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FIELD EFFICACY, PERSISTENCE, AND
ANTIBIOTIC PRODUCTION OF PSEUDOMONAS AUREOFACIENS

presented by

Philip Joseoh Dwyer, Jr.

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Botany 8: Plant Path.

 

 

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Date 5 May 1999

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FIELD EFFICACY, PERSISTENCE, AND ANTIBIOTIC PRODUCTION OF
PSEUDOMONAS A UREOFA CIENS

By

Philip Joseph Dwyer Jr.

A DISSERTATION

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

MASTER OF SCIENCE
Department of Botany and Plant Pathology

I999

ABSTRACT

FIELD EFFICACY, PERSISTENCE, AND ANTIBIOTIC PRODUCTION OF
PSEUDOMONAS A UREOFA CIENS

By

Philip Joseph Dwyer Jr.

Pseudomonas aureofaciens strain Tx-l is used as an effective biological control
bacterium for control of certain turfgrass diseases. The field efficacy and survivability of
Tx-l upon application to turf was studied. A bioactive mutant (Tn-IA) deficient in
production of the primary antibiotic, phenazine-l- carboxylic acid (PCA), was used to
determine if antibiotics other than PCA could be identified and correlated with control. In
separate studies, Tx-l was applied to turf at various concentrations and in combination
with fungicides to control the diseases dollar spot (Sclerotinia homoeocarpa) and pink
snow mold (Aficrodochium m‘vale). Significant control of both dollar spot and pink snow
mold was achieved by applying Tx-l at specific concentrations and intervals.
Survivability of Tx-I in turf was determined by monitoring populations over a two-year
period. Results showed Tx-l can overwinter and survive throughout the year in the
foliage, thatch, and soil of a turfgrass system. Strain Tn-IA was used in in vitro bioassays
and in the field to study its ability to control dollar spot. Tn-lA effectively controlled
dollar spot in both bioassays and in the field. The active compound from Tn-lA
fermentation broth was extracted, purified using various chromatographic techniques, and
identified using nuclear magnetic resonance (N MR) and mass spectral (MS) experiments.
The structural analysis of the active compound revealed the molecule to be C|0H6N202C12

(Pyrrolnitrin).

Copyright by
Philip Joseph Dwyer Jr.
1999

I dedicate this thesis to the lives of Bradley, Nicole, and Kevin Mohammad, may their
spirits live on by our actions today. God bless them, their families, and friends.

ACKNOWLEDGMENTS

I would like to express my gratitude and thanks to the many people who have
made my studies possible: to Dr. Joe Vargas, for his support and encouragement of my
research and learning, to Dr. Jim Crum who has been a helpful adviser since my time as
an undergraduate, and to Dr. Sheng Yang He for his input in my projects and for teaching
me about prokaryotes.

Special thanks to Drs. Muralee Nair and Russel Ramsewak for their help and
guidance in identifying the active compound pyrrolnitrin. Thanks also. to John
Ravenscroft for all ofhis help with the Tx-l bacterial persistence study. Thank you to
everyone in the Vargas lab who have helped me in research and enjoyment ofour day to
day experiences; Brandon, Ron, Nancy, Dave, and Jon for his previous work on Tx-l.

Thank you to my family (Mom. Dad, and Rosie) for all oftheir encouragement,
food, and love during my time here. Each ofthem have been so very helpful in providing
me with the tools to succeed in this and everything I have done.

Lastly, to two very special men who have inspired youth and in doing so have
succeeded in enhancing our world, thank you to Mr. Scott Puryis and Mr. Bruce Rae.

Congratulations on your retirements from Okemos High School.

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

INTRODUCTION

CHAPTER I LITERATURE REVIEW

Pseudomonas aureofaciens
Dollar Spot
Pink snow mold

CHAPTER II BIOLOGICAL CONTROL OF DOLLAR SPOT
FIELD STUDY

Introduction

Materials and Methods
Results

Discussion

CHAPTER III PERSISTENCE OF TX-I APPLIED TO TURF

Introduction

Materials and Methods
Results

Discussion

CHAPTER IV BIOLOGICAL CONTROL OF PINK SNOW MOLD

Introduction
Materials and Methods
Results and Discussion

CHAPTER V ANTOBIOTIC PRODUCTION OF PSEUDOMONAS
AUREOFACIENS

Introduction

Materials and Methods
Results

Discussion

CHAPTER VI EPILOGUE

vi

Page
viii

ix

1)
l7
I9
27

‘5

.31
32
34
38

41

41
42
42

45
46
49
52

APPENDICES

LITERATURE CITED

vii

58

61

LIST OF TABLES

Table
CHAPTER IV
Table 1. List of treatments and comparison of means as a
percent area of infection by pink snow mold.
CHAPTER V
Table 2. Plate bioassay, comparing fungal inhibition of
Tx-l to Tn-IA.
APPENDIX A
Table 3. 1997 treatment means from dollar spot study.
Table 4. 1998 treatment means from dollar spot study.

viii

Page

Figures
CHAPTER II
Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

CHAPTER III
Figure 7.
Figure 8.

Figure 9.

CHAPTER V
Figure 10.

Figure I I.

LIST OF FIGURES

Comparison of Tx-l treatments to the control
in 1997.

Comparison of Tx-I treatments to combinations
using Banner in 1997.

Comparison of Tx-I treatments grown for
different times in 1997.

Comparison of low concentrations of Tx-I
and Banner combinations in 1998.

Comparison of low concentrations of Tx-l
and the control in I998.

Comparison ofofo-I and Banner at high
concentrations in 1998.

Survival ofo-I in the foliage

Survival ofo-l in the foliage

Survival ofo-l in the foliage

Comparison ofo-l to Tn-IA in 1997

Comparison ofo-I to Tn-IA in 1998

Page

20

21

Ix)
IQ

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50

SI

INTRODUCTION

Biological control can be defined as using the disease suppressive preperties of an
organism to improve plant health. One mechanism of biological control is antibiosis in
which an organism produces antibiotics as their means of suppressing disease (15).
Pseudomonas aureofaciens is one such organism whose control mechanism is antibiosis.
Biological control occurs during a complex interaction between the plant, pathogen,
biological control organism, surrounding microbial community, and environment (15). It
is the understanding of this complex interaction in the rhizosphere, signal interchange,
and antibiotic expression which have led to P. aureofaciens being one of the best
understood biological control organisms (33). As the understanding of P. aureofacicns
increases in specific agricultural systems, the use of this organism for control will be
possible, as it has been in turfgrass disease management. The focus of this thesis research
is to increase our understanding of P. aureofaciens strain Tx-l in order to optimize the
effectiveness of its use as a tool in managing disease in turfgrass systems.

In recent times, commonly used disease management strategies involving
fungicides have come under question by both regulatory issues and public perception.
This has led to the elimination of certain fungicides and the possibility of losing others.
Another threat to current disease management techniques that is related specifically to the
turf grass pathogen dollar spot (Sclerotinia homoeocarpa) is the occurrence of fungicide
resistance (44). Of the systemic fungicides used to control dollar spot, resistance to the

benzimidazoles, dicarboxymides, and demethylation inhibitor (DMI) classes has been

reported (45)(9)(12). The loss of these important classes, in some cases, and a proactive
response to environmental concerns by the turfgrass industry has led researchers to
examine alternative disease management strategies.

The introduction of P. aureofaciens strain Tx-l for use in managing dollar spot
has been a successful addition for management of dollar spot on golf courses. A
commercial fermentation and delivery system was developed specifically for application
of this organism through the pre-existing irrigation system on a golf course. Turfgrass is
ideally suited for this type of application because delivery of Tx-I through irrigation
water allows the organism to be applied to all desired areas on a routine basis.
Application of Tx-I to turf will improve as research continues to discover ways of
optimizing its use.

These studies were done to investigate responses in the field using Tx-l and
various treatment combinations, determine Tx-I ’s ability to persist in a turfgrass system,
and identify the effect of a mutant with altered antibiotic production capabilities. The
field studies were done over two years in which Tx-I was used to control naturally
occurring dollar spot on a fairway height stand of annual bluegrass (Poa annua). A Tx-I
population study was conducted in the field over two years to monitor the organism’s
ability to persist in a turfgrass system at various times of the year. The results of this will
show population changes overtime, and may also expand future research into root
diseases by demonstrating that threshold bacterial populations can be achieved. It is
important for sufficient populations of a biological control organism to exist at the site of
pathogen infection to control disease, and also to persist in the environment where

released (47). The experiments done with a mutant strain of Tx-l sought to identify

production of an antibiotic other than PCA and correlate this antibiotic to disease

suppression in both the field and in vitro.

Chapter 1

LITERATURE REVIEW

Pseudomonas aureofaciens

Kluver first described the bacterium Pseudomonas aureofaciens in 1956 (20). P.
aureofaciens was isolated from clay soils that had been soaked in kerosene (3). The
bacterium is a fiagellated root colonizer, aerobic, and a member of the fluorescent
Pseudomonads (3). The name “aureofaciens” was given to it due to its ability to produce
yellow and orange pigments that are “made golden” (3). Haynes characterized these
pigmented antibiotics produced by P. aureofaciens into the phenazine class of antibiotics
in 1956 (16). Phenazines are heterocyclic,-N-containing molecules produced by bacteria
as secondary metabolites.

Phenazine producing strains of P. aureofaciens are found worldwide and are
associated with various plants and soils (33). These antibiotics have biological activity
against fungi, bacteria, and nematodes (33). The shikimic acid pathway has been
described as the likely site of phenazine synthesis (47). Pierson also found the product of
one phenazine biosynthesis gene shares homology with products from the shikimic acid
pathway (31). The primary antibiotics shown to be responsible for fungal inhibition are
phenazine-I- carboxylic acid (PCA), 2~hydroxy-phenazine carboxylic acid (2OHPCA),
2-hydroxy phenazine (2P2), 2,4-diacetylphIoroglucinol (Phl), and pyrrolnitrin (Pm)(33).

Hydrogen cyanide and siderophores are also produced by P. aurcofaciens (33). Several

hypotheses have been proposed to describe the primary mode of action by phenazines in
inhibiting fugal growth. These include disruption of normal membrane functions such as
active transport, inhibition of RNA synthesis, DNA replication and transcription
processes, and the uncoupling of electron transport and energy production (32).

Two decades after the discovery of P. aureofaciens, Cook and Rovira reported,
that a similar fluorescent Pseudomonad, Pseudomonasfluorescens, was possibly the
cause of a naturally occurring biological control of a fungal disease in wheat (7). They
distinguished general and specific antagonism as two types of disease suppressive soils.
General antagonism was characterized as a type of suppressiveness present in all soils,
not affected by moist heat, able to survive fumigation, and not transferable. Specific
antagonism was defined as disease suppressiveness, which was eliminated by moist heat
or fumigation, and this property was transferable from soil to soil. In defining a clear
distinction between properties of these two soil types, Cook and Rovira were then able to
correlate the presence and role of P. fluorescens with suppression. Certain soils were
found to become suppressive to the pathogen Gaeumannomyces graminis var. tritici that
causes the disease take-all in wheat (7). The phenomenon of “take-all decline” (TAD)
was noted when, after several years of wheat monocropping and occurrence of take-all,
natural suppression of the disease occurred. In searching for a factor that caused these
suppressive soils to have a specific antagonism, it was discovered that fluorescent
pseudomonads were found in these soils. Over 100 isolates of soil microbes from
diseased and protected wheat roots were used in bioassays to suppress take-all. Eight of
these microbes were able to suppress disease and all were pseudomonads, seven of which

were fluorescent. Treatments of moist heat and fumigation eliminated the specific

antagonism of these soils. This research correlated the presence of specific
pseudomonads to suppression of take-all.

Work by Weller and Cook verified that certain pseudomonads were able to
provide biological control of plant pathogen’s (46). Thomashow and Weller
demonstrated that disease control of take-all was from production of phenazine
antibiotics produced by P. fluorescens (47). Mutants defective in phenazine production
(th ') were compared to wild type (th +) strains to determine the effect of phenazines at
inhibiting take-all on wheat roots. th ' mutants did not inhibit take-all in vitro and were
significantly less suppressive on wheat seedlings compared to the th + strains. Genomic
DNA from the parent strain restored antibiotic biosynthesis and fungal inhibition to the
previously th ’ strain.

The phenazines produced by P. fluorescens strain 2-79 and P. aureofaciens strain
30-84 were shown to be responsible for take-all control on wheat roots (43). Direct
evidence of the role of phenazines was lacking because they were not recovered from
natural soils where disease control occurred. Thomashow et al. coated wheat seeds with
th + strains 2-79, 30-84, th ' mutants of each, and untreated controls. In both growth
chamber and field studies, PCA was recovered only from roots coated with th + strains.
PCA was not detected on roots from th ' mutant coated seeds or from the control. Roots
from which PCA was recovered had significantly less disease compared to the th ' and
control treatments. In detecting phenazines from protected roots, the effectiveness of
PCA was demonstrated due to the small amounts found.

Phenazines were shown to be the principal factors in disease suppression by P.

fluorescens strain 2-79 and P. aureofaciens strain 30-84. It was also thought that these

compounds played a role in the competitive fitness of the organisms. Mazzola et al.
introduced strains 30-84, 2-79, and th ' mutants into natural and pasteurized soils before
growing wheat seedlings from them (24). The population sizes of th ' strains declined
more rapidly than the th + strains in natural soils. In pasteurized soils, both th ' and
th + population levels were similar. It was thought that the th ' strains could not
compete with the indigenous microflora in the natural soils while the loss of competition
in steamed soils allowed more th‘ bacteria to survive. Mazzola et al. concluded the
production of phenazines by Pseudomonas spp. is a selective advantage to their survival
in the rhizosphere.

Research on P. aureofaciens and P. fluorescens demonstrated the role and
importance of phenazines as the mechanism by which the bacteria suppress fungal
pathogens. Subsequent research focused on identifying the genetics involved in the
biosynthetic pathways and regulation of production.

P. aureofaciens strain 30—84 was identified as producing PCA, ZOHPCA, and
ZHZPCA (33). Mutants capable of only PCA production provided less suppression than
strains producing all three phenazines. Mutants deficient in all phenazine production but
maintained pyoverdin and HCN production were no different from the control.
Researchers determined that production of all three phenazines as the primary mechanism
of take-all suppression by strain 30-84, and the other two metabolites play no major role
in suppression. A phenazine biosynthetic locus was identified in strain 30-84 by Pierson
and Thomashow that restored both phenazine production and fungal inhibition to th ’
mutants (29). When this locus and a functional promoter were introduced into E. coli,

expression of all three phenazines occurred.

Pierson et al. identified a regulator gene (pth) that activates expression of
phenazine biosynthesis in P. aureofaciens 30-84 (3 O). The [3th gene was identified as a
requirement for phenazine production. Its product, thR, regulates phenazine production
by activating a pth gene involved in biosynthesis. Inactivation of pth resulted in the
complete loss of phenazine biosynthesis. The amino acid sequence of the thR protein
has homology with several other bacterial transcriptional regulators suggesting it is a
member of the LuxR/Luxl family of activators. These activators regulate gene expression
in response to cell density signals. In recognizing thR as a member of this two
component system, researchers sought to identify a second gene whose product would be
a signal in the Luxl family of N-acyl-L-homoserine lactone synthases (N-acyl-HSL).
Wood et al. discovered the phzl gene whose product was a diffusable signal and member
of the Luxl family of N-acyl-HSL (48). Inactivation of phzl resulted in the loss of both
signal and phenazine production. To determine if [2th produces a diffusable signal, cell-
free supematants from E. coli containing the phzl gene and a control were added to strain
30-842. Strain 30-84Z contains a promoterless LacZ gene fused to the phenazine
biosynthesis gene, phzb, and cannot induce its own phenazine production. Signal
production was shown when cell-free supematants from cultures of E. coli containing
th I activated phzb expression in strain 30-84Z. The control supernatant, which did not
produce th I, failed to activate expression. Wood and Pierson identified a 5.7 kb region
from strain 30-84 which produced phenazines in E. coli when inserted with a promoter
(48). The nucleotide sequence of this fragment contained the open reading frames
(ORF’s) of five genes involved in phenazine biosynthesis. These ORF’s encode enzymes

found in the shikimic acid, entcrochelin, and tryptophan biosynthetic pathways. Future

understanding of the specific roles of each gene will help identify how bacteria form
phenazines.

Handelsman identified a minimum of three components that must be present for a
biological control organism to suppress disease (14). Interactions between the host plant,
infecting pathogen, and the biological control organism must occur for this phenomenon
to be successful. Pierson and Pierson hypothesized a likely scenario of how P.
aureofaciens interacts with a wheat plant and the pathogen G. gramim's var. tritici to
result in the decline of take-all in the field (32). The plant root provides a niche in the
rhizosphere where populations of P. aureofaciens survive off of root exudates. The
pathogen G. gramim's var. tritici seeks to infect the roots in seek of food and, in doing 50,
causes disease and death to the plant. The penetration of the root by the pathogen causes
a flush of nutrients to leak and are used by the pathogen and bacteria. The bacterial
population quickly increases due to the excess nutrient availability and, in turn, results in
an increase and accumulation of th I signals. The signals interact with the th R
transcriptional activator proteins that then activate the phz antibiotic biosynthesis genes.
The phenazines are produced in greater amounts and diffuse to the surroundings. The
presence of the phenazines results in the suppression andvbiological control of the fungal
pathogen and protection of the wounded plant from further infection.

The importance of and interest in PCA as a control mechanism is apparent in that
it is the most studied of the many antibiotics produced by P. aureofaciens. The antibiotic
pyrrolnitrin (Prn) was also studied due to its antifungal activity. Pyrrolnitrin was first
isolated and identified by Arima et al. in 1964 (I). The mode of action of pyrrolnitrin is

not fully understood, although it is thought to interfere with membrane functions of other

organisms. The addition of DL-tryptophans to P. aureofaciens culture increased
pyrrolnitrin production.

Much of the research involving the biosynthesis and regulation of pyrrolnitrin has
been done on P. fluorescens. Pfender et al. identified a genomic region that when cloned,
restored antagonism and pyrrolnitrin production to a mutant previously deficient in
pyrrolnitrin production (28). Hill et al. isolated a strain of P. fluorescens and identified
pyrrolnitrin as its mechanism of antagonism against the pathogen Rhizoctonia solam’.
Mutants deficient in pyrrolnitrin production did not inhibit the pathogen. Parental DNA
was transferred to the Prn' strains and resulted in both pyrrolnitrin production and
antagonism. (17). The global regulatory mechanism used by P. aureaofaciens for
pyrrolnitrin production was identified as LemA/GacA. Corbell et al. discovered a gene
closly related to lemA (8). Hammer et al. identified a 6.2 Kb region containing a cluster
of four genes required for pyrrolnitrin biosynthesis. When these genes were cloned by

PCR, fused to a toe promoter, and transferred to E. coli, pyrrolnitrin production resulted

(14).

Dollar spot

Dollar spot is a disease of turfgrass found throughout the world (41). It is
considered one of the most important diseases of high maintenance turf such as the type
found on golf courses (44). The causal agent of dollar spot is the fungus Sclerotim’a
homoeocarpa F .T. Bennet. This name is currently under contention due to the inability of
this organism to produce sclerotia, and instead producing a flat stroma. Current work by

Powell will likely result in the changing of its name to Rutstroemiafloccosum

10

(unpublished). Cultures of this fungus are characterized by a mat of fast-growing, fluffy
white mycelium that turns gray, brown and other colors as it ages. After 2-4 weeks of
growth, planes of dark stroma can be seen within culture media (41).

W

The common name of dollar spot is derived from the symptoms it produces on
turf, which resemble a silver dollar (44). The disease appears on greens and low mown
turf as small, straw colored patches, less than 6 cm and commonly 1-3 cm in diameter. In
taller grass, such as home lawns, these patches may reach 15 cm across. As disease
severity increases, the individual spots often coalesce into irregularly shaped patches,
destroying large areas of turf. Lesions on individual leaf blades are distinguished by a
chlorotic to bleached water soaked band, bound by a tan or reddish-brown margin. The
shape of this lesion takes on an hourglass shape across the leaf blade. During periods of
leaf wetness from morning dew, the active mycelium are visible as a white, cottony or

cobwebby growth (2)(4 l )(44).

Dollar spot is known to infect many species of turfgrass (41). In the northem US.
it is most damaging to creeping bentgrass (Agrostis palustris), annual bluegrass (Poa
annua), colonial bentgrass (A grostis tennis), and fine-leaf fescues (F estuca )(44). Dollar
spot can occur on turf from the latter part of the spring through the end of the fall. Most
epidemics occur in July and again in late August through early September (41).
Conditions most favorable to dollar spot are temperatures of 15-30° C with warm humid
days preceded by cool nights. Cooler night temperatures are conducive to dew formation
as nutrient rich guttation fluid is exuded from the plant. Guttation fluid is an ideal food

source for the fungus. Dollar spot is more severe under conditions of low nitrogen

11

fertility, dry soils, and/or water stress. Dissemination of the fungus on turf is by
mechanical movement such as infected leaf clippings spread by mowers, foot traffic, and
water. The fungus can survive as mycelia and stromata on leaf tissue when conditions for

infection are unfavorable.

Managing dollar spot

Dollar spot management combines the use of cultural, chemical, and biological
control techniques. Methods of cultural control are effective at reducing the level of
diseased turf and can reduce chemical inputs. Adequate nitrogen fertility, when applied at
times of heavy disease pressure, reduces disease (44). Maintaining proper soil moisture to
avoid plant stress also contributes to disease control. One of the most important cultural
control techniques is to remove dew from the leaf blades as early in the morning as

possible. Doing so inhibits fungal mycelial growth and subsequent spread of the disease.

In many cases, the use of fungicides is necessary for control of dollar spot.
Contact fungicides, like chlorothalonil, are effective at controlling dollar spot for shorter
periods of time. Resistance to contact fungicides has not been reported and is unlikely to
develop due to multi-site mode of action of these fungicides. Effective systemic
fungicides include prepiconazole, fenarimol, iprodione, and vinclozolin. Systemic
fungicides are desired for their longer control interval when compared to contact
fungicides. One drawback to systemics is there is a greater likelihood of resistance
occurring amongst dollar spot populations. Resistance can be defined as reduced efficacy
and shortened control interval of a previously sensitive fungal population. Since 1972,

resistance has been reported to all three of the major systemic fungicide classes which

12

includes the benzimidazoles (45), dicarboximides (9), and demethylation inhibitors

(DMI’s)(12).

Pink Snow Mold

Sjmptoms/Epidemiology

Pink snow mold, caused by the fungal pathogen Microdochium nivale, is a severe
disease in areas of prolonged cool (0-8 °C) and wet conditions (41). It differs from other
snow molds in that it does not require snow cover for infection to occur (41). This disease
is most severe in the Pacific Northwest and is present in most of the northern areas of the
United States. It occurs from early fall to late spring, and can exist year round if

conditions favor its development.

Pink snow mold spreads from leaf to leaf during snow and thawing cycles, and in
light rain. Foot traffic and equipment can spread conidia and infected tissue. Symptoms
appear as small, water-soaked, circular patches (5cm or less), and these change to a light
gray as the mycelium develops. Sunlight exposure induces sporulation and also changes
the mycelium to its characteristic pink color. As temperatures increase and drier
conditions occur, the pathogen becomes inactive and survives in the grass plants and dead
debris. Species most susceptible to infection by M. nivale include Poa annua and

A grostis spp.

Management techniques

 

Reducing fall nitrogen applications can help decrease the severity of this disease.
Excess nitrogen causes the grass plants to be succulent and more prone to infection. Poor
drainage, matted turf, and pockets of humidity are factors, which favor development of
this disease (41). Many fungicides exist for managing pink snow mold. Contact
fungicides are effective in areas without prolonged snow cover. In regions likely to
experience extended snow cover, the use of systemic fungicides provides longer control
intervals (44). Recent field trials have shown combinations of different fungicides to be

the most successful in controlling pink snow mold.

14

Chapter 2

BIOLOGICAL CONTROL OF DOLLAR SPOT FIELD STUDY

Introduction

The turfgrass disease dollar spot, caused by Sclerotim'a homoeocarpa, is
considered one of the most important foliar pathogens of low cut turf on golf courses
(44). The importance of this disease can be demonstrated by the fact that more money is
spent in managing dollar spot than any other disease (44). The most common means of
managing dollar spot has been through the use of cultural and chemical methods such as
proper irrigation, adequate nitrogen fertilization, and application of fungicides. The use of
biological controls, such as organic composted topdressings and antagonistic fungi, has
been reported as means of controlling dollar spot (13)(22). Perhaps the most successful
commercially available system for the biological control of dollar spot is the application
of the soil bacterium Pseudomonas aureofaciens strain Tx- 1. Other strains and species of
fluorescent pseudomonads have been correlated with disease suppression and biological
control (41). Sarniguet and Lucas correlated a higher population of antagonistic
fluorescent pseudomonads to a zone of disease remission in turfgrass infected by take all
patch, Gaeumannomyces gramim's var. avenae (40).

The purpose of this study was to further our understanding of Tx-l ’s ability to
control dollar spot in the field. The results will be used to optimize the current system and
be able to improve this technology to control the disease in different conditions. Showing
the effectiveness of Tx-l may encourage the acceptance of using Tx-l alone or as a

supplement to controlling dollar spot by turfgrass managers.

15

Previous field studies using Tx-l to control dollar spot were done on greens
height (5/15”) creeping bentgrass (A grostis palustris). The turf chosen for this study was
fairway height (‘lz”) annual bluegrass Poa annua). Fairway height annual bluegrass was
chosen for two reasons. Annual bluegrass is a species of low mown turfgrass comprises
large areas of low cut turf on some golf courses. The fairway height of cut accounts for
the largest area of intensively mown turf on a golf course. Fairways are valued differently
than greens and, in some situations, it may not be economically feasible to manage
disease on fairways to the high standard of greens. By determining the effectiveness of
using Tx-I on fairway height annual bluegrass, the application of Tx-l may make it
practical to manage dollar spot in these areas. Another objective of this study was to
determine if using fewer applications or lower concentrations of a fungicide in
conjunction with Tx-l could prolong the effective interval of the fungicide and allow for
reduced fungicide use. Such a reduction in fungicide use would be substantial due to the
large area fairways comprise.

A field study was done in August and September of I997 and 1998 using various
Tx-I concentrations, intervals, and combinations with fungicides to answer the above

questions and others related to the use of Tx-l on fairway height annual bluegrass.

16

Materials and Methods

Field Conditions

A field study was done in 1997 and 1998 to test the effectiveness of Pseudomonas
aureofaciens strain Tx-l in controlling dollar spot (Sclerotinia homoeocarpa) on annual
bluegrass (Poa annua). This study was done at Michigan State University’s Hancock
Turfgrass Research Center in East Lansing, Michigan. The stand of annual bluegrass was
maintained by mowing three times per week at '/2 of an inch. Irrigation was applied daily
at varied rates to maintain healthy turf. Fertility was applied at V2 lb. of N/ 1000 ftZ/month
during the study. The statistical error control design was a randomized complete block,
containing four replications of each treatment. Individual plot sizes were 4.5 by 2 feet,
separated by one foot buffer strips on all sides.

Treatments

In 1997 the study was initiated on August 15“” and terminated on September 25m;
the 1998 study was initiated on July 315-t and terminated on October 2'39. Termination
dates were determined when natural disease pressure in the control plots declined. A list
of treatments used in each year’s study is shown in the appendix, tables 3 and 4. In 1997
the contact fungicide Daconil Ultrex (chlorothalonil) and systemic fungicide Banner
(propiconazole) were both applied once on August 191”. In 1998 the 202. and 0.5 oz. rates
of Banner Maxx were applied once on August 33!. Banner Maxx was reapplied on
September 9Lh at a 102. rate to plots which had previously received the 0.5 02. rate. All
treatments involving Tx-l were applied either one or five times per week at the
concentrations stated in table I. Treatments were applied using a nitrogen powered

backpack sprayer calibrated to deliver rates of 2.2 gallons/ IOOOft2 for biologicals and 1.1

17

gallons/1000ft2 for chemicals. Tx-l treatments were applied after 4:30 pm. to lessen

mortality of bacteria from the suns ultraviolet radiation and reduce drift.

Bacterial Fermentation

Tx-l was grown for 24 to 30 hours before applying, the exception being the 8
hour growth treatment used in 1997. Seed cultures of 50 to 100 ml were used to inoculate
4L flasks containing 2L of Tryptic soy broth (Difco). Optimal growth was achieved by
aerating the culture with a constant supply of sterile forced air that was mixed into the
culture using a magnetic stir plate. Temperature was maintained at 28° C.

Tx-l was quantified daily for precise application. A spectrophotometer was used
to measure an absorbance by optical density. The absorbance value was used in a
previously derived fomiula, based on a growth curve of Tx-I (35).

The “heat-killed” treatment of Tx-l was killed by bringing the culture to a rolling
boil. This technique was shovm to be completely effective at lysing all cells since no
bacterial growth occurred when plated onto growth media. Thin layer chromatography
(TLC) was used to verify that the antibiotic PCA remained active after boiling. Boiled
and living cultures of Tx-l were extracted with chloroform, spotted onto a TLC plate, and
separated by a 1:1 chloroform:methanol solvent system. Similar Rf values, of 0.55 for
boiled and 0.57 for non-boiled treatments, were sufficient to conclude that PCA is not

destroyed by boiling.

18

Disease Ratings/Data Analysis

Disease ratings were taken every seven days unless delayed by inclement weather
conditions. The occurrence of disease was rated by counting individual infection sites.
Data was analyzed using the SAS (Statistical Analysis Software, Cary NC.) mixed
procedure slicing. Slicing allows for comparison of treatments overtime and also

comparison within treatments overtime.

Results

1_91

In 1997, the daily application of Tx-l at a rate of 2x107 CFU/cm2 provided
significant control of dollar spot as compared to the control (Figure 1). Significant control
by Tx-l applied weekly was observed on rating dates of September 2'”, 18m, and 250‘.
Results of Tx-l applied daily treatment show a significant drop in the dollar spot
population that correlates with Tx-I being able to curatively control this disease. Once
suppression occurred, Tx-l continued to prevent disease for the remainder of the study.
Tx-I applied daily showed significantly greater control than Tx-l applied one time per
week. The amount of control seen when one application of Tx-l per week was used was
not acceptable for managed turf on golf courses.

Tx-l, used in combination with the systemic fungicide, Banner, provided the
same amount of control as Tx-l used alone by the September 2"d rating. After the first
week, Tx-l alone provided more control than the combination. In the second week, the

combination was more effective than the contact fungicide Daconil Ultrex used alone but

was not different from Tx-l used alone. Figure 2 illustrates that the combinations of Tx—l

19

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and fungicides vs. fungicides alone work better at the September 2"d rating and also after
the September 9‘h rating. The Banner/Tx-l combination was more effective than the
Banner alone at the August 25‘h and September 2nd ratings.

The 8hr. growth and heat-killed treatments of Tx-l both provided significant
control by the August 25‘h rating (Figure 3). Compared to the Tx-l applied daily
treatment, the 8hr. treatment provided the same level of control throughout the study. The
heat killed treatment provided significantly less control than the Tx-l applied daily for
the first three rating dates.

1_98_

Comparisons amongst the lower concentrations of Tx-l and fungicides are shown
in Figure 4. The lower rate of Tx-I at 2x105 CF U/cm2 was similar to the control through
the September 12Lh rating. Significant control was obtained using the low rate from
September 19‘" through October 2'”. The low concentration of Tx-l used in combination
with the low rates (0.5 and 1 oz.) of Banner Maxx was similar to the fungicide used alone
during the entire study. Treatments with a low rate of the fungicide provided significant
control at the August ISmand August 21St ratings. After the fungicide was reapplied on
September 3”, control was observed from September 19‘h through October 2'”.

Tx-l applied lx/week at 2x107 CFU/cm2 provided significant control on August
15‘", August 21“, and from September 19‘h through October 2'”. The low concentration of
Tx-I applied 5x/week at 2x105 CF U/cm2 provided significant control from September
19“1 through October 2'”. The heat-killed treatment of Tx-l provided significant control
from 8/15 through 10/2 and was no different than the high rate of Tx-l at any rating date

during the study.

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Tx-l applied 5x/wk was similar to the control after the first week and significant
control was seen thereafter (Figure 6). Banner Maxx applied alone at the 2 02. rate was
more effective than Tx-l in the beginning of the study at the August 150‘ rating. The
combination of Banner Maxx, applied at the 2 02. rate and Tx-l applied 5x/week at 2x107
CFU/cmz, provided significantly greater initial control on August 7‘h and August 15‘“.
After that rating, there was no difference between the combination and Tx-I used alone,
which was similar to 1997 results. All data from both 1997 and 1998 are in Tables 3 and
4 found in the appendix.

Discussion

The results of two years of field studies show that applying Tx-l at a rate of
2x107 CF U/cm2 on a daily basis effectively controlled dollar spot on fairway height
annual bluegrass. It is possible that using Tx-l to manage dollar spot on fairways may
result in substantially less fungicide inputs here. This would be so because fairways are
the largest area of treated turf on golf courses.

Applying Tx-l at a rate of 2x107 CFU/cmz, 5x/week controlled dollar spot both
curatively and preventively. In both years the number of dollar spots declined
significantly when using this treatment, and did not significantly increase at any rating
date while Tx-l was being applied. The initial curative action was preceded by sustained
prevention of any significant increase in dollar spot, regardless of the disease pressure.

In 1997, Tx-l grown for 8 hrs. was compared to the standard 24hr.fermentation of
Tx-l to determine any differences in efficacy. This was compared because of concern
that PCA is produced in the greatest quantities during the stationary phase of growth,

which occurs after eight hours of fermentation. Throughout the study, the two treatments

27

were similar in their ability to control dollar spot and no significant difference was seen
between them. Similar results were likely due to the concentrations used. Both growth
period treatments were applied at a concentration of 2x107 CFU/cm2 and therefore the
growth period is not as great of a factor in control as concentration is.

To determine if less Tx-l would be efficacious, Tx-l at 2x105 CF U/cm2 Sx/week
and 2x107 CF U/cm2 lx/week were compared to 2x107 CF U/cm2 5x/week. Both 2x105
CFU/cm2 5x/week and 2x107 CFU/cm2 applied once per week provided significantly less
control than the higher rate of 2x105 CFU/cm2 5x/week in 1997 and 1998. When
compared to the control, the application of Tx-l once per week at the high rate provided
sustained control of dollar spot after September 10m in 1997 and again after September
12‘-’1 in 1998. Similarly, the low rate of 2x105 CFU/cm2 5x/week was effective compared
to the control after September 12g" in 1998. Although significant disease control was
achieved at these times by both treatments, the control afforded would not be acceptable
on maintained turf. Two possible explanations exist for the control seen after September
12"”. One reason is that a population of Tx-l built up over the period of application which
was effective at controlling the naturally declining dollar spot population. Another
possible explanation is that in the latter part of year (in September) the sun is setting
sooner after application. This may lessen the mortality of bacteria from ultraviolet
degradation and desiccation from heat, allowing more Tx-l to survive when applied.
Because control occurred in both years near the same rating date, and 1998 had more
total applications up to this time point, it is likely due to an increase in the Tx-l
population surviving from daily applications because of a decrease in mortality rather

than a buildup overtime. If effective control is due to greater numbers of bacteria

28

surviving daily then it could be recommended that Tx-l applied at night would have a
higher survivability and possibly greater efficacy.

The heat-killed Tx-l was effective afler the initial rating in both years. In 1997
the heat-killed treatment was less effective compared to the live Tx-l treatment for the
first three rating dates, and in 1998 the two treatments were similar at all rating dates.
This suggests that the active compound PCA is likely the means of fungal inhibition
rather than inhibition of pathogens by the living bacteria. Previous work by Powell
demonstrated that applying purified PCA to turf was sufficient for control (3 5).

Combined treatments of Tx-l and different fungicides and rates were tested to
determine if using a combination could be more effective than the Tx-l used alone. This
question was posed to develop a strategy for times of severe disease pressure in which
Tx-l alone might not provide adequate protection. During such times, a single application
of a fungicide may be used to increase control or extend the fungicide interval when used
as a combination. Our findings in comparing the high rate of Tx-l with a high rate of
fungicide versus the high rate of Tx-l alone showed the combination did not significantly
extend the required interval for fungicide application. The combination provided no
greater disease control than the Tx-l alone, demonstrating that all additional control is
from Tx-l. In 1998 lower concentrations of the systemic fungicide Banner Maxx were
used in combination with the lower rate of Tx-lxlOS. The fungicide was applied at a rate
of 0.5 oz./1000ft2 on August 3L4 and again at l oz./1000 ft2 on September 2M. At no time
was the combination any different than the fungicide alone. At several rating dates the
fungicide was more effective than the Tx-lxIOS used alone, which signifies all control

from the beginning of the study through September 12LIL was due to the action of the

29

fungicide. The lower rates of fungicides gave poor results as compared to the 202. rate.
The September 2fl application of loz.of Banner/1000ft2 is a rate commonly used and
recommended for the curative control of dollar spot. Since this rate performed poor in
comparison to what was expected, there is a possibility of fungicide resistance in this
population of dollar spot. Because of this possible resistance, it can not be determined
from this years results if a lower fungicide rate would reduce disease pressure enough to

increase the effectiveness of Tx-lx105 .

30

Chapter 3

PERSISTENCE OF TX-I APPLIED TO TURF

Introduction

Several factors are required for a biological control organism to be effective in the
field. Two such factors are the ability of the biological control agent to possess a
mechanism of controlling disease and, the ability to successfully colonize and persist in
the environment where control occurs (10). The ability of an introduced organism to
survive and colonize is based on factors such as mobility, growth rate, competition with
existing microflora, and ability to use organic acids as a food source (42). More often
than not, potential biological control organisms are unsuccessful when applied in the field
due to the organism’s inability to establish themselves in a particular cropping system
(48).

Previous studies were done involving fluorescent pseudomonads, in which the
organism’s success in control was determined by their ability to colonize the environment
where introduced (4). This study is the first to monitor populations of the biological
control organism Pseudomonas aureofaciens strain Tx-l applied to turf. The purpose of
this study was to determine if Tx-l applied to turf can survive in the phylloplane and
rhizosphere, and to measure these population changes in the system.

Measuring population sizes allows us to understand how a population changes
overtime and to determine the highest population of Tx-l attainable in a turf system. In
determining these factors, we can optimize applications to achieve successful control of

various turfgrass pathogens.

31

In this study Tx-l was applied daily for approximately two months in 1997 and
1998 and the populations were monitored at set intervals throughout both years.

Populations were measured in the foliage, thatch, and soil at depths of 1,2,and 4cm.

Materials and Methods

This study was done at the Hancock Turfgrass Research Center in East Lansing,
Michigan. The plots used were greens height (5/ 16 inch) creeping bentgrass (Agrostis
palustris)cu1tivar Penncross, established in 1981. The soil type was a modified loamy
sand composed of 83.5 % sand, 10.6 % silt, and 5.9 % clay (25). Tx-I was applied to five
plots, each of size 2 x 3 ftz, grown in full sun with daily irrigation to maintain healthy
turf. Applications were made from early August to the end of September totaling
approximately 40 applications per year. Tx-l was grown and applied five times per week
at a concentration of 2x107 CF U/cm2 by methods previously described on pg. 16 of this
thesis. All daily applications were made after 4:30 pm. The strain of Tx-l used was made
resistant to the antibiotic rifampicin to aid in its recovery and identification. Resistance to
rifampicin was achieved by spreading Tx-l colonies onto media amended with
rifampicin. Colonies that grew were selected because they had developed resistance to
the antibiotic.

Population samplings were taken at similar rating dates over two years. The first
sampling was taken in May (5/28/97 and, 5/13/98), after the turf had broken dormancy.
This sampling quantified the pepulation of Tx-l that had overwintered from the previous
year’s application. A sampling in late July (7/20/98) was taken to measure the population

after the summer months and prior to the beginning of the year’s application; the July

32

1997 sampling was lost due to error in sampling. A sampling was taken in late
September/early October after two months of applications to measure population while
Tx-l was being applied (10/ 19/97 and, 9/22/98). The final sampling was made
approximately one month after application had ceased (1 1/28/97 and, 11/5/98).

Three random samples were taken from each of the five plots at the specified
sampling dates. The soil probe used for this was surface-sterilized in a 10% bleach
solution and rinsed twice in dH20 between each sample taken. Samples were stored in a
4° C refrigerator for 2-4 hours until dissection began. The samplings on (10/19/97 and,
9/22/98) were taken 24 hours after application of the bacteria.

Each of the 15 total samples was dissected into five segments of 1cm sizes
comprising the foliage, thatch, 1, 2, and 4cm soil portions. Each segment was placed into
a 30ml. glass test tube containing 10mls. of buffered physiological saline solution, pH
7.0. The buffered solution was made by adding 3.9ml/L of solution from a stock made
from 2.8g NaHzPO4 in 100ml dHZO and 61va of solution from a stock made from 2.8g
NazHP04 in100ml deO. The buffered solution was added to all test tubes for dilution of
samples. Dilutions were made for each sample and 100111 aliquots were plated out at 103,
10", and 10.5 dilutions in four replications. The media used was potato dextrose agar
(PDA) (Difco, Detroit MI) amended with rifampicin at 50ug/ml to select for only
rifampicin resistant soil organisms. Plates were stored for 2-3 days at room temperature
for colony to growth. Individual colonies were counted on plates and numbers ranging

from 20-200 colony forming units (CFU’s) per plate were used for compiling data.

33

Results

This study demonstrated the ability of Tx—I applied to turf to overwinter and
persist throughout the year. Figure 7., depicts the change in Tx-l population in foliage
over two years. In comparing the 1997 time points 1,2, and 3 to 1998 points 5,6, and 7,
there is no significant difference between the pepulations in the two years. A direct
correlation of populations to times sampled is seen as the population increases when Tx-l
is applied and decreases after application. The foliage experienced the largest drop in
bacterial population in the month after application stopped in both years.

The population in thatch was measured by CFU/cm’and is represented by Figure
8. The May 1997 and 1998 pepulations were not significantly different from one another.
The two fall populations differed from one another with a higher population in 1997. In
both foliar and thatch layers, the largest populations of Tx-l were in October and the
lowest populations in the July sampling.

Measurements of Tx-l populations in the soil regions at 1,2, and 4cm depths
show the highest population in the 1cm region and the 4cm region has the lowest
population, (Figure 9). At no time did the mean populations of a region exceed a region
above it. A significant difference was observed between 1997 and 1998 at the 10/97 and
11/97 times. The populations in the three regions do not differ significantly from one
another at the May 1997 and 1998, July 1998 or November 1998 time points. At 10/97,
all three regions are different from one another, at November 1997 1cm and 4cm differ,

and at September 1998 1cm differs from 2cm and 4cm. The population at the 4cm region

34

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increased significantly from October 1997 to November 1997, which was after
application ceased. The 2cm region also increased at that time although not
significantly. The population from the 1cm depth had the largest population changes
throughout the two seasons. The 2 and 4cm populations were more stable and had the

same pattern of significant p0pulation increases and decreases over the two years.

Discussion

One of the initial objectives of this study was to determine if Tx-l applied to turf
had the ability to overwinter and persist for long periods of time. This study has shown
Tx-l does persist in the foliage, thatch, and soil regions of turf. Tx-l did not diminish to a
population of zero at any time. It may be possible that reestablishment of population
levels necessary for disease control may be achieved in less time by adding to a
population already present, rather than a non-existent population was at zero each time.

The foliage and thatch regions responded similarly over time by experiencing
significant increases and decreases in bacterial populations between the same rating
dates. In both years, Tx-l populations declined significantly in the fall one month after
application had ceased. This suggests less stability in these areas compared to the
populations in soil that did not experience as large a decline during this time.

Tx-l was detected on the foliage in July of 1998. Tx—l had not been applied since
the previous fall. Due to the daily removal of foliage by mowing, all Tx-l applied
previously would be removed. This shows that Tx-l can move to the phylloplane from
the soil and/or thatch. Lamb et al. demonstrated that P. aureofaciens can move from the

rhizosphere to aerial plant tissue on wheat and com (21). The bacteria was transported

38

internally through guttation drops to the outside of the leaf, and externally from the plant
emerging from the rhizosphere. It may be possible Tx-l is moving by one of these
mechanisms to the foliage.

Populations of Tx-l in the soil corresponded to depth, with the largest population
occurring at lcm and the smallest population occurring at the 4cm depth. The greatest
drop in soil populations occurred from November to the following May. The population
remained stable from the end of the application period to one-month post-application
time. During this time, a significant drop was seen only at the lcm depth in 1998, and in
1997, a significant increase was recorded at the 4cm depth at this time. The significant
increase seen after applications ceased may have occurred from the downward movement
of overlying populations. Percolating water plays an essential role in the passive
distribution of bacteria in the rhizosphere (27).

In this study, Tx-l was applied to an area where significant control of the foliar
pathogen dollar spot was occurring. The concentration of Tx-l applied daily to the plots
was 2x107 CF U/cmz. The threshold population measured during disease control was
4.2x10‘5 CFU/cm2 in 1997 and 3.5x10‘S CPU/cm2 in 1998; the value in both years was one
log factor lower than 107 which was being applied daily. In defining a threshold
population of Tx-l for control of dollar spot, a disease control model could be derived
based on this p0pulation. This type of p0pulation monitoring could be used in future
research to determine necessary Tx-l populations to control different diseases infecting
turf or being harbored in thatch and soil/roots. In identifying Tx~l threshold populations

for control of other diseases, it may be possible to predeterminc a control model which

39

would predict the number of applications needed to preventively control disease in both

turfgrass and other cropping systems.

40

Chapter 4

BIOLOGICAL CONTROL OF PINK SNOW MOLD

Introduction

Pink snow mold, caused by the fungus Microdochium nivale, is one of the most
severe diseases of turf in regions where prolonged cool and wet conditions exist (44). It
occurs on turf from early fall through late spring and, in some regions, it exists
throughout the year (41). One difficulty in controlling this disease with fungicides is that
the active infection period can be longer than the control period obtained with most
fungicides. During this time, prolonged periods of snow cover can prevent reapplication
of fungicides. In the past, mercury—based fungicides were used because of their
effectiveness and longevity (44). These fungicides are no longer labeled for use on turf,
and, since their removal, effective alternatives have been sought.

Pseudomonas aureofaciens strain Tx-l has been used successfully in the field for
controlling dollar spot (Sclerotim’a homoeocarpa) (3 5). Due to the success of Tx-l in
controlling dollar spot, it has been tested for its ability to control other diseases of turf.
The purpose of this study was to determine the effectiveness of Tx-I in controlling pink
snow mold. By applying Tx-l in the fall of the year prior to infection, it was thought Tx-
I might prevent this disease. We also sought to determine if a combination of a single
fungicide application used in conjunction with Tx-I could provide greater control than

either used alone.

41

Materials and Methods

This study was done at the Hancock Turfgrass Research Center in East Lansing
MI. The grass chosen for this study was the Pennlinks cultivar of creeping bentgrass
Agrostis palustris, mown at greens height (5/8 inch). Pennlinks was used because it is
more susceptible to pink snow mold than many other cultivars. The error control design
was a randomized complete block with four replications of each treatment. The seven
treatments are listed in Table 1. All treatments using Tx-l were applied a total of 17
times from 10/ 16/97 to 11/11/97. Tx-l was grown and applied using the same techniques
used in the dollar spot field study, pages 17-18, chap 2. The fungicide Fore (mancozeb)
was applied to treatments using fungicides on 10/31 and again on 11/10 to only one
treatment as noted in Table 1.

The occurrence of disease was rated as percentage of area infected, and data was
recorded 104 days after termination of applications. Data was analyzed using the
Statistical Analysis Software general linear model, differences in means were separated
by least significant differences.

Results and Discussion

In this study it was shown that all treatments were effective at controlling pink
snow mold on greens height creeping bentgrass as compared to the control. There was no
significant difference in control amongst any of the effective treatments. The results from

this field study are seen in table I.

42

Table 1. List of treatments and comparison of means as a percent area infected

 

 

 

 

 

 

 

 

 

 

 

 

y pink snow mold.
Treatment Mean P= .05

Control 43.75 A
Tx-l / 10‘, daily 15.50 B
Fore / 6oz./1000ft2 applied once 15.50 B
Tx-l / 10? daily 12.50 B
Tx-l / 107, daily + Fore / 6oz./1000ft2 9.75 B
applied once

Tx-l / 10‘, daily + Fore / 6oz./1000ftr 2.50 B
applied once

Fore / 6oz./1000ft2 applied weekly 2.00 B

 

The results of this study are the first to demonstrate Tx-l is effective at
controlling pink snow mold in the field. Because the low concentration of Tx-l provided
similar control to the high concentration, there would be no benefit from using the higher
concentration to manage this disease. One possible reason for the control seen using the
low 104 concentration is related to the time of year Tx-l was applied. During this study,
the sun would set shortly after application and a decrease in mortality due to UV
radiation and dessication would be likely. This differs from the summer dollar spot study
in which several hours of UV radiation and dessication could reduce the applied
population.

Tx-I provided a similar level of control as the fungicide mancozeb when it was
applied at recommended rates. If future research in varying conditions confirms the
effectiveness of Tx-l in managing pink snow mold, the use of such fungicides may be
reduced.

Many questions related to how Tx-l controls pink snow mold still exist. Several

possibilities might explain how Tx-l is working. It is not known if Tx-l remains active

43

throughout the season under snow cover to prevent infection when the pathogen is most
active. Studies on M. m'vale may reveal if the conidia it produces are prevented from
germinating by a fungistasis-type interaction with Tx-l, or if Tx-l kills the pathogen
prior to its infection of turf. Because of the few Tx-l applications made and low
concentration used, it is possible that the bacteria affected the pathogen at a time crucial
to the development of the pathogen. Once this is better understood, the use and

application timing of Tx-l may be Optimized.

44

Chapter 5

ANTIBIOTIC PRODUCTION OF PSEUDOMONAS AUREOFACIENS

Introduction

Pseudomonas aureofaciens has the ability to control plant pathogenic organisms
through production of several antibiotics. The first antibiotics reported were the
phenazines in 1956 by both Haynes and Cluver (16)(20). Pierson stated the most
important factors to determine the success of a biological control organism is in its ability
to disseminate and survive on the host, and for the organism to inhibit the pathogens
ability to cause disease. The primary factor responsible for P. aureofaciens control ability
is its production of antibiotics (15). Much research has been done to determine how
antibiotics are produced and regulated by P. aureofaciens. Work by Weller and
Thomashow showed that production of phenazines PCA was responsible for control by
the organism. Similar work has shown the antibiotics phloroglucinol and pyrrolnitrin also
to be produced and responsible for inhibition (36). MS research by Powell identified the
antibiotic PCA to be produced by strain Tx-l and correlated it to control in the field.
Powell was able to apply purified PCA from Tx-I to the field as a fungicide and obtained
significant control of dollar spot (35). Thomashow and Weller found that a mutant of P.
aureofaciens deficient in PCA production to be non-inhibiting compared to the PCA
producing parent strain 30-84 (42). Most work on this organism has focused on its
production and regulation of the phenazine PCA.

The objective of this study was to determine the importance of PCA by creating a

mutant deficient in PCA production. This mutant was evaluated in the field for control of

45

dollar spot and also was used for identification of other antibiotics produced by it. In
creating such a mutant, we could determine if other factors or antibiotics influence
control by P. aureofaciens. Understanding the effect of individual antibiotics may lead to
optimizing antibiotic production of these organisms. In this study a PCA' mutant strain
was effective in the field and the antibiotic responsible for control was identified as

pyrrolnitrin.

Materials and Methods

Mutagenesis. A random transposon mutagenesis was performed on the wild type
Tx-l to obtain a PCA' mutant. The donor strain, E. coli carrying a mini-TnS transposon,
was resistant to the antibiotic’s kanamycin (Km) and arnpicilin (Ap) and grown on Luria-
Bertani (LB) agar amended with 50 :1ng of Km and 100 ug/ml Ap. The recipient, P.
aureofaciens strain Tx-l , was resistant to rifampicin (Rif) and grown on LB plates -
amended with 100 ug/ml Rif. Both cultures were grown in amended LB broth shake
cultures overnight at 30° C. Bacteria were collected by centrifugation, and rinsed with
10mM MgClz buffer. The two cultures were mixed onto a LB plate and allowed to grow
overnight. The culture was resuspended in buffer and spread onto LB/Rif/Km plates to
select for transconj ugants of P. aureofaciens. Individual colonies were bioassayed against
Sclerolinia homoeocarpa. Mutants with any change in color or control ability were
chosen for further assay. Mutant Tn-IA was selected for use in in vitro bioassays, field
studies, and antibiotic identification. After mutagenesis, Tn-IA had lost the characteristic

orange pigment produced by Tx-l and also inhibited S. homococarpa differently then

46

Tx-l. Tn-lA was verified as P. aureofaciens by FAME (Fatty Acid Methyl Esterase)
analysis performed by Microcheck, Inc. (Northfield Falls, VT).

Plate Bioassay. Tx-l was compared to Tn-IA in a plate bioassay. A sterile loop
was used to streak a loopful of each culture lengthwise onto Potato Dextrose Agar (PDA)
Petri plates (Difco, Inc. Detroit MI). On both sides of each culture, a fungal plug of S.
homoeocarpa was placed to challenge the inhibitory action of the culture. The bioassay
used two growth times of 0 and 24 hours of bacterial growth before the fungal plugs were
added. Mycelial growth of the fungus towards the bacteria was measured 5 days after
each group of fungal plugs were added. Eight replications of each bacterium were
measured. Growth measurements were analyzed using SAS to separate means by least
significant differences.

Field Study. Tn-IA was used in the 1997 and 1998 Dollar spot Field Study. All
materials, methods, and statistical analysis used, were the same as described in pages 17
and 18, chapter 2, of this text.

Identification of Compound from Tn-lA

General Experimental Procedures. '11 (proton) and '3 C (carbon) NMR (nuclear
magnetic resonance) and DEPT spectra were recorded on a Varian INOVA 300 MHz
spectrometer. 'H NMR spectra were recorded at 300 MHz, while ‘3 C NMR spectra were
recorded at 75 MHz. Chemical shifts were recorded in CDCL3 and the values are in 5
(ppm) on the basis of the 6 residual of CHCL3_ 7.24, and CDCLg, 77.0. Coupling

constraints, J, are in hertz. The silica gel used for VLC was Merck Silica gel 60 (35-70

um particle size). TLC plates (GF Uniplate, Analtech, Inc. Newark DE), after developing

47

were viewed under UV light (254 and 366 nm). All organic solvents used were ACS
reagent grade (Aldrich Chemical Co., Milwaukee, WI) (36).

Fermentation. Seed cultures of Tn-IA were grown in TSB (Tryptic Soy Broth,
Difco, Inc., Detroit, MI). Batch fermentation was conducted using 2 L buffered flasks
containing 500 ml of autoclaved TSB. Each flask was innoculated with 5 m1 seed and
grown in a 27° C growth chamber for 5 days, in a 100 rpm shake culture at in the dark.

Extraction and Isolation. Fermentation broth was centrifuged on a Sorval
Instruments RC 5C centrifidge at 10,000 rpm, 4 °C, for 10 minutes. Pellets were collected
and added to 500 ml of a 4:1 CHC13: MEOH solution. The solution was homogenized at
2,000 rpm for 15 minutes; the chloroform soluble active fraction was separated using a
separatory funnel. The chloroform fraction was dried by rotoevaporation and dessicated
overnight. The dried extract was stored at -20 C. This extraction procedure yielded ~2 g
of crude extract/13 L of fermentation broth. Fractionation of this extract (20.0 g) was
carried out by vacuum liquid chromatography (VLC). Silica gel (240 g) was placed in a
sintered glass funnel (600 mL, 10-15 um mesh), hexane with increasing amounts of ethyl
acetate and lastly methanol was used as the eluting solvents. Five fractions, 1-5, were
collected: (1) 100% hexane, 1.05 g; (2) hexane-ethyl acetate, 8:1, 450 mg; (3) hexane-
ethyl acetate, 4:1, 540 mg; (4) hexane-ethyl acetate, 1:1, 560 mg; (5) 100% methanol,
7.87 g. Fraction 3 was biologically active at 250 ppm, and inhibited all growth of S.
homoeocarpa after seven days.

Fraction 3 was purified by prep. thin layer chromatography, (PTLC), and further
purified by high pressure liquid chromatography (HPLC) using a Jai LC-20 preperative

liquid chromatograph on two Jaigel S-343-15 ODS columns in series (id. 20 x 250 mm),

48

eluted with MeOH (70%) H20 (30%) at a flow rate of 3 mL/min, and detected using UV
of 210 nm. This yielded compound 1 (42 mg). Compound 1 was identified by 1H NMR,
'3 C NMR, and mass spectral experiments.

Compound 1. IH NMR: 8 6.78 (1H, m, H-2), 6.81 (1H, m, H-2), 7.42
(2H, m, H-4’ and H-6’) 7.51 (1H, m, H-S’) 8.34 (1H, bs, exchangeable with D20, -NH).
13C NMR: 5 111.70 (03), 115.24 (04), 116.45 (C-5)*, 117.21 (02)“, 124.77 (C-I’),
127.65 (C-3’), 128.55 (C-6’)*, 129.30 (C-2’), 130.12 (C-4’)*, 130.35 (C-5’)*; *
interchangeable. EI-MS: m/z 256 (M+), 229, 210, 201, 193, 183, 175, 166, 148, 140, 138,
113, 102, 87, 75, 63, 55. Compound 1 was identified as pyrrolnitrin, C lo H6 C12 N2 02, by
comparison of its lH, 13C NMR, and mass spectral data which corresponded to literature

values (5)(1 l)(1 8)(19).

Results

The plate bioassay comparing Tx-l to Tn—lA showed no significant difference in
control when bacteria were streaked onto plates at the same time fungal plugs were
added. Tn-IA provided significantly greater control than Tx-l when the bacteria were

allowed to grow for 24 hours before adding the fungal plug Table 2.

Table 2. Plate bioassay, comparing Tx-l to Tn-lA, grown for two time periods. Numeric numbers
represent mean growth in cm, and letter symbols separate means at P=.OS.

 

 

 

 

Time Tx-l Tn-lA
0 hours 8.96 A 8.09 A
24 hours 3.25 B 0.84 A

 

 

 

49

 

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51

After two weeks, there was a noted difference in mycelial development. The
mycelia on the Tx-l plates appeared thin and wispy, in comparison to the normally
developed mycelia on the Tn-lA plates. The S. homoeocarpa developed normally as
noted by the stromatized tissue in the agar, behind the Tn-lA zone of inhibition. The agar
in the Tx-l plates had an orange pigmentation as compared to Tn-lA that remained clear.

The two-year field study showed significant control of dollar spot by Tn-lA as
compared to the control. In 1997, Tn-lA provided significantly less control than Tx-l in
the first three ratings, (Figure 10). The last two ratings were similar. In 1998, there was
no significant difference between the two treatments at any time point throughout the
study, Figure 11.

Tn-IA was extracted to identify all active compounds produced by it. The
primary antibiotic produced by strain Tx-l is PCA. PCA was not produced by strain Tn-
1A. A total of 42 mg of pyrrolnitrin was extracted from 20 g of crude Tn-lA extract. In
plate bioassays, 250 ppm of purified pyrrolnitrin challenging a plug of S. homoeocarpa,
completely inhibited all growth for two weeks. There after, the mycelia from the plug

began to grow outward slowly.

Discussion

This study sought to identify if PCA was the only active compound or mechanism
responsible for control of dollar spot by P. aureofaciens strain Tx-l. This question was
answered by creation of a PCA' mutant for use in field and bioassay studies.

Results from the plate bioassay showed Tn-IA controls S. homoeocarpa similarly

to Tx-l. When given 24 hours to grow, Tn-IA was more effective than Tx-l. This may

52

have been due to a difference in time needed for antibiotic production of each strain. The
mycelium of S. homoeocarpa developed normally behind the zone of inhibition on the
Tn-lA plates, while mycelia ceased to develop on Tx-l plates. Normal development was
evident by the production of stromatized tissue. This indicates the antibiotic from Tn-lA
only inhibited grth rather than killing the organism. The type of inhibition seen
correlates to literature describing pyrrolnitrin as inhibiting growth rather than killing the
target organism (33).

The field studies showed greater control at some rating dates by Tx-l in 1997 than
Tn-lA, and no difference occurred between treatments in 1998. Although not always
significant, Tx-l plots maintained fewer dollar spot infections than Tn-lA. This may be
due to two factors. One is that the ecological competency and survivability in the field is
often reduced by manipulations to an organism (24). Another reason is in the time
required of each specific antibiotic being produced. PCA production is dependent upon
cell density, and PCA extracts can be produced and detected on TLC plates after a
fermentation period of 24 hours. It is said that Pyrrolnitrin is produced during starvation
and late into stationary growth phase. Our work showed optimal and maximum quantities
of pyrrolnitrin, being produced from five days of fermentation. Since both cultures were
grown for 20-24 hours, Tx-l may have had an advantage in quantity of antibiotic
produced daily.

Thin layer chromatography verified that Tn-IA did not produce PCA however it
is not known if Tx-l is producing pyrrolnitrin. Another unknown is if pyrrolnitrin
production by Tn-IA has been increased in quantity as compared to quantities possibly

produced by Tx-l. Work by Rodriguez and Pfender stated that an increase in one

53

antibiotic may cause a decrease or increase of other antibiotics (37). Salcher and Lingens
used mutagenesis to produce a mutant of P. aureofaciens that had a 30-fold increase in
pyrrolnitrin production (38). Our mutagenesis may also have affected normal pyrrolnitrin
production.

In understanding the role of various antibiotics produced by P. aureofaciens,
future research may fully utilize this organism for control of plant pathogens to improve
plant health. The results of this study are the first to show that a mutant of Tx-l
producing only pyrrolnitrin was suppressive to dollar spot in the field and similar to a

PCA-producing Tx-l.

54

Chapter 6

EPILOGUE

The four individual projects in my thesis were conducted to answer questions
regarding the biological control organism Pseudomonas aureofaciens strain Tx-l. The
answers to the questions posed by this research were used to increase the efficiency of
Tx-l as a biological control of diseases on Turf.

The dollar spot field study showed Tx-l to be effective at controlling dollar spot
on fairway height annual bluegrass. Since it will be used on golf courses, and applied at
lower concentrations, it must be determined how to make such applications effective.
One possible project would involve applying low rates of Tx-l at night or closer to the
time when the sun sets. This would show whether lower concentrations could be effective
by increasing the likelihood of lower mortality from UV radiation and dessication.
Another aspect to examine utilization of lower concentrations would be to apply Tx-l
preventively, before a large epidemic arises. In this study Tx-l was applied afier a large
initial infection had occurred. Perhaps less initial disease pressure would allow greater
sustained control when applying lower concentrations of Tx-l.

The use of Tx-l for control of pink snow mold was the first report of control of
this organism in the field. The effectiveness of the 104 CFU/cm2 application rate of Tx-l
is very promising for turf managers because such concentrations are attainable using the
current fermentation system. It may be possible that even lower concentrations of Tx-l
are also effective. Future research should determine when control is occurring. For

instance, is Tx-l actively inhibiting the pathogen during the season, or is precise

55

application timings inhibiting conidia before infection or normal grth of the fungi can
occur? Work could also be done to determine if Tx-l could control outbreaks in the late
spring.

The persistence of Tx-l in a turfgrass system has been shown. This is vital to
rebuilding a population capable of control in the spring or beginning of a new disease
epidemic. I believe the greatest applicability of this research will come in future studies
to determine threshold Tx-l populations necessary for control of different turfgrass
pathogens. It is likely that specific population sizes must be achieved in different regions
to control specific diseases. Sampling to determine populations present will allow for a
correlation of control or the lack of when population requirements are not met. Another
important study could be to determine the factor(s) most responsible for the mortality of
Tx-l over time. To do so may require a determination of population limit or carrying
capacities of Tx-l in turf. One aspect of the plating technique used for recovery and
enumeration is that it gives only an estimate of recoverable viable bacteria. This estimate
is not the total bacterial p0pulation. Modern molecular techniques using selective markers
such as IacZ Y have been widely used for quantifying recovery of bacterial populations
and perhaps such methods would prove more efficient or accurate.

The project involving mutant strain Tn-IA was important in demonstrating an
antibiotic other than PCA can effectively control disease in the field. Many studies could
be conducted to understand the genetics of this mutant as it compares to the wild type
strain. It is not known how PCA production was disturbed from mutagenesis and what
mechanisms if any have changed in the pathway regulating biosynthesis of pyrrolnitrin.

Understanding these pathways could lead to means of overexpressing specific antibiotic

56

production. The evolutionary role of multiple antibiotic production may be linked to
nutrient availability in the environment over time. Some antibiotics inhibit specific fimgal
pathogens differently or better than others. It would be interesting to determine if
Pseudomonas aureofaciens has developed separate antibiotics to compete with specific
groups of organisms within the microbial community, and at different times throughout
the year.

The use of Pseudomonas aureofaciens as a biological control in the field is still in
its infancy of understanding and optimally utilizing the naturally suppressive properties it
possesses. Future research will expand the knowledge needed to use this and other

microbes for protection of plants from phytopathogenic organisms.

57

APPENDICES

58

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59

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60

LITERATURE CITED

61

b.)

10.

11.

Literature cited

Arima, K., Imanaka, Masanobu, and Kousaka. 1965. Studies on pyrrolnitrin, a
new antibiotic. The Journal of Antibiotics 18: 201-204.

Beard, J ., Tani, T., 1997. Color atlas of Turfgrass Diseases, pages 141-147. Ann
Arbor Press and James Beard.

Bergey’s Manual of Systemic Bacteriology Vol. 1 pages 165-167....

Bull, C.T., Weller, D. M., and Thomashow, L. S., 1991. Relationship between
root colonization and suppression of Gaueumannomyces gramim’s var. tritici
Psuedomonasfluorescens strain 2-79. Phyt0pathology. 81, 954-959.

Carruthers, F. L., Conner, A. J., and Mahanty, H. K., 1993. Identification of a
Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition. Appl.
and Environ. Micro. 60: 71-77.

Communications. 1976. Application of Long-Range Spin-Spin Couplings in
Biosynthetic Studies. J. Org. Chem., 41: 2932-2933.

Cook, R. J ., and Rovira A.D., 1976. The Role of Bacteria in the Biological
Control of Gaeumannomyces graminis by Suppressive Soils. Soil Biol. Biochem.,
8: 269-273.

Corbell, N., and Loper, J. E., 1995. A Global Regulator of Secondary Metabolite
Production in Pseudomonasfluorescens Pf-S. J. Bacteriol. 177: 6230-6236.

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