LIBRARIES MICHIGAN STATE UNIVERSITY EAST LANSING, MICH 48824-1048
This is to certify that the thesis entitled
STRATEGIES FOR THE MANAGEMENT OF FUNGICIDE-RESISTANT RUTSTROEMIA FLOCCOSUM (SYN. SCLEROTINIA HOMOEOCARPA), THE CAUSAL ORGANISM OF DOLLAR SPOT
presented by
DAVID MURPHY GILSTRAP
has been accepted towards fulfillment of the requirements for the
DOCTOR OF degree in PLANT PATHOLOGY PHILOSOPHY
MSU is an Affirmative Action/Equal Opportunity Institution STRATEGIES FOR THE MANAGEMENT OF FUNGICIDE-RESISTANT RUTSTROEMIA FLOCCOSUM (SYN. SCLEROTINIA HOMOEOCARPA) , THE CAUSAL ORGANISM OF DOLLAR SPOT
By
David Murphy Gilstrap
A DISSERTATION
Submitted to Michigan State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Plant Pathology
2005 ABSTRACT
STRATEGIES FOR THE MANAGEMENT OF FUNGICIDE-RESISTANT RUTSTROEMIA 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 who believed that this would 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 1 INTRODUCTION , 1 DISCOVERY OF DOLLAR SPOT AS A DISEASE OF TURFGRASSES 2 TAXONOMIC CLASSIFICATION OF THE DOLLAR-SPOT PATHOGEN 3 RUTSTROEMIA FLOCCOSUM SIGNS - DOLLAR-SPOT SYMPTOMS AND EPIDEMEOLOGY 7 THE NATURE OF FUNGICIDE RESISTANCE 17 RUTSTROMIA FLOCCOSUM RESISTANCE TO FUNGICIDES 20
CHAPTER 2 EFFECTS OF REPEATED TREATMENTS OF DEMETHYLATION-INHIBITOR (DMI) FUNGICIDES ON DMI-SENSITIVE POPULATIONS OF RUTSTROEMIA FLOCCOSUM 41 LITERATURE REVI EW 42 The Ergosterol-Biosynthesis Pathway and Sterol- Biosynthesis Inhibitors 42 DMI Fungicides - Product Development 46 DMI-Fungicide Formulations and Efficacies 52 DMI Use on Dollar Spot 53 DMI-SENSITIVE R. FLOCCOSUM STUDY 53 Agronomic Practices and Site Assessment 54 Fungicide Treatments 58 Fungicide-Application Techniques 61 Sample Collection 61 In Vitro Sensitivity Assessments and Statistical Analysis 62 Sensitivity Differentiation Using AFLP Markers 63 Results and Discussion 64
CHAPTER 3 EFFECTS OF REPEATED TREATMENTS OF DEMETHYLATION-INHIBITOR (DMI) FUNGICIDES ON DMI-RESISTANT POPULATIONS OF RUTSTROEMIA FLOCCOSUM 105 LITERATURE REVIEW 106 Human Systerns 106 Animal Systems 108 Plant Systems 110
vii Turf Systems 112 INVESTIGATION OF REPEATED FUNGICIDE APPLICATIONS TO DMI-RESISTANT R. FLOCCOSUM 113 Experimental Area and Agronomic Practices 113 Sample Collection and Dates of Sampling 114 Treatments 115 Statistical Analysis 116 Resul ts 117 Discussion 119
CHAPTER 4 INVESTIGATION OF RUTSTROEMIA FLOCCOSUM SENSITIVITY TO BOSCALID 130 LITERATURE REVIEW 131 Product Development, Registration, and Formulation 131 Mode of Action and Activity 132 Carboximide Resistance 133 LABEL DIRECTIONS AND PRECAUTIONS 134 ALARMING REPORTS 135 INITIAL ASSESSMENTS IN VITRO 136 FIELD EXPERIMENT I 140 Materials and Methods 140 Resul ts 142 FIELD-EXPERIMENT II 145 Materials and Methods 145 Results 146 BROADENED DOSE-RESPONSE STUDY IN VITRO I 148 Materials and Methods 148 Resul ts 149 BROADENED DOSE-RESPONSE STUDY IN VITRO II 150 Materials and Methods 150 Resul ts 151 DISCUSSION 152 Benzimidazole Fungicides 152 QoI Fungicides 153 DMI Fungicides 156 Dicarboximide Fungicides 160 Chlorothalonil Fungicide 161 Boscalid Fungicide 162
CHAPTER 5 CONCLUSION 185 BROADER IMPLICATIONS 186 FUTURE RESEARCH 188
APPENDIX 191
Vlll LIST OF TABLES
Table 1.1. First reports of turf pathogens resistant to one or more fungicides 25
Table 2.1. Common names, chemical names, trade names, and current registrants of demethylation-inhibitor fungicides labeled for use on commercial turf in the U. S 76
Table 2.2. Average-percent-relative growth for each block of ten subsamples collected on 29 July 1994 and mean- percent-relative growth for each treatment shown along 2 with the fungicides, rates in g 100 m- , and intervals of applications for each treatment 77
Table 2.3. Application rates and timing intervals of treatments 78
Table 2.4. Number of propiconazole applications applied in 1994, 1995, 1996, and 1997 79
Table 2.5. Propiconazole amounts in g 100-2 applied in 1994, 1995, 1996, and 1997 79
Table 2.6. Average-percent-relative growth for each block of ten subsamples (or fewer where noted) collected on 14 October 1994 and mean-percent-relative growth for each treatment shown along with the fungicides, rates in g 2 100 m- , and intervals of applications for each treatment 80
Table 2.7. Average-percent-relative growth for each block of ten subsamples (or fewer where noted) collected on 14 July 1995 and mean-percent-relative growth for each treatment shown along with the fungicides, rates in g 2 100 m- , and intervals of applications for each treatment 81
Table 2.8. Average-percent-relative growth for each block of ten subsamples collected on 20 October 1995 and mean- percent-relative growth for each treatment shown along
lX 2 with the fungicides, rates in g 100 m- , and intervals of applications for each treatment 82
Table 2.9. Average-percent-relative growth for each block of ten subsamples collected on 12 July 1996 (or fewer where noted) and mean-percent-relative growth for each treatment shown along with the fungicides, rates in g 2 100 m- , and intervals of applications for each treatment 83
Table 2.10. Average-percent-relative growth for each block of ten subsamples collected (or fewer where noted) on 1 August 1997 and mean-percent-relative growth for each treatment shown along with the fungicides, rates in 2 g 100 m- , and intervals of applications for each treatment 84
Table 2.11. Average-percent-relative growth for each block of ten subsamples collected (or fewer where noted) on 21 August 1998 and mean-percent-relative growth for each treatment shown along with the fungicides, rates in 2 g 100 m- , and intervals of applications for each treatment 85
Table 2.12. Treatment means of percent-relative-growth values of four replications collected on 20 October 1995 86
Table 2.13. Treatment means of percent-relative-growth values of four replications collected on 1 August 1997 87
Table 2.14. Treatment means of percent-relative-growth values of four replications collected on 21 August 1998 88
Table 2.15. Number of chlorothalonil applications applied in 1994, 1995, 1996, and 1997 89
Table 2.16. Chlorothalonil amounts in g 100 m-2 applied in 1994, 1995, 1996, and 1997 9a
Table 2.17. An example of application rates and timing intervals of treatments for a study that would have had greater efficiency than the one conducted and presented in Chapter 2 91
x Table 3.1. Treatments with fungicides, rates, and intervals 121
Table 3.2. Mean-percent-relative-growth values of six treatments with three blocks at five observations with percent increase of final observation compared to first observation 122
Table 3.3 .. Slopes of linear trends over time by treatments and associated P-values 123
Table 4.1. Application rates and timing intervals of treatments 166
Table 4.2. Mean-dollar-spot counts of seven treatments with six fungicides representing different fungicide classes at five days after treatment (DAT) with least- significant-difference (LSD) comparisons (P ~ 0.05) of base-10 log-transformed counts 167
Table 4.3. Mean-dollar-spot counts of seven treatments with six fungicides representing different fungicide classes at 21 days after treatment (DAT) with least- significant-difference (LSD) comparisons (P ~ 0.05) of base-10 log-transformed counts 168
Table 4.4. Mean-percent-relative-growth values of fifteen isolates in three replications grown on PDA only and PDA amended with 40 pg ml-1 boscalid 169
Table 4.5. Percent-mean-relative-growth values of eight isolates grown on PDA only and PDA amended with three sets of different single-site fungicides with three replications 170
Table 4.6. Mean-percent-radial-growth values from three experiments at the Lochmoor Club from 1991-2004 with no statistical inferences noted or implied 171
Table 4.7 Mean-percent-relative-growth values of eight treatments with three replications at ten concentrations of bo scal id 172
Xl LIST OF FIGURES
Figure 2.1. Map of Block 4 with treatment numbers above 2 or below each 2.1 m by 8.4 m (17.6 m ) plot with 1.2 m 2 by 3 m (3.6 m ) sampling areas outlined and mowing directions indicated by parallel arrows in opposite directions 92
Figure 2.2. Mean-percent-relative growth of three treatments with 4 replications at 3 observation dates with each treatment applying chlorothalonil annually ..93
Figure 3.1. Treatment means for percent-relative growth by 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-percent relative-growth measurements of three replicates of isolate LF-7 at seven concentrations of boscalid .....173
Figure 4.2. Regression line of three replications of mean-percent-relative-growth measurements of isolate LF- 7 against the base-10 logarithms of five-fungicide concentrations ranging from 5 to 40 pg ml-1 173
Figure 4.3. Dollar-spot counts of the means of seven treatments with 4 replications (Field-Experiment I) ..174
Figure 4.4. Mean-dollar-spot counts of the means of three treatments with four replications at nine observations (Field Experiment 11) 175
Figure 4.5. Mean-dollar-spot counts of the means of three treatments 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 ten concentrations of boscalid expressed as base-10 logarithms 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. The reason for this is not well understood (133) 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. Iprodione and vinclozolin
19 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 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) Warren 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 to one or more fungicides. Year Pathogen Fungicide Reference
1 1966 R. floccosum cadmium succinate (76) cadmium chloride cadmium sulfate mercuric chloride
1971 R. floccosum anilazine (103)
1972 E. graminis2 benomyl (143 )
1973 R. floccosum benomyl (54 ) thiabendazole thiophanate-ethyl thiophanate-methyl
1982 M. nivale3 iprodione (13 ) vinclozolin
1983 R. floccosum iprodione (35 )
1984 M. nivale benomyl (49 ) thiophanate-methyl
4 1984 P. aphidermatum metalaxyl (113 )
1989 C. graminicola5 benomyl (34) thiophanate-ethyl thiophanate-methyl
1995 R. floccosum triadimefon (55 ) fenarimol propiconazole
2001 P. grisea6 azoxystrobin (145) trifloxystrobin
2003 C. graminicola azoxystrobin (5 ) trifloxystrobin
lRutstroemia floccosum (syn. Sclerotinia homoeocarpa F.T. Bennet), causal pathogen of dollar spot 2Erysiphe graminis DC, causal pathogen of powdery mildew 3Microdochium nivale (Fr.) Samuels & I. C. Hallett, causal pathogen of Microdochium patch 4pythium aphidermatum (Edson) Fitzpatrick, causal pathogen of Pythium blight 5Colletotrichum graminicola (Ces.) Wils., causal pathogen of anthracnose 6Pyricularia grisea (eke.) Sacc., causal pathogen of grey leaf spot
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