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Screening Genotypes of Bahiagrass (Paspalum notatum) for Resistance to Dollar Spot (Sclerotinia homeocarpa) Development

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Screening Genotypes of Bahiagrass (Paspalum notatum) for Resistance to Dollar Spot (Sclerotinia homeocarpa) Development
Creator:
WILLIAMS, BRANDY NICOLE
Copyright Date:
2008

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Ascomycota ( jstor )
Breeding ( jstor )
Diploidy ( jstor )
Diseases ( jstor )
Grasses ( jstor )
Inoculation ( jstor )
Lesions ( jstor )
Rice ( jstor )
Symptomatology ( jstor )
Tetraploidy ( jstor )
City of Pensacola ( local )

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University of Florida
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University of Florida
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Copyright Brandy Nicole Williams. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2006
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495636475 ( OCLC )

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SCREENING GENOTYPES OF BAHIAGRASS ( Paspalum notatum ) FOR RESISTANCE TO DOLLAR SPOT ( Sclerotinia homeocarpa ) DEVELOPMENT By BRANDY NICOLE WILLIAMS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Brandy Nicole Williams

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I dedicated this thesis to my parents, Bill and Denise Williams for their unselfish contributions and their faith in my ability to complete the requirements necessary to earn a Master of Science. Without their love a nd support, I would not have been able to accomplish my goals.

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ACKNOWLEDGMENTS I would like to thank my committee members, Dr. Rob Gilbert and Dr. Jim Miller, for their encouragement and suggestions throughout my research and the writing of this thesis. I would also like to thank my committee member, Dr. Lawrence Datnoff, for providing the use of his laboratory equipment and his time in initiating and developing an inoculation technique, which was a key component of my research. I would also like to thank Dr. Ken Quesenberry, the cochair of my committee, and Dr. Ann Blount, the chair of my committee, for their support and encouragement not only in completing my research and writing this thesis, but also in pursuing my educational and personal goals. A special word of thanks is due to my parents, Bill and Denise Williams, for their unwavering love, dedication and faith in my abilities. I would also like to acknowledge my sister, Rachel, who assisted me in editing this thesis and other presentations based on my research. I thank Micaela, my niece, for all her curiosity and frank questions that only a child would ask. I greatly appreciate my Uncle Tom for sharing his love and appreciation of the outdoors and wildlife. I would like to thank all my family for their understanding and encouragement throughout the pursuit of my education. I would like to acknowledge one very special person, Judy Dampier. She went above and beyond the call of duty and helped me to focus and push through especially towards the end. I also would like to mention three special friends who always had suggestions and words of encouragement, Aaron Hert, Matt Brecht, and Robin Oliver. iv

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Finally I would like to acknowledge a very special person in my life, Scott Millard. He has not only helped to edit this thesis but also spent his vacation helping to collect data in my greenhouse studies. He has been my rock throughout the highs and lows of my research, the writing of this thesis, and my life. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ...........................................................................................................viii LIST OF FIGURES ...........................................................................................................ix ABSTRACT .........................................................................................................................x CHAPTER 1 LITERATURE REVIEW.............................................................................................1 Bahiagrass (Paspalum notatum)...................................................................................2 Distribution in Southeastern United States............................................................2 Agronomic Attributes............................................................................................3 Introduction of Cultivars.......................................................................................4 Current Breeding Objectives.................................................................................6 Breeding Techniques.............................................................................................7 Foliar Plant Diseases.....................................................................................................8 Epidemiology........................................................................................................8 Isolation and Culture.............................................................................................9 Inoculation...........................................................................................................10 Dollar Spot (Sclerotinia homeocarpa)........................................................................10 Symptomology....................................................................................................11 Epidemiology......................................................................................................11 Inoculation...........................................................................................................13 Objectives of This Research.......................................................................................13 2 EVALUATION OF BAHIAGRASS UNDER NATURAL FIELD INFECTION....14 Introduction.................................................................................................................14 Materials and Methods...............................................................................................17 Experiment I........................................................................................................17 Experiment II.......................................................................................................18 Experiment III.....................................................................................................18 Experiment IV.....................................................................................................19 Results and Discussion...............................................................................................20 Experiment I........................................................................................................20 vi

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Experiment II.......................................................................................................22 Experiment III.....................................................................................................26 Experiment IV.....................................................................................................28 Conclusions.................................................................................................................30 3 PROTOCOL DEVELOPMENT FOR INOCULATION WITH SCLEROTINIA HOMEOCARPA.........................................................................................................32 Introduction.................................................................................................................32 Materials and Methods...............................................................................................33 Isolation and Culture...........................................................................................33 Preliminary Experiments.....................................................................................34 Inoculation Experiment.......................................................................................36 Results and Discussion...............................................................................................37 Isolation and Culture...........................................................................................37 Preliminary Experiments.....................................................................................37 Inoculation Experiment.......................................................................................39 Conclusions.................................................................................................................42 4 CONCLUSIONS........................................................................................................44 Field Evaluations........................................................................................................44 Greenhouse Evaluations.............................................................................................44 APPENDIX: INNOCULATION EXPERIMENT DATA.................................................46 LIST OF REFERENCES...................................................................................................51 BIOGRAPHICAL SKETCH.............................................................................................55 vii

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LIST OF TABLES Table page 2-1. ANOVA table for the bahiagrass nursery in Marianna, FL.......................................22 2-2. ANOVA table for the bahiagrass RCB experiment, Marianna, FL...........................24 2-3. ANOVA for the AUDPC’s of the epidemiology study in Marinanna......................28 2-4. Mean vigor rating and plant height for the sward epidemiology study .....................28 2-5. Pearson’s correlation table relating agronomic traits to dollar spot severity............28 2-6. ANOVA for the AUDPC’s of the clonal epidemiology study..................................30 3-1. Lesion expansion rate, AUDPC, and the R 2 values...................................................40 A-1. Data for greenhouse experiment, first run................................................................47 A-2. Data for greenhouse experiment, second run...........................................................49 viii

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LIST OF FIGURES Figure page 2-1. The bahiagrass nursery, Marianna, FL.....................................................................21 2-3. The bahiagrass RCB experiment, Marianna, FL.......................................................23 2-3. Monthly maximum and minimum temperatures.......................................................24 2-4. Monthly rainfall.........................................................................................................25 2-5. Disease progress curves for the epidemiology study................................................27 2-6. AUDPC’s for epidemiology study...........................................................................27 2-7. AUDPC’s for the clonal epidemiology study............................................................29 3-1 Dollar spot lesion development with an infested rice grain......................................39 ix

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SCREENING GENOTYPES OF BAHIAGRASS (Paspalum notatum ) FOR RESISTANCE TO DOLLAR SPOT ( Sclerotinia homeocarpa ) DEVELOPMENT By Brandy Nicole Williams August 2005 Chair: A. R. Blount Cochair: K. H. Quesenberry Major Department: Agronomy Bahiagrass (Paspalum notatum Flugge and P. notatum Flugge var. saure Parodi) is the predominant pasture grass species in Florida. In the United States, there are an estimated 5.5 million hectares of bahiagrass. In Florida alone, it is estimated that there are 2.5 million hectares. “Pensacola” bahiagrass, a diploid type (2x), occupies most of the acreage in the southern Coastal Plain. Sclerotinia homoeocarpa F.T. Bennett causes dollar spot, a leaf and crown disease of many turfgrasses. Disease symptoms include necrosis of the leaves, and in severe cases may result in the death of the entire plant. While outbreaks of dollar spot have been found in Florida, dollar spot disease has not been reported to be an extensive problem of pastures of bahiagrass or bermudagrass. However, in the spring and summer of 2001, a severe outbreak of dollar spot was reported on bahiagrass pastures in the x

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Florida panhandle with areas in the most severely affected pastures showing up to 95% plant loss. The objectives of this research were to 1) evaluate field response of bahiagrass cultivars and clones to dollar spot, and 2) establish a greenhouse methodology to reproduce the disease symptoms and select among bahiagrass genotypes for differences in response to the fungal disease. The response of ten bahiagrass cultivars and breeding lines to dollar spot under natural field infection conditions were tested at Marianna, FL, in 2003 and 2004. Standard fungal culture techniques were used to isolate and produce inoculum for greenhouse studies. Infested rice grains were used as the inoculum source on healthy plants maintained in a humidity chamber in the greenhouse. In three field experiments, there were consistent differences among bahiagrass populations and individual clones in response to dollar spot. Disease severity varied among years and may be related to different environmental conditions. The tetraploid bahiagrass cultivars had less disease severity than the diploid bahiagrass cultivars. The causative organism was isolated from diseased tissue, easily maintained in pure culture, used to inoculate greenhouse plants, and shown to induce the same disease symptoms, so proving Koch’s Postulates. A methodology using infested rice grains proved to be the most consistent inoculation technique. Although disease symptoms induced in the greenhouse were similar to those observed in the field, no differences in number of disease symptoms or in progression of disease was detected among the bahiagrass clones tested. Further research on conditions for development and spread of dollar spot on bahiagrass is needed. xi

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CHAPTER 1 LITERATURE REVIEW Bahiagrass (Paspalum notatum Flugge) is the predominant pasture grass species in Florida (Chambliss, 1996). This grass, originally from South America, is well adapted to the southern Coastal Plain region of the United States. Bahiagrass is widely grown and is used for grazing, hay, sod and seed production. In the United States, there are an estimated 2.2 million hectares of bahiagrass (Burton et al., 1997). In Florida alone, it is estimated that there are 1 million hectares (Chambliss, 1996). Bahiagrass is drought tolerant due to its deep fibrous root system, and its vast stolon and root system aids in regrowth of its foliage when over-grazed by livestock or when leaves die back due to cold weather. It produces moderate forage on low fertility soils, establishes easily, and has few pests or diseases, which explains its popularity with ranchers. Cultivar improvement in bahiagrass has been limited because of the apomictic nature of most bahiagrass germplasm collected in South America. Apomixis is a condition in which the seed of a plant are a clone of the mother plant. This apomitic condition limited early genetic manipulation efforts by plant breeders. ‘Pensacola’ bahiagrass reproduces sexually and is diploid. Pensacola bahiagrass occupies most of the acreage in the southern Gulf Coast, making it the dominant cultivar of bahiagrass in the southern Coastal Plain (Burton et al., 1997). Sclerotinia homoeocarpa F.T. Bennett. causes dollar spot, a leaf and crown disease of many turfgrasses. Disease symptoms include necrosis of the leaves and, in severe cases, may result in the death of the entire plant. Dollar spot typically develops when 1

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2 weather is mild and accompanied by frequent and prolonged rainy periods (Atilano et al., 1986: Freeman and Simone, 1988). This disease is found in North and Central America, Europe, and Australia. Dollar spot has a wide host range which includes warm and cool season, annual and perennial grasses, and is a well-known disease of turfgrasses (Atilano et al., 1986). Bahiagrass is also susceptible to dollar spot (Atilano et al., 1986). While outbreaks of dollar spot have been found in Florida, dollar spot has not been reported to be an extensive problem of pastures of bahiagrass or bermudagrass. However, in the spring and summer of 2001, there was a severe outbreak of dollar spot on bahiagrass pastures in the Florida panhandle. Most of the pastures with reported symptoms were planted with the cultivars Tifton 9 and Pensacola, but several Argentine bahiagrass fields were also mildly affected. Pasture symptoms ranged from mild tip necrosis of the leaves, to significant leaf death, and, in several cases, plant death. Severity was attributed, in part, to plant cultivar susceptibility. This led to the first publication documenting severity up to 95% plant loss in the most severely affected pastures (Blount et al., 2002). Bahiagrass (Paspalum notatum) Distribution in Southeastern United States Bahiagrass is an important warm-season perennial grass grown throughout Florida, the Coastal Plain and Gulf Coast regions of the southern United Sates (Chambliss, 1996). In Alabama, there are 1,340,000 estimated hectares of permanent pasture grasses, of which there are 896,000 hectares of perennial warm-season grasses of which 405,000 hectares are in bahiagrass. In Georgia, there are 919,000 estimated hectares of permanent improved pastures of which 202,000 hectares are in bahiagrass (Blount, 2000).

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3 In the state of Florida grasslands are estimated at 4,650,000 hectares. Pastureland accounts for 2,600,000 of those hectares, and is comprised of 1,200,000 hectares of native rangeland, and 1,400,000 hectares of improved pasture. It is estimated bahiagrass comprises about 70% of the 1,400,000 hectares of improved pasture. There is no recent available information on forages pertaining to hectares of individual plant species utilized for forage (Blount, 2000). Agronomic Attributes Bahiagrass has adapted well to the climate in Florida and the surrounding areas. It performs well when grown in a variety of soils. Bahiagrass grows on upland well-drained sands, as well as moist, poorly drained flatwoods soils, typical of peninsular Florida (Chambliss, 1996). It produces moderate yields on soils of very low fertility, with minimal fertilization (Chambliss, 1996). It is often used in crop rotation because it is known to suppress many plant parasitic nematodes and soil-borne diseases. Nearly all of the bahiagrass grown for seed is produced in the southern United States and supports a very viable bahiagrass seed industry (Chambliss, 1996). There are a few disadvantages associated with bahiagrass including seasonal forage shortages and certain disease and pest problems (Mislevy, 2001). Bahiagrass provides abundant forage of adequate nutritional value, for certain classes of livestock, from late spring through early autumn (Blount, 2000). Bahiagrass produces 86% to 90% of its annual yield between April and September, with only 10% to 14% of the total yield produced during the winter months (October-March) (Gates et al., 2001). This leads to a feed shortage in the late fall and early winter months. One of the goals of the University of Florida bahiagrass breeding program is to select for cultivars with improved late season forage production, and improvements have been accomplished in this area (Blount

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4 et al., 2001). Other than dollar spot, few pests or diseases cause significant problems on bahiagrass. Damage by the tawny mole cricket (Scapteriscus vicinus Scudder) and white grub (Leucopholis irrorata Chevrolat) have been occasional problems particularly in south Florida (Blount et al., 2001). Introduction of Cultivars Bahiagrass is native to South America and is widely distributed throughout Argentine, Uruguay, Paraguay, and Brazil (Chambliss, 1996). ‘Common’ bahiagrass was introduced into Florida in 1913 by the Florida Agricultural Experiment Station. It was thought to be a potentially improved pasture plant in Florida because it could be easily established on sandy soils. Common bahiagrass plants consist of short, broad, pubescent leaves. However, it was slow to establish, low in productivity and sensitive to the cold temperatures which sporadically occur throughout the state during the winter period and therefore, it was never adopted by Florida ranchers (Mislevy and Quesenberry, 1999). ‘Argentine’ bahiagrass (PI. no. 148996) was introduced into Florida in 1945 by the USDA and was released to producers in 1951(Killinger et al., 1951). This cultivar is a semi-erect tetraploid with wide leaves (Mislevy and Quesenberry, 1999). Leaves can range from glabrous to pubescent depending on the age of the plant, stage of growth, management and environment. It is not as cold tolerant as other bahiagrass and does not produce as much forage in late fall or early spring. It is popular in the sod industry because it has fewer seed heads. Argentine bahiagrass seed heads are affected by ergot (Claviceps paspali Stevens and Hall) which is toxic to cattle, and Fusarium spp. often is present on the seed heads. ‘Paraguay 22’ bahiagrass (PI no. 158822) was released in 1942 (McCloud, 1953). It has short, narrow, hairy leaves and is a much greater seed

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5 producer than common Bahiagrass. It is similar to Argentine in growth habit and cold tolerance, but unlike Argentine, it is reported as resistant to ergot (McCloud, 1953). Pensacola bahiagrass (PI no. 422024) was first distributed to producers 1943. Pensacola bahiagrass is believed to have originated in Argentina and was brought to Florida in the digestive tract of cattle shipped from the Sante Fe region of Argentina, to the Perdido dock near Pensacola. It was found growing in 1935 near the docks in Pensacola, FL, by the County Agricultural Agent, E. H. Finnlayson, who popularized the grass. It is a sexual diploid, is propagated by seed, and is more cold-tolerant than the more common apomitic types. Pensacola bahiagrass has long narrow leaves, taller seed stalks, and it flowers earlier than other cultivars. It has a fibrous root-system capable of growing to a depth of 7 feet or more. While Pensacola bahiagrass has some cold tolerance, top growth is killed by moderate frost. The grass has become the predominate cultivar grown in Florida. Pensacola can be used as a pasture grass for hay production (Killinger, 1959). Beginning in 1960, Dr. Glen Burton at the USDA-ARS Coastal Plain Experiment Station (CPES) at Tifton, Georgia used restricted recurrent phenotypic selection (RRPS) to develop improved bahiagrass populations from the diploid cultivar Pensacola. Tifton 9 (PI no. 531086), resulted from nine cycles of RRPS, is higher yielding than Pensacola and was released in 1987 (Burton, 1989). Tifton 9 has greater seedling vigor, develops longer leaves, and is 30% higher yielding than Pensacola. Eighteen cycles of RRPS breeding material resulted in the experimental population RRPS Cycle 18, which is higher yielding than both Pensacola and Tifton 9 but was not released by CPES because it lacked grazing persistence (Mislevy and Quesenberry, 1999).

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6 Wilimington is a tetraploid type with purported improved winter survival. It has narrow leaves of medium size. It is less productive than Pensacola bahiagrass and is a poor seed producer. Auburn University recently released “AU Sand Mountain” bahiagrass. AU Sand Mountain is the result of a natural selection in a plant introduction thought to have been planted at the Sand Mountain Substation some 30 years ago. This cultivar has narrow leaves, fine tillers and a short inflorescence. In the northern part of Alabama, this new cultivar has yielded more than Tifton 9 (Mislevy and Quesenberry, 1999). Current Breeding Objectives The current effort undertaken at the University of Florida is a multidisciplinary approach to bahiagrass improvement. This team emphasizes plant improvement in seedling vigor and establishment, cold tolerance, photoperiod response, seasonal distribution of forage production, forage quality, nematode and disease resistance. (Blount, et al., 2001) Increased forage digestibility and high yielding performance under heavy grazing pressure are also two other areas of program focus (Blount et al., 2001). The Crop Genetics and Breeding Research Unit at Tifton, Georgia is currently working to reduce the bahiagrass seed hardness and dormancy, and to increase early germination, seedling vigor and rapid stand establishment. For most forage production systems, control of foliar diseases by fungicides is not economically feasible, and the use of resistant grass cultivars is the most logical method of disease control (Waddington et al., 1992). A successful plant breeder has to be able to identify superior plants within a large, variable population. One method of identifying superior plants when dealing with a disease problem would be to inoculated and evaluate seedlings in the greenhouse. Generally, a grass resistant to a disease in the seedling stage

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7 will retain the resistance throughout its life cycle (Waddington et al., 1992). Efficient methods of inducing infection and disease development are key to a successful screening program. Pathogenic variation must be considered for an accurate evaluation of cultivar resistance (Cole et al., 1969). A selection may have considerable disease resistance in one location and limited resistance elsewhere, presumably due to pathogen strain differences (Cole et al., 1969). The most promising control strategy is to breed for resistance. Heritability is the proportion of the observed variation in a progeny that is inherited. High heritability of a particular trait implies that the trait can be rapidly improved with less intensive evaluation (Nyquist, 1991). Broad-sence heritability estimates include all genetic effects (additive, dominance, and epistatic) and can be calculated from variance components among clonal replicates by an analysis of variance (Poehlman and Sleper, 1995). Broad-sence heritability estimates are useful in determining the selection efficiency (Poehlman and Sleper, 1995). Burton and Devane (1953) devised a method to estimated broad-sence heritability from replicated clonal material that would separate the variation observed in a segregating population into genetic, environmental and genetic x environmental components. Steel and Torrie (1960) describe variance components and together with Burton and Devane’s (1953) method the broad-sence heritability estimates for field studies and greenhouse studies can be calculated. Breeding Techniques Naturally cross-pollinated grasses are usually heterozygous and are well suited to population breeding methods, such as mass selection. Frequently, such grasses carry a self-incompatibility mechanism that causes individual plants to be self-sterile but cross-fertile. Self-incompatibility allows for the production of hybrids without emasculation

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8 simply by bringing together two self-sterile, cross-fertile genotypes in the flowering stage. Floral induction is influenced by genetic and environmental conditions. Near the equator in South America, where a uniform 12-h day occurs, certain cultivars of bahiagrass rarely produce seedheads and are considered excellent turfgrasses (Waddington et al., 1992). When given a 13 to 14-h day, these grasses flower profusely throughout the long-day season. Cultural practices to induce flowering include timed burning of pastures, sod disturbance (disking), or fertilization timing (Waddington et al., 1992). With adequate fertilization, particularly with nitrogen, can induce heavy, prolonged flowering. Temperature also has a significant effect on floral induction (Waddington et al., 1992). Sexual, diploid bahiagrass may be crossed with other plants of its own polidy. Flowering culms may be placed in porous bags prior to anthesis. The bags must be porous enough to allow for moisture and gaseous interchange but tight enough to prevent pollen penetration. Because the culms are very fragile and can not support the weight of the bags, some mechanical support may be used such as wire flags or bamboo stakes. Culms may be cut close to the soil surface, placed immediately in tap water, and kept in the water until seeds mature. Self-incompatibility allows for the production of hybrids without emasculation simply by placing self-sterile, cross-fertile genotypes together at anthesis. Shaking the flowering culms daily (morning) will favor pollen movement and increase the number of hybrids produced (Waddington et al., 1992). Foliar Plant Diseases Epidemiology Evaluation of disease severity is a source of controversy in almost all areas of plant pathology. One must try to balance the time required against the accuracy of the

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9 evaluation. The complexity of evaluation methods must be justified by the desired results. The health and vigor of turf is usually evaluated on its aesthetic appearance, rate of leaf expansion, and root/rhizome mass. Epidemic evaluations are rated as percentage of area with foliar symptoms. The percent of necrotic foliage in a plot is sometimes rated using the Horsfall-Barratt rating scale or a modified Cobb scale (Waddington et al., 1992). Disease progress curves have been used to describe these epidemics using mathematical models to develop epidemiological theories and for use in disease forecasting. An epidemic is a biological process (a series of temporally and spatially related events) while symptoms are an instantaneous display of that epidemic. Monitoring epidemic development requires that important field data be collected. The pattern, number, density, spatial arrangement, and the size of the diseased areas, as well as the nutrient status and the age of the turf must be recorded. Instantaneous data describing the physical environment at the time of observation and preceding symptom appearance should be collected. Temporal data should be recorded for soil moisture, soil temperature, pesticide and fertilizer amendments, and cultivation practices. Disease severity is an instantaneous measure of disease, but not sufficient measure of an epidemic (Waddington et al., 1992). In general, disease problems develop due to changes in cultural practices, unusual weather conditions, or the genetic constitution of the grass plant. Nutrient levels are often important in establishing disease in field plots. For example, dollar spot is more pronounced at low nitrogen fertility levels (Waddington et al., 1992). Isolation and Culture Typically, the fungi that infect turfgrasses are quite easily isolated and grown in pure culture on standard nutrient media (Waddington et al., 1992). Because some fungi

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10 may change genetically or lose pathogenicity after repeated subculturing, it is useful to maintain original cultures in long-term storage as a reference collection (Waddington et al., 1992). Repeated subculturing of fungi in agar or liquid media can result in reduction or loss of aggressiveness or pathogenicity (Waddington et al., 1992). The pathogenicity and aggressiveness of an isolate can be maintained or restored by periodic inoculation and reisolation of the fungus from a host plant. (Waddington et al., 1992) Inoculation There have been several reports of infesting sterile grain, such as rye or oats, with the pathogen and then using a quantified amount of seed to inoculate potted plants or field plots. The inoculum can be scattered either whole or fragmented into the turf. The efficiency of the inoculum is increased by placing it at the infection court and protecting it from climatic exposure. Other reports suggested using sand, clay, or diatomaceous earth impregnated with molasses as a nutrient medium (Waddington et al., 1992). Dollar Spot (Sclerotinia homeocarpa) In Australia, North and Central America, and continental Europe a wide range of turf grasses are affected by dollar spot. Bennett (1937) examined isolates from British, American and Australian sources, and showed that the fungus existed in several distinct strains which differed in their capacity for conidial production in culture. The American and Australian isolates that he studied apparently were sterile but among the British isolates one strain yielded ascospores and conidia (Smith et al., 1989). Bennett (1937) identified the pathogen responsible for dollar spot as Sclerotinia homeocarpa. However, the inclusion of this pathogen in the genus Sclerotinia has been refuted based on examinations of apothecial anatomy (Jackson, 1937), stromatal anatomy, stromatal histochemistry (Kohn and Grenville, 1989: Novak and Kohn, 1991),

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11 and nuclear ribosomal internal transcribed spacer region 1 sequence data (Carbone and Kohn, 1993). The pathogen identified as S. homeocarpa is currently believed to belong to either the genus Lanzia, Moellerodiscus, or Rutstroemia (Carbone and Kohn, 1993). It has been suggested that dollar spot may not be caused by a single species, but rather by multiple species, or by a complex of species (Jackson, 1973: Kohn, 1979: Smith et al, 1989). A study by Powell and Vargas (2001) concluded that dollar spot is caused by a single species. Symptomology Dollar spot has been reported to be the most destructive disease of closely mowed turf, especially creeping bentgrass (Burton, 1989). Symptoms appear as bleached spots about the size of a silver dollar that may be so numerous that individual spots overlap to produce large irregular areas of sunken dead turfgrass (Burton, 1989). On higher-cut turfs the spots may be 4 to 6 inches in diameter (Burton, 1989). Individual leaves at the periphery of the spots typically have straw-colored bands across the blades with reddish brown borders (Burton, 1989). In many cases, the appearance of typical field symptoms requires a combination of environmental stresses involving nutrients, temperature, moisture and a complex interaction among pathogens, hosts, and other microorganisms (Waddington et al., 1992). Epidemiology The incidence of dollar spot disease shows a seasonal fluctuation in most years with most new infections occurring in the late spring and early summer, and again in autumn. From the onset of primary symptoms, build up of the disease may be rapid and once established it is quite persistent. Dollar spot incidence may be related to the presence of thatch. The fungus may persists in the thatch and be the source of inoculum

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12 when conditions are favorable contributing to seasonable outbreaks (Smith et al., 1989). Transport of infected plant parts serves to spread the disease to newly established turf areas. The fungus gains entry into the plant via cut leaf tips and through stomates (Waddington et al., 1992). Appressorial formation has been observed and these structures may aid in stomatal penetration. The fungus appears to produce a toxin that caused root necrosis on the seedlings of several grass species. The same type of root damage was observed in the field on turf with well-established foliar symptoms of dollar spot (Smith et al., 1989: Waddington et al.,1992). Cultural practices can be very effective for the management of dollar spot in turf. This disease is most severe on inadequately fertilized turfgrasses; besides regular fungicide use on greens, a major control measure is the implementation of an adequate nitrogen fertilization program (Turgeon, 2002). A suppression of dollar spot can be achieved when nitrogen is applied to turf (Cook et al., 1964; Markland et al., 1969; Watkins and Wit, 1995). Couch (1995) suggests that this is because nitrogen allows the grass to grow more quickly; and therefore the grass can be mowed more frequently which in turn removes more necrotic tissue. There is some indication that the nitrogen source may also influence the development of dollar spot. Several studies reported that when activated sewage sludge was used there was less evidence of symptoms as compared to the equivalent amount of inorganic nitrogen (Turgeon, 2002). The extra benefit obtained from the complex organic fertilizer was not explained. Other major turf nutrients, in particular phosphorous and potassium have little documented influence on dollar spot (Smith et al., 1989). Turf admendments containing microorganisms and nitrogen have

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13 shown promise for management of dollar spot; however, the role of microbes or nitrogen, or both, to disease suppression requires clarification (Walsh et al., 1999). Inoculation A method used by Cole (1969) suggested that the isolates of S. homeocarpa could be identified on the basis of mycelial characteristics and lesion induction on grass plants. They reported that none of the isolates produced conidia, hence they were maintained on potato dextrose agar under sterile mineral oil as mass transfers of mycelium. Cole and co-authors transferred the four S. homeocarpa isolates to autoclaved whole rye grain. The plot area was mowed immediately prior to inoculation and was maintained at a 2.5 cm cutting height throughout the experimental period all clippings were removed. Their plots were inoculated by distributing by hand the infected rye over the plot area. After inoculation, the plot area was irrigated for 10 min of each daylight hour during the next four days (Cole et al., 1969). Objectives of This Research This proposed research supports the efforts to improve bahiagrass response to dollar spot as part of the overall strategy to develop new cultivars of diploid bahiagrass. This research focuses on the response of the various cultivars and experimental breeding lines of bahiagrass to dollar spot development. The two specific objectives of this research were to 1) evaluate field response of bahiagrass cultivars and clones to dollar spot, and 2) establish a greenhouse methodology to reproduce the disease symptoms and select among bahiagrass genotypes for differences in response to dollar spot development.

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CHAPTER 2 EVALUATION OF BAHIAGRASS UNDER NATURAL FIELD INFECTION Introduction Bahiagrass (Paspalum notatum Flugge) is the predominant pasture grass found throughout the Southern Coastal Plain Region of the United States. While bahiagrass is native to several South American countries (Argentine, Brazil, Paraguay, and Uruguay), it has become widely naturalized to the Southern Coastal Plain Region. Bahiagrass is used for grazing, as a hay and sod crop, and for seed production (Chambliss, 1996). It produces well on low fertility soils, is easy to establish, and is drought tolerant, which makes it ideal for Florida’s sandy soils (Chambliss, 1996). Several cultivars of bahiagrass are grown in the United States, with ‘Pensacola’ and ‘Argentine’ being the two dominant cultivars (Chambliss, 1996). Several cultivars are grown in Florida. Argentine bahiagrass (PI no. 148996) was introduced into Florida in 1945 by the USDA and was released in 1951 (Killinger et al., 1951). It is popular in the sod industry because it has few seed heads. ‘Paraguay 22’ bahiagrass (PI no. 158822) was released in 1942 (McCloud, 1953). It is similar to Argentine in growth habit and cold tolerance. Pensacola bahiagrass (PI no. 422024) was first distributed to producers in 1943. Pensacola bahiagrass is believed to have originated in Argentina and was brought to Florida in the digestive tract of cattle shipped from the Sante Fe region of Argentina, to the Perdido dock near Pensacola. It was found growing in 1935 near the docks in Pensacola, FL by the County Agricultural Agent, E. H. Finnlayson, who popularized the grass. The grass became widely used as a pasture and 14

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15 meadow grass as well as for hay production in Florida (Killinger, 1959). It is a sexual diploid propagated by seed. Beginning in 1960, Dr. Glen Burton at the USDA-ARS Coastal Plain Experiment Station (CPES) at Tifton, Georgia used restricted recurrent phenotypic selection (RRPS) to develop improved bahiagrass populations from the diploid cultivar Pensacola. ‘Tifton 9’ (PI no. 531086), resulting from nine cycles of RRPS, is higher yielding than Pensacola and was released in 1987. Tifton 9 is more vigorous in the seedling stage, develops longer leaves, and can produce more forage than Pensacola (Burton, 1989). Eighteen cycles of RRPS breeding material resulted in the experimental population RRPS Cycle 18, which is higher yielding than both Pensacola and Tifton 9 but was not released by CPES because it lacked grazing persistence. ‘AU Sand Mountain’ is thought to originate from a natural selection of a plant introduction grown at the Sand Mountain Substation about 30 years ago. In the northern part of Alabama, this new variety has yielded more forage than Tifton 9. The apomictic tetraploids, Argentine and Tifton 7, are uniform populations; whereas the sexual diploids; Pensacola, Tifton 9, ‘WGP’, RRPS CYCLE 18, and ‘RRPS CYCLE 23’ are segregating populations (Mislevy and Quesneberry, 1999). Sclerotinia homeocarpa F.T. Bennett causes dollar spot which is a major foliar disease and has a wide host range that includes warm and cool season, annual and perennial grasses (Singh, 2003). Dollar spot typically develops when weather is mild and accompanied by frequent and prolonged rainy periods (Atilano et al., 1986). New fungal infections are usually seen in late spring and early summer (Singh, 2003). Once established, disease is quite persistent (Freeman and Simone, 1988).

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16 Outbreaks of dollar spot had not been reported as an extensive problem in pastures of bahiagrass until 2001, when a severe outbreak of dollar spot was reported in the Florida Panhandle. Plant symptoms ranged from mild tip necrosis, to significant leaf lesions, and in several cases, plant death. Differences in dollar spot severity were noticed among the bahaigrass germplasm suggesting that sources of resistance to this disease might be available (Blount et al., 2002). The most promising control strategy is to breed for resistance. Heritability is the proportion of the observed variation in a progeny that is inherited. High heritability of a particular trait implies that the trait can be rapidly improved with less intensive evaluation (Nyquist, 1991). Broad-sence heritability estimates include all genetic effects (additive, dominance, and epistatic) and can be calculated from variance components among clonal replicates by an analysis of variance (Poehlman and Sleper, 1995). Broad-sence heritability estimates are useful in determining the selection efficiency (Poehlman and Sleper, 1995). Burton and Devane (1953) devised a method to estimated broad-sence heritability from replicated clonal material that would separate the variation observed in a segregating population into genetic, environmental and genetic x environmental components. This method along with those proposed by Steel and Torrie (1960) will be used to calculate broad-sence heritability estimates for all of these field studies. Given the recent severity of dollar spot on bahaigrass, identification of germplasm susceptibility is a prerequisite for breeding for resistance. The purpose of this study was to examine different bahiagrass cultivars for their susceptibility to dollar spot under natural field conditions.

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17 Materials and Methods Experiment I The first study was conducted using bahiagrass breeding nursery at the North Florida Research and Education Center (NFREC) in Marianna, FL (30 46’ N, 85 14’W). Seven populations were maintained in this nursery: Argentine, Tifton 7, Pensacola, Tifton 9, and experimental breeding lines WGP, RRPS Cycle 18, and RRPS Cycle 23. Seeds for each of these bahiagrass populations were obtained as follows: Argentine, Dr. Paul Misley, Range Cattle Research and Education Center, Ona, FL (27 29’ N, 81 55’ W); Pensacola, Tifton 9, RRPS Cycle 18, and RRPS Cycle 23, Dr. G.W. Burton, USDA-ARS Coastal Plain Experiment Station, Tifton, GA. Individual seedlings from these populations were planted in the field on 0.6 m centers on 24 April 2000 and allowed to grow into swards. Soil analysis of field site was conducted prior to planting and fertilizers were applied based on this analysis. One hundred randomly selected plants in each population were rated for the presence of dollar spot severity. Populations were rated on 14 June 2003 using a rating scale of 0-9 (0=0% and 9=100% of the plant showing dollar spot symptoms). The Horsfall-Barrett scale (Horsfall and Barrett, 1945) was used to rate another 100 randomly selected plants on 15 June 2004, in each nursery. The ratings for this scale correspond to 1=0% and 12=97%-100% of plant exhibiting disease symptoms. The data from both years was converted to the percentage of plant exhibiting disease symptoms for statistical comparison. Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003). Differences among cultivar means were determined at P = 0.05 using Duncan’s New Multiple Range test. The data was managed and the graphics were produced using Microsoft Excel (Microsoft, 2004).

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18 Experiment II The second field rating study evaluated a bahiagrass cultivar trial at NFREC. It was planted in spring 2001 in a randomized complete block design with nine replications of seven cultivars. The cultivars examined in this study included Argentine, Tifton 7, Pensacola, Tifton 9, FL PCA Cycle 4 Forage, FL PCA Cycle 4 Turf, and Sand Mountain. Seed for Argentine, Pensacola, Tifton 9 was obtained in the same manner as in Experiment I. Tifton 7, FL PCA Cycle 4 Forage, and FL PCA Cycle 4 Turf seed was obtained from seed increases grown at NFREC, Marianna, FL and harvested in the fall of 2000. Seed of Sand Mountain was obtained from Bob Burdett, Alabama Seed Commission. Plants in each plot were arranged in plots consisting 6 x 4 plants on 0.6 m centers with 2 meter borders. The study was evaluated on 14 June 2003 and 15 June 2004 using a rating scale of 0-9 (0=0% and 9=100% of the plant exhibiting dollar spot symptoms 2003) and the Horsfall-Barrett scale (Horsfall and Barrett, 1945) (1-12 where, 1=0% and 12=97%-100% of plant exhibiting disease symptoms 2004). In both years, the mean dollar spot rating of the eight center plants for each plot was recorded and converted to percent disease for statistical comparison. Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003). Differences among population means were determined at P = 0.05 using Duncan’s New Multiple Range test. The data was managed and the graphics were produced using Microsoft Excel (Microsoft, 2004). Experiment III This experiment was planted at the Agronomy Forage Research Unit (AFRU), near Hague, FL (29 46’ N, 82 25’ W) on 1 April 2004 and at NFREC, near Marianna, FL on 22 April 2004 to evaluate the development of dollar spot on several bahiagrass cultivars and breeding lines. The study was arranged in a randomized complete block design with

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19 eight replications of eight genotypes: Argentine, Tifton 7, ‘Paraguay 22’, Pensacola, Tifton 9, ‘Rapid Germ Tifton 9’, FL PCA Cycle 4 Forage, and Sand Mountain. Argentine, Tifton 7, Pensacola, Tifton 9, FL PCA Cycle 4 Forage, and Sand Mountain were provided from sources referenced in Experiment I and II. Seeds of Paraguay 22 were obtained from C. M. Payne and Son, Sebring, FL and Rapid Germ Tifton 9 seeds were obtained from Dr. William Anderson, Coastal Plain Experiment Station, Tifton, GA. Plants were arranged in plots consisting of 6 x 4 plants each on 0.6 m centers with 2 m borders. This experiment was evaluated throughout the growing season using the Horsfall-Barrett scale (Horsfall and Barrett, 1945). These data were used to calculate areas under the disease progress curves (AUDPC) as described by Campbell and Madden (Campbell and Madden, 1990). The data was managed and the graphics were produced using Microsoft Excel (Microsoft, 2004). Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003). Differences among population means were determined at P = 0.05 using Duncan’s New Multiple Range test. On 14 July 2004 the NFREC experiment was evaluated for multiple agronomic traits; vigor (1-5 scale), plant area (cm 2 ), stem height (cm), leaf height (cm). Only the eight center plants in each plot were measured to eliminate border effects. Total plant height was calculated from the addition of stem height and leaf height. Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003). Agronomic trait means were separated at P = 0.05 using Duncan’s New Multiple Range test. These agronomic traits were correlated to disease severity ratings. Experiment IV This study was designed to examine the differences in selected Pensacola-derived clones in their response to dollar spot development under natural field conditions. On 15

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20 August 2003 at the NFREC, Marianna, FL individual plants were selected across all diploid populations of bahiagrass for extremes in reaction (susceptible or resistant) to dollar spot. Five plants were selected, across all nurseries, that appeared to have little or no dollar spot, and these were designated MR1 through MR5. Four plants were also selected that appeared to have extensive symptoms, and these were designated MS1 through MS4. These plants were propagated in the greenhouse at the University of Florida, Gainesville, FL, where they were divided into ten ramets, planted in four inch pots and maintained until the spring of 2004. Individual ramets were planted at AFRU, near Hague, FL on 1 April 2004 and at the NFREC, Marianna, FL on 22 April 2004 in a randomized complete block design with nine plant entries and eight replications. The nine plant entries were assigned the following unique identification: MR1, MR2, MR3, MR4, MR5, MS1, MS2, MS3, and MS4. Each plant entry was planted as a single spaced plant on 0.6 m centers. This experiment was evaluated throughout the growing season using the Horsfall-Barrett scale (Horsfall and Barrett, 1945). The AUDPC’s were calculated (Campbell and Madden, 1990) and subjected to analysis of variance (ANOVA) using PROC GLM of PC SAS (SAS Institute, 2003) and differences among entry means were determined at P = 0.05 using Duncan’s New Multiple Range test. The data was managed and the graphics were produced using Microsoft Excel (Microsoft, 2004). Results and Discussion Experiment I There was a significant interaction between year and cultivar, therefore cultivar responses were compared within each year (Table 2-1). Differences among cultivars were significant (P<0.05) for dollar spot severity in 2003 and 2004 (Fig. 2-1). In both

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21 years, the tetraploid cultivars, Argentine and Tifton 7, showed significantly (P<0.05) less disease development than the diploid cultivars. The disease severity rankings among the diploid types varied from 2003 to 2004. Argentine and Tifton 7 had mean dollar spot severity ratings of 15% in 2003. Mean ratings for diploid cultivars ranged from a low of 52% for WGP to a high of 54% for Tifton 9. In 2004, Argentine and Tifton 7 had similarly low means, 2% and 4%, respectively. Although as in the previous year, most diploid cultivars had significantly higher mean ratings than the tetraploids, the ranking of these cultivars for disease severity differed from that reported in 2003. The lowest diploid cultivar in 2004 was RRPS Cycle 18 with a mean of 7% while the highest was RRPS Cycle 23 with a mean of 19 percent (Fig. 2-1). 010203040506070Arg Figure 2-1. The bahiagrass nursery, Marianna, FL, evaluated in 2003 and 2004 under natural field conditions. Bars with the same letters do not differ significantly at P=0.05. Letters A-D correspond to 2003 ratings, and letters a-f correspond to 2004 ratings. ent ineTi 7PencolaTift 9WCC% dollar spot severit fton sa on PG 18 23 y 2003 2004 A B BC BC C a b D D c e d f f 2X 2X 2X 4X 2X 2X 4X

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22 Table 2-1. ANOVA table for the bahiagrass nursery evaluated in 2003 and 2004 under natural field conditions in Marianna, FL. Source df SS MS F Pr>F Year 1 302158 302158 3559.95 <.0001 Entry 6 200949 33492 394.59 <.0001 Year x Entry 6 79415 13236 155.54 <.0001 Error 1386 117639 84.88 2003 Entry 6 249168 41528 451.90 <.0001 Error 693 63684 91.89 2004 Entry 6 31195.88 5199.31 66.78 <.0001 Error 693 53956 77.86 The calculated broad sense heritability estimates (Steele and Torrie, 1960) for bahiagrass dollar spot response (completely random model, random effects) using individual year data for Exp. I were 82% and 40% for 2003 and 2004, respectively. The lower overall variance among entries can account for the reduced estimate in 2004. However, when data from both years were combined in an ANOVA, the large year and year x entry components of variance (Table 2-1) resulted in a heritability estimate of only 15%. These results confirm the need for multiple year evaluations of entries when identifying selections for further genetic improvement research. Experiment II As in Experiment I, there was a significant interaction between year and cultivar, so cultivars were compared within each year. Significant differences were seen among cultivars (P<0.05) for dollar spot development in 2003 and 2004 (Fig. 2-2). The tetraploid cultivars, Argentine and Tifton 7, had significantly lower dollar spot in both years in comparison to diploid types. This trend was consistent with that observed in Experiment I. Argentine and Tifton 7 had a mean dollar spot severity rating of 5% in 2003. In 2004, Argentine and Tifton 7 had similarly low means, 2% and 3%,

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23 respectively. Mean ratings for diploid cultivars (Pensacola, Tifton 9, Rapid Germ Tifton 9, FL PCA Cycle 4 Forage, and Sand Mountain) ranged from a low of 23% for FL PCA Cycle 4 Forage to a high of 45% for Tifton 9 and Pensacola in 2003 (Fig. 2-2). The lowest diploid cultivar in 2004 was Sand Mountain with a mean of 8% while the highest was FL PCA Cycle 4 Forage with a mean of 14 percent. The diploid cultivars appear to be more susceptible to dollar spot than the tetraploids, and this is consistent with Experiment I. 010203040506070ArgentineTifton 7PensacolaTifton 9RG T9FL ForageFL Turf% dollar spot severit y 2003 2004 A A A B B a a a a a C C b b Figure 2-3. The bahiagrass RCB experiment, Marianna, FL, evaluated for dollar spot severity under natural field conditions in 2003 and 2004. Bars with the same letters do not differ significantly at P=0.05. The letters A-C correspond to the 2003 ratings and the letters a and b correspond to the 2004 ratings. Broad sense heritability estimates (Steele and Torrie, 1960) for bahiagrass dollar spot response (randomized complete block model, random effects) using individual year data for Exp. II were 75% and 26% for 2003 and 2004, respectively. As in Exp. I, there was much lower overall total variability among entries and a greater proportion of 4X 4X 2X 2X 2X 2X 2X

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24 variance in the error term in 2004 resulting in the reduced heritability estimate for 2004. Also similar to Exp. I, a combined ANOVA over both years showed large year and year x entry components of variance (Table 2-1) resulting in a very low heritability estimate of only 9%. These low multi year heritability estimates suggest that some type of progeny testing breeding methodology will likely be needed to make progress in improving dollar spot resistance in bahiagrass. Table 2-2. ANOVA table for the bahiagrass RCB experiment, Marianna, FL, evaluated for dollar spot severity under natural field conditions in 2003 and 2004. Source df SS MS F Pr>F Year 1 9685.81 9685.81 158.04 <.0001 Rep 9 940.32 104.48 1.70 0.0965 Year x Rep 9 838.31 93.14 1.52 0.1499 Entry 6 11838.45 1973.07 32.19 <.0001 Year x Entry 6 4861.79 810.30 13.22 <.0001 Error 108 6618.80 61.29 2003 Rep 9 1320 146.67 2.03 0.0533 Entry 6 15357 2559 35.44 <.0001 Error 54 3900 72.22 2004 Rep 9 458.63 50.96 1.01 0.4421 Entry 6 1343.10 223.85 4.45 <.0001 Error 54 2718.80 50.35 -10010203040 50 JanFebMarAprMay J uneJulyAug S eptOctNovDecCelcius Max 2003 Max 2004 Min 2003 Min 2004 Figure 2-3. Monthly maximum and minimum temperatures for Marianna, FL 2003 and 2004.

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25 05101520253035JanFebMarApr M ay J uneJulyAug S eptOctNovDecRainfall (cm) 2003 2004 Figure 2-4. Monthly rainfall for Marianna, FL 2003 and 2004. The growth habit and vigor differences between the tetraploids and diploids may play a role in the degree of susceptibility to dollar spot development. Tetraploids typically exhibit slower growth, delayed flowering and larger plant parts such as leaves, flowers, and seeds (Singh, 2003) (Swanson et al., 1981). Tetraploid bahiagrass have a summer growth habit whereas; the diploid types tend to have a longer period of foliage growth, perhaps exposing the plants to a longer incubation period for potential infection, particularly in early spring when rain occurrence is frequent. Since lower disease ratings were noted for tetraploid cultivars compared to diploid cultivars in both years, it could be hypothesized that environmental components may be responsible for the difference between the two years. These components include but are not limited to temperature, rainfall, and soil fertility. Monthly minimum and maximum temperatures for both years were similar (Fig. 2-3). Rainfall was greater in 2003 specifically in March, May, July and August (Fig. 2-4). Since dollar spot development is favored by high humidity environments, greater rainfall may have contributed to a greater increase in the disease in 2003.

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26 Experiment III This study compared disease progression over time, and evaluated three tetraploid and five diploid bahiagrass cultivars or experimental lines. There was no dollar spot present at AFRU, Hague, FL. So the following results and discussion apply only to the NFREC, Marianna. The lowest disease rating for all populations was reported for the 23 June 2004 sampling date (Fig 2-5a). In general, ratings increased over the growing season, with all diploids reaching higher rating levels than tetraploids. The three tetraploid bahiagrass populations had similar ratings throughout the season. The progression of disease ratings of one of the diploid cultivars, Sand Mountain, was similar to the tetraploid cultivars. Argentine, Tifton 7 and Paraguay 22 showed a steady increase in disease progression from 23 June through 20 August, and then rapidly declined after that date. Ratings of the other diploid cultivars after 20 August, either increased, as with Tifton 9 and Rapid Germ Tifton 9, remained steady as with Pensacola, or showed a slight decrease as with FL PCA Cycle 4 Forage through the last observation, 10 September (Fig. 2-5). Analysis of the AUDPC’s in this study varied significantly (P <.05) for the various bahiagrass populations. The AUDPC’s for Pensacola (460) and Sand Mountain (360) were not significantly (P<0.05) different from the tetraploids, Argentine (260), Tifton 7 (440), and Paraguay 22 (390) (Fig. 2-6). Ploidy level appears to play a role in the differences observed in disease levels. The tetraploids tended to have lower disease severity values than the diploids which is consistent with the other two studies. However, Sand Mountain, a diploid cultivar, had levels of dollar spot that were similar to the tetraploids. A broad-sence heritability estimate of 39% was calculated as described by Steel and Torrie (1960).

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27 01234564-Jun11-Jun18-Jun25-Jun2-Jul9-Jul16-Jul23-Jul30-Jul6-Aug13-Aug20-Aug27-Aug3-Sep10-SepHorsfall-Barrett Disease Rating Argentine Tifton 7 Paraguay 22 Pensacola Tifton 9 RG T9 PICA 4 Sand Mt. Figure 2-5. Disease progress curves for the epidemiology study of bahiagrass cultivars and breeding lines in Marianna, FL 2004. 020040060080010001200140016001AUDPC for dollar spot development Argentine (4X) Tifton 7 (4X) Paraguay 22 (4X) Pensacola (2X) Tifton 9 (2X) RG T9 (2X) PICA 4 (2X) Sand Mt. (2X) A AB AB BC BC BC BC C Figure 2-6. AUDPC’s for epidemiology study in Marianna, FL 2004. Bars with the same letters do not differ significantly at P=0.05.

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28 Table 2-3. ANOVA for the AUDPC’s of the epidemiology study in Marinanna, FL 2004. Source df SS MS F Pr>F Rep 4 453594 113398 0.57 0.6897 Entry 7 5890465 841495 4.20 0.0028 Error 28 5614707 200525 Table 2-4. Mean vigor rating and plant height for the sward epidemiology study in Marianna, FL 2004. Entries with the same letters do not differ significantly at P=0.05. Cultivars and breeding lines Vigor Plant height Pensacola 3.26 C 32.08 DE Tifton 9 4.88 A 52.73 C Sand Mountain 2.96 D 29.58 EF FL PCA Cycle 4 Forage 4.89 A 65.00 A Paraguay 22 4.56 B 27.40 F Argentine 4.45 B 27.25 F Tifton 7 4.44 B 33.18 D Rapid Germ Tifton 9 4.65 AB 56.33 B The agronomic traits of each population showed significant differences (P<0.05) among populations for plant height and vigor (Table 2-1). Sand Mountain had the lowest vigor rating (Burton, 1989) and did not significantly differ from Argentine and Paraguay 22 in plant height (Table 2-1). The correlations between dollar spot ratings and each of the agronomic trait showed that plant height was the highest correlated trait (r = 0.67, P=0.05) (Table 2-2). Table 2-5. Pearson’s correlation table relating agronomic traits to dollar spot severity. Agronomic traits Dollar spot severity Pr > r Vigor 0.215 0.0001 plant area 0.217 <.0001 Stem height 0.514 <.0001 plant height 0.668 <.0001 leaf height 0.662 <.0001 Plant area x hgt 0.575 <.0001 Experiment IV There was no dollar spot present at AFRU, Hague, FL experimental plots; and so the following results and discussion applies only to the NFREC, Marianna experiment plots. The AUDPC’s for the Pensacola derived clonal study varied significantly (P<0.05)

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29 among clones in 2004. These AUDPC’s show that some of the entries responded differently than expected based on the disease severity when the clones were selected (Fig. 2-7). For example, MR4 (1500) was selected because it had very little disease symptoms, but it has the third highest AUDPC. This reversal from initial observation and response over the following growing season can also be seen with MR3 (860) and MS3 (550). These differences may be attributed to initial observations and selection being made towards the end of the growing season and the original plants being grown in a sward, not as single spaced plants. This also suggests that disease evaluations over time might provide better estimates of disease resistance as opposed to one or two visual observations especially at the end of the growing season. 0500100015002000250030003500 Figure 2-7. AUDPC’s for the clonal epidemiology study, Marianna, FL, 2004. Bars with the same letters do not differ significantly at P=0.05. A broad-sence heritability estimate of 76% was calculated as described by Steel and Torrie (1960). This estimate is relatively high compared to the other heritabilities 1 AUDPC for dollar spot development A B B B C CD CD CD D MS3 MR1 MR5 MR2 MS4 MS1 MR4 MS2 MR3

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30 calculated for the other studies because the variation within the replications is quite small (due to the fact that these were clones) compared to the variation within the entries. Table 2-6. ANOVA for the AUDPC’s of the clonal epidemiology study in Marianna, FL 2004. Source df SS MS F Pr>F Rep 7 2141164 305881 1.36 0.2374 Entry 9 56574629 6286069 27.98 <.0001 Error 63 14156205 224702 Conclusions Dollar spot severity of bahiagrass varied among the cultivars and experimental lines tested in the bahiagrass breeding nursery and the bahiagrass variety trials in 2003 and 2004. Both studies showed similar trends between years with less disease pressure occurring in 2004. The greatest contrast in disease susceptibility was seen between the tetraploid and diploid cultivars. The mean dollar spot rating for the tetraploid cultivars, Argentine and Tifton 7, were 4 to 5 times less than the mean for the diploid cultivars. The differences among years for dollar spot development could be attributed to differences in environmental conditions. The spring of 2003 had much greater rainfall than that of 2004. Experiment III allowed for the analysis of disease progress curves to be developed in 2004. Disease progress curves also recorded lower dollar spot ratings in the tetraploid types compared to the diploid types throughout the growing season. In 2003 and 2004, differences in dollar spot development between diploids and tetraploids were highly significant for the rating date in mid-July. When dollar spot was evaluated from June to September, the two diploids, Pensacola and Sand Mountain, did not differ (P<0.05) in their susceptibility to dollar spot compared to the tetraploids. Consideration should be

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31 given to seasonal progression of the disease when evaluating bahiagrass germplasm for resistance to dollar spot.

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CHAPTER 3 PROTOCOL DEVELOPMENT FOR INOCULATION WITH SCLEROTINIA HOMEOCARPA Introduction Sclerotinia homecarpa F. T. Bennett is the causal organism of dollar spot, a foliar disease on many grasses (Freeman and Simone, 1988). It has a wide host range which includes warm and cool season annuals and perennials (Atilano et al., 1986). S. homecarpa can be found in Europe, North and South America (Atilano et al., 1986). Only one report from Great Britain found cultures of this organism, which produced conidia (Bennett 1937). All other reported isolates only produced mycelium and pseudosclerotium (Bennett 1937). It is believed that this organism overwinters in the thatch of its host plant (Smith et al., 1989). Several techniques have been used to inoculate fungi on plants. The use of mycelial plugs was investigated as an inoculation technique for Sclerotinia sclertiorum on soybean (Voung et al, 2004). The colonized mycelial plugs were placed on the soybean plants and disease symptoms were produced (Voung et al, 2004). Yuanhong Han and colleagues (2003) used a conidial suspension was applied using a pressurized CO 2 hand sprayer for inoculating perennial ryegrass. Bonos and colleagues (2003) achieved successful field inoculation of dollar spot on bentgrass using infested Kentucky bluegrass seeds. An accurate and precise inoculation technique, which correlates plant responses to natural field infections, would be a valuable tool for plant breeders. This would aid them 32

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33 to develop a greenhouse procedure to rapidly evaluate genotypes in a breeding program fro response to dollar spot. Materials and Methods Isolation and Culture In order to collect isolates of S. homeocarpa, samples of Argentine and Pensacola bahiagrass were collected in different locations throughout Florida (North Florida Research and Education Center, NFREC, near Marianna (30 46’ N, 85 14’ W) and Quincy (30 35’ N, 84 35’ W); Agronomic Forage Research Unit, AFRU, near Hague (29 46’ N, 82 25’ W); Crystal River (28 54’ N, 82 36’ W); and Range Cattle Research and Education Center, RCREC, Ona (27 29’ N, 81 55’ W)). The samples were kept on ice and transported to the lab in Gainesville, FL. Each sample was sorted and the most characteristic dollar spot lesions were chosen. Characteristic lesions have a bleached center with a reddish-brown margin. The grass blades were trimmed to pieces approximately 3 cm in length. The remaining work was completed in a sterile air-flow hood. The trimmed grass blades were surface sterilized in a 5% bleach solution for 30 sec. They were then triple rinsed in distilled water for 30-45 sec. each time. Each grass piece was then dried with a sterile tissue and trimmed to approximately a 5 mm piece which included healthy tissues and the margin of the lesion. Five to six trimmed pieces were placed on water agar and placed in a growth chamber maintained at 20 C. The plates were examined within 48 hrs and characteristic white-gray fluffy mycelial growth was hyphal-tipped and placed on 25% potato-dextrose agar (PDA) and placed in the growth chamber. These isolates were then compared to a known culture obtained from Dr. J. Rollins, Professor in the Plant Pathology Department at the University of Florida and isolates verified by Dr. Rollins and Dr. Datnoff, Professor in the Plant Pathology

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34 Department at the Univerity of Florida, as S. homecarpa were maintained on 25% PDA by subculturing every 3 weeks. The first isolates were collected in August of 2001. These were isolated from Argentine and Pensacola samples obtained in Marianna, Quincy, and Gainesville, Florida. More collections were made and fresh isolates were obtained during the summer of 2002. These were collected from Marianna, Quincy, Gainesville, Crystal River, and Ona, Florida. No differences in colony morphology were noted among isolates from different locations or different source plants. Preliminary Experiments Several preliminary experiments were undertaken to determine an appropriate inoculation technique for use in a greenhouse study. Known field susceptible bahiagrass plants were used in these studies. They were clones that had been vegetatively propagated and maintained in pots under greenhouse conditions at the University of Florida campus in Gainesville, FL. Isolates of S. homeocarpa, were used to determine Koch’s postulates while developing a simple inoculation procedure several approaches were taken. The first method attempted was a mycelial plug. A 6mm x 3mm plug of agar with rapidly growing mycelia was placed in the whorl of a known susceptible bahiagrass plant. After misting the inside of a 3.8 l plastic bag, it was placed over the plant and secured with a rubber band around the lip of a 10 cm pot. The plants were monitored and notes were taken over a three week period. This was repeated five times on individual plants. The second technique attempted used infested rice grains as an inoculum source. The rice grains were prepared as followed: 1) 40 g of rice grains were place in a magenta

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35 box along with 20 ml of distilled water and autoclaved for 20 min; 2) the autoclaved rice grains were placed around the outer perimeter of a 25% PDA plate (approximately 12-15 rice grains plate -1 ); 3) a fresh plug of S. homeocarpa mycelium was placed in the center of each plate; 4) these plates were incubated in the growth chamber at 20 C. Variable incubation times were tested (1 wk, 2 wks, 3 wks, and 4 wks). After the rice grains were infested, they were placed in the whorl of a susceptible potted bahiagrass clone. After misting the inside of a 3.8 l plastic bag, it was placed over the bahiagrass plant and secured with a rubber band around the lip of a 10 cm pot. These pots were maintained in the greenhouse and monitored over a three week period. The third technique tested a mycelial slurry as the inoculum source. The slurry was prepared using 2 plates of S. homeocarpa mycelium which was 2 wks old blended with 100 ml of distilled water and 0.5 ml of molasses. This slurry mixture was sprayed onto potted bahiagrass plants with an aerosol sprayer (Crown Spra-Tool; Fisher Scientific Co., Pittsburgh). The plants were covered with a 3.8 l plastic bag (which had been misted inside) and the bag was secured with a rubber band around the lip of a 10 cm pot. The bahiagrass plants were maintained in the greenhouse and monitored over a three week period. This technique was repeated five times. A modification of the mycelial plug technique was also tested. The bahiagrass plants were wounded by trimming the leaves (with scissors) before the slurry mixture was sprayed. Everything else was performed as described previously. This modified technique was repeated four times. Due to unsatisfactory results, a humidity chamber was constructed in the greenhouse using polyethylene plastic sheeting. The humidity chamber measured 3 m x 1.75 m x 1 m. A misting system with two fogger nozzles (each nozzle produces 2 l water

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36 s -1 ) that were set to release 6 sec of water every 30 min was placed in the humidity chamber. The last preliminary experiment was designed as a randomized complete block design with two replications, four plant entries, and three treatments. Each plant entry replicate was by represented by clones. The four plant entries were the Pensacola derived clones MS4, MR4, and MS1 (which represent the three most susceptible clones from the field evaluation in 2004). Argentine was included to represent the least susceptible bahiagrass cultivar in the 2004 field trials. Three treatments were used: 1) no inoculum (control); 2) infested rice as the inoculum source (isolated from Argentine bahiagrass); 3) infested rice as the inoculum source (isolated from Pensacola bahiagrass). The experiment was maintained in the humidity chamber for ten days. The plants were evaluated daily for the presence of mycelium, number and size of lesions (measured to the nearest tenth of a millimeter using a digital micrometer). Inoculation Experiment This experiment was conducted twice and was designed as a RCB with six replications, and eight plant entries. The experiment was blocked from the north end to the south end of the humidity chamber. The eight plant entries included the diploid Pensacola-derived clones MR2, MR3, MR5, MS1, MS2, MS4, and the tetraploids Argentine and Tifton 7. All clones were infested with multiple grains of rice as described previously. All the rice grains were incubated for six days to provide optimal mycelia for infection of entries. Based on results from preliminary experiments which found that humidity was essential for lesion development, these experiments were maintained in the humidity chamber (6 sec of water was released every 40 min) for eight days. Lesion size and number were measured every 24 hours. The plant entries daily average lesion sizes

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37 were calculated. Each plant entries average lesion size was graphed over time and the line of best fit was calculated by linear regression. The slope of this line represents the lesion expansion rate (mm hr -1 ) for each plant entry. The area under the disease progress curve (AUDPC) was also calculated as described by Campbell and Madden (1990). Data were analyzed using PROC GLM of PC SAS (SAS Institute, 2003). Differences among cultivar means were determined at P = 0.05 using Duncan’s New Multiple Range test. The data was managed and the graphics were produced using Microsoft Excel (Microsoft, 2004). Results and Discussion Isolation and Culture Isolates of S. homeocarpa were easily obtained from samples of Argentine and Pensacola bahiagrass. The culture characteristics including growth rate and mycelial pigmentation appeared to be the same for isolates from both sources. There were no differences in colony morphology noted between isolates from different locations in Florida. Preliminary Experiments The first three preliminary inoculation techniques failed to produce characteristic lesions. Some entries of these first techniques showed leaf discoloration that eventually led to necrosis. Multiple symptomatic tissue samples were taken and plated onto selective media as described previously. No isolates of S. homeocarpa isolates were recovered. The discoloration and eventual necrosis of leaf tissue probably was due additional heat inside the plastic bag. The slurry mixture had more necrotic leaves produced than any other treatment, but since no characteristic lesions were produced this technique was no longer used. The leaf necrosis could be due to the components of the

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38 slurry mixture burning the leaves once they were placed under the plastic in the greenhouse. The infested rice that was used to inoculated plants showed visible mycelial growth along the leaf blades after 1 wk to a much greater extent than the infested rice held for longer periods of time. For the remainder of these experiments all the rice was infested for a period of 5 to 7 days, based on these results. After review of the literature concerning the onset of dollar spot under natural field conditions, it was determined that humidity was a necessary factor for disease development. The humidity chamber described in materials and methods was used for all further experiments. During the first experiment, plants were placed on shallow trays filled with water and enclosed in the chamber. No supplemental water was provided. There was no disease development observed on any entries that were inoculated and placed in the humidity chamber without supplemental moisture. Further investigation and personal communication with others working with S. homeocarpa led to the conclusion that higher levels of humidity were necessary. In personal communication with Dr. David Williams, Proffesor of Turfgrass Science in the Department of Plant and Soil Science at the University of Kentucky, he suggested that with S. homeocarpa on bentgrass extremely high humidity levels, to the point of moisture dripping off the plant, for extended periods of time (up to four days) were optimal for disease development. Once this irrigation system was installed in the humidity chamber, disease symptoms were observed. Our irrigation system released water for 6 sec every 40 min for the duration of the 10 d experiments. Lesions first appeared water soaked at approximately 40 h and then developed characteristic light centers with reddish-brown

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39 margins. These lesions were then cultured and S. homeocarpa was isolated from these lesions, proving Koch’s postulates. Inoculation Experiment The last preliminary experiment attempted to develop a system to characterize lesion development and differences in plant response. This experiment used only the infested rice as an inoculum source. Of the 24 plants in this experiment (3 of which were un-inoculated controls) only five plants developed lesions. Based on the amount of mycelial growth seen previously when the rice was used as the source of inculum these results were surprisingly low. One reason for these lower than expected results may have been due to the fact that water released from the fogger nozzles dislodged some of the rice grains. The five plants which were affected were MS4 (Rep 1 and 2), MR4 (Rep 1 and 2), and Argentine (Rep 1). On each of the five affected plants, number and size of lesions were recorded over time. The average expansion rate for each plant was calculated as described in materials and methods. (Figure 3-1a-c) A B C Figure 3-1 Dollar spot lesion development with an infested rice grain as the source of inoculum; A) after 45 hrs, B) after 62 hrs, C) after 72 hrs in the humidity chamber.

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40 The last inoculation technique experiment evaluated six Pensacola derived clones, Argentine and Tifton 7 using the infested rice (6 d incubation time) as the inoculum source. Lesions were observed 38 hrs after the experiment was initiated. The lesion number and size and any additional notes were recorded. (Table 3-1a-b) Some entries showed a difference in rate and number of lesion development, which may suggest different types of resistance being present in this population. Some entries had a few lesions which expanded rapidly, showed the least resistant. While others had a many lesions which never expanded very much, this reaction suggest a hyper-sensitive type of resistance. However, this difference in rate and number of lesion development was not consistent between replications. The analysis of variance of the expansion rate and AUDPC showed no significant differences among the plant entries when both runs where analyzed together. When the second run was analyzed separately, there were significant differences between the Pensacola-derived clones and Argentine and Tifton 7. This may suggest that the infested rice technique is not a sensitive enough technique to detect genetic differences among the plant entries for the population measured. The one week testing period also may be insufficient to see differences in plant canopy response as seen in the season long field evaluations (Chapter 2). This technique could be modified by changing the frequency, duration, and amount of humidity used and length of time for better disease development to better simulate field conditions. Table 3-1. Lesion expansion rate, AUDPC, and the R 2 values for each entry in the first and second run of the RCB inoculation technique experiment Entry Run Rep Expansion Rate AUDPC R 2 MR2 1 1 0.025 0.039 0.967 MR2 1 2 0.076 0.638 0.913 MR2 1 3 0.141 0.949 0.824 MR2 1 4 0.158 1.275 0.963 MR2 1 5 0.101 1.710 0.738

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41 Table 3-1. Continued. Entry Run Rep Expansion Rate AUDPC R 2 MR2 1 6 0.110 1.648 0.902 MR3 1 3 0.119 0.950 0.891 MR3 1 4 0.052 0.376 0.856 MR3 1 5 0.038 0.472 0.907 MR5 1 1 0.132 0.901 0.985 MR5 1 2 0.107 0.703 0.982 MR5 1 3 0.090 0.512 0.962 MR5 1 4 0.092 0.310 0.985 MR5 1 5 0.988 0.542 0.912 MR5 1 6 0.053 0.514 0.959 MS1 1 1 0.093 0.637 0.952 MS1 1 2 0.088 0.276 0.925 MS1 1 3 0.052 0.397 0.979 MS1 1 4 0.055 0.408 0.952 MS1 1 5 0.060 0.398 0.942 MS1 1 6 0.231 1.736 0.993 MS2 1 1 0.159 1.248 0.985 MS2 1 2 0.053 0.528 0.732 MS2 1 3 0.025 0.179 0.967 MS2 1 4 0.159 0.911 0.937 MS2 1 5 0.086 1.096 0.993 MS2 1 6 0.161 1.784 0.994 MS4 1 1 0.072 0.550 0.950 MS4 1 2 0.147 2.360 0.832 MS4 1 3 0.081 0.410 0.951 MS4 1 4 0.030 0.208 0.979 MS4 1 5 0.031 0.221 0.986 MS4 1 6 0.135 2.235 0.881 ARG 1 1 0.023 0.150 0.971 ARG 1 3 0.074 0.338 0.916 T7 1 2 0.068 0.455 0.979 T7 1 4 0.056 0.273 0.969 MR2 2 1 0.100 0.299 0.947 MR2 2 2 0.110 0.481 0.924 MR2 2 3 0.070 0.325 0.991 MR2 2 4 0.050 0.098 0.999 MR2 2 5 0.190 1.118 0.985 MR2 2 6 0.090 0.521 0.989 MR3 2 2 0.060 0.250 0.926 MR3 2 3 0.190 0.822 0.960 MR3 2 4 0.140 0.563 0.960 MR3 2 6 0.140 0.294 0.980 MR5 2 1 0.090 0.187 0.997 MR5 2 2 0.180 1.711 0.974 MR5 2 3 0.190 0.792 0.996 MR5 2 4 0.120 0.658 0.940

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42 Table 3-1. Continued. Entry Run Rep Expansion Rate AUDPC R 2 MR5 2 5 0.190 0.666 0.983 MR5 2 6 0.050 0.096 0.996 MS1 2 1 0.230 1.910 0.998 MS1 2 2 0.080 0.503 0.972 MS1 2 4 0.120 0.702 0.953 MS1 2 5 0.250 2.110 0.948 MS1 2 6 0.020 0.003 0.781 MS2 2 1 0.035 0.182 0.916 MS2 2 2 0.030 0.177 0.957 MS2 2 3 0.060 0.415 0.928 MS2 2 4 0.030 0.153 0.971 MS2 2 5 0.180 0.360 0.993 MS2 2 6 0.120 0.426 0.981 MS4 2 1 0.130 0.550 0.947 MS4 2 2 0.060 0.463 0.834 MS4 2 3 0.100 0.290 0.940 MS4 2 4 0.070 0.364 0.970 MS4 2 6 0.010 0.035 0.932 ARG 2 1 0.040 0.185 0.928 ARG 2 3 0.030 0.096 0.962 T7 2 1 0.060 0.065 0.958 T7 2 3 0.020 0.029 0.983 Conclusions The preliminary inoculation technique experiments proved that sufficient humidity was essential for disease development. The use of fogger head nozzles which release 2 l water s -1 on an irrigation system which releases 6 s of water every 40 min was the amount and frequency of irrigation used in these experiments. Further research is needed to determine the optimal humidity for dollar spot development on bahiagrass. Of the three inoculation techniques attempted here, the rice seed colonized by mycelia of S. homeocarpa was chosen as the preferred technique. It was easy to prepare, inoculate the plant, and had the most consistent results. To overcome the problem of some rice grains falling off, multiple tillers were inoculated on each plant. The optimal

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43 time period to allow rice grain colonization by S. homeocarpa was 5 to 7 days. This may be due to the fact that S. homeocarpa may be in an actively growing state. There were no significant differences among plant entries when infested rice was used as an inoculum source. This technique may not be sensitive enough to detect difference among different Pensacola-derived clones. Testing a wider range of germplasm may allow difference to be seen using infested rice as an inoculum source. With further modifications this technique may prove useful in screening for resistance to dollar spot in bahiagrass. Because there were no differences in response to dollar spot seen among the Pensacola-derived clones, no heritability estimate could be calculated from these inoculation experiments.

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CHAPTER 4 CONCLUSIONS Field Evaluations There were differences among bahiagrass cultivars and breeding lines in dollar spot severity when tested in the field at Marianna in 2003 and 2004. All the field evaluations, the tetraploid cultivars Argentine and Tifton 7 had less dollar spot severity than the diploid bahiagrass cultivars and breeding lines. There was also a difference in the severity ratings between year 2003 and 2004, which could be related to the greater rainfall in the spring of 2003. The two field evaluations in 2004 allowed for the analysis of disease progress curves. When the dollar spot severity was evaluated from June to September, two diploid cultivars were not different in their susceptibility than the two tetraploids. When dealing with a pasture grass, consideration should be given to seasonal progression of the disease especially when this grass is being grown as a hay crop. Greenhouse Evaluations From the preliminary inoculation experiments it can be concluded that sufficient moisture is necessary for lesion development. These studies used a misting system in a humidity chamber to achieve the level of moisture needed. Further research is needed to determine the optimal amount and duration of humidity. The most reliable inoculation technique of those tested was the rice grains infested with mycelium placed in the whorl of the plant. The optimal incubation period for infesting the rice grains was found to be 5 to 7 days. 44

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45 When the Pensacola-derived clones that had shown variable response to dollar spot in the field were evaluated with this technique in the greenhouse humidity chamber, their response was not different when the inoculum source was infested rice grains. It may be that a continual presence of a fungal disease source growing on the rice grains may have overcome any differences in responses due to plant genetics that were manifested under field conditions. Other researchers with cool season grasses have also shown that dollar spot is a difficult disease to replicate field plant response under greenhouse conditions. With further research and modifications this technique may prove useful for screening for resistance to dollar spot in bahiagrass.

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APPENDIX INNOCULATION EXPERIMENT DATA

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Table A-1. Data for greenhouse experiment, first run. Hours after Inoculation 38 53 59 77 83 107 125 Reps Expansion rate y-intercept R 2 MS2 1 3.5 5.1 7.3 11.0 12.0 15.0 17.4 0.159 -2.220 0.985 3 1 2 6 2 6.3 9.8 10.7 11.7 11.9 13.0 13.1 0.053 0.732 0.732 3 4.2 4.7 4.8 5.3 5.4 5.9 6.1 0.025 3.303 0.967 4 10.1 10.9 11.6 12.2 15.5 20.4 0.159 0.937 0.937 5 7.4 7.7 9.9 10.4 13.5 0.086 2.942 0.993 6 7.2 8.7 10.8 11.9 18.2 0.161 -1.300 0.994 MR2 1 7.0 8.7 6.4 6.8 -0.013 8.443 0.136 2 3.2 6.0 6.2 7.0 8.4 8.5 11.0 0.076 1.271 0.913 3 6.0 11.0 11.3 17.0 18.0 18.0 19.0 0.144 3.170 0.808 4 8.0 11.0 12.8 15.0 17.0 18.0 23.0 0.158 2.732 0.963 5 3.6 7.0 9.2 9.2 11.0 12.0 0.087 2.375 0.766 6 7.0 9.0 8.1 11.0 12.0 0.062 4.509 0.720 MR 3 2.9 5.7 10.3 10.8 11.2 13.5 0.119 -0.640 0.891 4 6.6 7.8 9.2 9.1 10.4 0.052 3.898 0.856 5 3.9 4.8 6.0 0.038 1.304 0.907 MS4 1 4.1 6.1 7.0 8.3 8.0 9.9 10.8 0.072 2.159 0.950 2 3.0 8.9 10.3 12.3 12.6 15.5 0.126 1.287 0.807 3 5.4 6.2 6.5 7.0 10.0 11.1 0.081 0.915 0.951 4 4.8 4.9 5.4 5.7 5.9 6.8 7.2 0.030 3.543 0.979 5 4.6 4.9 5.4 5.7 5.9 6.8 7.2 0.031 3.407 0.986 6 3.2 7.2 7.3 10.9 11.0 12.9 0.106 1.039 0.841 MR5 1 5.7 7.7 9.4 10.7 11.8 15.8 16.9 0.132 0.869 0.985 2 6.8 7.2 8.3 10.6 11.6 13.5 15.6 0.107 2.202 0.982 3 4.1 4.8 5.6 6.4 6.8 10.2 11.6 0.090 0.122 0.962 4 10.4 11.1 12.8 15.0 0.092 3.342 0.985 5 7.0 8.2 11.4 11.5 14.2 0.104 1.127 0.873 6 5.1 5.4 5.8 7.2 7.3 0.032 3.613 0.742 47

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Table A-1. Continued. Hours after Inoculation 38 53 59 77 83 107 125 Reps Expansion rate y-intercept R 2 MS1 1 7.5 8.3 9.6 11.1 11.3 12.5 16.2 0.093 3.752 0.952 2 6.9 7.2 7.2 8.4 10.6 0.050 3.628 0.818 3 5.4 7.0 6.6 7.7 8.5 9.2 10.2 0.052 3.752 0.979 4 2.6 4.8 3.6 5.4 5.6 7.1 7.4 0.055 0.951 0.952 5 5.8 6.9 8.9 0.060 1.513 0.942 6 9.4 8.5 7.1 11.5 12.2 21.0 0.152 0.609 0.868 48

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Table A-2. Data for greenhouse experiment, second run. Hours after Inoculation 39 48 63 91 113 133 Reps Expansion rate y-intercept R 2 MS1 1 0.0 9.1 12.6 19.5 23.7 28.3 0.270 -6.290 0.943 2 2.7 3 4.9 5.4 8.3 9.5 10.9 0.080 0.248 0.972 4 4.8 6.1 7.9 9.5 12.6 17.3 0.120 -0.059 0.953 5 5.9 14.1 24.1 28.6 31.4 0.250 -0.728 0.948 6 5.4 4.1 5.5 4.4 4.5 5.0 0.002 4.975 0.021 MR2 1 6.9 8.6 10.2 15.0 0.090 1.851 0.811 2 4.2 7.0 6.7 11.7 13.5 0.110 -0.886 0.924 3 8.0 11.2 9.4 12.6 14.7 0.070 4.504 0.991 4 3.8 4.9 5.8 0.050 -0.685 0.999 5 7.1 10.8 13.9 17.8 26.0 59.0 0.450 -14.075 0.781 6 4.4 5.9 7.4 9.4 12.2 13.4 0.090 1.159 0.989 MR 1 2 2.8 6.2 4.8 7.4 7.6 0.060 0.827 0.926 3 5.3 5.5 6.7 12.0 16.5 22.9 0.190 -3.643 0.960 4 6.3 10.0 7.6 15.3 18.3 0.140 -1.413 0.960 5 6 5.3 7.7 11.4 0.140 -8.114 0.980 MS4 1 4.0 4.1 6.3 11.1 16.5 0.150 -4.645 0.897 2 6.4 5.3 4.9 5.3 7.2 11.2 0.050 2.929 0.545 3 4.3 3.6 4.2 7.6 7.4 8.2 0.050 1.721 0.871 4 4.4 4.8 7.1 8.6 10.2 11.0 0.070 1.833 0.970 5 6 2.0 2.4 2.0 2.6 0.010 1.601 0.932 MS2 1 3.2 3.5 3.8 4.1 5.7 6.6 0.035 1.662 0.916 2 3.6 4.3 4.8 5.1 5.7 6.3 0.030 2.897 0.957 3 3.5 5.0 5.8 6.3 7.6 10.0 0.060 1.657 0.928 4 4.1 4.5 4.8 5.2 6.4 6.8 0.030 3.008 0.971 5 7.5 10.9 15.0 0.180 -8.881 0.993 6 5.3 9.2 5.6 12.3 15.0 0.120 -1.234 0.981 49

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50 Table A-2. Continued. Hours after Inoculation 39 48 63 91 113 133 Reps Expansion rate y-intercept R 2 MR5 1 7.9 9.8 11.8 0.090 -0.598 0.997 2 5.3 10.0 11.2 19.1 23.8 0.180 -0.174 0.974 3 8.7 11.5 17.4 20.7 25.6 0.190 -0.730 0.996 4 3.2 6.2 6.5 11.9 13.4 14.2 0.120 -0.331 0.940 5 8.4 16.2 9.0 20.7 24.0 0.190 -2.015 0.983 6 2.5 4.6 5.2 5.9 0.040 0.738 0.980 Arg 1 3.2 4.7 5.2 5.9 6.6 0.040 1.926 0.928 2 3 2.6 3.2 4.4 4.8 0.030 0.962 0.962 T7 1 3.6 4.1 4.8 5.1 0.060 3.838 0.958 2 3 5.7 6.5 9.7 9.8 11.0 0.020 2.162 0.983

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52 Carbone, I., and L.M. Kohn. 1993. Ribosomal DNA sequence divergence within internal transcribed spacer 1 of the Sclerotiniaceae. Mycologia 85:415-427. Cole, H., J.M. Duich, L.B. Massie, and W.D. Barber. 1969. Influence of fungus isolate and grass variety on Sclerotinia dollarspot development. Crop Sci. 9:567-570. Cook, R.N, R.E. Engel, and S. Bachelder. 1964. A study of the effect of nitrogen carriers on turfgrass disease. Plant Disease Report 48:254-255. Couch, H.B. 1995. Disease of turfgrasses. 3 rd ed. Krieger Publ., Malabar, FL. Freeman, T.E., and G.W. Simone. 1988. Turfgrass diseases and their control. Florida Coop. Ext. Ser. Cir. 221-H. Gates, R.N, P. Mislevy, and F.G. Martin. 2001. Herbage accumulation of three bahiagrass populations during the cool season. Agron. J. 93:112-117. Han Y., S.A. Bonos, B.B. Clarke, and W.A. Meyer. 2003. Inoculation techniques for selection of gray leaf spot resistance in perennial ryegrass. USGA Turfgrass and Environmental Research Online v2, no. 9. Horsfall, F.G., and R.W. Barrett. 1945. An improved grading system for measuring plant diseases. Phytopathology 35:655. Jackson, N. 1973. Apothecial production in Sclerotinia homeocarpa F. T. Bennet. Journal Sports Turf Res. Inst. 49:58-63. Killinger, G.B. 1959. Pasture herbage changes in Florida during the past two decades (1939-1959) Soil Crop Sci. Soc. Fla. Proc. 19:162-166. Killinger, G.B., G.E. Ritchey, C.B. Blickensderfer, W. Jackson. 1951. Argentine bahia grass. Agricultural Experiment Station Annual Report. Univ. of Florida, Gainesville, FL. Kohn, L.M. 1979. Delimitation of the economically important plant pathogenic Sclerotinia species. Phytopathology 69:881-886. Kohn, L.M. and D.J. Grenville. 1989. Anatomy and histochemistry of stromatal anamorphs in the Sclerotiniaceae. Can. J. of Bot. 67:371-393. Markland, F.E., E.C. Roberts, L.R. Frederick. 1969. Influence of nitrogen fertilizers on Washington creeping bentgrass, Agrostis palustris Huds. II. Incidence of dollar spot, Sclerotinia homeocarpa, infection. Agron. J. 61:701-705. McCloud, D.E. 1953. Forage and cover plant introduction by the Florida Agricultural Experiment Station. Soil Crop Sci. Soc. Florida Proc., 13:32-38.

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53 Mislevy, P. 2001. Management practices for consideration with perennial grasses. The Florida Cattleman and Livestock Journal. July 2001. Mislevy, P. and K.H. Quesenberry. 1999. Development and short description of grass cultivars released by the University of Florida (1892-1995). Soil and Crop Soc. of Fla. Proc. 58:12-19. Novak, L.A., and L.M. Kohn. 1991. Electrophoretic and immunological comparisons of developmentally regulated proteins in members of the Sclerotiniaceae and other sclerotial fungi. Appl. Environ. Microbiol. 57:525-534. Nyquist, W 1991. Estimation of heritability and prediction of selection response in plant populations. Crit. Rev. Plant Sci. 10:235-322. Poejlaman, J.M., and D.A. Sleper. 1995. Breeding field crops. Iowa State University Press, Ames, Iowa. Powell, J.F. and J.M. Vargas, Jr. 2001. Vegatative compatibility and seasonal variation among isolates of Sclerotinia homeocarpa. Plant Dis. 85:377-381. Singh, R.J. 2003. Plant cytogenetics, 2 nd Ed. CRC Press. Urbana, Illonions. Smith, J.D., N. Jackson, and A.R. Woolhouse. 1989. Fungal diseases of amenity turf grasses. E & F.N. Spon, New York, New York. Steel, R.G. and J.H. Torrie. 1960. Principles and procedures of statistics with special reference to the biological sciences. McGraw-Hill Book Co., New York, New York. Swanson, C.P., T. Merz, and W.J. Young. 1981. Cytogenetics: The chromosome in division, inheritance and evolution. Prentice-Hall. Englewood Cliffs, New Jersey. Turgeon, A.J. 2002. Turfgrass management. 6 th ed. Prentice Hall, Upper Saddle River, New Jersey. Vincelli, P., J.C. Doney, Jr., and A.J. Powell. 1997. Variation among creeping bentgrass cultivars in recovery from epidemics of dollar spot. Plant Dis. 81:99-102. Vuong, T.D., D.D. Hoffman, B.W. Diers, J.F. Miller, J.R. Steadman, and G.L. Hartma. 2004 Evaluation of soybean, dry bean, and sunflower for resistance to Sclerotinia sclerotiorum. Crop Sci. 44:777-783. Waddington, D.V., R.N. Carrow, and R.C. Shearman. (editors). 1992. Turfgrass. ASA, CSSA, and SSSA. Madison, WI. Walsh, B., S.S. Ikeda, and G.J. Boland. 1999. Biology and management of dollar spot (Sclerotinia homeocarpa); an important disease of turfgrass. HortScience 34:13-21.

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54 Watkins, J.E. and L.A. Wit. 1995. Effect of nitrogen rate and carrier on the suppression of dollar spot symptoms on Penncross bentgrass. Plant Dis. 10:37

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BIOGRAPHICAL SKETCH Brandy Nicole Williams was born in Pahokee, FL, on March 27, 1979, to Bill and Denise Williams. She was home-schooled from first through twelfth grade. She received her GED in 1997, and was awarded a 2-year scholarship based on her high GED score. She also received the 4-year Bright Fu tures Academic Scholar ship. She attended Palm Beach Community College and graduated with an Associate of Arts in 2000. In 1999 and 2000, she worked as a summer intern at the USDA-ARS in Canal Point, FL, which cemented her goals of pursuing a career in agriculture. She attended the University of Florida beginning in Fall 2000 with a major in plant pathology and graduated with a Bachelor of Science from the College of Agricultural and Life Sciences in 2002. She began her graduate studies in 2003 in the Agronomy Department with a major in plant breeding and a minor in plant pathology. She graduated from the University of Florida with her Master of Science in 2005. 55