LIBRARIES MICHIGAN STATE UNIVERSITY EAST LANSING, …archive.lib.msu.edu/tic/thesdiss/gilstrap2005a.pdf · 2006. 10. 16. · Weigel, and Dr. Terry Warren. During this ordeal I have

LIBRARIESMICHIGAN STATE UNIVERSITY

EAST LANSING, MICH 48824-1048

This is to certify that thethesis entitled

STRATEGIES FOR THEMANAGEMENT OF FUNGICIDE-RESISTANT

RUTSTROEMIA FLOCCOSUM(SYN. SCLEROTINIA HOMOEOCARPA),

THE CAUSAL ORGANISMOF DOLLAR SPOT

presented by

DAVID MURPHY GILSTRAP

has been accepted towards fulfillmentof the requirements for the

DOCTOR OF degree inPHILOSOPHY

PLANT PATHOLOGY

MSU is an Affirmative Action/Equal Opportunity Institution

STRATEGIES FOR THE MANAGEMENT OF FUNGICIDE-RESISTANTRUTSTROEMIA FLOCCOSUM (SYN. SCLEROTINIA HOMOEOCARPA) ,

THE CAUSAL ORGANISM OF DOLLAR SPOT

By

David Murphy Gilstrap

A DISSERTATION

Submitted toMichigan State University

in partial fulfillment of therequirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Plant Pathology

2005

ABSTRACT

STRATEGIES FOR THE MANAGEMENT OF FUNGICIDE-RESISTANTRUTSTROEMIA FLOCCOSUM (SYN. SCLEROTINIA HOMOEOCARPA) ,

THE CAUSAL ORGANISM OF DOLLAR SPOT

By

David Murphy Gilstrap

Dollar spot is an important disease of turfgrasses

worldwide. The pathogen, Rutstroemia floccosum, has

developed resistance to three classes of systemic

fungicides: the benzimidazoles, the dicarboximides, and

the demethylation inhibitors (DMI). Two multiyear studies

assessed changes in DMI sensitivities over time using DMI

and non-DMI fungicides at different rates applied alone,

in alternation, or in combination with each other.

The first experiment involved a DMI-resistant

population of R. floccosum resident to a mixture of

creeping bentgrass and annual bluegrass maintained as a

golf-course fairway. Isolates taken at five time points

were grown into pure culture and then assayed using

relative comparisons of their radial growth on PDA and PDA

amended with 2 pg ml-1 triadimefon (DMI). A similar

experiment was conducted on a DMI-sensitive population of

R. floccosum from another site. In both studies, the

pathogen's resistance to DMI fungicides increased with all

treatments that involved exposures to DMI fungicides. A

positive relationship was shown between the number of DMI-

fungicide applications and the rates of increase in DMI

resistance. An AFLP analysis of a selection of DMI-

resistant and -sensitive isolates failed to distinguish

differences among those isolates.

A final investigation was conducted at the DMI-

resistant R. floccosum site above where unsatisfactory

dollar-spot control had occurred with a first-time use of

boscalid, a new dollar-spot fungicide of the carboximide

class. In a field experiment, significant numbers of

dollar spots appeared at three days after treatment (DAT)

with boscalid compared to a treatment with chlorothalonil

only. The dollar spots had disappeared at 8 DAT. In a

second experiment, the dollar spots began appearing at 4

DAT and had disappeared by 14 DAT. The number of dollar

spots in the bcscalid treatment was significantly greater

than the chlorothalonil treatment at 9 and 12 DAT.

Isolates were collected from the transient dollar-spots

during the second experiment and found to have

significantly greater in vitro resistance to boscalid

compared to isolates of five different strains collected

in other locations in Michigan.

To all of those whobelieved that thiswould eventually

be completed

lV

ACKNOWLEDGEMENTS

Over the twelve years it has taken to complete my

terminal degree, I have received substantial assistance,

support, and encouragement from numerous individuals.

First and foremost, I thank Dr. Joe Vargas, Jr., for being

my major professor and mentor. I appreciate the advice

and patience of the rest of my committee: Drs. Larry

Olsen, Mary Hausbeck, Willie Kirk, and especially Ray

Hammerschmidt whose timely and detailed editing played a

key role in the completion of this thesis. Winfred

Motherwell served as an additional proofreader. Drs.

Sasha Kravchenko and Oliver Schabenberger provided crucial

assistance with my statistical analysis.

I thank Michael Jones for graciously providing

research sites and taking data at the Lochmoor Club.

Substantial assistance and advice was given by the

research technicians in the Vargas lab: Nancy Dykema, Ron

Detweiler, and Danielle McMahon. Drs. Rob Golembiewski,

Jon Powell, Brandon Horvath, and Phil Dwyer helped me to

better understand plant pathology while they were fellow

graduate students. Mark Collins at the turf center was

invaluable. Two student workers who helped me out

significantly were Greg Elliot and Trevor Thorp.

v

The chairpersons of the Department of Crop and Soil

Sciences where I am employed have given me their full

support for this endeavor: Drs. Eldor Paul, Boyd Ellis,

Taylor Johnston, Doug Buhler, and Jim Kells. Fellow turf

faculty who did the same were Drs. Paul Rieke, Bruce

Branham, Jim Crum, and Trey Rogers. Other CSS faculty who

inspired me were Drs. Bernie Knezek, Russ Freed, Karen

Renner, and Larry Copeland. Within the college I received

particular encouragement from Drs. Fred Poston, Ian Gray,

Cliff Jump, Eunice Foster, Rick Brandenburg, Bridget Behe,

Bob Schutzki and Weijun Zhao. Office professionals who

were always helpful included Donna Ellis, Kathy Bedford,

Linda Colon, Joan Gilliland, Carol Fosburg, Beverly

Riedinger" and Darlene Johnson.

Timely encouragement, advice, and a fellowship were

provided by Harriet and Dr. Jim Beard, my major professor

for my M. S. at Texas A&M. Others who heartened me along

the way included Jim Epolito, Dan Taylor, Mel Lanford, Jim

Weigel, and Dr. Terry Warren. During this ordeal I have

enjoyed the support and encouragement of my lovely wife,

the Honorable Paula Manderfield, and my children:

Madeline, Harrison, Katherine, and Rudy. My parents,

Bette and Chena Gilstrap, and my two brothers, Frank and

Randy, believed ln me, as well.

Vl

TABLE OF CONTENTS

LIST OF TABLES ix

LIST OF FIGURES Xl.l.

CHAPTER 1INTRODUCTION , 1

DISCOVERY OF DOLLAR SPOT AS A DISEASE OF TURFGRASSES 2TAXONOMIC CLASSIFICATION OF THE DOLLAR-SPOT PATHOGEN 3RUTSTROEMIA FLOCCOSUM SIGNS - DOLLAR-SPOT SYMPTOMS

AND EPIDEMEOLOGY 7THE NATURE OF FUNGICIDE RESISTANCE 17RUTSTROMIA FLOCCOSUM RESISTANCE TO FUNGICIDES 20

CHAPTER 2EFFECTS OF REPEATED TREATMENTS OF DEMETHYLATION-INHIBITOR

(DMI) FUNGICIDES ON DMI-SENSITIVE POPULATIONS OFRUTSTROEMIA FLOCCOSUM 41

LITERATURE REVIEW 42The Ergosterol-Biosynthesis Pathway and Sterol-

Biosynthesis Inhibitors 42DMI Fungicides - Product Development 46DMI-Fungicide Formulations and Efficacies 52DMI Use on Dollar Spot 53

DMI-SENSITIVE R. FLOCCOSUM STUDY 53Agronomic Practices and Site Assessment 54Fungicide Treatments 58Fungicide-Application Techniques 61Sample Collection 61In Vitro Sensitivity Assessments and Statistical

Analysis 62Sensitivity Differentiation Using AFLP Markers 63Results and Discussion 64

CHAPTER 3EFFECTS OF REPEATED TREATMENTS OF DEMETHYLATION-INHIBITOR

(DMI) FUNGICIDES ON DMI-RESISTANT POPULATIONS OFRUTSTROEMIA FLOCCOSUM 105

LITERATURE REVIEW 106Human Systerns 106Animal Systems 108Plant Systems 110

vii

Turf Systems 112INVESTIGATION OF REPEATED FUNGICIDE APPLICATIONS TO

DMI-RESISTANT R. FLOCCOSUM 113Experimental Area and Agronomic Practices 113Sample Collection and Dates of Sampling 114Treatments 115Statistical Analysis 116Resul ts 117Discussion 119

CHAPTER 4INVESTIGATION OF RUTSTROEMIA FLOCCOSUM SENSITIVITY TO

BOSCALID 130LITERATURE REVIEW 131

Product Development, Registration, andFormulation 131

Mode of Action and Activity 132Carboximide Resistance 133

LABEL DIRECTIONS AND PRECAUTIONS 134ALARMING REPORTS 135INITIAL ASSESSMENTS IN VITRO 136FIELD EXPERIMENT I 140

Materials and Methods 140Resul ts 142

FIELD-EXPERIMENT II 145Materials and Methods 145Results 146

BROADENED DOSE-RESPONSE STUDY IN VITRO I 148Materials and Methods 148Resul ts 149

BROADENED DOSE-RESPONSE STUDY IN VITRO II 150Materials and Methods 150Resul ts 151

DISCUSSION 152Benzimidazole Fungicides 152QoI Fungicides 153DMI Fungicides 156Dicarboximide Fungicides 160Chlorothalonil Fungicide 161Boscalid Fungicide 162

CHAPTER 5CONCLUSION 185

BROADER IMPLICATIONS 186FUTURE RESEARCH 188

APPENDIX 191

Vlll

LIST OF TABLES

Table 1.1. First reports of turf pathogens resistant toone or more fungicides 25

Table 2.1. Common names, chemical names, trade names, andcurrent registrants of demethylation-inhibitorfungicides labeled for use on commercial turf in theU. S 76

Table 2.2. Average-percent-relative growth for each blockof ten subsamples collected on 29 July 1994 and mean-percent-relative growth for each treatment shown alongwith the fungicides, rates in g 100 m-2, and intervals ofapplications for each treatment 77

Table 2.3. Application rates and timing intervals oftreatments 78

Table 2.4. Number of propiconazole applications appliedin 1994, 1995, 1996, and 1997 79

Table 2.5. Propiconazole amounts in g 100-2 applied in1994, 1995, 1996, and 1997 79

Table 2.6. Average-percent-relative growth for each blockof ten subsamples (or fewer where noted) collected on 14October 1994 and mean-percent-relative growth for eachtreatment shown along with the fungicides, rates in g100 m-2, and intervals of applications for eachtreatment 80

Table 2.7. Average-percent-relative growth for each blockof ten subsamples (or fewer where noted) collected on 14July 1995 and mean-percent-relative growth for eachtreatment shown along with the fungicides, rates in g100 m-2, and intervals of applications for eachtreatment 81

Table 2.8. Average-percent-relative growth for each blockof ten subsamples collected on 20 October 1995 and mean-percent-relative growth for each treatment shown along

lX

with the fungicides, rates in g 100 m-2, and intervals ofapplications for each treatment 82

Table 2.9. Average-percent-relative growth for each blockof ten subsamples collected on 12 July 1996 (or fewerwhere noted) and mean-percent-relative growth for eachtreatment shown along with the fungicides, rates in g100 m-2, and intervals of applications for eachtreatment 83

Table 2.10. Average-percent-relative growth for eachblock of ten subsamples collected (or fewer where noted)on 1 August 1997 and mean-percent-relative growth foreach treatment shown along with the fungicides, rates ing 100 m-2, and intervals of applications for eachtreatment 84

Table 2.11. Average-percent-relative growth for eachblock of ten subsamples collected (or fewer where noted)on 21 August 1998 and mean-percent-relative growth foreach treatment shown along with the fungicides, rates ing 100 m-2, and intervals of applications for eachtreatment 85

Table 2.12. Treatment means of percent-relative-growthvalues of four replications collected on 20 October1995 86

Table 2.13. Treatment means of percent-relative-growthvalues of four replications collected on 1 August1997 87

Table 2.14. Treatment means of percent-relative-growthvalues of four replications collected on 21 August1998 88

Table 2.15. Number of chlorothalonil applications appliedin 1994, 1995, 1996, and 1997 89

Table 2.16. Chlorothalonil amounts in g 100 m-2 applied in1994, 1995, 1996, and 1997 9a

Table 2.17. An example of application rates and timingintervals of treatments for a study that would have hadgreater efficiency than the one conducted and presentedin Chapter 2 91

x

Table 3.1. Treatments with fungicides, rates, andintervals 121

Table 3.2. Mean-percent-relative-growth values of sixtreatments with three blocks at five observations withpercent increase of final observation compared to firstobservation 122

Table 3.3 .. Slopes of linear trends over time bytreatments and associated P-values 123

Table 4.1. Application rates and timing intervals oftreatments 166

Table 4.2. Mean-dollar-spot counts of seven treatmentswith six fungicides representing different fungicideclasses at five days after treatment (DAT) with least-significant-difference (LSD) comparisons (P ~ 0.05) ofbase-10 log-transformed counts 167

Table 4.3. Mean-dollar-spot counts of seven treatmentswith six fungicides representing different fungicideclasses at 21 days after treatment (DAT) with least-significant-difference (LSD) comparisons (P ~ 0.05) ofbase-10 log-transformed counts 168

Table 4.4. Mean-percent-relative-growth values of fifteenisolates in three replications grown on PDA only andPDA amended with 40 pg ml-1 boscalid 169

Table 4.5. Percent-mean-relative-growth values of eightisolates grown on PDA only and PDA amended with threesets of different single-site fungicides with threereplications 170

Table 4.6. Mean-percent-radial-growth values from threeexperiments at the Lochmoor Club from 1991-2004 with nostatistical inferences noted or implied 171

Table 4.7 Mean-percent-relative-growth values of eighttreatments with three replications at ten concentrationsof boscalid 172

Xl

LIST OF FIGURES

Figure 2.1. Map of Block 4 with treatment numbers aboveor below each 2.1 m by 8.4 m (17.6 m2) plot with 1.2 mby 3 m (3.6 m2

) sampling areas outlined and mowingdirections indicated by parallel arrows in oppositedirections 92

Figure 2.2. Mean-percent-relative growth of threetreatments with 4 replications at 3 observation dateswith each treatment applying chlorothalonil annually ..93

Figure 3.1. Treatment means for percent-relative growthby observation dates [from Gilstrap et. al (8)] 124

Figure 3.2. Estimated linear trends by treatment 125

Figure 4.1. Dose-response curve for the average-percentrelative-growth measurements of three replicates ofisolate LF-7 at seven concentrations of boscalid .....173

Figure 4.2. Regression line of three replications ofmean-percent-relative-growth measurements of isolate LF-7 against the base-10 logarithms of five-fungicideconcentrations ranging from 5 to 40 pg ml-1 173

Figure 4.3. Dollar-spot counts of the means of seventreatments with 4 replications (Field-Experiment I) ..174

Figure 4.4. Mean-dollar-spot counts of the means of threetreatments with four replications at nine observations(Field Experiment 11) 175

Figure 4.5. Mean-dollar-spot counts of the means of threetreatments with four replications at five observations(Field Experiment II) 176

Figure 4.6. Dose-response curves for the percent-mean-relative-growth values of eight isolates at tenconcentrations of boscalid expressed as base-10logarithms 177

Xll

CHAPTER 1

INTRODUCTION

1

DISCOVERY OF DOLLAR SPOT AS A DISEASE OF TURFGRASSES

Dollar spot is a prominent and serious turfgrass

disease in most parts of the world. Symptoms of the

disease were noted first probably by Piper and Coe who

investigated brown spots "varying from a few inches to a

foot or more in diameter, which appeared more or less

abundantly scatterered" about on golf-course-putting

greens near Philadelphia in 1913 (106). Every brown area

on putting greens at that time was termed "sun scald"

(107), and in this instance it was concluded that the

cause was primarily poor drainage (106). In 1914, similar

brown spots appeared on turf being experimented upon by

Mr. Fred Taylor at his home near Philadelphia who reported

"a fine white cobwebby covering could be seen on the

newly, formed patches in the early morning" (107). A

biotic malady was suspected because of the "definiteness

of the spots and their concentric growth." However,

investigations by several pathologist failed to identify a

causal organism (106).

Brown spots could be found abundantly on golf courses

in the Washington, D. C., area by 1916. Piper and Coe

reported that "early in the morning a fine white mycelium

could be seen covering new spots" that were "a few inches"

in diameter (106). Larger spots with two-foot diameters

2

were observed actively growing and having edges "sharply

marked by a narrow zone of dark smoky green where the

grass leaves are dying." Small black sclerotia were

collected along these edges and placed in culture. The

dark mycelia produced were examined by Piper and Coe and

deemed to be that of Rhizoctonia solani Kuhn. The

possibility of the presence of two causal organisms

causing two different turfgrass diseases was not

addressed.

TAXONOMIC CLASSIFICATION OF THE DOLLAR-SPOT PATHOGEN

The differences in relative-spot diameters led to the

connotations of "large brown patch" and "small brown

patch", which was described by Monteith and Dahl in 1932

(95). They proposed that it be called "dollarspot", a

name already being used for the disease. They suggested

that its pathogen was a fungus and probably a species of

the Rhizoctonia DC genus since Piper and Coe had already

identified R. solani as causing "large brown patch".

Monteith and Dahl proposed shortening this name to

"brownpatch".

In 1937, Bennett collected and cultured isolates from

dollar spots in Britain, America, and Australia (9). Only

rudimentary, sterile apothecia grew on any of the American

3

or Australian isolates. One group of British samples

produced ascospores, and another produced ascospores and

conidiospores. He considered his collections as

representing three strains of the same specles;

differences in pathogenicity were not reported. Bennett

described the pathogen as a perfect fungus based on his

observations of the isolates that produced both ascospores

and condiospores, and he named it Sclerotinia homoeocarpa.

Unfortunately, those isolates no longer exist (121).

Findings were similar in later attempts to duplicate

Bennett's work except none of the British isolates

produced conidiospores (6,77,124). Efforts with solely

American isolates have yielded nothing more than aborted

or sterile apothecia (43,44). Natural occurrences of

sclerotia or conidiospores have not been reported

(97,124). Newell and Baldwin, in 1990 (100) and 1992 (6),

reported finding fertile apothecia on both diseased and

healthy fescue grasses (Festuca L. sp.) growing in

England. However, they were unable to confirm

pathogenicity using Koch's Postulates. Couch stated that

ascospores and conidiospores appeared to be of minor

importance in the epidemiology of the disease (16).

Whetzel, In 1945 (154), excluded S. homoeocarpa from

the family Sclerotiniaceae because it did not produce true

4

sclerotia. A year later he suggested that it was probably

Rutstroemia sp. and synonymous with a previously described

fungus Ciboria armeriae Von Hohnel (155). Jackson also

concluded that the organism most likely belonged in the

genus Rutstroemia (77). He also suggested that the

symptoms of dollar spot may have been caused by more than

one species. Kohn concluded that the organism would more

appropriately fit into either the Lanzia Sacco and/or

Moellerodiscus Henn. genera (83). Many publications

concerning dollar spot list Lanzia and Moellerodiscus but

not Sclerotinia as keywords (6,11,57,64,79,100). Kohn

agreed with Jackson that more than one species might be

involved (84). The term "dollar spot syndrome" was

suggested by Smiley (120). Mycologists using traditional

taxonomy for over fifty years failed to distinctly

identify and reclassify the organism(s) responsible, and

its epithet remained S. homoeocarpa F. T. Bennett (Ill).

Kohn and Grenville found S. homoeocarpa to be unlque

ln a series of studies that compared anatomical,

histochemical, and ultrastructural properties of selected

stromatal and sclerotial fungi grown in vitro (84,85).

The first reports of studies with the DNA of the pathogen

began in 1993 when Carbone and Kohn (12) analyzed some of

its isolates as well as others classified as members of

5

the Sclerotiniaceae. They placed S. homoeocarpa in close

relations with four Rutstroemia sp. based on phylogenetic

comparisons using nuclear ribosomal internal transcribed

spacer-region 1 (ITS1) sequencing. No comparisons were

made to any isolates of Moellerodiscus or Lanzia, (110)/

both genera that Kohn had supported earlier as being

possibilities where it should belong (83). Holst-Jensen

et al. sequenced ITS1 and ITS2 regions plus part of the

nuclear ribosomal small subunit (18S rDNA) regions of a

broader spectrum of isolates. Their analysis showed S.

homoeocarpa in a cluster with several Rutstroemia species

and apart from a sole Lanzia specie (67).

Powell and Vargas in 1999 showed that the causal

organism most closely resembled Rutstroemia cuniculi

(Boudier) Elliott and R. henningsianum (Plottn.) T.

Schumach. & L. M. Kohn. using parsimony analysis of ITS1

sequencing as a basis (110). They proposed a new name,

Rutstroemia festucae, for the causal organism of dollar-

spot disease of British origin. Rutstroemia floccosum was

proposed as a new name for the corresponding pathogen ln

North America, Australia, and the Netherlands, (109).

This name appears as such in recently published monographs

(8,141,142); and, it will be used throughout the balance

of this dissertation.

6

RUTSTROEMIA FLOCCOSUM SIGNS - DOLLAR-SPOT

SYMPTOMS AND EPIDEMEOLOGY

Both warm-season- and cool-season-turfgrass species

and cultivars are susceptible to dollar spot in varying

degrees (7,16,66,92,98,130,147,156). Mycelia of R.

floccusum can survive long periods of inactivity as

stromata upon or embedded in above-ground-turfgrass

tissue, primarily the leaves (59). It may exist for brief

periods in plant debris as a facultative saprophyte (138).

Hyphae elongate via terminal-cell division under favorable

conditions and enter the plant through stomata and cut-

leaf tips (95). Appressoria have also been observed

suggesting direct penetration (38). Harman et al. stated

that the pathogen demonstrates two distinct periods of

growth: a slow-growing phase where small colonies lay ln

a quiescent state and a rapidly growing phase in which

copious amounts of mycelium are produced (60).

Environmental conditions favorable for disease

activity vary among isolates from different geographical

areas (133). Bennett found that the optimum temperature

range for in vitro growth of British isolates was 20 to 30

C, and that growth occurred very slowly at 0-1 C. None of

the American or Australian isolates grew at that

7

temperature, but both grew optimally at 30 C and 25 C

respectively (9). Endo found that 32 C was the maximum

temperature at which any of his collection of isolates

could survive under controlled conditions (37).

Conditions favorable for R. floccusum are generally cool-

humid nights with temperatures as low as 15 C with

extended periods of leaf wetness and warm days with

temperatures as high as 32 C (141). The pathogen has

demonstrated its adaptability to proliferate at the higher

end of this temperature range in warmer climates (46,47)

and at the lower end of this temperature range in cooler

regions (131,134).

Puffy aerial mycelium may be seen growlng from

infected leaves before the dew dries and bridging to

adjacent, healthy leaves so that further infections may

occur (69,131). Movement of the pathogen beyond leaf-to-

leaf transmission occurs in a passive manner via infected

tissue in plant debris, mainly leaf clippings (15). Means

of transport across a sward include wind, rain (122),

equipment, as well as human traffic and animal activities

(124). Migration over longer distances occurs on shoes,

equipment (76), and probably on clothing, wildlife, and

domesticated dogs, particularly those whose owners have

been contracted by golf courses to frighten geese and

8

other waterfowl away from their premises. No evidence of

dollar spot as a seed-borne disease has been reported (1),

therefore dissemination via seed as a vector lS

improbable. Hsiang speculated that wind could have

transported mycelia into southern Ontario from the United

States (70), but this is unlikely since the pathogen is

not known to produce airborne spores.

Kerr reported that R. floccosum mycelia, introduced

into the soil, grew vigorously for a short period of time

and produced a mycotoxin that inhibited root growth of

several cereals without penetrating them (82). Endo and

his co-investigators assayed stunted roots of creeping

bentgrass (Agrostis stolonifera L.) symptomatic for dollar

spot. They isolated galactose from cultures of R.

floccosum and identified the sugar as being toxic to

bentgrasses (39,40,41,89). Couch stated that R. floccosum

can colonize such undersized roots (16).

Infection centers are distinct necrotic spots that

sink slightly and may increase in diameter to

approximately 20-35 romwhere the turf is being maintained

at a cutting height of less than 13 rom (133,141). These

circular spots reach some finite diameter and do not

enlarge further.

understood (133)

The reason for this is not well

These circles are less distinguishable

9

and may grow into diameters of approximately 150 mID. where

the turf is allowed to grow taller (121). Couch stated

that blighted areas could reach a width of nearly four

meters where the turf is mowed infrequently (16,153).

Adjacent infection centers may coalesce regardless of the

cutting height (16,141). Harman inoculated creeping

bentgrass with 27 isolates from different areas and hosts.

His efforts to re-isolate the pathogen from the hub areas

of infection centers were futile (61).

Other symptoms of the disease are small leaf-lesions

that are first chlorotic, then watersoaked, and lastly

necrotic (121). The lesions have smooth edges as opposed

to the irregularly-shaped symptoms caused by R. solani. A

necrotic band may form across the leaves that can be light

or dark, depending upon the species of the host. These

bands often have an hourglass shape (121), more so on

cool-season grasses than on warm-season grasses (30).

These bands may be outlined with a reddish-brown border.

An exception is on annual bluegrass (poa annua L.) upon

which the bands have no borders (141). Entire leaves may

become blighted and shrivel within a few days (29).

Damaged turfgrasses usually recover completely when

environmental conditions become unfavorable for pathogen

growth if an adequate supply of nitrogen and water is

10

available. This is especially true during cool weather ln

the fall. However, disease symptoms may persist

throughout the winter if turfgrass-growing conditions are

poor (133). Its symptoms can be quite unrelenting once

the disease is well established (95,124). Jackson

reported symptoms persisting on "sea marsh turf" in

western England from 1958 through 1960 (71,72,73) during

which time the winters were mild, and additions of

nitrogenous fertilizers were quite low (N. Jackson,

personal communication). Smith reported that in "sea-

marsh turf" the primary species and apparently sole host

of the disease was slender creeping red fescue (Festuca

rubra L. subsp. litoralis [Meyer] Auquir) mixed with

creeping bentgrass, annual bluegrass (poa annua L.) and

pearlwort (Sagina procumbens) (127). The disease can be

active year around on warm-season turfgrasses (91).

CONTACT FUNGICIDES FOR THE CONTROL OF DOLLAR SPOT

The first fungicide used on turf was a combination of

copper sulfate and hydrated lime known as Bordeaux

mixture. By 1919, it was generally used to control brown

patch, or "large brown patch", as the disease was then

known (95). It was ineffective on "small brown patch" and

its use on turf was abandoned later due to a phytotoxic

11

accumulation of copper in the soil (132). In 1927,

Monteith reported that mercury compounds were effective on

"small brown-patch" (93,94). A shortage of mercury during

World War II dictated the need for alternative fungicides

(96). The use of thiram on dollar spot had mixed success

in the 1940s and early 1950s (3,4,58). It is generally

believed that this era marked the real beginning of plant-

fungicide technology (65).

In 1949, the first National Cooperative Turf

Fungicide Trial was conducted in which eleven-turf

fungicides were tested at twelve locations (19). These

were located in eight states and one Canadian province.

The number of dollar spots with each treatment was counted

at trial sites in California, Iowa, Massachusetts,

Indiana, and two locations in Rhode Island. The data were

assimilated by Rowell at the Rhode Island Agricultural

Experiment Station, and the results were circulated as

twelve-page mimeographs (112). Davis interpreted these

findings as demonstrating the superiority of the cadmium-

containing products in suppressing R. floccosum (19) He

confirmed this in subsequent reports (18,20,21,22).

In 1951, Smith was appointed turfgrass pathologist at

the Sports Turf Research Institute (STRI) in Bingley,

England. Several of his annual reports demonstrated the

12

high efficacy of cadmium and mercury compounds

(123,124,126,128). The antibiotic griseofulvin was

reported as having effective, early-season control of

dollar spot (125) but less than adequate efficacy after

that (126).

Jackson replaced Smith in 1958 and continued the

fungicide-research program at STRI. Subsequently, he

reported dollar-spot control with Ortho Lawn and Turf

Fungicide, a combination product containing 66% folpet,

10% thiram, and 5% cadmium carbonate. A cadmium- and

urea- based product developed earlier by Smith (129) was

also effective (74).

Cadmium-containing fungicides were banned in the U.

K. in 1965 (132) and shortly thereafter in the U. S. (90)

due to their toxicological and carcinogenic properties.

Field research during this time showed adequate control of

dollar spot using folpet, quintozene, and three mercury-

based fungicides (75). Quintozene, or

pentachloronitrobenzene (PCNB) (150), had much better

control when applied as a spray rather than as a dust

(75) .

Cycloheximide, anilazine, and chlorothalonil became

available in the late 1960s and proved to be effective

dollar-spot fungicides (58,133). Cycloheximide is an

13

antibiotic with fungicidal properties. It was marketed as

Actidione (23) and offered acceptable dollar-spot control

up until the mid 1980s (28,45). The product registration

was cancelled in 1988 due to its high toxicity (157).

Anilazine is a substituted-aromatic triazine (151)

and one of the first organic compounds used as a fungicide

(65) . It was marketed as Dyrene by Miles Inc. and offered

broad-spectrum control of many plant diseases. The

product registration was voluntarily cancelled by the

manufacturer due to its detection in groundwater (104)

Chlorothalonil is a substituted-aromatic nitrile fungicide

(99) that is one of the most widely used fungicides in the

world.

Grace-Sierra Crop Protection voluntarily cancelled

Calo-Chlor and Calo-Gran in 1993, primarily due to

concerns about mercury bio-magnification (105). These

were the last mercury-based turf fungicides sold in the U.

S. These products were used primarily to control the snow-

mold diseases Typhula blight and Microdochium patch on

golf course tees and greens. The products could be

lawfully sold until mid 1994 (137). Use of these products

is still permitted and have been reported being commonly

used in Minnesota as recently as 1999 (42). Small

dwindling stockpiles still exist in northern Michigan and

14

surely in other locales, as well, where snowfall lS

significant.

All of the above are contact fungicides, which can

also be referred to as being multi-site fungicides because

they affect several of the pathogen's critical metabolic

pathways (78,151) Contact fungicides are also known as

protectant fungicides because when applied they form a

protective-surface barrier that inhibits spore germination

(99). Contact fungicides must be applied as prophylactics

(118) and have should adhere well to foliage (116).

Turfgrass-shoot growth occurs from the base of the plant,

and therefore an appreciable amount of any leaf surfaces

covered with a contact fungicide are removed when mowed.

Also, their exposure to light, moisture, and other

environmental factors leads to erosion and degradation of

these compounds (151). Accordingly, contact-fungicide

effectiveness is relatively short-lived.

SYSTEMIC FUNGICIDES FOR THE CONTROL OF DOLLAR SPOT

A systemic fungicide enters the plant and moves

within the plant to some degree as opposed to a contact

fungicide (99). The compound or its derivative(s) may

move short distances in pyrenchyma tissue or be

transported relatively long distances via the xylem, the

15

phloem, or both. The degree to which such movement occurs

depends upon the chemistry of the fungicide and the health

status of the plant. A systemic fungicide is no longer

exposed to environmental erosion and degradation once it

is inside the plant. Therefore, they have residual

effects that are generally two to three times longer than

the contact fungicides.

Systemic fungicides can also be referred to as being

single-site fungicides because they affect a pathogen's

metabolism by altering one vital process, or very few

closely-related processes (151). Systemic fungicides are

grouped into classes. Fungicides within a common class

use the same physiological mode of action to control

pathogens. Most systemic fungicides are fungistatic

rather that fungicidal since often the pathogen recovers

and lS able to reproduce as the chemical dissipates (10).

The first group or class of systemic fungicides, the

benzimidazoles, was introduced in 1968 (26). Products

from two more classes, the dicarboximides (108), and the

demethylation inhibitors (115) began to appear in the

1970s. They were all highly effective on a broad spectrum

of diseases and became widely used. Each of these classes

partially or completely lost their effectiveness on

16

certain pathogens over time as populations became

resistant to each of them.

THE NATURE OF FUNGICIDE RESISTANCE

A fungal strain is a group of clonally related

isolates (63). Resistance to fungicides is the heritable

ability of a strain to completely or partially overcome

the effects of single or repeated exposures to those

particular or similar chemicals (25). The resistance can

be qualitative or quantitative (51,86) In genetic terms,

qualitative resistance is when a single-major gene within

a pathogen confers complete resistance to a fungicide.

Fungal strains sensitive to a fungicide are termed

common or "wild-type" strains (26,138). Those that can

survive and reproduce under natural conditions are said to

be environmentally fit (68). The degree of this fitness

can vary, and the relative fitness of sensitive and

resistant strains is of particular importance (24,158)

Fungicide-resistant strains that can continue to exist in

competition with wild-type strains must have pathogenic

aggressiveness (141).

The presence of a fungicide, where resistant isolates

exist, effects the divergence of a population into sub-

populations consisting of either resistant or sensitive

17

strains that are distinct genotypes (88,158). This

pattern of development is termed disruptive selection

(51) . Repeated applications of the fungicide will

continue to eliminate sensitive strains and have no effect

upon the resistant ones. The use of highly effective and

persistent fungicides (25) applied with thorough coverage

(51) is the most efficient way to eliminate sensitive

strains. Further applications of such a fungicide or

another member of its class are often uneconomical when a

qualitatively-resistant strain dominates a sward (27)

A resistant isolate arises from a spontaneous

mutation (36,99). There is no definite evidence of a

systemic fungicide as a mutagenic, although Hastie

reported benomyl-induced instability with Aspergillis

nidulans Link (62). Attempts to select for resistant

isolates using repeated exposures to sub-lethal doses of

fungicides have been unsuccessful (48). Koller stated

that fungicide resistance has developed where resistant

isolates were present prior to any fungicide applications,

and that the resulting population shift was due to

fungicide selection and not to a mutagenic effect of the

toxicant itself (86).

Strains resistant to the benzimidazoles are at least

as environmentally fit as the common strains (27). The

18

resulting population shift lasts for years once fungicide

selection eliminates the sensitive strains (26), if not

permanently and irreversibly (27). A dicarboximide-

resistant strain often has reduced environmental fitness

so that dicarboximide applications can be effectively

resumed when the resistant strain dies off to the extent

that a sensitive strain again dominates the population

Quantitative resistance is controlled by multiple

genes within the pathogen (65). Resistance is not

complete but rather intermediate to some degree.

Repeated-fungicide-selection pressure favors the strains

having the greatest resistance. Disease control becomes

intermediate as well and can only be improved by either

increasing the application rate or shortening the spray

interval (51). Quantitative resistance to the DMls has

been demonstrated with many pathogens (87). This

condition is also known as reduced sensitivity as opposed

to complete resistance (99,144). This pattern of

development is termed directional selection (88).

Cross-resistance is a condition in which a pathogen

is also resistant to fungicides of the same class (141).

A benzimidazole-resistant pathogen is resistant to both

benomyl and thiophanate-methyl.

19

Iprodione and vinclozolin

are similarly ineffective on a dicarboximide-resistant

strain. Cross-resistance to the DMIs includes resistance

to triadimefon, fenarimol, propiconazole, cyproconazole,

tebuconazole, and myclobutanil. A fungicide may be

ineffective even if it has never been used before at that

particular location if cross-resistant strains are

present.

Multiresistance lS a condition in which a pathogen lS

resistant to more than one fungicide class (141).

Examples of this would be a strain that is resistant to

both the benzimidazoles and the dicarboximides, or a

strain that is resistant to the benzimidazoles and the

DMIs. The term multiresistant could be conferred to a

strain that is resistant to three or more classes as well.

RUTSTROMIA FLOCCOSUM RESISTANCE TO FUNGICIDES

In 1964, Jackson found evidence that R. floccusum was

resistant to cadmium fungicides at the Rhode Island

Agricultural Experiment Station. His subsequent field

experiments showed no control using Cadminate, a cadmium

succinate fungicide, at six times the recommended rate

(76) . Five mercury fungicides also proved ineffective.

He confirmed this cross-resistance and multiresistance

using in vitro response tests and reported it in 1966

20

(76). Widespread occurrences on the east coast were

confirmed by Cole et. al in 1968 (14).

In 1970, Nicholson investigated instances of

suspected R. floccosum resistance to anilazine. These

Warren

incidences were isolated and not widespread (101).

Nicholson (102) and Nicholson et al. (103) documented this

resistance in 1971.

The first report of benomyl-resistant strains of R.

floccusum was by Goldberg and Cole in 1973 (54)

et al. documented cross-resistance with other

benzimidazoles a year later (152). A cross-resistant,

multiresistant strain to the benzimidazoles and the

dicarboximide classes was reported by Detweiler et. al in

1983 (35). Golembiewski et al. in 1995 (56) characterized

a strain that was cross-resistant to triadimefon,

fenarimol, and propiconazole. Strains of R. floccusum

that are multiresistant to the benzimidazoles,

dicarboximides, and DMIs, and also cross-resistant within

each class, now exist (136,139,141).

Rutstroemia floccosum was the first turfgrass

pathogen reported to be resistant to a fungicide (76)

Table 1.1 lists turfgrass pathogens to which fungicide

resistance has been reported. It is of note that R.

floccusum, Microdochium nivale (Fr.) Samuels & I. C.

21

Hallett, and pyricularia grisea (Cke.) Sacco are

facultative saprophytes, and Erysiphe graminis DC is an

obligate parasite (141). These pathogen types live all or

most all of the time as parasites (2). Fungicides applied

during these times are particularly effective on the

common strains (141). This makes them easily selected

against, i. e., eliminated, so that shifts toward

fungicide-resistant populations can occur as described

earlier.

The other types of plant-pathogenic fungi are the

facultative parasites that can survive long periods and

reproduce as saprophytes when not causing disease (2).

Fungicides are less likely to be applied to control these

pathogens when they are not causing disease.

Consequently, common strains usually have ample

opportunity to repopulate an area unimpeded by fungicides.

This ability to "escape" exposure to fungicides is the

single most important factor in determining a pathogen's

propensity to develop fungicide resistance (80).

Sensitive strains have to be reduced in numbers via

directional-selection pressure caused by the use of a

fungicide class before a new strain resistant to that

class can begin to dominate the population (141).

22

The remaining two pathogens shown in Table 1 are

facultative parasites. pythium aphidermatum (Edson)

Fitzpatrick has shown resistance to metalaxyl (113).

However, less than 100 of the more than 10,000 U. S. golf

courses have been verifiably affected during more than 20

years of metalaxyl use. Most of these instances occurred

where a combination of high rates and frequent

applications were made (141). Colletotrichum graminicola

(Ces.) wils. lS a true exception, however, in that

resistance has happened after only a limited number of

applications with a QoI fungicide (5,141).

Dollar spot is the most common (16,31), expensive

(144), and economically important (33) disease to manage

on U. S. golf courses. The exception is in portions of

the Pacific Northwest where Microdochium patch is more

problematic than dollar spot (13,58). Dollar-spot

presence In Oregon and Washington was not reported until

1991 and 1992 respectively (135).

Fungicide applications are usually warranted where

dollar spot undesirably affects the functionality or

aesthetic value of a turf and environmental conditions

favorable to the development of the disease persist

(39,133,141). Dollar spot is becoming increasingly more

difficult to control (32,81), and some of the resistant

23

strains appear to be more aggressive than the sensitive

ones that they displaced (140).

Fungicide resistance to R. floccusum has forced turf

managers to stop using the benzimidazoles, to periodically

withhold the dicarboximides, to apply DMIs at increased

rates and/or reduced intervals, and to resort to greater

or sole use of a contact fungicide. It would be in the

best interest of manufacturers and end-users alike to stop

or delay the advent of fungicide-resistant R. floccusum.

The prevailing concepts are that fungicide resistance can

be managed by applying contact and systemic fungicides

either in combination or by alternating them

(16,17,119,133,146), despite a lack of research

(117,141,149). This thesis will clarify these theories as

they relate to dollar spot. Chapters 2 and 3 will detail

studies involving R. floccosum exposure to repeated

applications of DMIs, dicarboximides, and chlorothalonil,

some of which has been previously reported (52,53,114).

Chapter 4 concerns an investigation of boscalid, the first

of a new class of fungicides used on dollar spot. The

broader implications of these studies and suggested future

research are contained in Chapter 5.

24

Table 1.1. First reports of turf pathogens resistant toone or more fungicides.

Year Pathogen Fungicide Reference1966 R. floccosum1

1971 R. floccosum

1972 E. graminis2

1973 R. floccosum

1982 M. nivale3

1983 R. floccosum

1984 M. nivale

1984 P. aphidermatum4

1989 C. graminicola5

1995 R. floccosum

2001 P. grisea6

2003 C. graminicola

cadmium succinatecadmium chloridecadmium sulfatemercuric chloride

anilazine

benomyl

benomylthiabendazolethiophanate-ethylthiophanate-methyl

iprodionevinclozolin

iprodione

benomylthiophanate-methyl

metalaxyl

benomylthiophanate-ethylthiophanate-methyl

triadimefonfenarimolpropiconazole

azoxystrobintrifloxystrobin

azoxystrobintrifloxystrobin

(76)

(103)

(143 )

(54 )

(13 )

(35 )

(49 )

(113 )

(34)

(55 )

(145)

(5 )

lRutstroemia floccosum (syn. Sclerotinia homoeocarpa F.T. Bennet),causal pathogen of dollar spot

2Erysiphe graminis DC, causal pathogen of powdery mildew3Microdochium nivale (Fr.) Samuels & I. C. Hallett, causal pathogen

of Microdochium patch4pythium aphidermatum (Edson) Fitzpatrick, causal pathogen of Pythium

blight5Colletotrichum graminicola (Ces.) Wils., causal pathogen of

anthracnose6Pyricularia grisea (eke.) Sacc., causal pathogen of grey leaf spot

25

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LIBRARIES MICHIGAN STATE UNIVERSITY EAST LANSING, …archive.lib.msu.edu/tic/thesdiss/gilstrap2005a.pdf · 2006. 10. 16. · Weigel, and Dr. Terry Warren. During this ordeal I have