantly
higher in mole percentage in the 2x a.m. rolled treatment than in the untreated control (P
S 0.10). These included the straight chain saturated fatty acids 12:0, 14:0, 15:0, 18:0,
19:0cy, 20:0, 9:0 (bacterial indicators) and the monounsaturated fatty acid 18:1w9t
(actinomycete indicator).

Other signiﬁcant differences in individual PLFAs existed between the 1x a.m.
treatment and the control (P S 0.10). These included cyclopropane fatty acids 17:0cy and
19:0cy, straight chain saturated fatty acids 18:0, 20:0, and 9:0, and the monounsaturated
fatty acid 18:1 w7c (all bacterial indicators); the polyunsaturated fatty acid 18:2(06c
(saprophytic fungi indicator); and the methyl branched fatty acid 117:] and

monounsaturated fatty acid 18:1(o9t (both actinomycete indicators).

64

Table 2.01. Relative abundance (mol %) of PLFA indicators in turfgrass root zones.

 

PLF A

12:0
14:0

114:0 30H

14:1(05e
15:0
a15:0
115:0
16:0
[16:0
116:1 I-I
16:1m5c
16:1(o7c
16:1(o9c
17:0
(117:0
17:0cy
117:0
17:1m8c
18:0
18:10)5c

18:10)9c
18:1(n9t

18:3cu6c
19:0

19:0cy
20:0

9:0

117:1
18:206c
18:1037c

Rolling Treatment

 

Control

0.7446
0.9251
0.3279
0.2097
0.4878
1.1085
2.4031
15.949
0.5792
0.5080
36.410
2.6360
0.4481
0.1997
0.3455
0.5650
0.3455
0.4281
0.6418
0.4178

7.5882
0.2344

0.5733
0.1188

0.5445
0.2255
0.2318
1.2652

3.6361
4.9095

1x a.m.

0.7797
1.0461
0.4073
0.2225
0.5458
1.4009
2.9951
15.599
0.7681
0.5875
31.143
3.0052
0.5341
0.2736
0.4717
0.77704 "'
0.4717
0.5226
0.9294 *
1.2252

7.4477
0.29724 *

0.6938
0.1500

0.8611 **

0.33765 **

0.28886 *
1.6838 *

4.0150
6.0582 *

1x p.m.

0.8962
1.0315
0.3889
0.2189
0.5477
1.3757
2.8863
15.540
0.7288
0.6872
32.753
2.9218
0.5392
0.2506
0.4321
0.7137
0.4321
0.6033
0.7805
0.8376

7.5651
0.2694

0.6054
0.1235

0.7305
0.2714
0.2576
1.5674

3.1897
5.2525

2): a.m

1.0465 **
1.1277 **
0.4011
0.1428
0.5591 "'
1.3671
2.9857
15.714
0.7313
0.4279
31.896
2.9093
0.5150
0.1780
0.4319
0.7324
0.4319
0.5518
0.8558 **
0.4048

8.2416
0.3214 *"‘

0.6865
0.1483

0.7731 *
0.3068 *
0.2904 *
1.5392

3.6111
5.8397

PLFA Biomarker't

Bacteria

Bacteria
Unknown

(Gram -) bacteria
Bacteria

(Gram +) bacteria
(Gram +) bacteria
Bacteria and fungi
(Gram +) bacteria
(Gram -) bacteria
Arbuscular mycorrhizae
Gram- bacteria
Unknown
Bacteria

(Gram +) bacteria
Gram - /anaerobes
(Gram +) bacteria
(Gram -) bacteria
Bacteria
Unknown
Saprophytic or
ectotrophic fungi
Actinomycetes
Saprophytic/
ectotrophic fungi
Bacteria

Gram - / anaerobic
bacteria
Bacteria

Bacteria
Actinomycetes
Saprophytic fungi
(Gram -) bacteria

*, " Means within the same row are signiﬁcantly higher than the control according to Dunnett’s test

at the P S 0.10 and 0.05 probability levels respectively.

1' PLFA biomarker indicators according to: Bossio et al., 1998; Ratledge and Wilkinson, 1988; Zogg
et al., 1997; Frostegérd and Baath, 1996; Myers etal., 2001; Vestal and White, 1989; Turpeinen et al.,
2004; T. Balser, personal Communication, 2009.

65

 

The 1x p.m. rolling treatment resulted in no signiﬁcant differences in any of the
30 indicator PLFAs when compared to the control, but remained higher in relative
abundance than the non-rolled control in most of the bacterial indicators. The 1x p.m.
treatment also did not stray statistically from the other rolling treatments in any of the
individual PLF A relative abundances. Conversely, the untreated control had signiﬁcantly
higher proportions of the PLFA 16:1m5c (Arbuscular mycorrhizae indicator) compared to
the 1x a.m. rolling treatment.

Commonly used combinations of PLFAs were analyzed and used to represent
taxonomic groups of interest in the root zone soil samples and make comparisons among
rolling treatments, PLFA combinations are reported in Table 2.02. These combinations
rendered signiﬁcant differences among treatments as indicated in Figure 2.01. Gram-
negative bacterial PLF A relative abundances were signiﬁcantly higher (P < 0.10) in plots
rolled 1x a.m. (10.70%) compared to the control (8.66%), with a standard error (SE) of
0.8941. Treatments 1x p.m. and 2x a.m. resulted in 0.963% and 1.599% higher gram
negative bacteria PLFA abundances than the control respectively. Gram-positive
bacterial PLFAs were not statistically different in any treatment but were slightly higher
in all rolled treatments than the control.

Total bacterial PLF A mole percentages were signiﬁcantly higher (P < 0.10) in the
1x a.m. rolled treatment compared to the non-rolled control. A trend towards higher total
bacterial PLF A indicators existed in all rolled treatments when compared to the control

with a difference of 2.15% and 2.82% in the 1x p.m. and 2x a.m. treatments respectively.

66

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67

Figure 2.01. PLF A group abundance (mol %)T in turfgrass root zones subjected to different rolling treatments.

 

 

g Gram-negative bacteria

5 Fungi

 

I Total Bacteria

 

 

7 El Gram-postive bacteria

 

 

 

 

 

*+_

 

 

 

 

 

(0/0 [0111) oouepunqv analog

68

2xa.m.

1x p.m.

3.1!].

Control

Rolling Treatment

in relative abundance ﬁ'om the control using Dunnet’s test (P S 0.10).

a signiﬁcant difference

1' Vertical bars represent standard error (SE) of mean.

* Indicates

Total ﬁmgal PLFA mole percentages were not signiﬁcantly different among
treatments; additionally, no trends towards higher fungal PLFAs were observed in rolled
treatments.

Results from a principal component analysis (data not shown) were insigniﬁcant
when discriminating rolling treatments by PLFA proﬁle. No deﬁnite shifts in microbial
community structure or patterns with regard to particular PLFAs under different rolling

treatments were observed.

CONCLUSIONS

PLF As are widely accepted as biomarkers that indicate viable components of soil
microbial biomass and can provide a microbial community “ﬁngerprint” (Liang et al.,
2008). PLFA analysis can provide more detailed information of the “active” soil
microbial community compared with traditional culture methods (Vestal and White 1989;
Yao et al., 2000; Liang et al., 2008). Unlike nucleic acid proﬁling where there is high
species speciﬁcity, PLFAs largely proﬁle functional groups.

Rolled treatments resulted in signiﬁcantly higher relative abundance of particular
PLFAs across a range of fatty acid indicators. With most PLFAs being statistically
similar among treatments, increases in the saturated fatty acids 9:0, 12:0, 14:0, 15:0, 18:0,
and 20:0 in rolled treatments suggests a potential increase in soil bacteria, as these are all
listed as being common bacterial indicators. Additionally, increases in the
monounsaturated fatty acid 18:1(o7c and the cyclopropane fatty acid 17:0cy, both listed
as being common in gram-negative bacteria, suggest further increases in bacterial

abundance. However, care should be taken when connecting individual PLFAs to

69

speciﬁc microbial groups; it is, perhaps, better to look at the trends in groups of fatty
acids.

Combining PLFAs into common taxonomic indicator groups such as gram-
negative bacteria, gram-positive bacteria, total fungi and total bacteria allows for a more
representative analysis as opposed to individual fatty acid comparisons. There was
evidence of an increased relative abundance of total bacterial PLFAs in rolled treatments
when compared to the not rolled control. This general trend towards higher bacterial
proportions could be due to the aforementioned increase in volumetric water content in
rolled plots (see chapter 2 of this thesis). Low water availability can inhibit microbial
activity by lowering intracellular water potential, thus reducing hydration and activity of
enzymes. In solid matrices such as soil, low water content may also reduce microbial
activity by restricting substrate supply (Stark and Firestone, 1995). Therefore, higher soil
water contents could be contributing to higher bacterial proportions and activities.

Fungal PLFAs were not signiﬁcantly different among treatments, indicating
consistent PLF A extraction among samples and, therefore, low sampling error. Fungi are
ﬁlamentous, enabling them to bridge air gaps between the thin water ﬁlms that occur in
soil pore spaces under conditions of soil desiccation (Killham, 1994). It would follow
then that, fungi would be expected to better withstand lower soil water contents compared
to bacteria, thus possibly contributing to the similarities in ﬁmgal relative abundance
among treatments with different volumetric water contents under different rolling
treatments. Additionally, dollar spot incidence has been found to be more severe in soils

exhibiting lower moisture contents (Couch and Bloom, 1960; Liu et al., 1995), adding to

70

 

the notion that fungal pathogens, such as R. ﬂoccosum, are better suited to dehydrated
conditions than bacteria.

Table 2.03 includes bacterial organisms found to have biological control qualities
on turfgrass pathogens. While extensive, this table is only a small portion of bacterial
species isolated from soil with biological control capabilities on plant patghogens.
Biological control mechanisms can be thought of as a series of traits expressed
synchronously, in a controlled sequence. Many non-pathogenic soil microorganisms can
effectively colonize foliage as well as roots in soil and allow protection of these tissues
from pathogen infection (Nelson, 1992). Of the traits most common to soil microbes,
ﬁve have been consistently linked with biocontrol: 1) production of metabolites such as
antibiotics or inhibitory volatiles, 2) microbial resource competition, 3) hyperparasitism,
4) induction of systemic plant resistance, 5) Rhizosphere competence (Nelson, 1998).
Increased volumetric water contents in turfgrass root zones continually rolled throughout
the season may be contributing to a favorable environment which is more suited for
bacterial populations than dryer soils. These bacteria may, perhaps, be having profound
effects on dollar spot incidence by inhibiting the grth and proliferation of R. ﬂoccosum
in the turfgrass root zone via the aforementioned mechanisms. The mechanisms
discussed above are all subject to further investigation and discussion with regard to
dollar spot reductions and bacterial populations.

Preliminary research has indicated possible increases in bacterial relative
abundance in turfgrass root zones subjected to continual season long rolling regimens.

While this groundwork has elucidated interesting trends related to disease reductions,

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72

much research is still required in order to draw any substantial conclusions. PLF A
proﬁles can be used to ﬁngerprint the structure of soil microbial communities and
measure their biomass (Bossio and Scow 1998; Bossio et al., 1998). These methods are
free of the distortion associated with the requirements for quantitative removal of
microbes from surfaces or the selectivity associated with growth on artiﬁcial media
(White 1988; Bossio & Scow 1998). PLFA measurement can provide detailed
information about the structure of the active microbial community because only lipids
from living organisms are measured (Vestal & White 1989). As a result, PLFA proﬁles
can be useful in predicting and manipulating physical and chemical factors in soils to
sustain long-term pathogen suppression (Chen et al., 1988).

It must be noted that microbial populations, particularly biomass and metabolic
activities, in soil environments are extraordinarily dynamic, dependent on factors such as
temperature, moisture, radiation, and atmosphere, and can change drastically throughout
the growing season and even over the course of one day (F. Dazzo, personal
communication, March 1, 2010). Population ﬂuxes occur between areas within close
proximity to one another as well. While the study area examined was maintained
relatively consistently throughout (with exception to rolling treatments), many spatial
discrepancies can result with regard to the abovementioned environmental factors. For
this reason, relative abundance was chosen for comparative analysis in order to limit the
misinterpretation of population estimates. By measuring the amount PLFAs belonging to
particular taxonomic groups in relation to the total amount of PLF As extracted,
conclusions could be drawn regarding population affects due to rolling with minimal

concern to variations due to sampling parameters.

73

Perhaps, future rolling studies can implement multiple soil sampling dates for
PLFA analysis during a growing season. Additionally, incorporating molecular-based
techniques by which community diversity and qualiﬁcation can be elucidated should be
considered. Identifying key microorganisms associated with cultural practices such as
rolling and microbial disease suppression is crucial in understanding the intricacies in
disease control mechanisms. This initial study is meant to serve as a foundation for future
projects aimed at elucidating soil microbial characteristics linked to turfgrass disease

suppression.

74

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81

CHAPTER THREE
DOLLAR SPOT REDUCTION THROUGH LIGHTWEIGHT ROLLING ON
CREEPING BENTGRASS PUTTING GREENS
ABSTRACT

Hypovirulent and virulent isolates of Rutstroemia ﬂoccosum were compared for
mycelial growth, oxalic acid and cell wall degrading enzyme production. Virulence of
isolates VCG-B, VCG-C, and CS was conﬁrmed while a known hypovirulent strain,
Sh12B, was unable to cause disease symptoms. Isolates were grown for 14 days on
potato dextrose agar (PDA) amended with the acid indicator dye bromophenol blue. All
isolates were capable of changing the media from purple to yellow indicating acid
production, with VCG-B showing statistically less of a color change than the three other
isolates. Oxalic acid (0A) concentrations were measured via HPLC in culture ﬁltrates
grown in potato dextrose broth (PDB) for 15 days. Virulent isolates produced
signiﬁcantly higher amounts of OA than isolate ShIZB. The cell wall degrading enzymes
cellulase and pectin methylesterase were assayed in culture ﬁltrates grown on isolated
creeping bentgrass cell walls. All isolates exhibited pectin methylesterase activity, with
isolate ShIZB displaying the highest activity relative to mycelia dry weight. Cellulase
activity was highest in isolate Sh12B as well relative to mycelia dry weight. Mycelia]
grth of the hypovirulent isolate was less than the virulent isolates in all experiments;
however enzyme activity and oxalic acid production remained steady. These results
suggest that oxalic acid and extracellular enzymes are produced by hypovirulent strains
of R. ﬂoccosum, indicating that virulence may not be solely determined by the production

of these particular compounds.

82

 

INTRODCTION

Association between oxalic acid (0A) production and pathogenicity has been
reported for a number of phytopathogenic fungi (Bateman and Beer, 1965; Kritzman et
al., 1977; Magro et al., 1984; Marciano et al., 1983; Godoy et al., 1990). Extensive
research has documented the importance of OA production, particularly by Sclerotinia
spp. as a factor in pathogenesis. However, the role of additional factors such as
extracellular enzymes remains debated (Callahan and Rowe, 1991). Mutants of
Sclerotinia sclerotiorum that are deﬁcient in the ability to synthesize 0A are non-
pathogenic, whereas revertant strains that regain their OA biosynthetic capacity exhibit
normal virulence (Godoy et al., 1990.)

Mechanisms by which 0A secretion might enhance virulence of certain pathogens
have long been investigated. Oxalic acid appears to be involved in pathogenesis by
lowering pH in advance of infected tissues to enhance the activity of extracellular
enzymes produced by the pathogen (Hodgkinson 1977; Dutton and Evans 1996). Other
theories center around OA being directly toxic to the plant, thus weakening the plant,

facilitating further invasion (Noyes and Hancock, 1981). This occurs by the chelation of

cell wall Ca2+ by the oxalate anion which is thought to compromise the function of Ca2+-

dependent defense responses (Bateman and Beer, 1965).

Variation in production of 0A in liquid medium by hypovirulent and virulent
strains of Cryphonectria parasitica (Murr.) Barr has been reported (McCarroll, 1978).
Virulent strains produced twice as much calcium oxalate as hypovirulent strains.
Hypovirulence is a phenotype associated with reduced virulence in fungal plant

pathogens, and has been associated with several mechanisms of action (Bharathan and

83

Tavantzis, 1990; Boland, 1992; Elliston, 1982; Zhou and Boland, 1997). The dollar spot
pathogen Rutstroemia ﬂoccosum (Syn. Sclerotinia homoeocarpa) produces oxalic acid in
vitro, though to a much lesser extent than other Sclerotinia members such as S.
sclerotiorum and S. trifoliorum (Beaulieu, 2008). Very little is known regarding the
pathogenesis of R. floccosum, and whether or not 0A is an important factor related to
turfgrass infection. Understanding the mechanisms of action by which hypovirulence
inﬂuences the fungal pathogen with regard to 0A and extracellular enzyme production,
may give valuable insight into the role that oxalic acid plays in the infection process.
Elucidating the role that OA plays in R. ﬂoccosum pathogenicity, allows for
‘ investigations into novel methods of dollar spot control related to plant defense
mechanisms. For instance, the expression of oxalate oxidase in transgenic plants has
been shown to be successful in conveying resistance to a diverse array of pathogens
particularly those that produce OA (Thompson et al. 1995; Liang et al., 2001; Hu et al.,
2003). Since many pathogens produce CA as a toxin (as mentioned above), and it is
thought to play a major role in pathogenesis, the overall breakdown of CA by oxalate

oxidase may be detrimental to the pathogen, as well as beneﬁcial to the plant with the
generation of H202 possibly playing a role in defense signaling.

Within the context of this thesis, different mechanisms related to dollar spot
reduction as a result of lightweight rolling were investigated. One hypothesis not
discussed in these chapters centered on the idea that rolling simulates a wounding effect
in turfgrass plants, which subsequently induces an enzymatic response. Two enzymes of
interest (oxalate oxidase, and peroxidase) were assayed for in grass tissues from various

rolling treatments (data not shown). Unsuccessful attempts investigating plant oxalate

84

oxidase activity led to an assessment of pathgoenicity factors in the dollar spot pathogen
R. ﬂoccosum. The purpose of this study, therefore, was to examine in vitro oxalate
production by virulent and hypovirulent strains of the ﬁmgus and to clarify the effect of
hypovirulent agents on DA and enzyme production. The goal of this research was to
examine whether oxalic acid and extracellular secreted cell wall degrading enzymes are

important in the infection of creeping bentgrass (A grostis palustris) by R. ﬂoccosum.

MATERIALS AND METHODS
Fungal Isolates.

Isolates of R. ﬂoccosum used in this study are summarized in Table 3.01. All
isolates were grown on potato dextrose agar (PDA) (Becton Dickinson, Sparks, MD), and
subcultured onto fresh PDA plates using colonized agar plugs (5 mm diam.) 7-10 days
prior to each test. The hypovirulent strain ShIZB, was obtained from Dr. Greg Boland at
the University of Guelph, ON, Canada. The presence of dsRNA and hypovirulent

characteristics in strain ShIZB is discussed in Zhou and Boland (1996).

Table 3.01. Rutstroemiafloccosum isolates studied and their source of obtainment.

 

Isolate Source

 

Vegetative compatibility strain B (VCG-B) Dr. Joseph M. Vargas Jr., Michigan State
University. East Lansing, MI

 

Vegetative compatibility strain C (V CG-C) Dr. Joseph M. Vargas Jr., Michigan State
University. East Lansing, MI

 

 

 

Common Strain (CS) Dr. Joseph M. Vargas Jr., Michigan State

University. East Lansing, MI
Hypovirulent Sclerotinia homoeocarpa Dr. Greg J. Boland, University of Guelph.
(Sh12B) Guelph, Ontario

 

 

 

85

Isolate Virulence Testing

Virulence for each isolate was tested under greenhouse conditions by inoculating
9 cm diameter cups of 10 wk old creeping bentgrass (Agrostis palustris cv. Crenshaw)
with 6 mm colonized agar plugs of actively growing R. floccosum isolates. Each
virulence trial was replicated four times for each of the four isolates tested. Inoculated
plants were kept in enclosed chambers surrounded by plastic in a greenhouse maintained
at approximately 26-28 C with an approximate 13 hr photoperiod in order to encourage
humidity; cups were arranged in a completely randomized design.

Disease ratings were taken by measuring the advancing fungal mycelium and
subsequent infected area on each plant on a daily basis. Advancing mycelium radiated
from the center inoculation point, causing subsequent infection of turfgrass in a circular
pattern. Infected turfgrass was rated by measuring the area of bleached, water soaked
patches on the inoculated cups. Mean disease measurements were used for statistical
comparisons among dollar spot isolates.

Oxalic Acid production on Bromophenol Blue amended Potato Dextrose Agar.

In order to compare different strains of R. ﬁocossum and their ability to produce
acid when grown in vitro, a slightly revised method from Steadman et al. (1994) was
used. Fungal isolates growing on potato dextrose (PDA) agar for 10 days were
transferred to PDA amended with 0.05g/l bromophenol blue (BB) adjusted to pH 6.0 with
1M NaOH. Bromophenol blue is an indicator of acid production due to its rapid and
visible response indicated by a characteristic yellow color change when pH is below 3.
Approximately 20 ml of BB amended PDA (BBPDA) was aseptically pipetted into 100 x

15 mm polystyrene Petri-plates. One plug (5mm) of agar containing each isolate was

86

inoculated onto the center of a BBPDA plate and placed under ﬂuorescent light
(photoperiod of 12 h) at room temperature (20-22 C), and observed for oxalic acid
production for 14 days. Each strain grown on BBPDA was replicated 4 times and the
experiment was repeated twice. Measurement of fungal radial growth was taken daily,
along with color change measurements taken visually against a 5000K ﬂuorescent light
Porta-Trace (Gagne Inc.). Color change ratings were given on a scale of 0-5 with 0
indicating no color change and 5 indicating a complete yellowing of the plate. Mean
color change and fungal radial growth measurements were used for statistical
comparisons.

Growth and acid production in potato dextrose broth medium

Mycelial growth and production of oxalic acid were evaluated by culturing
individual isolates in potato-dextrose broth (PDB). One 5 mm diameter agar plug from
actively growing margins of 7-day-old colonies was transferred to 50 ml liquid medium
in 125-ml Erlenmeyer ﬂasks. Inoculated ﬂasks were incubated on a rotary shaker at 100
rpm at 20-22 C.

Mycelial dry weights were determined by vacuum ﬁltration of culture contents
through a layer of pre-weighed Whatrnan No.1 ﬁlter paper in order to separate mycelium
from culture ﬁltrates. Dry weight of mycelium was determined after ﬁlter paper with
mycelium was dried in a 80 C oven for 72 h.

Approximately 3 m1 of culture ﬁltrate from each treatment was collected and
measured for pH on ﬁve different sampling days, collected samples were maintained at -
20 C until analysis. Concentrations of oxalic acid in PDB ﬁltrates were determined after

15 days of grth in PDB. Extracts were analyzed on a Waters XBridge C18 3.5 um

87

 

 

column (Waters Corp., Milford, MA) high pressure liquid chromatograph (HPLC)/ mass
selective detector (MSD) for oxalic acid. The column diameter and length were 3/0x50 mm,
and the system was run at ambient temperature. The mobile phase was run at 0.25 ml/min
which started at 80 % 0.1 % formic acid (A) and 20 % 0.1 % formic acid in acetonitrile
(B) and was held there for one minute. A gradient began moving to 10 % A at four
minutes. This was held for 5 minutes and then back to original ﬂow. The MSD was
monitoring ions 46 and 45 daltons in electrospray positive mode. 10p] from each sample
was used in analysis, and values are reported as pg oxalic acid ml sample".

Growth and production of extracellular enzymes on isolated creeping bentgrass cell
walls.~

In order to investigate the fungal secretion of cell wall degrading enzymes by R.
floccosum, cell walls from creeping bentgrass (Agrostis palustris cv. ‘Crenshaw’) were
isolated and used as the sole carbon source in a minimal growing medium for R.
ﬂoccosum isolate inoculation.

Cell walls were prepared according to English et al., (1971) by grinding the
frozen tissue to a ﬁne powder in liquid nitrogen with the aid of a mortar and pestle. This
frozen powder was then ground in approximately 2.5 volumes (v/w) of cold 100 mM
postassiurn phosphate buffer, pH 7.0. The insoluble material was collected on Whatman
No. 1 ﬁlter paper by suction ﬁltration. The residue was resuspended in 1 volume of cold
buffer, before re-ﬁltration. This washing procedure was repeated four times, using 1
volume of buffer each time. Buffer washes were followed by one wash with 1 volume of
cold distilled water to remove salts. The residue was then suspended in 2.5 volumes of a

cold mixture of chloroform and methanol (1:1 v/v) and ground with a mortar and pestle.

88

 

The insoluble material was collected on Whatman No. 1 ﬁlter paper, washed three times
with 1 volume of the chloroform-methanol mixture at room temperature, then washed
three times with 1 volume of acetone at room temperature. The residue remaining after
the acetone extraction, which constitutes the cell walls used in this study, was air dried
and stored at room temperature. Immediately preceding use, the cell walls were placed in
a dessicator to remove residual water.

The medium for liquid culture was prepared according to a modiﬁed protocol

from Anderson (1978). In one liter the medium contained 0.25g K2HPO4, 0.25 g

NH4NO3, 0.1 g MgSO4'7H20, 0.15 ml 2% FeCl. In 125 ml Erlenmeyer ﬂasks 50 ml of

medium was added along with 0.5 g of cell wall material, ﬂasks were then autoclaved at
121 C for 20 min.

Cell wall amended media ﬂasks were inoculated with one colonized agar plug (5
mm diam.) ﬁ'om 7-10 day old isolates growing on PDA. Inoculated ﬂasks were incubated
on a rotary shaker at 100 rpm at 20-22 C for 14 days. Filtrates were obtained from the
liquid cultures by passage of the suspensions through Whatman No. 1 ﬁlter paper via
suction ﬁltration. Mycelia was weighed after drying at 80 C for 72 h. Isolates were
replicated four times, and liquid ﬁltrates were stored at -20 C until enzyme assays were
performed.

Enzyme assays were performed on culture ﬁltrates for cellulase and pectin
methylesterase activities following modiﬁed procedures described in Downie et al.
(1998) Hagerrnan et al. (1985) and Taylor and Secor (1988). Pectin-containing medium
was prepared by mixing 0.1% (w/v) pectin, and 1% (w/v) Type II agarose (Sigma

Chemical Co., St. Louis, MO) in 0.2 M phosphate buffer adjusted to pH 5.3. The mixture

89

 

 

was dissolved by heating to a boil while stirring. 18 m1 of the hot mixture was aseptically
dispensed into 100 X ISO-mm sterile petri dishes using a sterile 25 ml pipette.

Carboxymethylcellulose (CMC) containing medium was prepared using the same
procedure but substituting 0.1% CMC for the pectin. Once agar plates cooled, a cork
borer was used to punch 3 holes, 5 mm in diameter and approximately 1.7 cm apart, in
the solidiﬁed medium. The holes were arranged in three rows, in a 3 x 3 x 3 pattern and
the wells were ﬁlled with (35 ul) with standard, control, or ﬁltrate solutions. The assay
was incubated at 28 C for 17 hours.

Pectin containing gels were developed after incubation by ﬂooding the assay plate
with 10 ml of 0.05% (w/v) ruthenium red (Sigma Chemical Co.) in water for 30 min at
25 C. Excess dye was removed by washing the plate several dimes with deionized water.
The diameters of the resulting rings or halos of activity were measured against a 5000K
ﬂuorescent light Porta-Trace (Gagne Inc., Johnson City, NY).

The cellulose containing plates were developed in a similar fashion using 1%
(w/v) Congo red (Sigma Chemical Co.) in water to stain the plates. The stain was
removed after 15 min and plates were de-stained for 15 min with 1 M NaCl in 0.2 M
phosphate buffer. Enzyme activity was assessed by measuring ring diameter similar to
the pectin containing plates, and values were standardized by dividing halo diameter by
mycelia dry weight from each isolate. Assays for each isolate were replicated three times,
and experiments were repeated twice. Mean ring diameters were used for statistical

comparisons among isolates using the PROC GLM procedure in SAS.

RESULTS AND DISCUSSION

90

Virulence.

Isolates VCG-B, VCG-C, and CS initiated dollar spot symptoms on cups of
creeping bentrgrass ranging from 7.10 to 8.00 cm in diameter after 16 days of incubation
(Table 3.02). Some differences existed among these isolates with VCG-C displaying
signiﬁcantly higher virulence ratings than the other isolates at 6, 8, 11, 13, and 16 days
after inoculation (DAI). Isolate Sh12B failed to cause disease symptoms, and resulted in
no signiﬁcant difference from the control at any DAI. These results conﬁrmed the If
hypovirulence phenotype of the strain Sh12B. Mean virulence measurements resulted in

no statistical differences among isolates VCG-B, VCG-C, and CS. However, all three of

 

the aforementioned isolates resulted in statistically higher virulence ratings (P < 0.05) p

than the control and isolate Sh12B; neither isolate resulted in any disease observation.

Table 3.02. Disease ratings of R. ﬂoccosum isolates on swards of creeping bentgrass
(Agrostis palustris cv. Crenshaw)*.

 

 

 

Days After Inoculation
Disease Rating (cm)

IsoLrte 5T 6 8 11 13 16 Mean
VCG-B 1.07 a 1.17 b 2.90 b 4.03 b 5.02 b - 7.10 b 3.55 a
VCG-C 1.27 a 1.43 a 4.70 a 5.37 a 6.37 a 8.00 a 4.52 a
CS 1.07 a 1.17 b 3.40 b 4.17 b 4.70 b 7.43 b 3.67 a
ShIZB 0.00 b 0.00 c 0.00 c 0.00 c 0.00 c 0.00 c 0.00 b
Control 0.00 b 0.00 c 0.00 c 0.00 c 0.00 c 0.00 c 0.00 b

 

* Means are average disease ratings from four replications.
‘1' Means followed by different letters are signiﬁcantly different according to Fisher’s LSD (P < 0.05).
Oxalic acid production on bromophenol blue amended PDA.

Acid production in vitro was measured by observing the characteristic color

change of PDA amended with the acid indicator dye bromophenol blue. All isolates

91

displayed some degree of acid production, with evident color changing of the media from
purple to yellow. Figure 3.01 reports mean color change ratings for the four dollar spot
isolates tested after 15 days of growth on BBPDA. Isolate VCG-B resulted in
signiﬁcantly lower color change ratings after 15 days of growth compared to all other

isolates tested (P < 0.05).

t
Figure 3.01. Mean media color change ratingsf from acid production of four R.

ﬂoccosum isolates grown on bromophenol blue-amended potato dextrose agar for 15
days.

5
G?
é4
'00
~83
a
8.
g2
.12:
O
.531
O
U
0

 

VCG-B VCG-C

Isolate

"‘ Values are means of 8 replications ﬁ'om two separate assays.

1' Color change ratings were given on a scale from 0-5 with 0 indicating no color change, and 5
indicating a uniform change of color in the media ﬁ'om purple to yellow.

1 Means followed by different letters indicate a signiﬁcant difference according to Fisher’s LSD (P <
0.05). Error bars represent the standard error (SE) of the mean

92

 

Growth and acid production in PDB

Oxalic acid production was measured in vitro by growing dollar spot isolates in
potato dextrose broth. Culture pH was measured throughout the duration of the
experiment every three days. Table 3.03 gives mean culture pH measurements. All
isolates displayed similar culture pH after 3 days of growth, however after 6 days, VCG-
B had signiﬁcantly lower pH than both the control and isolate Sh12B. After 15 days of
growth, both VCG-B and VCG-C cultures were signiﬁcantly lower in pH than the

control; while VCG-C was signiﬁcantly lower than isolate Sh12B as well (P < 0.05).

*
Table 3.03. Mean culture pH measurements of four R. ﬂoccosum isolates growing in
potato dextrose broth for 15 days.

 

 

 

 

Days After Inoculation
Culture pH
Isolate 3 6 T 9 12 15
VCG-B 6.22a 6.02 c 5.64 c 5.29 b 5.07 bc
VCG-C 6.21 a 6.03 bc 5.67bc 5.17 b 4.92 c
CS 6.22 a 6.09 abc 5.81 be 5.69 ab 5.76 abc
Sh12B 6.17a 6.10 ab 5.93 ab 6.11 a 5.97 ab
Control 6.20a 6.15 a 6.16 a 6.16 a 6.20 a

 

"' Each pH measurement represents the mean of four replications.
T Means followed by different letters indicate a signiﬁcant difference in pH according to Fisher’s LSD (P
< 0.05).

Oxalic acid levels were measured in culture ﬁltrates after 15 days of growth. All
isolates displayed some level of OA production in liquid broth media. VCG-B displayed
the highest levels followed by the other virulent isolates VCG-C and CS (Figure 3.02).

The hypovirulent isolate Sh12B displayed variable levels of oxalic acid production

93

 

among replications, yet was able to produce CA at levels signiﬁcantly lower than the
other isolates (Figure 3.02). When OA production was standardized to the dry weight of
fungal mycelia in each ﬂask, hypovirulent isolate ShIZB produced signiﬁcantly higher
levels of OA than VCG-B (ﬁgure 3.03). While not statistically signiﬁcant, ShIZB had
higher OA production relative to mycelial grth than isolates VCG-C and CS as well

(ﬁgure 3.03).

Figure 3.02. Oxalic acid production* of R. floccosum isolates grown in potato dextrose
broth for 15 days.

 

 

 

 

50.0 — t
45.0 ~‘ 41:9 a
400 ‘1 :::: 22::
33:-5;; 28.8 b
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3 3.3.3.5.} 145° ICS
g 15.0 1 §:§:§:§:§ lControl
g“ 10.0 — 5:323:33
5.0 ~ zzszszszs 0" d
0.0 J .....

 

 

 

 

 

 

Isolate

* Means represent the average of 4 replications
'1' Means followed by different letters are signiﬁcantly different according to Fisher’s LSD (P < 0.05).
Error bars represent the standard error (SE) of the mean.

94

Figure 3.03. Oxalic acid production related to mycelial dry weight* of R. ﬂoccosum
isolates grown in potato dextrose broth for 15 days.

mg oxalic acid mycelia dry wt.

450
40*

35 "
30 ‘r
25 ___..-..
20 ‘m

15‘

10 ‘“'

 

 
   
 

El VCG-B
‘22? VCG-C
-— -~— cs
i 2,-_ 8 ShIZB
I Control

17,46 ab 15.60 ab

ooooo

Isolate

* Means represent the average of 4 replications
1' Means followed by different letters are signiﬁcantly different according to Fisher’s LSD (P < 0.05).
Error bars represent the standard error (SE) of the mean.

Growth and production of extracellular enzymes on isolated creeping bentgrass cell

walls.

Isolates were assayed for their ability to produce pectolytic and cellulolytic

enzymes in vitro and were compared. In vitro activities of pectin methylesterases (PME)

and cellulase from ﬁltrates of 14 day old developing hyphae growing on creeping

bentgrass cell walls are reported in ﬁgures 3.04 and 3.05. Levels of PME and cellulase

activity were adjusted with respect to dry weight of mycelia from cultures.

95

 

up
Figure 3.04. Pectin methylesterase activity of four R. ﬂoccosum isolates grown in liquid

culture with creeping bentgrass (Agrostis palustris) cell walls.

 

 

 

 

 

80‘2"" "“' " r ' r
E’
78 ‘*
Z‘
'6
g 76 ‘*' ** **
‘53
O: _A 74 7
v '2
e __-
7' a 72
5 a
Q 70 ‘"
>
’5
g 68 ""“
E”
m 66 r
64 _ .

Isolate

* Means are the average of 12 measurements from 4 replications.

 

EJ VCG-B
VCG-C
CS

8 Sh12B

1' Means followed by different letters are signiﬁcantly different according to Fisher’s LSD (P < 0.05).

Error bars represent the standard error (SE) of the mean.

When PME levels were adjusted with respect to mycelia dry weight, isolate

Sh12B exhibited signiﬁcantly greater activity than the other three isolates (P < 0.05). For

cellulase activity, adjusted means showed Sh12B to have signiﬁcantly higher enzyme

activity than all other strains with respect to mycelium production (P < 0.05), while

VCG-C had the lowest cellulase activity among all isolates.

96

:3:
Figure 3.05. Cellulase activity of four R. ﬂoccosum isolates grown in liquid culture with
creeping bentgrass (Agrostis palustris) cell walls.

 

 

140—2
3
r: 120»
a
a
:5; 100—
3E
.2 3 80“
‘5»?
IE; 60*"
a
$5 402-
.2
g 20—
0
U

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118.42 a

  

..... El VCG-B
323:3:313 VCG-C

5:13:52? , cs
In ShIZB

Isolate

* Means are the average of 12 measurements from 4 replications.
‘1' Means followed by different letters are signiﬁcantly different according to Fisher’s LSD (P < 0.05).
Error bars represent the standard error (SE) of the mean.

CONCLUSIONS

In order to investigate whether oxalic acid plays a crucial role in pathogen

virulence, four isolates of R. ﬂoccosum were subjected to multiple assays related to OA

production and extracellular enzyme activity. The isolate Sh12B, a known hypovirulent

strain of R. ﬂoccosum, was evaluated and compared to three other known virulent

isolates.

97

 

Virulence testing conﬁrmed prior ﬁndings by Zhou and Boland (1997) that isolate
Sh12B is indeed unable to maintain virulence on swards of creeping bentgrass. All other
isolates displayed typical dollar spot disease symptoms on inoculated creeping bentgrass
with bleached, circular spots, growing outward from the inoculation point, containing
evident ﬂuffy mycelium near the infected margins. Other studies involving
hypovirulence and the production of 0A have shown hypovirulent ﬁmgal strains
exhibiting reduced or delayed accumulation of 0A. They hypovirulence phenotype in
isolate ShIZB of R. ﬂoccosum was previously associated with a reduced growth rate,
atypical colony morphology, and reduced virulence, as well as the presence of double
stranded RNA.

If pathogenesis is associated with the production of OA and/or extracellular
enzymes as proposed by many authors in the past (Hancock, 1967; Lumsden, 1969, 1976,
1979; Hancock, 1967; Marciano et al., 1983; Noyes and Hancock, 1981), one could
hypothesize that the hypovirulent isolate ShIZB should be deﬁcient in both acid and cell
wall degrading enzyme production.

Contrary to the initial hypothesis, isolate ShIZB displayed marked acid
production when grown on BBPDA plates, displaying a high degree of color change in
the indicator medium. Cultures of ShIZB tended to grow at about half the rate of the
other virulent isolates, however, after 15 days of growth, the hypovirulent isolate Sh12B
exhibited a higher degree of color change compared to VCG-B, and similar color change
ratings to isolates VCG-C and CS. This initial examination of acid production in vitro
indicated a possible delayed, yet sufﬁcient production of acid by the hypovirulent strain

when compared to the other R. ﬂoccosum isolates. The virulent isolate VCG-B displayed

98

signiﬁcantly less color change ratings in the medium, indicating that overall acid
production in vitro may not be directly associated with virulence on creeping bentgrass.

In order to conﬁrm the production of 0A, and make comparisons among fungal
isolates culture ﬁltrates 0A levels were measured via HPLC/MS. The results conﬁrmed
the increased production of CA by virulent isolates compared to the hypovirulent
counterpart. These results coincide with Callahan and Rowe (1991) and Zhou and Boland
(1999) in that reduced or delayed accumulation of oxalic acid in hypovirulent isolates
appears to be associated with reduced disease severity. However, the production of OA
way relative to mycelial production was signiﬁcantly higher in the hypovirulent isolate,
indicating marked acid production even when fungal growth is at a minium. With these
results, oxalic acid does not appear to be a sole pathogenic determinant in R. ﬂoccosum.
In vivo studies should be done as well as the investigation into oxalic acid deﬁcient
mutants in R. ﬂoccosum to truly identify the importance of 0A in pathogenesis.

Isolate Sh12B produced degradative enzymes (PME and cellulase) at levels equal
to, or higher than the virulent strains evaluated. These hydrolytic enzymes have been
associated with infection of tissue by Sclerotinia spp. (Hancock, 1966; Lumsden, 1979).
However, these results coincide with Godoy et al. (1990), who found that non-pathogenic
mutants of Sclerotinia sclerotiorum produced equal or greater amounts of pectolytic and
cellulolytic enzymes when compared to wild type isolates. This could be an indication
that factors other than pectolytic or cellulolytic enzymes are factors in the virulence of R.
ﬂoccosum. In vivo studies involving extracellular cell wall degrading enzymes should be
conducted in order to draw further conclusions regarding their importance in R.

ﬂoccosum virulence and pathogenesis. This initial study set out to investigate differences

99

among virulent and non-virulent (hypovirulent) strains, and whether oxalic acid and/or
degradative hydrolytic enzymes were essential in host/pathogen interactions. Isolate
Sh12B may retain mechanisms necessary for the production of these enzymes for
saprophytic survival purposes. If the isolate is to survive in nature by the acquisition of
nutrients via other organisms, these degradative enzymes most likely are needed in the
breakdown of detritus.

Oxalic acid is thought to play a major role in the pathogenesis of many plant
pathogens, but the importance of 0A production by the dollar spot pathogen R. floccosum
has yet to be deﬁned. Investigations into the importance of 0A in pathogen virulence and
infection could potentially lead to novel methods of disease control or reduction. The
previously mentioned breakdown of OA via plant derived oxalate oxidase has been
characterized as an option in transgenic plant production (Thompson et al. 1995; Liang et
al., 2001; Hu et al., 2003). This same enzyme has been shown to be induced in certain
grass species after wounding, and when subjected to heavy metal and temperature stress
(Valentovicova et al., 2009; Le Deunff et al., 2004).

Prior chapters in this thesis pertained to the reduction of dollar spot disease
through lightweight rolling of creeping bentgrass. A proposed means in disease
suppression could be related to oxalate oxidase activity increasing in response to the
rolling treatment. Initial research investigating this hypothesis proved sporadic and
inconclusive (Appendix). However, without a basic knowledge of the role that oxalic
acid plays in R. ﬂoccosum pathogenesis, it is difﬁcult to make presmnptions on whether
0A degradation by oxalate oxidase could be having part in dollar spot disease

suppression. The hypovirulent strain ShIZB which was unable to produce disease

100

 

 

symptoms on swards of creeping bentgrass, still exhibited marked acid and cell wall
degrading enzyme production in vitro. These ﬁndings suggest that while oxalic acid and
extracellular enzyme production may be contributing factors in R. floccosum
pathogenesis, alone or in tandem, their production does not necessarily translate to
disease development. It is likely that oxalic acid plays a diminutive role in the
pathogenicity and disease development of R. ﬂoccosum, however, in vivo studies on the

production of 0A and potential cell wall degrading enzymes should be done.

101

LITERATURE CITED

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103

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104

APPENDIX

 

from different

eping bentgrass tissue

 

 

A

Figure A.1. Oxalate oxidase speciﬁc activity* in cre

rolling treatments.

we we 28% £28 case one? 0335

throughout the growing

'1' Means followed by the same letter are statistically similar according to Fischer’s LSD. Bars

‘Means represent the average of 6 different assays performed on tissue
represent the standard error of the mean.

seasons of 2008 and 2009

105

 

 

from different

eping bentgrass tissue

 

\\\\\\\\\\\\\\\\

m\\\\\\\\

 

1x a.m. 1x p. m. 2x a.m.

Control

m... m m. m m. m m 0
Wm. p.m.: 35381338 case 032:2

Rolling treatment

sue throughout the growing

* Means represent the average of 4 different assays performed on tis

seasons of 2008 and 2009

‘1' Means followed by the same letter are statistically similar according to Fischer’s LSD. Bars

represent the standard error of the mean.

106