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Population and Identification of Mycorrhizal Fungi in St. Augustinegrass in Florida and Their Effect on Soilborne Pathogens

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PAGE 1

POPULATION AND IDENTIFICATION OF MYCORRHIZAL FUNGI IN ST. AUGUSTINEGRASS IN FLORIDA AND THEIR EFFECT ON SOILBORNE PATHOGENS By WHITNEY COLLEEN ELMORE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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DEDICATION This dissertation is dedicated to my family in the memory of my father, Malcome Elmore.

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iii ACKNOWLEDGMENTS I would like to thank my parents, Malcome and Donna Elmore, for their loving support and my sister, Emilee. I would also like to acknowledge a very special person, LaVette Burnette, for all of the patience and caring attention she has sh own me for many years. I would also like to thank Dr. James Kimbrough and his wife, Jane, for their support, both emotionally and spiritually. I would also like to thank Drs. Jim Graham and Kevin Kenworthy for agreeing to serve on my graduate committee a nd for their willingness to offer advice on my studies. I also owe Dr. Vertigo Moody a big “thank you” for motivating me to finish my Ph.D. as well as for his technical support in writing. Additionally, I would like to say a big “thank you” to Dr. Gerald Benny both for serving on my committee and for his attention in the lab. Dr. Benny is always ready to help with research, or simply listen to my ramblings about research and politics which I appreciate greatly. I would like to extend a personal “thank yo u” to the Department of Plant Pathology staff, Gail Harris, Lauretta Rahmes, and Donna Perry. These ladies always have a smile ready and a helping hand for students. I would also like to thank Eldon Philman and Herman Brown for their assistance in experi mental studies at the greenhouse complex. They seem to always have a good solution or answer to any problem or question. Finally, I would like to extend my sincerest appreciation to the Department of Plant Pathology, namely Dr. Gail Wisler, at the Univer sity of Florida and to the Institute of Food and Agricultural Sciences for financial and technical support in

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iv this endeavor. I would not have been able to fulfill my dreams without the help and support from all of these people.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. iii LIST OF TABLES ................................ ................................ ................................ ............ vii LIST OF FIGURES ................................ ................................ ................................ .......... viii ABSTRACT ................................ ................................ ................................ ...................... xii CHAPTER 1 GENERAL INTRODUCTION ................................ ................................ .................... 1 Mycorrhizal Types and Phylogeny ................................ ................................ .............. 2 Arbuscular Mycorrhiza Physiology ................................ ................................ ............. 4 Arbuscular Morphology ................................ ................................ ............................... 5 Mycor rhizal Colonization ................................ ................................ ............................ 6 Mycorrhizal Rhizosphere Interactions ................................ ................................ ......... 8 Effects of Abiotic Factors on Mycorrhiza ................................ ................................ 12 Effects of Seasonality on Mycorrhiza ................................ ................................ ........ 14 Mycorrhizas in Grasses ................................ ................................ .............................. 16 2 POPULATION AND IDENT IFICATION OF ARBUSCULAR MYCORRHIZAL FUNGI IN ST. AUGUSTINEGRASS ................................ ......... 24 Materials and Methods ................................ ................................ ............................... 25 Results ................................ ................................ ................................ ........................ 29 Discussion ................................ ................................ ................................ .................. 34 3 THE EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI ON GAEUMANNOMYCES GRAMINIS VAR. GRAMINIS AND RHIZOCTONIA SOLANI COLONIZATION OF ST. AUGUSTINEG RASS SOD IN NORTH CENTRAL FLORIDA SOILS ................................ ................................ ................... 57 Materials and Methods ................................ ................................ ............................... 63 Results and Discussion ................................ ................................ .............................. 65

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vi 4 EFFECT OF GLOMUS INT RARADICES ON THE EXT ENT OF DISEASE CAUSED BY GAEUMANNOM YCES GRAMINIS VAR. G RAMINIS AND RHIZOCTONIA SOLANI I N ST. AUGUSTINEGRASS ................................ ......... 74 Material and Methods ................................ ................................ ................................ 77 Direct Experiments ................................ ................................ ............................. 77 Indirect Experiments ................................ ................................ ........................... 82 Res ults ................................ ................................ ................................ ........................ 84 Direct Experiments ................................ ................................ ............................. 84 Discussion ................................ ................................ ................................ .................. 86 Results ................................ ................................ ................................ ........................ 87 Indirect Experiment ................................ ................................ ............................ 87 Discussion ................................ ................................ ................................ .................. 88 5 SUMMARY AND CONCLUSI ONS ................................ ................................ ...... 100 APPENDIX A SELECTIVE MEDIA RECI PES FOR ISOLATION OF G. GRAMINIS VAR. GRAMINIS AND R. SOLANI FROM PLANT TISSUE ................................ .......... 104 B NUTRIENT SOLUTION (2 0 0 20) USED IN DIRECT AND INDIREC T TRIALS DESCRIBED IN CHAPTER 4 ................................ ................................ .. 105 C RHIZOCTONIA SOLANI AND G. GRAMINIS VAR. GRAMINIS INOCULUM PRODUCTION PROTOCOLS ................................ ................................ ................ 106 D ADDITIONAL DATA ANAL YSIS RESULTS REFEREN CED IN CHAPTER 4 DIRECT EXPERIMENTS ................................ ................................ ....................... 107 E ADDITIONAL DATA ANAL YSIS RESULTS REFEREN CED IN CHAPTER 4 INDIRECT EXPERIMENTS ................................ ................................ ................... 110 F ANALYSIS OF VARIANCE TABLES FOR CHAPTERS 2, 3, AND 4 ............... 115 LIST OF REFERENCES ................................ ................................ ................................ 133 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 150

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vii LIST OF TABLES Table page 2 1. Species of AMF positively identified at each sod farm location from pot cultures of sorghum sudangrass within a combination of field and sterile, low P soil. ......... 45 2 2. Evaluation of analysis of variance data for spore density data from each sod farm location by date. ................................ ................................ ................................ ........ 48 2 3. Pe arson correlation coefficients (r) for AMF spore density and soil moisture and temperature. ................................ ................................ ................................ .............. 50 2 4. Evaluation of analysis of variance data for percent root length colonized from each sod far m location. ................................ ................................ ............................. 54 2 5. Chemical characteristics of soils sampled for AMF at three north central Florida sod farm locations during January, April, August, and November 2005. ................ 56

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viii LIST OF FIGURES Figure page 2 1 A C. ‘Floratam’ St. Augustinegrass sod farms located at (A) Fort McCoy (Marion County), (B) Lake Butler (Union County), and (C) Starke (Bradfo rd County) in north central Florida. ................................ ................................ .............. 40 2 2. Sorghum sudangrass pot cultures containing 50% (w/w) field soil combined with 50% sterile, low P soil. ................................ ................................ ..................... 41 2 3. Spore extract from field soil following the wet sieving procedure. .......................... 42 2 4 – 2 7. Stained arbuscular mycorrhizal structures observed within ‘Floratam’ St. Augusti negrass. ................................ ................................ ................................ ........ 43 2 8 – 2 11. Stained arbuscular morphology types found within ‘Floratam’ St. Augustinegrass …………………………………………………………….. ...... 44 2 14 – 2 19. Arbuscular mycorrhizal fungal spores identified at the Lake Butler sod farm location ……………………………………………………… ....... 46 2 20 – 2 28. Arbuscular mycorrhizal funga l spores identified at the Fort McCoy sod farm location …………………………………………………… ......... 47 2 29. Spore density with increasing soil moisture levels over a 12 month period at the Starke sod farm lo cation. ................................ ................................ .......................... 51 2 30. Spore density with increasing soil moisture levels over a 12 month period at the Fort McCoy sod farm location. ................................ ................................ ................ 51 2 31. Spore density with increasing soil moisture levels over a 12 month period at the Lake Butler sod farm location. ................................ ................................ ................. 52 2 32. Spore density with increasing soil temperatures over a 12 m onth period at the Starke sod farm location. ................................ ................................ .......................... 52 2 33. Spore density with increasing soil temperatures over a 12 month period at the Fort McCoy sod farm location. ................................ ................................ ................ 53

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ix 2 34. Spore density with increasing soil temperatures over a 12 month period at the Starke sod farm location. ................................ ................................ .......................... 53 3 1. ‘Floratam’ St. Augustinegrass sod mat infected with Gaeumannomyces graminis var. graminis Insert in bottom right hand corner depicts underside of a mat with rotting roots. ................................ ................................ ................................ ..... 68 3 2 – 3 3. Comparison of healthy ‘Floratam’ St. Augustinegrass sod mat and sod affected by brown patch. ................................ ................................ .......................... 69 3 4. Deeply lobed hyphopodia isolated from Gaeumannomyces graminis var. graminis in ‘Floratam’ St. Augustinegrass sod samples. Scal e bar = 40 m. ......... 70 3 5. Medium isolation plate depicting a Gaeumannomyces graminis var. graminis colony isolated from ‘Floratam’ St. Augustinegrass sod samples. Arrow points to colony. ................................ ................................ ................................ .................. 70 3 6. Rhizoctonia solani hyphae isolated from ‘Floratam’ St. Augustinegrass sod exhibiting diagnostic 90 o branching at constriction points and characteristic septa. Scale bar = 40 m. Arrow poi nts to branching pattern. ............................... 71 3 7. Medium isolation plate depicting light brown Rhizoctonia solani colony isolated from ‘Floratam’ St. Augustinegrass sod samples … … … … … … … … … ............ ..... 71 3 8. Mean percent of Rhizoctonia solani colonization of 'Floratam' St. Augustinegrass in north central Florida. ................................ ................................ 72 3 9. Mean percent of Gaeumannomyces graminis var. graminis colonization of 'Floratam' St. Augustinegrass in north central Florida. ................................ .......... 73 4 1. Rhizoctonia solani isolate (PDC 7884) colony used to prepare inoculum in direct and in direct experiments. ................................ ................................ .......................... 90 4 2. Gaeumannomyces graminis var. graminis isolate (JK2) used to prepare inoculum in direct and indirect experiments. ................................ ................................ ........... 90 4 3. Conetainers filled with low P soil and ‘Floratam’ St. Augustinegrass sprigs inoculated in trial 1 of the direct experiment. ................................ ........................... 91 4 4. Glomus intraradices isolate (FL 208 A) used in direct and indirect assays to inoculate ‘Floratam’ St. Augustinegrass sprigs. ................................ ..................... 91 4 5. Photo showing nylon sleeves and plastic clips used in direct and indirect experiments to cle ar and stain root segments from treatment replicates. ................. 92 4 6. Photo of mycorrhizal St. Augustinegrass root with arbuscules and intraradical hypha of Glomus intraradices stained with 0.05% try pan blue from the direct experiment G. intraradices inoculated control sprigs. ................................ .......... 92

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x 4 7. ‘Floratam’ St. Augustinegrass sprigs after inoculation with Rhizoctonia solani depicting disease seve rity rating scale (1 6). ................................ .......................... 93 4 8. ‘Floratam’ St. Augustinegrass sprigs after inoculation with Gaeumannomyces graminis var. graminis depicting disease severity rating scale (1 6). .................... 94 4 9 – 4 10. Photo depicting re isolation plates of the two pathogenic isolates used to challenge Glomus intraradices in both the direct and indirect experimental trials. 95 4 11. Photo of the indirect experimental trial 3 conetainers arranged in a randomized complete block design with four replicates per treatment. ................................ ....... 96 4 12. Photo showing a close up view of the experimental units of the indirect experimental trial 1 depicting the split root assay. ................................ ................... 96 4 13. Photo showing the split root assay of the indirect exper imental trial 2 after inoculation with ryegrass seeds inoculated with Gaeumannomyces graminis var. graminis (JK2). ................................ ................................ ................................ ....... 97 4 14. The direct effect of G. graminis var. graminis on St. Augustinegr ass take all root rot disease severity without G. intraradices ................................ .................... 98 4 15. The direct effect of G. graminis var. graminis on St. Augustinegrass take all root rot disease severity with G. intrar adices ................................ ......................... 98 4 16. The indirect effect of R. solani without G. intraradices on St. Augustinegrass brown patch disease severity in an adjacent split sprig system. ..... 99 D 1. The direct effect of G. intraradices colonization on take all root rot disease severity in ‘Floratam’ St. Augustinegrass. ................................ ........................... 107 D 2. The relat ionship between R. solani colonization and brown patch disease severity in ‘Floratam’ St. Augustinegrass ................................ ........................... 108 D 3. The relationship between R. solani colonization and G. intraradices on brow n patch disease severity in ‘Floratam’ St. Augustinegrass. ................................ ..... 109 E 1. Photograph depicting a conetainer used in the indirect experiment with drilled hole and cut to allow for sprig to be inser ted without tissue damage. .................... 110 E 2. The indirect effect of G. graminis var. graminis on take all root rot diease severity in ‘Floratam’ St. Augustinegrass without G. intraradices ..................... 111 E 3. The effect of Glomus intraradices colonization on brown patch and take all root rot disease severity in ‘Floratam’ St.Augustinegrass on plants in the split sprig assay. ................................ ................................ ................................ .................... 112

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xi E 4. The indirect effect of R. solani on disease severity in ‘Floratam’ St. Augustinegrass with G. intraradices on an adjacent split sprig system .............. 113 E 5. The indirect effect of G. graminis var. graminis on disease severity in ‘Floratam’ St. Augustinegrass with G. intraradices ................................ ........... 114 F 1. Analysis of variance tables for spore densi ty and percent colonization data in Chapter 2, and Pearson’s product moment correlation coefficients for attempted correlations between variables and soil chemical characteristics and soil moisture and soil temperature. ................................ ................................ ............... 120 F 2. Analysis of variance tables for Rhizoctonia solani percent colonization data in Chapter 3. ................................ ................................ ................................ ............... 122 F 2. Analysis of variance tables for Gaeumannomyces gramin is var. graminis percent colonization data in Chapter 3. ................................ ................................ ............... 126 F 4. Analysis of variance tables for the direct assay in the split sprig challenge including Gaeumannomyces graminis var. graminis and Rhizoctonia solani data in Chapter 4. ................................ ................................ ................................ ........... 129 F 4. Analysis of variance tables for the indirect assay in the split sprig challenge including Gaeumannomyces graminis var. graminis and Rhizoct onia solani data in Chapter 4. ................................ ................................ ................................ ........... 132

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xii Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy POPULATION AND IDENTIFICATION OF MYCORRHIZAL FUNGI IN ST. AUGUSTINE GRASS IN FLORIDA AND THEIR EFFECT ON SOILBORNE PATHOGENS By Whitney Colleen Elmore August, 2006 Chair: James W. Kimbrough Major Department: Plant Pathology Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of more than 90 % of all land plants. Mycorrhizae are documented in many crops as positive associations with roots of plants that help reduce disease severity soilborne pathogens and increase nutrient and water uptake while lowering plant stress and ultimately management costs. However, there is no information concerning the effects of AMF colonization in St. Augustinegrass. In Florida, St. Augustinegrass sod production contributes hundreds of millions of dollars to the economy annually while supplying a product to homeowners and commercial entities with great aesthetic value. The use of AMF in St. Augustinegrass sod production has many potential benefits to the sod industry and the environment including lowered management costs pesticide use and pollution. In these studies, a survey of S t. Augustinegrass sod farms in north central Florida revealed a moderate level of AMF colonization as well as a diverse population of AMF species. Direct and indirect

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xiii pathogen challenges with the ubiquitous AMF, Glomus intraradices in St. Augustinegrass plants suggested a limited role for AMF in lowering disease severity in two of the more devastating diseases of St. Augustinegrass in Florida, brown patch and take all root rot. While no positive correlation was observed between AMF colonized St. Augusti negrass plants and the soilborne pathogens Rhizoctonia solani or Gaeumannomyces graminis var. graminis, effective assays for mycorrhizal St. Augustinegrass evaluation s were developed and foundation information concerning the association between St. Augusti negrass and AMF provided valuable data, which may help in the development of f uture AMF evaluations in St. Augustinegrass field trials and with other AMF species. These results were the first to suggest an association between AMF and St. Augustinegrass a nd to evaluate their potential effects on disease severity.

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1 CHAPTER 1 GENERAL INTRODUCTION “Mykorrhizen” was a term first applied by the German forest pathologist, A.B. Frank, who described structures in plant roots as “ fungus roots ” (1885). Harley (1989) described them as a mutualistic symbiosis in which a fungu s and host exist as one. Despite minuscule differences in description, mycorrhizas are recognized by scientists as economically important in most agricultural crops. In fact, the mutually beneficial relationships are actually three way associations in wh ich the soil, plant root, and fungus interact to produce symbiotic effects. In 1879, de Bary defined symbiosis as “the living together of differently named organisms,” which included both parasitic and beneficial relationships. Later, Raymer (1927), co mmenting on the nature of symbionts, acknowledged such partnerships, but did not provide functional information concerning the fungi involved. However, after many years of advanced research throughout the 1960’s and 70’s, the meaning of the relationship was refined to refer to naturally beneficial relationships exclusively. Most likely, organisms co existing became symbiotic as a result of selection pressures exerted over the course of time (Remy et al., 1994). In fact, it is possible that the movement of plants from water to land could not have occurred without mycorrhizal associations (Nicolson, 1975; Pirozynski and Malloch, 1975). It is now recognized that mycorrhizas are the norm and not the exception within the Kingdom Planta With ancient lineage s stretching across evolutionary history, Bryophytes Angiosperms Pteridophytes and some Gymnosperms all possess these associations

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2 (Fitter, 1991), while members of the Brassicaceae seem to evade infection by any type of mycorrhizal fungi (Gerdemann, 19 68), even in close proximity to mycorrhizal plants. Involved in mycorrhizal symbiosis are members of the fungal taxa Ascomycotina Basidiomycotina Zygomycotina Deuteromycotina and Glomeromycota (Schssler et al., 2001; Srivastava et al., 1996). Infreq uently found living as saprobes, most of these fungi are widespread across various soil types with strong biotrophic host dependence (Smith and Read, 1997). Mycorrhizal Types and Phylogeny Types of mycorrhizae are divided based on their fungal associations extent of root penetration, presence or lack of an external mantle and/or sheath, as well as the intra and intercellular structures produced inside of the host root (Srivastava et al., 1996). Presently, seven types of mycorrhizae are recognized by taxo nomists (Bagyaraj, 1991). The types of mycorrhizae include: Ecto mycorrhizae Ectendo mycorrhizae Arbutoid Monotropoid Ericoid Orchidoid and Endo mycorrhizae or the vesicular arbuscular mycorrhizae (Bagyaraj, 1991). Endo mycorrhizae also known as vesic ular arbuscular mycorrhizae or VAM, were taxonomically placed within the Order Glomales of the Phylum Zygomyco ta based on morphological features of asexual spores resembling sexual reproductive structures of the Zygomycota Six genera are recognized withi n the Glomales: Glomus Sclerocystis Gigaspora Scutellospora Acaulospora and Entrophospora (Morton and Benny, 1990). In 2001, Schussler et al., using information provided by small subunit rRNA gene sequences, proposed a new Phylum, to separate arbuscu lar mycorrhizal fungi from other fungal groups in a monophyletic clade. Schussler et al. (2001) suggested that they be removed from the Zygomycota and placed into a newly erected Phylum Glomeromycota Small subunit rRNA gene sequencing also

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3 placed Geosip hon pyriformis a n endocytobiotic fungus, which is a distant relative of the arbuscular mycorrhizal fungi, within this new Phylum (Schussler et al., 2001). Within the same article, Schussler et al. (2001) also suggested that the Glomus genus be emended to include the termination – er aceae with the family named Glomeraceae and the higher taxon names reflecting this change with Glomerales Furthermore, Schussler et al. (2001) suggested three new orders, mostly diverged from the Ascomycetes and Basidiomycete s, be recognized as well. These are the Archaeosporales Diversisporales and the Paraglomerales Based on a combination of molecular, ecological, and morphological characteristics, these fungi can now be separated from other fungal groups. The use of m olecular techniques such as small subunit rRNA sequencing has led to the recent introduction of other species within the genus Glomus Walker et al. (2004) and Rani et al. (2004) also used this technology to add Glomus hyderabadensis from India, and a new genus Gerdemannia to the growing list of arbuscular mycorrhizal fungi collected and speciated around the world. Based on their distinct molecular differences from the Zygomycota and placement into a new phylum, Goto and Maia (2005) recently suggested th at spores of the arbuscular mycorrhizal fungi be referred to as glomerospores. Indeed, these spores are not chlamydospores, conidia, or azygospores, so differentiation based on molecularly distinct features is pertinent. Forming vesicles and arbuscules within cortical root cells, fungi of the Glomeromycota produce aseptate hyphae without the presence of a sheath or mantle. Gigaspora and Scutellospora produce arbuscules only within roots and vesicles only within the soil, and, therefore, the vesicular a rbuscular mycorrhizal term has been emended to simply read as arbuscular mycorrhizae. The name was amended simply

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4 because arbuscules are the most basic and one of the few commonalities between the members of the group (Morton and Benny, 1990). Taylor et al. (1995) proposed that Glomites be included as a new fossil genus of Glomales, and two years later, Wu and Lin (1997) added another genus, Jimtrappea However, these two genera are not widely accepted. Currently, there are about 150 recognized species described within the Glomales of which only a few have been carefully studied and recognized as endo mycorrhizal (Morton and Bentivenga, 1994; Morton and Benny, 1990; Morton et al., 1992; Pirozynski and Dalpe, 1989; and Stuessy, 1992). Glomeromycota are not known to produce sexual reproductive spores and, therefore, are characterized and classified by their resting structures. These structures vary in wall characteristics, size, shape, and color (Morton et al., 1992; Morton and Bentivenga, 1994; and Mort on and Benny, 1990). Arbuscular Mycorrhiza Physiology The most widespread of the mycorrhizae, both geographically and among species, the arbuscular mycorrhizae occur frequently in the top 15 30 cm of cultivated soil (Bagyaraj, 1991). Arbuscular mycorrh izae forming fungi colonize and form associations with most agriculturally and horticulturally important plant species, from fruit and forest trees to shrubs and grasses. Unlike other mycorrhizae, these associations do not typically lead to noticeable ext ernal morphological changes in plant roots, and they cannot be observed easily without staining procedures (Phillips and Hayman, 1970). In most cases, plants which have formed associations with other types of mycorrhizal fungi, such as basidiomycetes and ascomycetes, do not form relationships with arbuscular mycorrhizae. From the standpoint of the fungus, host specificity exists while the opposite view would be held about the host due to the wide host range of most of the arbuscular mycorrhizal fungi (Ger demann, 1955). Their limited capacity to be grown from spores, vesicles, or

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5 hyphae from root residue has led to special methodologies in order to maintain strains and for taxonomic evaluation. Typically, single spore types are cultivated in “pot cultures ” on plant roots so that characteristics of spores, their mode of colonization, and effects on plant growth can be studied (Smith and Read, 1997). Arbuscular Morphology Named by Gallaud (1905) for the structures formed inside cortical root cells, arbusc ules are similar to branched haustoria, which form early on in the association between plant root and the repeatingly branched fungal hyphae. Baylis (1975) and St. John (1980) suggested that the form of the root system is a defining factor in the extent t o how plants react, nutritionally, and in growth to mycorrhizal colonization. Evolving across phylogenetic lines many times, it appears that dicotyledons have a large incidence of associations with fungal species which form mycorrhizal associations, with very few being non mycorrhizal in nature (Trappe, 1987). In comparison, the lines of monocotyledons studied by Cronquist (1981) are heavily mycorrhizal, with arbuscular mycorrhizas predominating except in the Orchidaceae which have mycorrhizas formed by Basidiomycetes. In plants forming primarily magnolioid type roots, with wide diameters up to 1.5 mm, slow growth habits, and little root hair development, mycorrhizas are usually well accepted and form greatly receptive relationships. On the other hand, roots that are primarily fine and rapidly growing with long root hairs lack the same responsiveness (Baylis, 1975; St. John, 1980). Mycorrhizal relationships were first described by the type of colonization patterns, referred to as either Arum or Paris t ype (Gallaud, 1904). In fact, there appears to be a continuum between the two forms, with intermediate types along the way.

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6 The Arum type, which was considered the most common association, develops primarily within cultivated crops and consists of inte rcellular hyphae and arbuscules. In contrast, the Paris type of symbiosis – involving intercellular hyphae, arbusculate coils, and hyphal coils, typically develops within forest trees and herbs (Dickson, 2004). In surveys of mycorrhizal plants and trees from both natural and cultivated environments, it appears that most plant families are dominated by only one symbiotic type (Smith and Smith, 1997). There are, however, a few plant families that appear to possess intermediate forms of the colonization typ es, including the Poaceae (Smith and Smith, 1997). In an extensive survey of various plant families and mycorrhizal fungi, eight distinct classes of colonization types were found along a continuum ranging from the Paris to Arum type (Dickson, 2004). Mos t researchers agree that one fungus can form either type of arbuscular colonization with most of the specificity in structure dependent upon the host plant (Barrett, 1958; Gerdemann, 1965). Brundett and Kendrick (1988) commented on the presence of interce llular spaces within the host root cortex as being the main factor influencing arbuscular type. Conversely, in tomato, Cavagnaro et al. (2001) suggested that the colonization type was dependent on both the host and fungus involved. Mycorrhizal Colonizat ion In mycorrhizal colonization, the host plasmalemma is invaginated with the encroaching arbuscules. These are physiologically active sites for nutrient translocation, for 4 6 days, within the roots (Bracker and Littlefield, 1973; Brundett et al., 1984). Arbuscules are important sites for P exchange for plants under deficient conditions (Simth and Read, 1997). The vesicles, which are small and usually dark, globular or spherical structures, form later in the association and arise from swelling of termin al and intercalary hyphal cells. Vesicles act as storage sites for lipids (Srivastava et al., 1997).

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7 Transversing long distances of soil beyond nutrient depletion zones and reaching areas untouched by growth limited root hairs, the external hyphae absorb nutrients such as P and make it available to plants, rendering these plants more equipped to survive nutrient competitions (Nicolson, 1967). Once the fungal hyphae and plant roots become closely associated in space, a functionally and structurally comple x symbiotic relationship is formed between the compatible organisms. Formed only on unsuberized root tissue, certain areas of the root are more readily colonized even though mycorrhizae can develop on any portion of young root tissue (Brundett and Kendr ick, 1990). Based on mathematical and geometrical models, root tissue directly behind the meristematic area is considerably more susceptible to penetration and colonization when compared to other root segments (Garriock et al., 1989; Bonfante Fasolo et al ., 1990). This area of discrete colonization was described earlier as the mycorrhizal infection zone by Marks and Foster (1973), who considered the area to be “non static,” thus growing with the root. Furthermore, Brundett and Kendrick (1990) found that the fungus penetrates and colonizes root cells with little or no suberin deposition, which has been shown to occur just prior to or after fungal penetration. Usually, epidermal and outermost cortical cell colonization is minimal with the intercellular hyp hae formed in the inner cortex and the majority of the colonization is deep within the cortex where arbuscules are formed (Srivastava et al., 1997). With the aid of cellulolytic and pectinolytic enzymes produced by the fungus, direct penetration of the outermost cell wall is the preferred mode of hyphal entry (Jarvis et al., 1988). Physiochemical aspects of the epidermal cell wall seem to be the primary reasons for preferential site penetration (Jarvis et al., 1988). After cell to cell contact

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8 between fungus and host, the external mycelia swell to form defined appresoria (20 40 m in length). Within these appresoria, infection hyphae are formed and penetrate host cell walls (Garriock et al., 1989). Once penetration has occurred via mechanical and enzy matic interactions, the host’s plasmalemma appears to extend around the fungus (Bracker and Littlefield, 1973). Arbuscule formation takes between 4 5 days after which extramatrical hyphae occurs promoting new penetration sites (Brundett et al., 1984). Ar buscules are major contributors to the transfer of nutrients, in particular sugars, between the plant to fungus and inorganic materials, mainly P, from the fungus to the plant (Smith and Gianinazzi Pearson, 1988). Mycorrhizal Rhizosphere Interactions A ne cessary component of plant life, the macro element P, occurs as part of DNA and RNA nuclei and as part of plant membranes as phospholipids (Griffiths and Caldwell, 1992; Smith and Read, 1997). Present in high amounts within active meristematic regions as part of nuclear proteins and as part of ADP, ATP, NADP, and NAD, P is partly responsible for oxidation reduction reactions such as respiration, nitrogen and fat metabolism, and photosynthesis, which are necessary for life (Beever and Burns, 1980; Munns and Mosse, 1980). Symptoms of deficiency often include purple or red leaf pigmentation, dead and/or necrotic leaves, petioles, and fruits, premature leaf drop, stunting, and poor vascular tissue development (Srivastava et al., 1997). An important aspect of arbuscular mycorrhizal associations is the increase in P uptake by the plant. The importance of arbuscular mycorrhizal fungi for P absorption was first suggested by Baylis (1959) and then Gerdemann (1964). Later, Baylis (1967), Daft and Nicolson (1966), Holevas (1966), and Murdoch et al. (1967) provided advanced

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9 information showing the close association between mycorrhizas and P nutrition of the host. Interestingly, Mosse (1973) once remarked that more than one quarter of mycorrhizal text is devoted to P research. In fact, Sanders and Tinker (1973) stated that “the value of these mycorrhizas for the phosphate nutrition of plants in deficient environments may rival that of Rhizobium in nitrogen.” Obviously, such a strong statement must be supported by a n abundance of research. As mycorrhizal research progressed during the last three decades, P research remained an important topic. For instance, in 1986, Gianinazzi Pearson and Gianinazzi studied the kinetic associations between P concentration in soil s olutions and its effect on root and shoot tissues, while Young et al. (1986) evaluated the effect of arbuscular mycorrhizal fungi inoculation on soybean yield and P utilization in tropical soils. Later, Koide (1991) determined that it is the variation amo ng plant species in phenological, morphological, and physiological traits that influence P demand and supply which are directly connected to potential response of mycorrhizal associations. Once absorbed, P is allocated for plant functions or stored for la ter use (Cox and Sanders, 1974). Since P deficiency is caused by both P availability and plant demand, mycorrhizal associations can have various effects based on the plant species (Koide, 1991). In low P soils, mycorrhizal plants have an advantage over non mycorrhizal plants with root to shoot ratios lowered and shoot fresh weight to dry weight ratios higher in mycorrhizal plants (Tinker, 1978). The plant’s growth rate is influenced by interactions in mycorrhizal colonization such as nutritional, and n on nutritional, physiological effects, such as pH, temperature, microbial turnover, phosphatase activity, soil and plant moisture, and/or iron (Fe) or aluminum (Al) chelate concentration (Nye and Tinker,

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10 1977; Rusell, 1973). In P deficient soils, studies have shown that plant species with few root hairs are strongly mycorrhizal, providing evidence that root anatomy has a strong correlation to mycorrhizal colonization (Crush, 1974; Baylis, 1975). Smith and Read (1997) wrote “the focus (of current research) is on P uptake, as well as on the uptake of other nutrients for which there is now unequivocal evidence of mycorrhizal involvement.” Furthermore, they noted that “there is excellent evidence to demonstrate that external hyphae of VA mycorrhizal fungi abs orb non mobile nutrients (P, Zn, Cu) from soil and translocate them rapidly to the plants, thus overcoming problems of depletion in the rhizosphere which arise as a consequence of uptake by roots.” Throughout the 1960’s, reviews of the occurrence of arbus cular mycorrhizal colonized plants and anatomy were the norm in mycorrhizal research (Smith and Read, 1997). There had been little mention of mineral nutrition until Mosse (1957) released details of an experiment with apple seedlings which provided eviden ce for increased amounts of potassium (K), iron (Fe), and (copper) Cu in mycorrhizal plant tissue versus noninoculated control plants. Other researchers such as Gerdemann (1964) established that P tissue concentrations were also higher in mycorrhizal plan ts, although the mechanisms were not yet clearly understood. Mosse (1973) reported a shift in mycorrhizal research from pot experiments to study the anatomy of arbuscular mycorrhizal fungi to that of plant growth and P uptake. Now, the mechanisms underly ing the mycorrhizal effect on P uptake are coming to light including extraradical hyphae growing into soil not already colonized by roots; hyphae that are more effective than roots, due to size and spatial distribution, in competing with free living microo rganisms or mineralized or solubilized P; the kinetics of P uptake into hyphae may differ from

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11 roots; and that mycorrhizal roots can use sources of P in soil that are not plant available (Smith and Read, 1997). Hyphal pathways between plants may offer lin ks for soil derived nutrient transfer, as is the case with plant derived carbon (C), which can have important roles in the inter plant and species competition in the environment (Smith and Read, 1997). Enzymes are not the only substances produced by arbus cular mycorrhizal fungi. An Iron containing glycoproteinaceous substance called glomalin, produced by these fungi, is deposited in soils (Rilling et al., 2003). Glomalin is considered to be linked to soil Carbon storage due to its effect on soil aggregat ion (Rilling et al., 2003). Consistently correlated with soil aggregate water stability, glomalin is involved in C and N content as well as being useful as a potential land use change indicator (Rilling et al., 2003). After many years of taxonomic resear ch with proteins and soil stability, micronutrient uptake research has increased following studies by Mosse (1957), Daft et al. (1975), and Gildon and Tinker (1983) where uptake of Cu and zinc (Zn) were observed in apples and maize when inoculated with arb uscular mycorrhizal fungi. The uptake of other micronutrients is not well documented, however, Marschner and Dell (1994) observed that the uptake of manganese (Mn) is usually reduced by mycorrhizal associations. Occasionally, instances of increased K con centrations in plant tissues have been reported, which is to be expected given the immobility of the K ion within the soil matrix (Srivastava et al., 1997). Conversely, with increased P uptake as well as other nutrients in mycorrhizal plants comes the ris k of accumulating toxic elemental levels. With improved P nutrition and plant growth, the uptake of heavy metals per plant is greatly increased as demonstrated

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12 by El Kherbawy et al. (1989) on alfalfa inoculated with arbuscular mycorrhizae in various soil pH levels with and without rhizobia. Effects of Abiotic Factors on Mycorrhiza Many climatic and physiochemical or abiotic features of the soil influence arbuscular mycorrhizal establishment, growth and benefit. For instance, light, which is not directly required by mycorrhizas in some cases, is essential for the host to thrive and translocate photosynthates to the root, which in turn provides a home for mycorrhizal fungi. In other cases, arbuscular mycorrhizal fungi are stimulated by light to increase r oot colonization and spore production as well as plant response to mycorrhizal colonization (Furlan and Fortin, 1973; Hayman, 1974). The rate of photosynthesis and translocation of its products are heavily influenced by air temperature (Furlan and Fortin, 1973; Hayman, 1974). By increasing air temperature to 26 o C an increase in plant growth is typical (Hayman, 1974). Soil temperatures also influence mycorrhizal development at all stages: spore germination, hyphal penetration, and proliferation within co rtical root cells (Schenck and Schroder, 1974; Smith and Be, 1979). Optimal temperatures vary for spore germination between species and other stages in development. The ability of the arbuscular mycorrhizal spores to survive following host death or harve st is also dependent on soil temperature, though also affected by soil texture (Bowen, 1980). Soil pH is an additional determinant factor in mycorrhizal growth and development. The efficiency of the mycorrhizae is directly determined by its ability to adapt to soil pH. Soil pH affects both spore germination and hyphal development (Angle and Heckman, 1986; Green et al., 1976). The interaction of soil pH and mycorrhizal development is difficult with soil type, plant and fungal species and P forms involv ed.

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13 Typically, mycorrhizas are able to colonize and grow well in soils of pH 5.6 to 7.0, but not in soils of pH 3.3 to 4.4, as reported by Hayman and Mosse (1971). Generally, mycorrhizas are not found within aquatic conditions, due to a reduction in colo nization, however, some aquatic plants are commonly mycorrhizal, such as Lobelia dortmanna L. and Eichhornia crassipes [Martius] Solms (Read et al., 1976). Conversely, most plants found within drought are typically mycorrhizal, which aids in their surviva l in harsh conditions (Sondergaard et al., 1977). Arbuscular mycorrhizal colonization of roots affects many mechanisms in plant water determination. Root hydraulic conductivity, leaf gas exchange and expansion, phytohormone regulation, and leaf conductan ts are all affected by interactions with arbuscular mycorrhizas (Gogala, 1991; Hardie and Leyton, 1981; Koide, 1985; Nelson, 1987; Auge et al., 1986). Fungal mycelium is involved in the transport of water especially at low soil potentials, which has made arbuscular mycorrhizae colonization and development a hot research topic in arid and tropical landscapes (Faber et al., 1991). Mycorrhizal roots and organic matter content play important roles in arbuscular mycorrhizal survival and development as well. O rganic root debris may act as a reserve for soil inocula (Warner and Mosse, 1980), while in arid areas contact between susceptible plant roots and colonized root residue is considered by Rivas et al. (1990) to be the most important means for mycorrhizal di ssemination when little water is available for spore transport. Soil structure, pH, water, and nutrient availability are all affected by organic matter content, thus influencing mycorrhizal associations (Khan, 1974; Daniels and Trappe, 1980; Johnston, 194 9). For instance, Johnston (1949) suggested that organic materials such as manures can enhance tropical soil mycorrhizas in cotton stands. And,

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14 Sheikh et al. (1975) reported that spore population and organic matter content were positively correlated in s oils with 1 2% organic matter, but low in soils with 0.5% organic matter or less. Organic matter and root residue are important ecologically as part of the three way soil, plant and fungal mycorrhizal relationship. Effects of Seasonality on Mycorrhiza Se asonality is another abiotic contributor to arbuscular mycorrhizal colonization. Seasonality has been shown to affect spore production as a function of host and climate (Hetrick, 1984), while seasonal patterns can be correlated with P availability and soi l water potential in combination with host growth stages, other biotic and abiotic factors, and management practices such as fertilization (Cade Menun et al., 1991; Yocums, 1985). Hayman (1975) demonstrated that fertilizers such as P and Nitrogen (N) coul d potentially reduce spore number and fungal colonization with N having a more detrimental effect than P. Despite the possibility for soil chemical treatment injury, arbuscular mycorrhizae can be found in fertile soils, which Hayman et al. (1976) contribu ted to other factors such as host species, soil type, and management practices influencing fungal survival and development. As previously mentioned, management practices such as pesticide applications, in particular, fungicides, may inhibit the effect o f arbuscular mycorrhizal fungal sporulation and colonization (Nemec and O’Bannon, 1979; El Giahmi et al., 1976). Rhodes and Larsen (1979) examined arbuscular mycorrhizae of turfgrasses in field and greenhouse conditions. The researchers discovered that w hen fungicides were applied to bentgrass, infection averaged 9 to 17%, however, in non treated field plots, the roots were infected at a rate of 40 60 percent. The same observation was reported in the greenhouse evaluations, with one fungicide, PCNB, tota lly eliminating mycorrhizae (Rhodes and

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15 Larsen, 1979). Conversely, DBCP, a nematicide, has actually been reported by Bird et al. (1974) to enhance arbuscular mycorrhizal development. It is imperative to mention that mycorrhizal interactions lie along a continuum from mutualistic to parasitic based on the cost to benefit ratio colonization. Obviously, mycorrhizal associations can be mutualistic, but they can also be parasitic, commensal, amensal, and even neutral in nature (Johnson et al., 1997). Where along this continuum the association will fall, depends on a complex hierarchy mediated by biotic and abiotic factors within the rhizosphere and ecosystem being affected. No doubt, this range of mycorrhizal associations is greatly affected by time and s pace. The complexity of mycorrhizal investigations is ultimately confounded by the fact that the plant and fungal perspective on costs to benefits differs greatly from situation to situation (Johnson et al., 1997). With this in mind, Ryan and Graham (2 002) presented the point of view that arbuscular mycorrhizal fungi do not play such a vital role in production agricultural systems, in relation to nutrition and growth, simply because the high cost of energy from the plant to support the fungal invader ou tweighs the benefits of that association. This outcome is not beneficial in terms of crop production and may, in fact, be detrimental. Nonetheless, those production systems not considered to be within a natural or traditional cultivated production system such as sod, still need much attention where mycorrhizal symbiosis is concerned before a definitive yes or no can be applied to functional use of mycorrhizal fungi. Conversely, in 1997, Srivastava et al. concluded that “there is little doubt that vesicu lar arbuscular mycorrhizae fungi will emerge as a potential tool for improving crop plants in the years to come.” These opinions, in conjunction with the

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16 increased concern for environmental quality and sustainable technologies warrants an examination of m ore specific research reports in agricultural crops. In this review, the concentration is on turfgrass research. Mycorrhizas in Grasses There has been a considerable amount of research on mycorrhizal fungi associated with grasses (Hetrick et al., 1988, 1991; Trappe, 1981; Bethlenfalvay et al., 1984). Though much of the work conducted on grasses was begun in the 1970’s, Nicolson (1955) examined mycotrophic nature in grasses and later (Nicolson, 1956) with mycorrhizae in both grasses and cereals. These first studies in grasses and cereals were mainly concentrated on the ecological aspects of mycorrhizal infection. In fact, it was not until Nicolson (1956) showed diagrammatically that external hypha penetrate the root hairs or epidermal cells and spread throughout the cortex of grasses. Additionally, Nicolson noted that arbuscules form later in the inner cortical layers, which was valuable information in the study of grasses and their mycorrhizal partners. In experiments on fescue ( Festuca ovina L ), cocksfoot ( Dactylis glomerata L.), sand fescue ( Festuca rubra var. arenaria L.), and marram grass ( Ammophila arenaria L.: Link), Nicolson (1956) found that mycorrhizal infection was prevalent throughout a wide range of different habitats and soil types, al though the incidence of infection varied greatly between habitats and communities. With a lull in ecological studies throughout the 1960’s, environmental issues surpassed many of the more basic research topics. In 1979, Rhodes and Larsen examined the eff ects of fungicides on mycorrhizal development in cool season turfgrasses. Again, Rhodes and Larsen (1981) conducted a similar study, where the effects of fungicides on bentgrasses and the mycorrhizal fungus, Glomus fasciculatus were explored. Arbuscular mycorrhizas of ‘Penncross’ creeping bentgrass

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17 ( Agrostis palustri s Huds. ) were studied in greenhouse experiments to evaluate popular fungicides, such as, chloroneb and maneb, which did not affect mycorrhizal development. However, foliar applications of PC NB, chlorothanil, bayleton, anilazine, benomyl, and chloroneb at various weeks after inoculation with Glomus fasciculatus resulted in significantly reduced mycorrhizal colonization, thus limiting their beneficial effects. Later, studies of mycorrhizas i n turfgrasses seemed to swing back toward ecological studies with the introduction of seasonal and edaphic variation of arbuscular mycorrhizal infection (Rabatin, 1979). In a population survey, Rabatin (1979) sampled for Glomus tenuis infection in Panicum virgatum L., Poa compressa L., Poa pratensis L., Poa palustris L., Phleum pratense L., and Festuca etalior L., all cool season meadow grasses. Rabatin (1979) determined that the greatest percentage of root infection by this fungus occurred in grass roots from dry, P deficient fields. Moreover, the percent of infection was lowest in the cool, wet months of the spring. Thus, Rabatin (1979) concluded that mycorrhizal infection tends to be greater in drier, P deficient soils versus wet or flooded conditions Bagyaraj et al. (1980) concluded that a study of the spread of mycorrhizas from the site of infection along the root to deeper soil layers was necessary to provide important information for plant inoculations. This was done in grasses since the roots g row out of the inoculated sites quickly. Researchers collected root samples from various depths and found that roots at 3 4 and 8 9 cm were mycorrhizal at 45 days after inoculation. However, when roots were collected from deeper layers, the roots wer e only mycorrhizal after 75 days. The research lead Bagyaraj et al. (1980) to conclude that mycorrhizal infection of warm season grasses such as Sudangrass ( Sorghum bicolor L.:

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18 Moench), was spread to deeper layers by mycelial growth through the root, whic h was helpful information when researching inoculation methodologies important in such experiments as population surveys where pot cultures are a necessary to speciate the fungi collected. In an attempt to determine the distribution and occurrence of myco rrhizal fungi in Florida’s agricultural crops, Schenck and Smith (1981) examined bahiagrass ( Paspalum notatum Flgge) and digitgrass ( Digitaria decumbens Stent ) among 30 Cucurbitaceae Legumino sae Solanaceae and Vitaceae crops. In a population survey, t he authors found that mycorrhizal fungi in Glomus occurred most frequently in Florida, with species of Gigaspora found regularly in central and south Florida and Entrophospora collected only once (Schenck and Smith, 1981). Furthermore, Acaulospora was fou nd in the highest frequency in the grasses evaluated. In this instance, there was no correlation among species or genera occurrence and the available soil P or soil pH. In another study, endomycorrhizas and bacterial populations were examined in three cool season grasses. Agrostis tenuis Sibth., Deschampsia flexuosa L.: Trin., and Festuca ovina L., were collected and examined by Lawley et al., (1982) for mycorrhizal associations. In this case, the researchers noticed that mycorrhizal abundance was low est when Agrostis species were partnered with other plants and highest when partnered with Festuca Finally, Sylvia and Burks (1988) began working with grasses other than those only found in cool season climates. Beach erosion in coastal areas became a major economic concern in the late 1980’s; beach grasses such as sea oats ( Uniola paniculata L.) were often utilized to restore southeastern beaches to slow loss of sand. It was

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19 unclear whether or not these grasses relied on arbuscular mycorrhizal associ ations for survival in the harsh climate. Sylvia and Burks (1988) found that isolates of Glomus deserticola and G. etunicatum significantly increased the dry mass, height, and P content of the sea oats, while other isolates had little or no effect. In the search for a better host for inoculum production, compared to the traditional bahiagrass, Sreenivasa and Bagyaraj (1988) evaluated seven grasses for their ability to quickly produce large masses of mycorrhizal spores for inoculations. Grasses such as guinea grass ( Panicum maximum Jacq. ) and rhodes grass ( Chloris gayana Kunth) were studied and all were found to be mycorrhizal. However, the highest root colonization was observed in the rhodes grass, as well as the highest production of spores and infect ive propagules. Studies on other warm season grasses such as St. Augustinegrass ( Stenotaphrum secund atum [Walt.] Kunze), Centipedegrass ( Eremochloa ophiuroides [Munro] Hack.), or even bermudagrass ( Cynodon dactylis L.: Pers.) have not been identified. In studies of the difference in responses of C 3 and C 4 grasses to P fertility and mycorrhizal symbiosis, Hetrick et al. (1990) showed that warm season grasses such as big bluestem ( Andropogon geradii Vitm.) and indian grass ( Sorghastrum nutans L.: Nash), res ponded positively to mycorrhizae or P fertilization, or mycorrhization in cool season grasses, such as perennial ryegrass ( Lolium perenne L.). In warm season grasses, there was a positive relationship between root colonization and dry weight, with an inv erse relationship between mycorrhizal root colonization and P fertilization. The evaluation provided evidence that the C 3 and C 4 grasses display profoundly different nutrient acquisition strategies (Hetrick et al., 1990b).

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20 The effect of mycorrhizal symbio sis on regrowth of rhizomes of big bluestem was assessed as a function of clipping tolerance (Hetrick et al., 1990a). Mycorrhizal clipped plants were larger than nonmycorrhizal clipped plants, but the effect diminished with successive clippings as did myc orrhizal root colonization. This information on clipping tolerance indicates that mycorrhizal turfgrasses respond similarly when clipped or mowed under constant turf management. Hetrick et al. (1991) compared the root architecture of five warm and five cool season grasses in an attempt to evaluate whether mycorrhizal symbiosis confers a greater tolerance to drought, soilborne disease, vigor, and yield through direct or indirect improved nutritional status of the host plant. The cool season grasses had significantly more primary and secondary roots than the warm season grasses and the diameter of those roots was smaller than that of the warm season grasses. The mycorrhizas did not affect the number or diameter of cool season grass roots, however, the wa rm season grasses did respond to mycorrhizal inoculation. Additionally, the root length was significantly increased in the warm season grasses with mycorrhizal infection when compared to the cool season grasses. Through the aid of topological analysis of root architecture, mycorrhizal symbiosis was shown to inhibit root branching in warm season grasses, but had no effect on cool season grass rooting (Hetrick et al., 1991). The researchers concluded that mycorrhizal dependent warm season grasses have uniq ue root architecture, allowing energy to be conserved for root development, while the less dependent cool season grasses do not exhibit the same benefits of mycorrhizal infection. In studies designed to determine the dependence of warm season grasses on a rbuscular mycorrhizae and relationships between mycorrhizae and P availability and

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21 plant density, Brejda et al. (1993) and Hetrick et al. (1994) evaluated sand bluestem ( Andropogon geradii var. paucipilus Nash ), switchgrass ( Panicum virgatum L.), and Canad a wild rye ( Elymus canadensis L. ). The popular cool season grasses, creeping bentgrass ( Agrostis stolonifera L.) and Kentucky bluegrass ( Poa pratensis L.) were evaluated in relation to the impact of arbuscular mycorrhizae and P status on plant growth (Ch arest et al., 1997). The authors revealed that as mycorrhizal infection increased in the grasses, root colonization increased to more than 40% with lowered P fertilization. This information could be particularity helpful in warm season grasses where P ma y have a major impact in soils, such as those found throughout Florida. The researchers of this study concluded that arbuscular mycorrhizal symbiosis could be considered as a potential fertilizer reduction agent (Charest et al., 1997). More recently, m ycorrhizal symbiosis and fertilizer relationships have dominated arbuscular mycorrhizal research; however, the majority of this work has concerned cool and warm season prairie grasses. The emphasis of molecular technologies has resulted in less applied ty pes of research being performed with grasses and mycorrhizas. Using terminal restriction fragment length polymorphism (T RFLP), Vandenkoornhuyse et al. (2003) assessed the diversity of arbuscular mycorrhizal fungi in various cool season grasses, which co occurred in the same research plots. Based on a clone library, the level of diversity was consistent with past studies; showing that mycorrhizae fungal host plant preference exists, even between grass species. Obviously, there is limited information on warm season turfgrasses when compared to the warm season prairie and cool season meadow grasses. In the Southeast,

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22 warm season turfgrasses are highly valued for their drought resistance, aesthetic importance and generally low maintenance on some home law ns, golf courses, soccer, and football fields. Species such as bermuda, St. Augustinegrass, seashore paspalum ( Paspalum vaginatum Swartz ) zoysia ( Zoysia sp.) bahia, and centipede are used in landscapes throughout Florida. St. Augustinegrass is dominant residential species in Florida (Trenholm, 2004). Haydu et al., ( 2002 ) estimated that 36% of the total lawn acreage in Florida, or 1.5 million acres, was comprised of St. Augustinegrass in 1996. Valued for its shade tolerance, ability to adapt to various soils, and color, St. Augustinegrass cultivars such as ‘Floratam’, ‘bitterblue’, ‘Raleigh’, and ‘Floratine’ became popular with home owners. Chinch bug resistant ‘Floratam’ quickly became the number one cultivar upon its release in the 1970’s. St. August inegrass is a desirable species home lawn, however problems with disease susceptibility can be devastating. Two examples are brown patch ( Rhizoctonia solani K hn ) and take all root rot ( Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. graminis ). T o date, research evaluating the potential benefit of mycorrhizae in St. Augustinegrass has been neglected such as reduced fertilizer use and production cost. The method of production of St. Augustinegrass may result in limited benefits of mycorrhizal rese arch. St. Augustinegrass is produced vegetatively as sod throughout the southeast. Once or twice a year, the sod is harvested leaving “ribbons” or strips of grass behind. These ribbons are responsible for re growth, through stolons, of the sod field. H arvesting cycles would make lengthy mycorrhizal studies difficult. An extensive survey of this plant system in relation to the arbuscular mycorrhizal fungi is warranted.

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23 The overall objective of this research is to investigate the impact of mycorrhizal fungi on warm season turfgrasses in Florida. A survey of the population and identification of arbuscular mycorrhizal fungi associated with St. Augustinegrass roots in Florida sod is provided in Chapter II. In Chapter III, a survey of root pathogens is e xplored in relation to arbuscular mycorrhizal colonization in sod production fields. Chapter IV includes studies designed to determine whether or not arbuscular mycorrhizal fungi affect root disease caused by pathogenic isolates of R. solani and G. gramin is var. graminis and if potential affects are direct fungal interactions or indirect systemically acquired mechanisms of resistance. In Chapter V, a general summary and conclusions concerning arbuscular mycorrhizal fungi in St. Augustinegrass in Florida are provided.

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24 CHAPTER 2 POPULATION AND IDENT IFICATION OF ARBUSCU LAR MYCORRHIZAL F UNGI IN ST. AUGUSTIN EGRASS There is no information regarding arbuscular mycorrhizal fungi (AMF) in the popular warm season St. Augustinegrass ( Stenotaphrum secundatum ). In Florida, St. A ugustinegrass sod is a valuable commodity in home lawns and commercial landscapes. ‘Floratam’ the most common and widely adaptable cultivar is extensively used across the state. It is also the primary cultivar grown in Florida for sod. In north central Florida, sod production is increasing and growers are eager to increase production and lower pesticide and fertilizer inputs. No information exists about mycorrhizas in this species. The information is potentially useful in sod management to reduce disea se severity, chemical usage, and other production costs. In most cases, AMF populations are decreased by agricultural practices are associated with conventional farming. St. Augustinegrass sod production is unique in that it is not a traditional or natur al plant system. Currently, no information is available to growers to make informed decision about inoculation with these fungi. The feasibility of inoculation studies for nutrient acquisition, pesticide, and disease management can be performed using myc orrhizal fungi more efficiently in the future once St. Augustinegrass is determined to be mycorrhizal. Of current interest to mycorrhizal researchers is the ecology of mycorrhizal populations and their benefit to both organic and more conventional cropp ing systems. Information from less natural and conventional systems like St. Augustinegrass

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25 sod is timely and could shed light on a little known ty cropping method. Mycorrhizal systems and those interactions within it are complex and require extensive e valuation, especially in crops not yet known to possess such associations. This evaluation may supply valuable answers about mycorrhizal ecology. The objective of this study is to determine if AMF colonize St. Augustinegrass, to what extent, and to ident ify the colonizing fungi. Materials and Methods Sampling ¦¦ ‘Floratam’ St. Augustinegrass plant roots and associated soil were collected monthly from three sod farms in three counties (Marion, Bradford, and Union) in north central Florida from December 200 4 through December 2005 with the exception of July. Each of the sod farms had been cropped with ‘Floratam’ St. Augustinegrass for 12 years or more (Fig. 2 1 A C). Ten subsamples of soil were taken from three (3 m 2 ) plots per sod farm with a 1.27 cm dia meter soil probe to a depth of approximately 15 cm as suggested by Brundrett et al. (1995). Root samples from each plot were extracted with a small hand trowel. Subsamples of roots and soil from each plot were pooled, resulting in three separate composit e plot samples per location. Root samples were placed into plastic ziplock bags separate from soil samples and stored at room temperature for approximately 1 d prior to spore extraction and root manipulation for mycorrhizal evaluation. Approximately 200 g of field soil from each plot were combined with 200 g of a low P, low organic matter soil mined from the UF/IFAS Plant Science Research and Education Unit in Citra, Florida. This soil was then potted into 10 cm clay pots sown with sorghum sudangrass hyb rid seed ( Sorghum bicolor [L.] Moench x Sorghum sudanens e ) cv. Summergrazer III. Low P

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26 soil was used in pot cultures to enhance sporulation of potentially cryptic species in order to facilitate their recovery and identification (Fig. 2 2). The cultures were incubated for 60 d at 20 25 C with 12 h artificial light (day/night). The seed was surface sterilized using a 10% sodium hypochlorite and deionized water solution for 30 sec and rinsed for 1 min with sterile deionized water prior to planting. The po t cultures received a Peter’s 20 0 20 (Spectrum Group, St. Louis, MO) nutrient solution, devoid of P, every two weeks. Approximately 90 d later, single spores from the field soil pot cultures were selected from spore extracts (Fig. 2 3). This process was accomplished by wet sieving, decanting (Gerdemann and Nicolson, 1963), and 40% sucrose (v/v) centrifugation (Jenkins, 1964). These spores were used to inoculate sterile, low P soil (Citra, Florida) and sorghum sudangrass hybrid seed for spore production and subsequent identification of the sporulating AMF as suggested by Gerdemann and Trappe (1974). The soil was sterilized twice for 90 min at 121 C at 15 psi for two consecutive days. Samples of field soil were also submitted to the IFAS Extension Soil T esting Laboratory in Gainesville, Florida on a tri monthly basis for soil nutrient composition and pH testing. Soil pH, from all three fields, ranged from 5.6 to 7.0 during the 12 month sampling period. Phosphorous levels ranged from 5 to 119 ppm. Root p reparation ¦¦ Young, healthy appearing fibrous roots were rinsed in tap water and separated with a scalpel from the plant crown and/or seminal roots. Selected roots were cut into 1 2 cm long segments and cleared of cell and wall components in 10% KOH (w /v) under pressure in an autoclave for approximately 20 min (Brundrett et al., 1996). The root segments were cooled, then rinsed in tap water, and placed into hot 0.05% trypan blue with glycerol overnight to stain mycorrhizal structures ( Beve ge, 1968; 2 2

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27 Phi llips and Hayman, 1970; Kormanik and McGraw, 1982 ). Excess stain was rinsed from the root segments with tap water and then mounted in water on glass slides to view vesicles and arbuscules. Slight pressure applied to the cover slip, with occasional heatin g over an alcohol burner, aided in flattening the root segments adequately for microscopic evaluation of mycorrhizal structures in root cells. One hundred root segments were evaluated per sample for intensity of colonization and to identify any variation s in arbuscular morphology which might exist. Mycorrhizal structures on glass slides were viewed with a Nikon Optiphot compound microscope at 200, 400, and 1000x magnifications, and photographs were taken with a Nikon CoolPix 990 digital camera. In order to judge the amount of mycorrhizal root colonization, the grid line intersect method was used to estimate the total root length colonized by AMF (Newman, 1966; Tennant, 1975; Giovannetti and Mosse, 1980). Spore extractions ¦¦ Mycorrhizal spores were ext racted by wet sieving and decanting by mixing 100 g of air dried sample soil with 300 ml of tap water, blending at low speed in a commercial Waring blender for 1 min, and then allowed to settle for 1 min. The supernatant was then passed through a series o f Tyler 250, 125, and 38 m mesh sieves (Daniels and Skipper, 1982). The remaining fraction was rinsed with tap water to remove sediment and any organic materials left behind. The fraction was decanted into 50 ml centrifuge tubes containing a 40% sucrose /deionized water solution (w/v) (Jenkins, 1964). The tubes were centrifuged for 3 min at 2,000 rpm in a Dynac III centrifuge. The supernatant, containing the spores, was decanted off the top of the tube into a 38 m mesh sieve and rinsed to remove the su crose. The extracted spores were collected in a 9 cm Petri dish with tap water rinse and viewed with a Zeiss dissecting scope.

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28 Mycorrhizal spore densities were enumerated by using an ocular field method described in the International Culture Collectio n of (Vesicular) Arbus cular Mycorrhizal Fungi for high spore densities (Morton, 2005) Intact and parasite free spores were selected using a Gilson 20 l pipetman. These spores were used to inoculate 10 cm diameter clay pots containing the low P, sterile soil (as described above) and planted with surface sterilized sorghum sudangrass hybrid seed. The monocultures were kept at 20 25 C for approximately 60 d. At that point, any spores that had been produced as a result of the inoculations were extracted a s previously mentioned, and used to inoculate another crop of sorghum sudangrass in sterilized, low P soil. The second generation of monocultures were then maintained for 60 90 d and processed for spore extraction and mycorrhizal identification. Arbusc ular mycorrhizal fungi identification ¦¦ Identification of the mycorrhizal fungi associated with St. Augustinegrass was accomplished by selecting healthy, single spores with a 20 l Pipetman and mounting in either sterile, deionized water or (1:1 v/v) PVLG (polyvinyl alcohol lactic acid) + M elzer’s reagent (Khalil et al., 1992). The spores were then viewed at 200, 400, and 1000x using a Nikon compound microscope and identified. Using arbuscular mycorrhizal descriptions by Schenck and Prez (1988), a tentative determination to genus was made based on the average measurement of 20 similar spores per pot. The species was determined based on taxonomic descriptions from the INVAM Species Guide (Schenck and Prez, 1988). Identifying characteristics of the monocultured spores, such as spore wall number and width, hyphal appendages, the presence or absence of germ shields, approximate overall spore diameter and color in reagents, were used as described by Schenck and Prez (1988).

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29 Statistical analysis. ¦¦ Spore density and percent colonization da ta were analyzed using the General Linear Model procedure (SAS Institute, Version 9.0, 2004) (Appendix F 1). The survey was performed using a random model in a randomized complete block design with multiple samplings at multiple locations. The percent ro ot colonization data were transformed with the arcsine square root transformation prior to an analysis of variance due to distribution of propagules within soil being highly variable resulting in a non normal frequency of distribution points (St. John and Hunt, 1983; Friese and Koske, 1991). Spore density data were transformed to their natural log prior to analysis of variance to prevent violation of the assumption of normal distribution. Significant interactions were separated using Tukey’s Studentized R ange Distribution test. Correlations between percent colonization or spore density data, with soil nutrient composition, and percent colonization to spore density were done in SAS using Pearson product moment correlation coefficients. Regression analyses also were performed with the regression procedure in SAS. Results Root Evaluation ¦¦ Roots, collected from sod fields evaluated in this survey revealed the first evidence of an interaction between AMF and St. Augustinegrass. In stained roots mounted on glass slides, AMF structures such as internal vesicles, intra and extraradical hypha, and an assortment of arbuscular types were observed. Bulbous appressoria (Fig. 2 4) were noted at inoculation points along the length of the root, giving rise to carbohy drate storage vesicles of various shapes within cortical root cells (Figs. 2 5, 2 6). Copious amounts of intra and extraradical hypha were observed within and along the outer surface of root tissue (Fig. 2 7). Most notably, a variety of arbuscular types were observed within the cortical root cells. Arbuscules, or haustoria like structures,

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30 have been categorized into two morphological types (Gallaud, 1904); Arum and Paris types. These intercellular mycorrhizal structures are the presumed active fungal s ites of nutrient translocation between host and fungus (Bracker and Littlefield, 1973; Brundett et al., 1984). In this study, field grown plant roots were found to contain both the Arum and Paris type of arbuscules along with a variety of intermediate A rum morphologies. Intermediate forms of the Arum type found in cortical root cells of St. Augustinegrass sod plants ranged from a typical “feathery” form (Fig. 2 8) extending from intracellular hypha to a “dense compact” form between cells of conjoined intercellular hyphae (Fig. 2 9). A “grainy” form (Fig. 2 10) was also found in cortical root cells on several occasions. This could be a collapsing arbuscule instead of an intermediate arbuscular form. The Paris type arbuscule found in St. Augustinegras s plant roots shows a typical arbusculate coil (Fig. 2 11) in the root cell, while intermediate forms were not observed. An unusual structure was found along intercellular hyphae that resembled a hyphal mat with a mantle like appearance often found in con junction with certain types of ectomycorrhizas (Fig. 2 12). This may be a new arbuscular form found in the Poaceae This structure was only observed once in St. Augustinegrass plants harvested in April 2005 at the Fort McCoy location. Spore density evalu ation ¦¦ Further evidence supporting an interaction between AMF and St. Augustinegrass was observed outside the root within the rhizosphere. AMF spores clinging to epidermal tissue on roots were frequently observed in field samples and in pot cultures us ing field soil from each farm location and sorghum sudangrass as the trap plant. The three sod farms sampled in this survey have been cropped solely in

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31 ‘Floratam’ St. Augustinegrass sod for more than 12 years. Weeds are heavily controlled with herbicides at each location. The AMF spores recovered from field soil are entirely dependent upon the St. Augustinegrass plants because they are obligate heterotrophs. The limited availability of other plant species at each location, and the availability of numero us spore types for pot culturing and subsequent AMF identification, provides adequate evidence of AMF colonizing St. Augustinegrass plants in North Central Florida soils. Additional mycorrhizal structures such as auxiliary cells were frequently observe d in slide mounts of spores from both pot cultures and field soil (Fig. 2 13). Selected single spores that appeared non parasitized and viable, were chosen under light microscopy for culturing in sterile, low P soil in order to obtain consistent spore str uctures compatible with identification procedures. Spores, retrieved from pot cultures were used as sieved soil sub cultures to produce another generation of spores capable of being readily identified from their morphological structures according to Schen ck and Prez (1988). Table 2 1 lists the species of AMF positively identified from sub cultures of soil from each location over a year long period. Species of Glomus were the most commonly encountered AMF in north central Florida soils at each location At the Lake Butler location, Glomus species included: G. etunicatum Becker & Gerdemann (Fig. 2 14), G. intraradices Schenck & Smith (Fig. 2 15, 2 16), G. reticulatum Bhattacharjee & Mukerji (Fig. 2 17, 2 18), and G. aggregatum Schenck & Smith (Fig. 2 19 ). Glomus species isolated at the Fort McCoy location included : G. ambisporum Smith & Schenck (Fig. 2 20), G. formosanum Wu & Chen

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32 (Fig. 2 21), G. macrocarpum Tulasne & Tulasne (Fig. 2 22), G. gerdemannii Rose, Daniels & Trappe (Fig. 2 23) G. intraradice s and G. etunicatum Acaulospora spinosa Walker & Trappe (Fig. 2 24) a nd an unidentified species of Scutellospora were isolated at Lake Butler. Additional AMF genera were found at Fort McCoy including: Entrophospora infrequens [Hall] Ames & Schneider ( Fig. 2 25), A. denticulata Sieverding & Toro (Fig. 2 26), A. lacunosa Morton (Fig. 2 27), and Scutellospora minuta [Ferr. & Herr.] Walker & Sanders (Fig. 2 28). The Starke location was unusual in species diversity with only 3 species isolated: Glomus etun icatum G. intraradices and Scutellospora minuta One unique spore type was found at the Fort McCoy location, but could not be grown in a pot culture successfully. The unidentified spore type was observed on two occasions during the late spring of 2005 in very sma ll numbers and appeared to be either a species of Acaulospora or Entrophospora based on morphology. W ithout a sufficient number of cultivated spores for microscopic evaluation positive identification of the species was not possible. Sieving f ield soil from each location not only yielded spores for pot culturing, but also enabled a numerical count of spore density, which is a good indicator of the infectivity of the AMF in the soil and their level of activity in the rhizosphere. The total spor e density at the three locations ranged from 78 to 2,132 spores per 100 g of dry soil (non transformed data). Spore density but did not vary among or within sod farm locations (P < 0.0001), indicating that variations in soil factors did not significantly affect AMF spore production between locations from December 2004 through December 2005 (Table 2 2). Spore production did vary significantly (P < 0.0001) between monthly sampling, which suggested a possible seasonal influence on spore production. Greater

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33 spore density totals occurred in soils collected during the warmer summer and fall months, as compared to, lowered spore production occurring in the cooler months of winter and spring. Total spore density in December 2004 was significantly lower when comp ared to December 2005. This might be explained by increased rainfall, prior to the sampling period, in north central Florida during the 2004 hurricane season. With spore densities varying between dates, analysis of variance for these points showed a si gnificant date by location interaction (P < 0.05) indicating that seasonal effects and unknown variations in site related effects might measurably influence the total spore density. In this survey, rainfall and soil moisture where positively correlated to spore density (Table 2 3). Based on the regression equations, a quadratic response was generated in total spore density to soil moisture at each location. Spore density at the Starke location increased at soil moisture levels between 0 and 2 cm, but d eclined until soil moisture levels reached 6 cm where another increase was observed (Fig. 2 29). Above 9 cm a decrease in spore density occurred (r=0.73). The same general response to soil moisture was noted at the Fort McCoy location except where soil m oisture declined to approximately 8 cm (r=0.61) (Fig. 2 30). At the Lake Butler location, spore density increased slightly until soil moisture levels reached 7 to 8 cm when a slight decline in spore density was observed (r=0.68) (Fig. 2 31). This lends c redibility to the theory that excessive rainfall during the hurricane season of 2004 lowered spore production in December of that year. A quadratic response was also produced in total spore density to temperature at each location. Spore density at the S tarke location (r= 0.60) (Fig. 2 32) decreased from

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34 15 C until the temperature reached 20 C. Between 20 C and 28 29 C a gradual increase in spore density was observed until the temperature reached 30 C. At that point there was another gradual decrease in spore density, which seemed to level off near 35 C. At the Fort McCoy location (r=0.84) a gradual increase in spore density was observed until the temperature was approximately 28 29 C, then a decline was noted (Fig. 2 33). At the Lake Butler location (r=0.59) a slight increase in spore density occurred across all temperature ranges (Fig. 2 34). Based on these data, it appears that soil temperatures above 28 30 C have a detrimental effect on the AMF. In addition, this temperature range might also dama ge host root tissue. Percent colonization evaluation ¦¦ Percent root length colonized by AMF yielded no significant difference among or within location differences, but there was a significant date interaction (P < 0.0001). Colonization was generally highest in the cooler months of winter and spring, with l ower colonization occurring in the warmer summer and fall months except in December 2005, when colonization was the lowest. The amount of root length colonized ranged from 13 to 39% across the sampling dates (non transformed data). No correlation was fou nd between temperature and soil moisture in relation to percent root length colonized (Table 2 4). Discussion Dickson (2004) suggested an Arum Paris continuum of mycorrhizal symbioses in a survey of 12 colonized plant families, with arbuscule formation d ependent on the fungus as well as the host plant. Most mycorrhizal angiosperms were once thought to only produce the Arum type of arbuscule, which consists of both intercellular hyphae and arbuscules, while most angiosperms and bryophytes were thought to only produce the Paris type with intercellular hyphae and arbuscular coils (Dickson, 2004). The majority

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35 of scientific research has been conducted on flowering plants versus trees and bryophytes causing these fallacies to be argued as fact until Smith and Smith (1997) produced a comprehensive list of plant families that included their arbuscule types. The list showed that the Paris type is in fact most common among all plant families and that, “intermediate” or transitional arbuscular morphotypes were obs erved in some plant species. One genus ( Ranunculus ) forms both types within the same plant (Smith and Smith, 1997). Experiments on maize ( Zea mays ) and the tuliptree ( Liriodendron tulipfera ), among many others, revealed that AMF can form either type of arbusculate structure based on the host plant (Barrett, 1958; Gerdemann, 1965). In a field experiment using tomatoes ( Lycopersicon esculentum ) and other annual crops, investigators found that arbuscule morphology is actually dependent on intercellular spa ces in cortical root cells ( Brundrett and Kendrick, 1988; Cavagnaro et al., 2001). Intermediate forms of the Arum and Paris type arbuscules are common in certain plant families such as those described in three cultivars of flax ( Linum usitatissimum ), whic h Dickson et al. (2003) referred to as arbuscules “in pairs in adjacent longitudinally arranged cortical cells arising from a single, radial intercellular hyphae.” On rare occasions, both arbuscule types ( Arum and Paris ) occur in the same plant species, which Smith and Smith (1997) noted in the family Poaceae The Paris and Arum types were found in millet, ryegrass, and wheat. In addition, a series of intermediate forms between the two main types of arbuscules were also observed. The same can be sai d for St. Augustinegrass plants in relation to AMF colonization. In field studies, environmental effects may interact to influence fungal and plant response to the

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36 mycorrhizal interaction. Sylvia et al. (1993) suggested that even in the presence of high amounts of soil P, water stress and pesticide applications can have extensive effects on mycorrhizal response. Rabatin (1979) noted that soil moisture may have the greatest effect on the degree of infection of Glomus species in field situations. Furtherm ore, the stages of plant development (Saif and Khan, 1975) as well as temperature (Giovannetti, 1985; Schenck and Kinloch, 1980; Smith & Smith, 1997; Sylvia, 1986) all play a major role in mycorrhizal activity. In this survey, AM fungi preferred warmer m onths for spore production and cooler months for colonization of St. Augustinegrass plants. In the north central region of Florida, St. Augustinegrass does not usually go completely dormant in cooler temperatures, and there is usually some plant activity during the winter months especially in the roots where AMF colonization occurs. This increase in colonization during cooler temperatures may be an effort to preserve valuable carbon and energy reserves for future spore production. Subsequent proliferatio n in the warmer months, while the plant host is most active, would provide more carbohydrates from a symbiotic interaction (Johnson et al., 1997). It is also possible that AMF are actually acting as a parasite in the winter months when colonization is hig hest while the plant is less active. During less than optimal winter growing conditions, the St. Augustinegrass plant is less able to defend itself against infection and colonization due to lowered metabolic activity. Johnson et al. (1997) suggested a m ycorrhizal continuum ranging from mutualistic to parasitic in some managed habitats where humans unknowingly altered the association through management regimes. Another possibility is environmentally

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37 induced parasitism due to morphological, phenological, and physiological differences in the symbionts which may influence the mycorrhizal association (Johnson et al., 1997). Conversely, in natural habitats, mycorrhizal associations have evolved over many years to encourage fitness in the plant and the fungu s making the interaction continually mutual (Johnson et al., 1997). St. Augustinegrass sod systems are not traditional cropping systems needing continual management inputs from man, nor are they a natural, non impacted habitat. St. Augustinegrass sod cou ld be referred to as a non conventional cropping system due to minimal inputs after harvesting where ribbons of grass are left behind for re growth. Cloned host plants are in constant supply in sod fields providing the AMF with a dependable host, but when the plant is semi dormant throughout the winter months the fungi may actually pose a threat to the health of the plant because net costs in carbon might then exceed net benefits in some situations. For example, during instances of lowered metabolic activ ity in the winter, plants lower photosynthetic ability and subsequent output and will not benefit from the added benefits of a mutual interaction. Acquisition of nutrients and water is less important during these times, but St. Augustinegrass may be harme d by the loss of stored carbon to AMF. Throughout the year, there are potential times when the interaction between plant and AMF is such that the symbiosis might actually be neutral in nature (Johnson et al., 1997). An attempt was made in this survey t o correlate spore density to the percent root length colonized, but no correlation was found. Some researchers have reported a correlation between the two variables (Giovannetti, 1985; Miller et al., 1979) while others have observed no such relationship ( Giovannetti and Nicolson, 1983; Hayman and Stovold, 1979). This is most likely due to the vast variations observed in soils, plant

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38 species and their developmental stage, and fungal specificity. Many mycorrhizal studies suggest a significant interaction w ith soil P where spore production or colonization is lowered by increasing levels of P. Correlations between soil chemical characteristics such as P content to spore density and percent root colonization have been reported in grasses ( Brejda et al., 1993 ) Others suggest that mycorrhizal ecology plays less of a role. P content in south Florida soils had no effect on AMF in tropical forage legume pastures (Medina Gonzalez et al., 1988), nor did potassium or pH in studies of cultivated soils (Abbott and Ro bson, 1977; Hayman, 1978). In this survey, soil samples from each location were evaluated during the months of January, April, August, and November 2005 in an attempt to correlate soil Mg, Ca, K, P, soil pH, and organic matter percentage to spore density and/or percent root length colonized, but a correlation was not observed (Table 5). One theory to explain the lack of correlation between AMF and P content, in this case, might be explained by asexual organisms, without the cost of sexual reproduction and consequently no genetic variability, and having scores of mutations that accumulate over a long period of time (Helgason and Fitter, 2005). The Glomeromycota possess ancient asexual lineages (Gandolfi et al., 2003). This apparent genetic isolation would presumably cause mutations to allow for some adaptations such as P tolerance. In AMF the coenocytic mycelium is multinucleate providing a set of mutations within the DNA of all nuclei (Helgason and Fitter, 2005). Reductions in fitness due to a lack of g enetic variability due to asexual reproduction may never be noticed in AMF because mutated, non functional genes from one nuclear lineage might be subjugated by functional alleles on another nucleus (Helgason and Fitter, 2005).

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39 Arbuscular mycorrhizal fun gi in these sod fields are secluded, thus reducing genetic variability, so it is possible that the ancient fungi are capable of evolving and adapting through mutations to tolerate large amounts of added nutrients like P. P is widely used in large amounts in St. Augustinegrass to promote root growth and health for winter survival and spring green up. Through years of isolation in sod fields and large applications of P on a frequent basis, these fungi might have evolved a mechanism through spontaneous mutat ion to tolerate elevated P levels. This is speculation, but the lack of spore density and percent colonization variable correlation to P levels could be due to genetic mutation in the fungi within these fields leading to a significant adaptation and evolu tionary event. Overall root colonization and spore density were low to moderate, which suggests that the AMF populating St. Augustinegrass sod production soils are moderately active. This situation might lend itself to field inoculation where AMF could potentially provide a level of root disease protection, which might lower pesticide use and cost. It could also lead to increased and more efficient P acquisition and use when combined with more conducive management strategies. On the other hand, inocul ation with AMF might be ineffective in situations where genetic isolation combined with perennial cropping and moderate to heavy fertilizer inputs are unavoidable for proper management.

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40 Figure 2 1 A C. ‘Floratam’ St Augustinegrass sod farms located at (A) Fort McCoy (Marion County), (B) Lake Butler (Union County), and (C) Starke (Bradford County) in north central Florida. A B C

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41 Fig. 2 2. Sorghum sudangrass pot cultures containing 50 % (w/w) field soil combined with 5 0% sterile, low P soil. 2 3

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42 Fig. 2 3. Spore extract from field soil following the wet sieving procedure.

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43 Figs. 2 4 – 2 7. Stained arbuscular mycorrhizal structures observed within ‘Florata m’ St. Augustinegrass. Fig. 2 4. Bulbous appressoria found originating from extraradical hypha. Bar = 40 m. Fig. 2 5. Circular type of AMF vesicle stained with trypan blue. Bar = 40 m. Fig. 2 6. Oblong type of AMF vesic le stained with trypan blue. Bar = 40 m. Fig. 2 7. Extraradical hyphae observed with light microscopy infecting and colonizing roots. Bar = 20 m. 2 5 4 6 7 5

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44 Figs. 2 8 – 2 11. Stained arbuscular morphology types found wi thin ‘Floratam’ St. Augustinegrass. Fig. 2 8. Feathery form of the Arum type arbuscule morphology, stained with trypan blue, within cortical root cells. Bar = 40 m. Fig. 2 9. Dense and compacted Arum type arbuscule morpholog y stained with trypan blue. Bar = 40 m. Fig. 2 10. Grainy or collapsing Arum type arbuscule morphology stained with trypan blue. Bar = 40 m. Fig. 2 11. Paris type coiled arbuscule, stained with trypan blue, within co rtical root cells. Bar = 40 m. Fig. 2 12. Net like AMF structure observed in roots across adjacent cortical root cells. Bar = 20 m. Fig. 2 13. Auxiliary cells of an AMF observed in spore extracts from field soil. Bar = 40 m. 8 9 10 11 13 12

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45 Table 2 1. Species of AMF positively identified at each sod farm location from pot cultures of sorghum sudangrass within a combination of field and sterile, low P soil. Location AMF Species Lake Butler Acaulospora spinosa Lake Butler Glomus et unicatum Lake Butler G. intraradices Lake Butler G. reticulatum Lake Butler G. aggregatum Lake Butler Scutellospora sp. Fort McCoy A. denticulata Fort McCoy A. lacunose Fort McCoy Entrophospora infrequens Fort McCoy G ambisporum Fort McCoy G e tunicatum Fort McCoy G formosanum Fort McCoy G gerdemanii Fort McCoy G intraradices Fort McCoy G macrocarpum Fort McCoy Scutellospora minuta Starke G etunicatum Starke G intraradices Starke S minuta

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46 Figs. 2 14 – 2 19. Arbuscular mycorrhizal fungal spores identified at the Lake Butler sod farm location. Fig. 2 14. A spore of G lomus etunicatum stained in Melzer’s reagent. Bar = 20 m. Fig. 2 15. A spore of G. intraradices in de ionized water. Bar = 20 m. Fig. 2 16. Spore wall morphology of G. intraradices spore stained in Melzer’s reagent (arrows point to cell wall layers). Bar = 40 m. Fig. 2 17. A spore of G. reticulatum in deionized water. Bar = 20 m. Fig. 2 18. Spore wall morphology of G. reticulatum in deionized water (arrows point to cell wall layers). Bar = 40 m. Fig. 2 19. A broken spore of G. aggregatum in Melzer’s reagent. Bar = 20 m. 14 15 16 17 18 19

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47 Figs. 2 20 – 2 28. Arbuscular mycorrhizal fungal spores identified at the Fort McCoy sod farm location. Fig. 2 20. A spore of Glomus ambisporum stained in Melzer’s reagent. Bar = 20 m. Fig. 2 21. A spore of G. formosanum stained in Melzer’s reag ent. Bar = 20 m.. Fig. 2 22. A spore of G. macrocarpum stained in Melzer’s reagent. Bar = 20 m. Fig. 2 23. A spore of G. gerdemannii stained in Melzer’s reagent. Bar = 20 m. Fig. 2 24. A spore of Acaulospora spinosa stained i n Melzer’s reagent. Bar = 20 m. Fig. 2 25. A spore of Entrophospora infrequens stained in Melzer’s reagent. Bar = 20 m. Fig. 2 26. A spore of A. denticulata stained in Melzer’s reagent. Bar = 20 m. Fig. 2 27. A spore of A. lacunosa stained in Mel zer’s reagent. Bar = 20 m. Fig. 2 28. A spore of Scutellospora minuta stained in Melzer’s reagent. Bar = 20 m. 20 22 21 25 24 23 26 27 28

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48 Table 2 2. Evaluation of analysis of variance data for spore density data from each sod farm location by date. Date Location Tota l Spore Density (spores/100g air dried soil) Dec. '04 Fort McCoy 5.06† Lake Butler 4.82 Starke 5.13 mean 5.00 d‡ Jan '05 Fort McCoy 5.42 Lake Butler 5.17 Starke 5.54 mean 5.38 cd Feb '05 Fort McCoy 5.80 Lake Butler 5 .78 Starke 5.56 mean 5.71 bcd March '05 Fort McCoy 5.53 Lake Butler 6.33 Starke 5.30 mean 5.72 bcd April '05 Fort McCoy 6.48 Lake Butler 6.84 Starke 6.32 mean 6.55 a May '05 Fort McCoy 6.72 Lake Butler 6.54 S tarke 6.94 mean 6.73 a June '05 Fort McCoy 6.90 Lake Butler 6.78 Starke 6.29 mean 6.66 a Aug '05 Fort McCoy 5.90 Lake Butler 7.01 Starke 5.88 mean 6.26 ab Sept '05 Fort McCoy 6.36 Lake Butler 6.13 Starke 5.66 mean 6.05 abc

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49 Oct '05 Fort McCoy 6.65 Lake Butler 6.08 Starke 5.70 mean 6.14 abc Nov '05 Fort McCoy 6.22 Lake Butler 6.54 Starke 6.76 mean 6.51 a Dec '05 Fort McCoy 5.81 Lake Butler 5.80 Starke 6.36 mean 5.99 abc †Each value is the average of three sample plots/location (10 sub samples/plot). ‡Means followed by the same letter are not significantly different according to Tukey’s (HSD) Studentized Range Test (P = 0.0001).

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50 Table 2 3. P earson correlation coefficients (r) for AMF spore density and soil moisture and temperature. Sporeden† Rainfall† Soiltemp† Percolon† 0.007 0.14 0.02 Sporeden 0.45*** 0.48*** Rainfall 0.61*** *** Significant at P = 0.0001, respectively. † Percolon = percent root length colonized; Sporeden = spore density; Rainfall = amount of rainfall in month preceding sampling date; Soiltemp = soil temperature for sampling date.

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51 y = -0.0264x 4 + 0.4465x 3 2.3561x 2 + 4.4331x + 3.4988 R 2 = 0.7319 0 1 2 3 4 5 6 7 8 0 2 4 6 8 10 Soil Moisture (cm ) Spore Density (# spores/100g soil) Fig. 2 29. Spore density with increasing soil moisture levels over a 12 month period at the Starke sod farm location. y = -0.0067x 4 + 0.1113x 3 0.6217x 2 + 1.5001x + 4.6121 R 2 = 0.6136 0 1 2 3 4 5 6 7 8 0 2 4 6 8 10 Soil Moisture (cm ) Spore Density (# spores/100g soil) Fig. 2 30. Spore density with increasing soil moisture levels over a 12 month period at the Fort McCoy sod farm location.

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52 y = 0.0118x 4 0.195x 3 + 0.9987x 2 1.4413x + 5.8091 R 2 = 0.6888 0 1 2 3 4 5 6 7 8 0 2 4 6 8 10 Soil Moisture (cm ) Spore Density (# spores/100g soil) Fig. 2 31. Spore density with increasing soil moisture levels over a 12 month period at the Lake Butler sod farm location. y = -3E-06x 6 + 0.0004x 5 0.028x 4 + 0.9465x 3 17.252x 2 + 160.12x 583.17 R 2 = 0.6062 0 1 2 3 4 5 6 7 8 15 20 25 30 35 Soil Temperature (C) Spore Density (# spores/100g soil) Fig. 2 32. Spore density with incr easing soil temperatures over a 12 month period at the Starke sod farm location.

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53 y = 0.0002x 4 0.0206x 3 + 0.8144x 2 13.691x + 88.104 R 2 = 0.8455 0 1 2 3 4 5 6 7 8 15 20 25 30 35 Soil Temperature (C) Spore Density (# spores/100g soil) Fig. 2 33. Spore density with increasing soil temperatures over a 12 month period at the Fort McCoy sod farm location. Fig. 2 34. Spore densit y with increasing soil temperatures over a 12 month period at the Starke sod farm location. y = 1E-05x 4 + 0.0009x 3 0.1124x 2 + 3.1385x 20.969 R 2 = 0.5939 0 1 2 3 4 5 6 7 8 15 20 25 30 35 Soil Temperature (C) Spore Density (# spores/100g soil)

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54 Table 2 4. Evaluation of analysis of variance data for percent root length colonized from each sod farm location. Date Location %Colonization (GIM) Dec. 04 Fort McCoy 27.226† Lake Butler 25.47 Starke 25.69 mean 26.13 ab‡ Jan '05 Fort McCoy 28.28 Lake Butler 28.05 Starke 30.69 mean 29.01 a Feb '05 Fort McCoy 24.86 Lake Butler 29.91 Starke 31.79 mean 28.85 a March '05 Fort McCoy 26.96 Lake Butler 24.01 Starke 23.58 mean 24.84 abc April '05 Fort McCoy 26.98 Lake Butler 30.03 Starke 28.74 mean 28.58 a May '05 Fort McCoy 24.62 Lake Butler 28.35 S tarke 27.21 mean 26.73 ab June '05 Fort McCoy 25.25 Lake Butler 29.54 Starke 22.97 mean 25.92 ab Aug '05 Fort McCoy 23.00 Lake Butler 22.76 Starke 22.14 mean 22.63 bcd Sept '05 Fort McCoy 23.81 Lake Butle r 18.27 Starke 22.96 mean 21.68 bcd Oct '05 Fort McCoy 21.13 Lake Butler 18.87 Starke 18.51 mean 19.50 cd Nov '05 Fort McCoy 18.08

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55 Lake Butler 20.94 Starke 20.71 mean 19.91 cd Dec '05 Fort McCoy 18.87 Lake Butler 19.90 Starke 17.29 mean 18.68 d †Each value is the average of three sample plots/location (10 sub samples/plot). ‡Means followed by the same letter are not significantly di fferent according to Tukey’s (HSD) Studentized Range Test (P = 0.0001).

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56 Table 2 5. Chemical characteristics of soils sampled for AMF at three north central Florida sod farm locations during January, April, August, and November 2005. Soil Nutrient Levels Date Location P/g soil Ca K Mg pH† OM‡ Jan '05 FM 9 883 13 46 5.5 1.57 Jan '05 LB ** 112 455 75 28 5.8 2.31 Jan '05 Starke 38 306 117 38 5.7 2.02 April '05 FM 12 830 16 47 7.0 1.70 April '05 LB 91 418 91 26 5.8 2 .53 April '05 Starke 27 359 103 44 5.7 2.01 Aug '05 FM 35 260 37 30 5.9 1.34 Aug '05 LB 88 1065 97 91 6.3 2.97 Aug '05 Starke 56 903 87 87 6.2 2.08 Nov '05 FM 55 392 82 27 5.4 1.99 Nov '05 LB 47 414 91 37 5.7 2.07 Nov '05 Starke 45 370 83 30 5.4 1.9 3 †Soil pH, nutrient level, and organic matter con tent based on the mean of three composite samples/location. ‡OM = Organic matter content. *FM = Fort McCoy location. **LB = Lake Butler location.

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57 CHAPTER 3 THE EFFECT OF ARBUSC ULAR MYCORRHIZAL FUN GI ON GAEUMANNOMYCES GRAMI NIS VAR. GRAMINIS AN D RHIZOCTONIA SOLANI COLONIZATION OF ST. AUGUSTINEGRAS S SOD IN NORTH CENTR AL FLORIDA SOILS Take all root rot and brown patch are two of the more common and devastating diseases of St. Augustinegrass sod throughout Florida. Take all root rot, caused by Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. graminis is a disease of both grasses and cereals ( Nilsson, 1969 ; Huber and McCay Buis, 1993). Take all root rot was first described in Sweden in the early 1800’s infecting grasses ( Mathre, 1992 ). It is one of several G. graminis varieties which infect many important crops worldwide (Rovira and Whitehead, 1983). This particular variety of the fungus infec ts all cultivars of St. Augustinegrass (Elliott, 1995 ; Datnoff et al., 1997). In the late 1980’s, large, chlorotic patches of St. Augustinegrass were observed on sod farms in South Florida and were confirmed as the first disease symptoms of G. graminis va r. graminis infection observed in this species (Elliott, 1993). The disease was found in St. Augustinegrass throughout Alabama, Florida, and Texas (Fig. 3 1) and it is notably more severe in the summer and fall months, especially during periods of increas ed precipitation (Elliott, 1993). Early studies suggested that the fungus preferred alkaline or high pH soil, mild winters, thatch accumulation and frequent light irrigation, however the conditions that predisposed the stand to disease or prompted disease escape are not known (Guyette, 1994). Management recommendations included elimination of low areas where water accumulates, watering only when needed, and the use of pH decreasing

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58 fertilizers in the fall, as well as thatch prevention and aeration (Guyett e, 1994). Fungicides were recommended as preventative but not curative treatments, which limited management options to growers (Guyette, 1994). The effect of systemic fungicides on G. graminis var. graminis infection and colonization of turfgrasses was e valuated; but results indicated that preventative and/or curative rates of fungicides did not limit take all root rot disease or increase turfgrass quality (Elliott, 1995). Biological controls were explored in an attempt to decrease take all root rot in w heat and turfgrasses. The effects of bacterial isolates, actinomycetes, and fluorescent pseudomonads on the roots of wheat were evaluated as antagonists against G. graminis var. tritici (Sivasithamparam and Parker, 1978). These organisms make up a large portion of the microbial community of soils and researchers expected their production of antibiotics or toxic metabolites would inhibit take all in wheat in suppressive soils. While combinations of these microorganisms reduced disease, none were successfu l alone (Sivasithamparam and Parker, 1978). To date, no effective curative or preventative controls for take all root rot are recognized for use in St. Augustinegrass. In order to determine the impact of arbuscular mycorrhizal fungi (AMF) on take all roo t rot in St. Augustinegrass sod, it is necessary to accurately diagnose G. graminis var. graminis and determine its population within the field. The diagnosis of take all root rot involves several characteristics and diagnostic tools for isolation and ide ntification. The pathogen is somewhat elusive and may be easily confused with other fungi if the scientist is not familiar with the morphology of the fungus and patterns of infection. The ascomycete, G. graminis var. graminis is classified in the order Diaporthales because it produces ascospores in black, flask shaped, ostiolate perithecia, which are fully enclosed

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59 and lined with hyaline periphyses (Landschoot, 1997; Walker, 1973). The perithecia are typically 200 400 m x 150 300 m in length, with the neck portion 100 400 m in length and 70 100 m wide (Landschoot, 1997). The asci, clavate in shape, are unitunicate, are formed in a hymenium, and range in length from 80 140 m and 10 15 m in width. The apex of the ascus, which has a refractive apica l ring, is generally yellowish en masse. Each ascospore is typically 70 110 m in length, 2 4 m in width and they usually contain 3 8 septa, but there may be 11 or 12 septa produced. The anamorphic state, which is rarely observed, is a Philaphora specie s that produces conidia 5 14 m in length x 2 4 m in width. The use of conidia as taxonomic criterion is not recommended due to variation between isolates and their non descript morphology. In culture, mycelia range from short to aerial, white to gray, green to brown, or black (Landschoot, 1997). Dark runner hyphae are typically observed on and around the crown portion of the plant, with extension onto the stem and stolons. The roots usually have relatively fewer dark surface runner hyphae, compared t o the foliar portion of the plant, which may remain green. Instead of dark runner hyphae, the roots are often covered with dark brown to black lesions and subsurface hyaline hyphae. The cortical browning of roots is thought to be a host defense mechanism while the discoloration of shoots is a necrotic symptom of disease (Penrose, 1992). The name “take all root rot,” implies that the roots are the first plant parts to be severely affected whether facilitated by feeding damage from nematodes or mole crick ets, mechanical damage from sod production, cultural techniques, or through natural openings.

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60 After the initial invasion, the seminal roots are colonized internally by more hyaline and infectious, secondary hyphae usually right behind the root tip (Henso n et al., 1999; Gilligan, 1983), which is were AMF usually colonize root tissue. Pathogenic colonization causes an occlusion of vascular tissues resulting in the characteristic gradual decline in plant health and potential death. Dark runner hyphae may c ontinue up the plant in search of more juvenile and susceptible tissue while producing deeply lobed and melanized hyphopodia. The hyphopodia are considered by most as superficial hyphal structures (Henson et al., 1999) since they originate from the hypha e, however they behave much in the same way as appressoria, which develop from the germ tube of germinating fungi providing infection pressure and anchoring the fungus to plant tissue (Agrios, 2004). Hyphopodia cluster and develop into an infection cushio n which provides the added structural stability while helping to maintain the turgor pressure required for colonization (Henson et al., 1999). The force of exertion of G. graminis var. graminis is associated with reduced cell wall permeability, turgor, an d wall rigidity (Bastmeyer et al., 2002). The deeply lobed hyphopodia are unique to G. graminis var. graminis and may exist to allow the fungus to overcome plant resistance mechanisms. Plants of St. Augustinegrass may benefit from AMF colonization in the presence of Gaeumannomyces graminis var. graminis But, it is possible for AMF to have a negative impact on plants in some situations, or they may even be neutral in nature (Johnson et al., 1997). Brown patch or Rhizoctonia blight, caused by Rhizoctoni a solani Khn (Figs. 3 2, 3 3), is most active in St. Augustinegrass from November to May when temperatures average

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61 25 C and below (Elliott and Simone, 2001). Brown patch is typically worse in periods of excessive rainfall or irrigation, or when grass lea ves remain wet for more than 48 hours (Elliott and Simone, 2001). In the field, small chlorotic patches of sod gradually turn brown as infected leaf blades die, hence the name brown patch (Elliott and Simone, 2001). As patches expand, they may coalesce i nto large rings of yellow brown sod with dark and wilted margins. It is not uncommon for sod to appear green and healthy in the center of the rings. Grass blades are killed near the crown due to restriction of water and nutrient transport, which creates a dark rot near the base of the blade. Infected blades can easily be pulled from the leaf sheath due to the soft rot (Elliott and Simone, 2001). Most usually the stolons and leaves are affected more than the roots themselves. A barrage of chemical contr ols, such as azoxystrobin, fluotanil, and mancozeb offer effective brown patch control when used as preventatives. Cultural controls include irrigating only when necessary between 2 and 8 AM and removal of mower clippings from the site. However, the use of quick release nitrogen during periods of R. solani activity seems most beneficial (Elliott and Simone, 2001). The use of chemicals in sod production has been controlled in recent years and these restrictions will continue according to state and federal regulations. Effective disease prevention strategies including the use of biological controls, such as AMF, are essential research objectives in an industry where quality is of utmost importance to buyers and growers. Brown patch was first described in St. Augustinegrass in the 1980’s (Hurd and Grisham, 1983; Martin and Lucas, 1984) as an aerial type of pathogen common to a variety of crops including corn, soybean, and rice (Sneh et al., 1991). Other pathogenic species of Rhizoctonia affecting St. Augu stinegrass include R oryzae Ryker & Gooch

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62 and R zeae Voorhees which cause a sheath rot or spot, but the two species are rare (Martin and Lucas, 1984; Haygood and Martin, 1990). The telomorph, Thanatephorus cucumeris Frank, is assigned to the Basidiomyco ta (Ainsworth et al., 1973). Mycelia of R. solani appear buff to dark brown in culture with irregularly shaped light to dark brown sclerotia (Sneh et al., 1991). R hizoctonia solani is identified by its characteristic right angle (90 o ) branching between t he primary and secondary hypha (Duggar, 1915) with branches forming acute (45 o ) angles to main hypha (Butler and Bracker, 1970). Identification is made easier by the presence of a septum at the branches near hyphal constrictions at the base of right angle s (Duggar, 1915). Additionally, the older, main runner hypha of R. solani are more than 7 m in diameter with more than two nuclei per cell (Sneh et al., 1991). Arbuscular mycorrhizal fungi have been associated with increased nutrient and water acquisit ion in plants for many years. Mycorrhizal symbiosis often results in increased plant vigor and the use of AMF has been studied in many crops as potential antagonists to root pathogens (Schenck, 1987; Sylvia and Williams, 1992; Smith and Read, 1997; Yao et al., 2002). Glomus etunicatum Becker & Gerdemann and G. intraradices Schenck & Smith are two of the more common AMF species investigated as potential biological controls and chemical alternatives against R. solani in crops such as potato (Yao et al., 200 2) and species of Fusarium in tomato crops and alfalfa (Caron et al., 1986; Hwang et al., 1992). In several cases, G. intraradices provided significant control of soilborne pathogens (Niemira et al., 1996; Khalil et al., 1994; Viyanak and Bagyaraj, 1990). Newsham et al., (1995) reported that mycorrhizal fungi are capable of protecting annual grasses from soilborne fungi. In other surveys, researchers found that

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63 G. intraradices significantly reduced take all root rot caused by G. graminis var. graminis in cool season bentgrasses on greens with low soil P levels (Koske et al., 1995). Reductions in take all disease severity in mycorrhizal wheat may be due to increased P uptake, increased root cell wall lignification, pathogen exclusion, production of antag onistic compounds, or altered root exudates (Graham and Menge, 1992). However, baseline information concerning pathogen colonization and potential effects of AMF on disease in the field is necessary before experiments concerning mechanisms of resistance a nd inoculation can be undertaken. The objective of this survey was to determine the extent of R. solani and G. graminis var. graminis colonization in production fields of ‘Floratam’ St. Augustinegrass sod in north central Florida and to determine whether populations of AMF are having any effect on disease incidence in the field. Many researchers may feel that the effects of AMF in turfgrass systems may be outweighed by the benefits of added nutrients, pesticides, and irrigation. However, in St. Augustin egrass sod systems where inputs are limited, AMF may serve a greater role in plant resistance to soilborne pathogens or soil suppressiveness. Materials and Methods Root Pathogen Sampling – ‘Floratam’ St. Augustinegrass stolons and roots were collected o n a bimonthly basis from the three north central Florida sod farms described in chapter 2 in January through December 2005. The roots and stolons were surveyed for take all root rot and brown patch. From each of the three (3 m 2 ) plots described in chapte r 2, ten subsamples of root and stolon tissue (1 5 cm above the crown) were randomly dissected from collected plants and cut into 100 pieces of tissue 2 5 cm in length, in order to quantify the extent of root rot disease and to isolate and identify the

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64 cau sal organisms. The pieces were washed, surface sterilized for 1 min in a 10% sodium hypochlorite and deionized water solution, rinsed twice for 1 min with sterile deionized water, and blotted dry. Pathogen Identificatio n Forty pieces of tissue from e ach of the 100 segments/plot were randomly selected for isolation of G. graminis var. graminis and forty for isolation of R. solani and aseptically plated into selective agar media (Appendix A) in 15 x 100 mm Petri dishes. Selective media (Appendix A) wer e used to isolate the pathogens from tissue and to slow growth of other soilborne fungi not associated with diseased tissue. The Petri dishes were incubated at 24 C under a 12 h diurnal cycle. Fungal growth was monitored by light microscopy for 5 8 d or until opportunistic fungal growth required colony transfer to sterile media, in order to isolate the desired root pathogens. Samples of fungal colonies suspected of being R. solani or G. graminis var. graminis were mounted in water on glass slides and vie wed with a Nikon Optiphot compound microscope to identify fungal structures microscopically. Gaeumannomyces graminis var. graminis colonies were readily identified in media by the presence of deeply lobed hyphopodia ( Fig s. 3 4, 3 5) within melanized mycel ium (Landschoot, 1997). Rhizoctonia solani colonies ( Fig s. 3 6, 3 7 ) were identified based on the auburn to light brown color and 90 o branching of the mycelium (Sneh et al., 1991). Pathogen Quantification and Statistical Analysis – The number of coloni es of G. graminis var. graminis and R. solani observed emerging from root or stolon pieces were used to quantify the amount of infection of these root pathogens at each sod farm location (Figs. 3 5, 3 6). The mean colonization data were expressed as the percentage of sampled root or stolon pieces colonized by G. graminis var. graminis or R. solani on

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65 selective agar media (Appendix A). The survey was performed using a random model in a randomized complete block design with multiple samplings at multiple l ocations. The percent colonization data were analysed using the Generalized Linear Model (SAS Institute, Version 9.0, 2004) (Appendix F 2; Appendix F 3). Arbuscular mycorrhizal fungi sampling data, as described in chapter 2, were used in this survey sinc e root pathogen sampling occurred simultaneously in the same plot locations as the survey of AMF in the previous chapter. Significant interactions (P < 0.05) were separated using Tukey’s Studentized Range Distribution test, and correlations between AMF pe rcent colonization and spore density to percent colonization of each root pathogen were done in SAS using Pearson product moment correlation coefficients. Results and Discussion No correlation between AMF spore density or percent colonization in relatio n to R. solani or G. graminis var. graminis colonization were found Additionally, no location effects were detected in the analysis of variance among or within the sampling months (P < 0.001). However, pathogen colonization did vary significantly betwee n sampling months (P < 0.001), which suggested a seasonal influence on pathogen activity in north central Florida soils at each sod farm location. Mean values of root colonization by R. solani were greatest in December 2004 at 24.40 % and lowest in June 20 05 at 10.71 percent (Fig. 3 8 ). The warmer months of June and August had the lowest R. solani colonization percentages but the values were not significantly different from values in March, January, or October. The cooler months of December and April had the highest percentages of R. solan i al though the April mean was not significantly different (P < 0.05) from Oct ober January, or March (Fig. 3 8 ). This finding is not surprising since R. solani has optimal growth below 26 C therefore it is typically mo re

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66 active in cooler weather (Elliott and Simone, 2001). Inter estingly, as noted in chapter 2 AMF spore density (Table 2 2) was generally lowest during the cooler months of December, January, and April and highest during warmer weather, with percent colon ization highest during the cooler months when R. solani is most active in these soils (Table 2 4). Mea n values of root colonization by G. graminis var. graminis were highest in the warme r months of August 2005 at 20.01 % and lowest in De cember 2004 at 5. 35 percent (Fig. 3 9 ). The months of August, June, and October had the highest percentages of G. graminis var. graminis colonization, with the lowest mean values occurring in December, January, March, and April. However, there were no significant differ ences (P < 0.05) between mean values in June and October, or October, April, March, and January. Again, this finding is not surprising because G. graminis var. graminis is most active in warm, markedly wet conditions where there is excessive thatch accumu lation (Elliott, 1993; Guyette, 1994). During the warm, humid days of summer, St. Augustinegrass sod is often heavily irrigated and mowed, which produces favorable growth conditions for G. graminis var. graminis because of surplus moisture and accumulatin g clippings which add to thatch layers. In this survey, the pathogen is most active during periods when AMF percent colonization is lowest suggesting a limited role for AMF in take all root rot disease suppression in these soils. More controlled studies might shed light on potential AMF effects on soilborne pathogens which may be confounded during field evaluations due to rhizosphere variability and environmental effects. If these criteria can be evaluated under less variable conditions, beneficial AMF e ffects could be evaluated and

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67 perhaps manipulated for optimal disease suppression and concurrent decreases in pesticide use.

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68 Fig. 3 1. ‘Floratam’ St. Augustinegrass sod mat infected with Gaeumannomyces gramin is var. graminis Insert in bottom right hand corner depicts underside of a mat with rotting roots. 1

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69 Figs. 3 2 – 3 3. Comparison of healthy ‘Floratam’ St. Augustinegrass sod mat and sod affected by brown patch. Fig. 3 2. Healthy ‘Floratam’ St. Augustinegrass sod mat. Fig. 3 3. ‘Floratam’ St. Augustinegrass sod mat infected with R. solan i causing brown patch 6 3 7 2 3

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70 Fig. 3 4. Deeply lobed hyphopo dia isolated from Gaeumannomyces graminis var. graminis in ‘Floratam’ St. Augustinegrass sod samples. Scale bar = 40 m. Fig. 3 5. Medium isolation plate depicting a Gaeumannomyc es graminis var. graminis colony isolated f rom ‘Floratam’ St. Augustinegrass sod samples. Arrow points to colony. 4 5

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71 Fig. 3 6 R hizoctonia solani hyphae isolated fro m ‘Floratam’ St. Augustinegrass sod exhibiting diagnostic 90 o branching at constri ction points and characteristic septa. Scal e bar = 40 m. Arrow points to branching pattern. Fig. 3 7. Medium isolation plate depicting light brown R hizoctonia solani colony isolated from ‘Floratam’ St. Augustinegrass sod samples Arrows point to colonies. 6 7

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72 0 5 10 15 20 25 30 Dec 04 Jan 05 April 05 March 05 June 05 Aug 05 Oct 05 Date Mean Percent R. solani Fig. 3 8. Mean percent of Rhizoctonia solani colonization of 'Floratam' St. Augustinegrass in north central Florida. Means followed by the same number are not significantly different according to the Tukey’s mean separation test (P < 0.05). The percent colonization is based on the mean number of colonies where R. solani was recovered. a abc ab bc c c abc

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73 0 5 10 15 20 25 Dec 04 Jan 05 April 05 March 05 June 05 Aug 05 Oct 05 Date Mean Percent G. graminis var. graminis Fig. 3 9. Mean percent of Gaeumannomyces graminis var. graminis colonization of 'Floratam' St. Augus tinegrass in north central Florida. Means followed by the same number are not significantly different according to the Tukey’s mean separation test (P < 0.05). The percent colonization is based on the mean number of colonies where G. graminis var. gramin is was recovered. c b ab ab a a ab

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74 CHAPTER 4 EFFECT OF GLOMUS INT RARADICES ON THE EXT ENT OF DISEASE CAUSE D BY GAEUMANNOMYCES GR AMINIS VAR. GRAMINIS AND RHIZOCTONIA SOLANI IN ST. AUGUST INEGRASS Arbuscular mycorrhizal fungi (AMF) are widespread symbionts in the majority of plant species; and are associated with increased plant vigor via improved nutrient uptake, especially P, and increased water acquisition (Smith and Read, 1997). The beneficial effects of AMF on crop yield have been thoroughly documented (Harley and Smith, 1983). There is much debate on whether or not AMF alter plant resistance to pathogens by an indirect mechanism or simply interact directly with the pathogens themselves. When AMF act as pathogen antagonists, there are likely one or more mechanisms of resistance. For ex ample, AMF may be deterring pathogen infection by increasing plant vigor through improved nutrient acquisition, the AMF themselves may be producing anti microbial metabolites, or the AMF may be stimulating the plant’s own natural defense response to coloni zation by increasing phytoalexin production (Schenck, 1970). Previous studies have indicated that AMF symbiosis greatly improves plant resistance to abiotic pressures such as water stress (Sylvia and Williams, 1992) and transplant shock (Menge et al., 197 8) in various crops. AMF have also been evaluated as biological controls against biotic stresses such as bacterial pathogens (Weaver and Wehunt, 1975), parasitic nematodes ( Baltruschat et al., 1973; Schenck and Kellam, 1978), viral pathogens (Daft and Oku sanya, 1973; Giannakis and Sanders, 1989), and soilborne fungal pathogens (Jeffries, 1987; Schenck, 1987; Hooker et al., 1994; Linderman, 1994; Azcn Aquilar and Barea, 1996).

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75 The vast majority of evaluations concerning the effects of AMF on disease se verity involve fungal pathogens (Schenck and Kellam, 1978). The first report of an interaction between mycorrhizal fungi and fungal pathogens involved soybean ( Glycine max L. Merr) and Phytophthora root rot, where the mycorrhizal plants actually had highe r rates of disease versus the nonmycorrhizal plants (Ross, 1972). In other reports, AMF had no effect on disease at all (Ramirez, 1974; Sherinkina, 1975). Depending on the stage of host plant development, plant and mycorrhizal fungal species, and the com plexities between biotic and abiotic rhizosphere factors, there is evidence that mycorrhizal interactions lie along a continuum ranging from mutualistic to parasitic, commensal, amensal, and potentially even neutral (Johnson et al., 1997). However, there are many reports of mycorrhizal colonization reducing disease severity in many plant systems such as pea, tomato, soybean, wheat, and peanut involving such fungal pathogens as Fusarium solani Mart. (Sacc.), G. graminis (Sacc.) Arx & Olivier var. tritici J. Walker, Sclerotium rolfsii (Sacc.), Pythium spp., Phytophthora parasitica Dastur, and R. solani Khn (Graham and Menge, 1992; Dehne, 1982; Krishna and Bagyaraj, 1983; Zambolim and Schenck, 1983; Hedge and Rai, 1984; Vigo et al., 2000; Yao et al., 2002). In fact, the effects of mycorrhizal colonization on disease severity is potentially so important that Newsham et al. (1995) suggested that the benefits of AMF to disease suppression may be as important as the nutritional benefits derived from the symbios is in some instances. For example, in temperate grasslands, the effects of a direct AMF interaction with root pathogens reduced disease severity and increased plant vigor and fecundity greatly (Newsham et al., 1995). Soilborne pathogen suppression by AMF includes both physical and physiological mechanisms (Sharma et al., 1992). Physical

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76 plant defense responses against pathogen penetration are: increased lignification (Dehne and Schoenbeck, 1978), greater mechanical strength and nutrient flow within vascu lar systems (Schoenbeck, 1979), and direct competition with the pathogen for cortical infection courts and resources (Graham, 2001). Becker (1976) observed that pathogen penetration of root cells was directly reduced by the presence of AMF and not indirec tly by a systemic plant resistance based on thickening cell walls. In some cases the direct influence of AMF may be the only reason for observations of disease resistance. It is important to establish whether or not particular plant systems benefit, suff er, or remain unaltered by mycorrhizal colonization. If the relationship appears to be beneficial, Gerdemann (1975) remarked that the effect of mycorrhizal fungi on disease should be determined whether resistance is due to direct or indirect mechanisms. The host pathogen relationship can be greatly influenced by indirect or physiological effects of AMF through increased P nutrition, enhanced mycorrhizal root growth which aids in disease escape, or up regulation of pathogenesis related proteins (Gianinazzi Pearson and Gianinazzi, 1989; Blee and Anderson, 2000; Graham, 2001). AMF may also be responsible for lowering disease severity in complex reactions involving host physiology such as the production of rhizosphere leachates from mycorrhizal plant roots. These leachates have been observed to substantially limit the production of zoospores and sporangia of Phytophthora cinnamomi Ronds in sweet corn and chrysanthemum (Meyer and Linderman, 1986). There appears to be no information concerning the effects of AMF, if any, on disease severity in St. Augustinegrass. If there is a direct or indirect beneficial effect of AMF on disease severity of St. Augustinegrass in relation to brown patch or take all root

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77 rot, several questions will remain concerning the actu al mechanism of observed resistance. However, without basic information and techniques to differentiate between direct and indirect effects and to determine what extent disease severity may or may not be lowered, further evaluations would not be warranted The economic importance of AMF in soils of north central Florida St. Augustinegrass sod fields may be considerable where diseases such as brown patch and take all root rot reduce harvestable hectares. Arbuscular mycorrhizal fungi can stimulate plan t vigor and possibly interact directly or indirectly with soilborne pathogens to limit disease. AMF have been observed colonizing St. Augustinegrass (see Chapter 2), and they might benefit sod production. The potential AMF benefits to sod growers include reduced loss of sod and revenue to soilborne pathogens, and lowered management costs through reduced fungicide use. The potential advantages of AMF inoculation or field manipulation with specialized techniques may also benefit the environment by decreasi ng soil and water pollution through reduced of fungicide use. For these reasons, it is prudent to evaluate the potential benefits of AMF to disease resistance whether by direct or indirect mechanisms in St. Augustinegrass sod. As part of ongoing research on the effect of AMF on disease severity in St. Augustinegrass, the objective of this study was to determine the effect of G intraradices on St. Augustinegrass in disease development by challenging it both directly and indirectly with G. graminis var. g raminis or R. solani Material and Methods Direct Experiments St. Augustinegrass Sprig Propagation and Stock Plants ‘Floratam’ St. Augustinegrass sprigs having no apparent signs or symptoms of disease were obtained from Hendrick’s Turf Farm (Lake Butle r, Florida). The sprigs were rooted in flat, plastic

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78 nursery trays or 18 cm clay pots in a sterilized Arrodondo fine sand medium supplemented with a nutrient solution (Appendix B) every three weeks. The sprigs were grown and maintained in a growth chambe r at 25 27 C under cool white fluorescent bulbs with irradiance at 25 E/m 2 /s and a 15 h photoperiod/day. Sprigs were watered every other day throughout the experimental period with water adjusted to pH 6.0 6.5. After approximately 6 weeks of propagation selected sprigs, not in direct contact with soil, were excised from the edge of the flat trays and replanted as sterile stock plantlets. These sub cultured plants were maintained as described above until additional sprigs, not touching the soil and hang ing from the edge of the tray, were collected for experimentation. R. solani Inoculum Production A virulent strain of R. solani (PDC 7884) (Fig. 4 1) isolated from diseased St. Augustinegrass submitted by a homeowner in Leon County, Florida was provided by the Plant Disease Clinic (Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida). The isolate was cultured at 4 C and stored on potato dextrose agar (Difco Laboratories, Inc., Detroit, Michigan) for approximately 2 we eks. An oat ( Avena sativa L.) inoculum was prepared according to Sneh et al. (1991) and Gaskill (1968) with modifications (Appendix C) and inoculated with agar plugs from actively growing R. solani (PDC 7884) mycelium or with sterile agar plugs (control). The inoculum substrate was incubated at 21 C with a 12 h photoperiod for 4 weeks and shaken 2 3 times/week to prevent packing of the oat seeds. The inoculated seeds were then air dried, sealed in plastic zip lock bags, and stored at room temperature unt il use.

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79 G. graminis var. graminis Inoculum Production A virulent strain of G. graminis var. graminis (JK2) was collected and identified from diseased St. Augustinegrass (Fig. 4 2) from the lawn of Dr. James Kimbrough (Gainesville, Florida) and isolated o n selective media amended with antibiotics (Appendix A). Actively growing G. graminis var. graminis mycelium from a single Petri dish was chopped and combined with sterilized ryegrass seed as described by Datnoff and Elliott (1997) with modification (Appe ndix C). The inoculated flasks of sterile ryegrass seed substrate and uninoculated control flasks were incubated in total darkness at 21 C for 4 weeks prior to use. The flasks were shaken 2 3 times/week to prevent packing of the inoculated ryegrass seed. Mycorrhization of ‘Floratam’ St. Augustinegrass Sprigs Sprigs of ‘Floratam’ St. Augustinegrass were selected from the edge of sterile stock plants in flat trays, as previously described. Sprigs were inspected visually for any signs or symptoms of pot ential pathogens or diseases, and if healthy, were selected for experimental use. The sprigs were then planted into 6.8 cm wide by 18 cm deep conetainers (Steuwe and Sons, Inc., Corvallis, Oregon) filled with a sterilized low P soil, as mentioned in Chapt er 2 (Fig. 4 3). The sprigs were then placed in a controlled growth room with a 15 h photoperiod/d at 21 25 C, watered daily with pH adjusted 6.0 6.5 deionized water, and maintained for approximately 3 weeks to allow root development to occur and transpla nt shock to subside. After the 3 week growth period, the sprigs, with approximately 8 cm of root length, were inoculated with approximately 20 spores of G. intraradices (FL 208A) (Fig. 4 4) obtained from the INVAM Culture Collection (Morgantown, West Virg inia) or noninoculated water controls. The FL 208A isolate was selected because it was first isolated in a citrus grove in central Florida, near Orlando, in 1978 in 7.0 7.5 pH soil,

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80 which is similar to that of the sod fields in north central Florida. The sprigs were then acclimatized for approximately 4 weeks in the growth room to allow the AMF time to colonize the sprig roots, which was determined at 2 and 4 weeks in extra experimental units. Pathogen Inoculation The AMF colonized sprigs were inocula ted with either R. solani (PDC 7884) or the G. graminis var. graminis (JK2) isolate or uninoculated as controls by gently pushing the soil aside to expose a portion of the roots near the crown of the sprig. Approximately 3 5 infected seeds of either the R solani inoculated oat substrate or G. graminis var. graminis inoculated ryegrass seed substrate were placed equidistant from the crown in each conetainer at a 1 2 cm distance from the plant. The soil was carefully replaced following inoculation. Inocul ated sprigs were maintained in the growth room for approximately 4 weeks with a 15 h photoperiod/d at 21 25 C. Each cone was supplied with a nutrient solution devoid of P on two occasions at 50 ml/conetainer (Appendix B). Plants were watered daily with 5 0 ml water/conetainer adjusted to 6.0 6.5 pH. Mycorrhizal Evaluation Roots from the sprigs were rinsed in tap water and separated with a scalpel from the plant crown Selected roots were cut into 1 2 cm long segments, put into porous nylon sleeves, ins erted in small, plastic clips (Fig. 4 5), and the cell and wall components cleared in 10% KOH (w/v) under pressure in an autoclave for approximately 20 min at 121 C psi (Brundrett et al., 1996). The root segments were cooled, then rinsed in tap water, and placed into 0.05% trypan blue in 25% glycerol overnight to stain mycorrhizal structures ( Bevenge, 1968; Phillips and Hayman, 1970; Kormanik and McGraw, 1982 ). Excess stain was rinsed from the root segments with tap 2 2

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81 water and then the roots were mounted i n water on glass slides to view vesicles, intraradical hyphae, and arbuscules (Fig. 4 6). Root segments from each replicate were pooled from each treatment, and evaluated for intensity of colonization. Mycorrhizal structures on glass slides were viewed with a Nikon Optiphot compound microscope at 200, 400, and 1000x magnifications, and photographs were taken with a Nikon CoolPix 990 digital camera. In order to judge the amount of mycorrhizal root colonization, the grid line intersect method was used to approximate the total root length colonized by AMF (Newman, 1966; Tennant, 1975; Giovannetti and Mosse, 1980). Direct Experiment Disease Assessment Disease severity (root and shoot rot) was rated at the conclusion of a 3 week growth period on both the AM F inoculated, pathogen inoculated, and control sprigs. Disease severity was assessed using an arbitrary disease scale from 1 to 6 with 1 = no symptoms of disease; 2 = 1 25% disease; 3 = 26 50% disease; 4 = 51 75% disease; 5 = 76 100% disease; and 6 = plan t death (Figs. 4 7; 4 8). The presence of either the R. solani or G. graminis var. graminis pathogens on each infected sprig was confirmed by re isolation of each pathogen (Figs. 4 9; 4 10) on selective media (Appendix A). For each sprig, the percent col onization of the pathogen and/or AMF was recorded as described in Chapters 2 and 3. Direct Experiment Design and Statistical Analysis The experiment was performed using a factorial arrangement (1 cultivar of St. Augustinegrass) x (1 AMF + uninfected pa thogen control) x (1 R. solani infected + 1 AMF) x (1 R. solani infected – AMF) and ( 1 G. graminis var. graminis infected + 1 AMF) x (1 G. graminis var. graminis – AMF) and (uninfected pathogen control + uninoculated AMF control) in a randomized complete

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82 b lock design with four replicates/treatment (Fig. 4 11). Regression analyses were performed with the regression procedure in SAS (SAS Institute, 2004) (Appendix F 4). All data presented are the means of four replicates. As there were no differences betwe en trials based on the ANOVA, all data presented were combined for the purpose of presenting the results and discussion more easily. Indirect Experiments St. Augustinegrass sprigs were produced and maintained in the same manner as described above in the D irect Experiment section as were mycorrhization and pathogen inoculum production, inoculation, and quantification. However, in this experiment, the potential effects of indirect AMF interactions with soilborne pathogens were evaluated instead of the poten tial direct impacts of mycorrhization. Instead of a direct challenge between AMF and pathogen in one container, indirect effects were investigated using a split root assay. Indirect AMF Challenge Split Root Assay Sterile, 4 week old ‘Floratam’ St. Augus tinegrass sprigs with approximately 8 cm of healthy root tissue were placed into two adjacent conetainers with one rooted end of the sprig in one conetainer and the other rooted end in another conetainer (Fig. 4 12). Holes (1 cm in diameter) were drilled 2.5 cm from the top of each 6.5 cm wide by 18 cm deep conetainer (Steuwe and Sons, Inc., Corvallis, Oregon) prior to planting, on one side of the conetainer (Appendix E 1). A cut was made from the top of the drilled hole to the top of each conetainer to a llow the sprig to be inserted into the hole without tissue damage. Sprigs were planted into conetainers filled with sterile low P soil as previously described and maintained in the growth room for 3 weeks to limit transplant shock and acclimatize the spri gs. Sprigs were then inoculated with the G. intraradices isolate (FL 208A) as described in the direct

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83 experiment above, or a control substrate in one conetainer, with either the G. graminis var. graminis isolate (JK2) or R. solani isolate (PDC 7884) inocu lated or an uninoculated control substrate in the adjacent conetainer occupied by the other rooted end of that same sprig (Fig. 4 13). The conetainers were watered daily with 50 ml water/conetainer adjusted to pH 6.0 6.5 and supplied with a nutrient solut ion on two occasions (Appendix B). The sprigs were maintained for 3 weeks in the growth chamber at 21 25 C with a 15 h photoperiod. The sprigs were visually inspected every 2 3 d for the presence of invading pathogenic mycelia along the stolon portion of the sprig to prevent cross contaminiation. The presence of the pathogen used to inoculate one conetainer was not observed in any of the adjacent experimental units (conetainers) based on the lack of recovery of the pathogen from adjacent conetainers by s elective media isolation (Appendix A). The stolon portion spanning the distance between the two adjacent conetainers was approximately 5 cm in length. Percent G. intraradices colonization was measured using the gridline intersect method described in the previous section. Indirect Experiment Design and Statistical Analysis The experiment was performed using a factorial arrangement (1 cultivar of St. Augustinegrass) x (1 AMF + uninfected pathogen control) x (1 R. solani infected + 1 AMF) x (1 R. solani infected – AMF) and ( 1 G. graminis var. graminis infected + 1 AMF) x (1 G. graminis var. graminis – AMF) and (uninfected pathogen control + uninoculated AMF control) split root assay in a randomized complete block design with four replicates. The entire e xperiment was setup three times from January – May 2006. Regression analyses were performed with the regression procedure in SAS (SAS Institute, 2004) (Appendix F 5). All data presented

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84 are the means of four replicates/treatment. No differences were fou nd between trials based on the ANOVA, therefore, data were pooled for analysis. Results Direct Experiments Mycorrhizal Colonization In the direct experiment, mean values of root colonization by the AMF, Glomus intraradices were 10% for the R. solani infect + AMF treatment, 11.3% for the AMF inoculated control treatment (no pathogen), and 11.7% for the G. graminis var. graminis infected + AMF treatment, respectively, after mycorrhizal inoculation. Root colonization of AMF was not significantly affect ed by the direct presence of either pathogen nor did the AMF control treatment (no pathogen) have any direct effect, either positive or negative, on disease severity itself (Appendix D 1). In this study, the colonization of plants by AMF, G. intraradices apparently had a neutral effect on the St. Augustinegrass plants without the direct presence of either pathogen nor did the AMF affect plant growth. Disease Development The direct effect of G. intraradices on brown patch (caused by R. solani ) disease severity was evaluated by first investigating the relationship of the R. solani infected control (no AMF) treatment (Appendix D 2) to disease severity. The mean percent colonization of the R. solani infected control treatment was 60%, but the disease seve rity (mean = 3.8 on a scale of 1 to 6) was not significantly correlated with the mean colonization percentage of R. solani using the regression procedure in SAS (SAS Institute, 2004). Since there was no definitive relationship between plant disease sever ity and the percentage of R. solani colonization with this treatment, there was no need to assume that G. intraradices in the R.solani infected + AMF treatment would have a beneficial effect on disease severity. This was supported by the regression analys is

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85 comparing the relationship of disease severity to percent R. solani colonization (mean colonization = 57%) in the R. solani infected + AMF treatment (Appendix D 3) where disease severity (3.3 on a scale of 1 to 6) was not correlated to the mean percenta ge of AMF colonization (mean colonization = 18%). In this study, the AMF treatments had no effect on disease severity in the direct presence of R. solani regardless of the mean colonization of the pathogen or AMF. The direct effect of G. intraradices o n disease severity was also evaluated in this study for take all root rot caused by G. graminis var. graminis Based on regression, the relationship between disease severity and the G. graminis var. graminis infected control (no AMF), it appears that the pathogen (mean colonization = 42.8%) had a significant relationship (r 2 = 0.65) with disease severity (2.4 on a scale of 1 to 6). This model shows that as disease severity increases so does G. graminis var. graminis percent colonization in a direct pathog enicity challenge ( Fig. 4 14 ). This finding suggests that the AMF could potentially have a direct effect on disease severity and that the relationship could be evaluated since the percent colonization of G. graminis var. graminis had a measurable effect o n disease severity. The regression analysis of disease severity (mean = 3.3 on a scale of 1 to 6) to the G. graminis var. graminis infected + AMF inoculated treatment revealed a highly correlated relationship between the treatment and disease severity (r 2 = 0.81). As disease severity increased according to this treatment, so did the percent colonization of G. graminis var. graminis even in the direct presence of AMF (mean = 8.6%) ( Fig. 4 15 ). There was no apparent reduction or increase in disease severit y. Therefore, the AMF have no direct beneficial effect on take all root rot disease severity. Additionally, the AMF treatment alone could not be correlated to a reduction in percent

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86 G. graminis var. graminis colonization (data not shown) nor did the trea tment have a direct effect on lowering take all root rot disease severity since the disease severity trend did not differ from that of the G. graminis var. graminis infected – AMF treatment. Since disease severity was not affected by G. intraradices in t he G. graminis var. graminis infected + AMF treatment or correlated to the percent of G. graminis var. graminis colonization in the control uninoculated with AMF, it appears that the AMF colonization had no direct negative or positive impact on the pathoge n or disease severity. In this study, the interaction between AMF and the plant in the direct presence of the pathogens, G. graminis var. graminis and R. solani would thus be considered neutral in nature. Discussion More importantly, this study demonstr ates that mycorrhization with the AMF, G. intraradices did not reduce development of R. solani or G. graminis var. graminis in direct contact nor did the AMF treatment reduce or increase disease severity of brown patch or take all root rot in ‘Floratam’ S t. Augustinegrass, as has been observed in other mycorrhizal studies ( Ross, 1972; St. Arnaud et al., 1994; Mark and Cassells, 1996) Arbuscular mycorrhizal fungi have been associated with increased disease severity in some instances with R. solani so ana lysis based on this assumption was as necessary as assuming the AMF treatment would lower disease severity (Ramirez, 1974; Sherinkina, 1975; Johnson et al., 1997; Y ao et al., 2002 ). No beneficial effects of AMF inoculation on take all root rot or brown pa tch disease severity in St. Augustinegrass were observed This is perhaps due to the relatively low levels of mycorrhizal root colonization. Possibly AMF inoculation would be more beneficial to plants with a higher level of mycorrhizal colonization

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87 In summary the results show that the purported beneficial effects of direct AMF interactions with plant roots such as increased cell wall lignification or the production of antagonistic mycorrhizal root exudates did not play a role in this study ( Becker, 197 6; Dehne and Schoenbeck, 1978; Graham, 2001). Thus, inoculation with G. intraradices will not improve disease severity or reduce disease development. The effects of such an interaction within field trials could potentially yield contradictory results, an d the microbial and environmental variability within the rhizosphere would make such experiments difficult at best. Results Indirect Experiment In order to th o roughly evaluate the potential effects of AMF on disease severity and/or soilborne pathogen d evelopment, another series of studies involving a more indirect method was performed simultaneously with the direct experiment described above. This assay was designed to isolate potential systemic resistance responses from mycorrhization which have been documented (Gianinazzi Pearson and Gianinazzi, 1989 ; Blee and Anderson, 2000; Graham, 2001). In this assay, the R. solani control (no AMF) treatment revealed a significant correlation between pathogen colonization and disease severity (Fig. 4 16 ). In t his instance, as percent colonization of R. solani (mean = 54.9 %) increased so did disease severity (mean = 3.5 on a scale of 1 to 6; r 2 = 0.75 ). Since there was a significant relationship between the pathogen and disease severity, the regression procedur e in SAS was also used to analyze the indirect effects of the R. solani + G. intraradices treatment on disease severity. The combination of this pathogen and AMF in an indirect assay, where one conetainer was inoculated with R. solani and the other coneta iner containing

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88 the other end of that same sprig was inoculated with G. intraradices showed no correlation (r 2 = 0.33) betwee n AMF (mean colonization = 10.2 %) and the pat hogen (mean colonization = 35.4 %) on disease severity ( mean = 2.4 on a scale of 1 to 6 ) (Appendix E 4). As in the direct experimen t, described above the percent of G. intraradices (mean colonization = 6.75 %) did not have an impact on disease severity of take all root rot (mean = 1.33 on a scale of 1 to 6 ) or brown patch as a treatment alo ne (r 2 = 0.34) (Appendix E 3). In this assay, the indirect effect of the AMF, G. intraradices (mean colonization = 9.7%) had no significant effect on take all root rot disease severity (mean = 2.33 on a scale of 1 to 6 ) nor did the G. graminis var. grami nis infected + AMF treatment (r 2 = 0.33) have an impact on pathogen coloniz ation (mean colonization = 22.9 %) (Appendix E 5 ). In the G. graminis var. graminis infected control treatment (no AMF), there was no significant correlation (r 2 = 0.32) based on th e regression analysis between the percent of G. graminis var. graminis colonization (mean = 59.6%) and take all root rot disease severity (mean = 3.0 on a sca le of 1 to 6) (Appendix E 2 ). Discussion Based on this indirect assay and on the direct challen ge between the AMF, G. intraradices and R. solani or G. graminis var. graminis there is no correlation between AMF colonization and disease severity. Dise ase severity does not increase or decrease, which is important considering that mycorrhizal benefits lie along a continuum ranging from mutualistic to parasitic (Johnson et al., 1997). If there is an interaction between this AMF and either of these two pathogens in St. Augustinegrass sod field soils, it is most likely neutral in nature. Based on these results, AMF colonization of St. Augustinegrass

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89 in north central Florida soils are n either harmful nor benefi cial to the plants when infected with these pathogens In summary, these results provide a foundation for future field trials in relation to dir ect and indirect impacts of AMF in St. Augustinegrass sod. This is the first study that attempt s to correlate take all root rot or brown patch disease severity to potential direct or indirect AMF effects. Since no influences were observed in either exper iment, the proposed mechanisms of direct resistance or indirect systemic resistance were not examined.

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90 Fig. 4 1. Rhizoctonia solani isolate (PDC 7884) colony used to prepare inoculum in direct and indirect experiments. Fig. 4 2. Gaeumannomyces gra minis var. graminis isolate (JK2) used to prepare inoculum in direct and indirect experi ments. 2 1

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91 Fig. 4 3. Conetainers filled with low P soil and ‘Floratam’ St. Augustinegrass sprigs inoculated in trial 1 of the direct experiment. Fig. 4 4. Glomus intraradices isolate (FL 208 A) used in direct and indirect assays to inoculate ‘Floratam’ St. Augustinegrass sprigs. Bar scale = 40 m. 4 3

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92 Fig. 4 5. Photo showing nylon sleeves and plastic clips used in direct and indirect experiments to clear and stain root segments from treatment replicates. Fig. 4 6. Photo of myco rrhizal St. Augustinegrass root with arbuscules and intraradical hypha of Glomus intraradices stained with 0.05% trypan blue from the direct experiment G. intraradices inoculated control sprigs. Arrows pointing to A = arbuscule; Arrow pointing to IR = in traradical hypha. Bar scale = 4 0 m. IR A A 6 5

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93 Fig. 4 7. ‘Floratam’ St. Augustinegrass sprigs after inoculation with Rhizoctonia solani depicting disease severity rating scale (1 6). Respective numbers below each sprig signify the disease severity rating of that sprig. 7 3 2

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94 Fig. 4 8. ‘Floratam’ St. Augustinegrass sprigs after inoculation with Gaeumannomyces graminis var. graminis depicting disease severity rati ng scale (1 6). Respective numbers below each sprig signify the disease severity rating of that sprig. 8 4 1

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95 Figs. 4 9 – 4 10. Photo depicting re isolation plates of the two pathogenic isolates used to challenge Glomus intraradices in both the direct and indirect experimental trials. Fig. 4 9. Rhizoctonia solani (PDC 7884) re isolation plate with sele ctive medium from the R. solani infected without Glomus intraradices treatment in the indirect experimental trial 2. Fig. 4 10. Gaeumannomyces graminis var. graminis (JK2) re isolation plate with selective medium from the G. graminis var. graminis infected with Glomus intraradices treatment in the direct experimental trial 2. 10 9

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96 Fig. 4 11. Photo of the indirect experimental trial 3 conetainers arranged in a randomized complete block design with four replicates per treatment. Fig. 4 12. Photo showing a close up view of the experimental units of the indirect experimental trial 1 depicting the split root assay.

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97 Fig. 4 13. Phot o showing the split root assay of the indirect experimental trial 2 after inoculation with ryegrass seeds inoculated with Gaeumannomyces graminis var. graminis (JK2). Arrow points to the inoculum.

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98 y = 0.0333x + 1 R 2 = 0.6485 1 2 3 4 5 6 0 20 40 60 80 100 Mean Percent Colonization of G. graminis var. graminis Mean Disease Severity (1-6 Scale) Fig. 4 14. The direct effect of G. graminis var. graminis on St. Augustinegrass take all root rot disease severity without G. intraradices y = 0.042x + 0.3 R 2 = 0.8167 1 2 3 4 5 6 0 20 40 60 80 100 Mean Percent Colonization of G. graminis var. graminis Mean Disease Severity (1-6 Scale) Fig. 4 15 The direct effect of G. graminis var. graminis on St. Augustinegrass take all root rot disease severity with G. intrar adices

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99 y = 0.0632x + 0.0968 R 2 = 0.752 1 2 3 4 5 6 0 20 40 60 80 100 Percent Colonization of R. solani G. intraradices Mean Disease Severity (Scale 1-6) Fig. 4 16 The indirect effect of R. solani without G. intraradices on St. Augustinegrass brown patch disease severity in an adjacent split sprig system.

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100 CHAPTER 5 SUMMARY AND CONCLUSI ONS From Frank’s (1885) initial report of “fungus roots” in the forests of Germany there has been great interest in the potential benefits of arbuscular mycorrhizal fungi colonization in a vast array of crops. Many documente d evaluations suggest a positive role for AMF in the reduction of disease severity and increased uptake of limited nutrients and water which all contribute to improved vigor and fecundity ( Newsham et al., 1995 ). However, there are also a number of reports suggesting a parasitic role for AMF in plant disease (Ross, 1972; Graham and Menge, 1992; Dehne, 1982; Krishna and Bagyaraj, 1983; Zambolim and Schenck, 1983; Hedge and Rai, 1984; Vigo et al., 2000; Yao et al., 2002). In fact, there appears to be a range of mycorrhizal effects from positive or negative to neutral, commensal, or amensal (Johnson et al., 1997). The impact of AMF must be evaluated, whether the result is positive or negative, within each plant system so that further research can be undertake n to determine the best strategies for maximizing their benefits or for minimizing their damage in the ecology of the cropping system (Gerdemann, 1975). Prior to these studies, there was no information concerning St. Augustinegrass and the role of AMF in sod production, or even if there was a mycorrhizal association between the two types of organisms. In Chapter 1, an overview of past and present research objectives concerning AMF and their role in various hosts was highlighted for the purpose of detaili ng their potential effects and to report on the vast amount of information from previous research studies. In Chapter 2, a survey of three St.

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101 Augustinegrass sod farms in north central Florida revealed a moderate level of AMF colonization as well as a div erse population of arbuscular mycorrhizal fungi. There was no correlation between AMF spore density and percent colonization of the St. Augustinegrass plants, or to soil P levels, as previously documented in other crops (Hayman and Stovold, 1979; Giovanne tti and Nicolson, 1983; Medina Gonzalez et al., 1998). In these soils, there was a correlation to soil moisture and temperature. Spore density and percent colonization fluctuated in relation to soil moisture with spore density tending to decrease at tem perature above 28 C and soil moisture levels above 7 cm. The overall trend of percent AMF colonization was to decrease during warmer months increase in cooler weather; however, there was no highly correlated response to soil moisture levels or temperature observed. Based on this survey, AMF prefer warmer months for spore production and cooler months for colonization in these soils, perhaps due to physiological effects of seasonal change on the host plant that leave the plant more susceptible to colonizati on during less than optimal growing conditions. These results suggest a potentially harmful role for AMF in St. Augustinegrass based on the continuum of mycorrhizal symbiosis proposed by Johnson et al. (1997) and the fact that AMF colonization is highest when St. Augustinegrass is least active increasing carbon depletion in the relationship. In Chapter 3, a survey of the amount of Rhizoctonia solani and Gaeumannomyces graminis var. graminis colonization in St. Augustinegrass was documented in an effort t o highlight the importance of evaluating the potential benefits of AMF on disease severity in this plant system. In the field, no correlation was observed between AMF spore

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102 density or percent colonization of the plants in relation to either of the pathoge ns. However, disease severity did vary greatly for each pathogen based on seasonal variations. Results suggest R. solani colonizes St. Augustinegrass at higher rates in cooler weather, as do the arbuscular mycorrhizal fungi in the field soils surveyed in Chapter 2. This observation suggests a greater potential role for AMF in lowering brown patch disease severity since both the beneficial fungi and the pathogen are active during the same seasons, which happens to be the time when St. Augustinegrass is un der the most seasonal stress. Conversely, the rate of G. graminis var. graminis colonization was highest in the survey during warmer months. During this time period AMF spore density was highest, but percent colonization of the plant was lowest. This fi nding suggests less of a potentially beneficial role for AMF in lowering take all root rot disease severity since the AMF and pathogen are not most active in the same season. Based on the findings presented in Chapters 2 and 3, it was pertinent to evalua te the potential effects of AMF on brown patch and take all root rot disease severity in a more controlled environment in order to evaluate the interaction more thoroughly. Any role that AMF might have in soilborne pathogen disease severity whether positi ve or negative is important to document since the mycorrhizal interaction might be manipulated in a field situation to lower disease severity and possibly fungicide use and cost. In Chapter 4, both direct and unique indirect assays were designed to invest igate the role of AMF in controlled growth room experiments where R. solani and G. graminis var. graminis were challenged by the common AMF, Glomus intraradices In the direct experiments, no correlation between both pathogens and G. intraradices were obs erved, which suggests limited impact of AMF in a direct interaction. Apparently, AMF were

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103 not producing antimicrobial metabolites, occupying infection courts, or improving plant health enough to reduce brown patch or take all root rot disease severity in a highly controlled environment as suggested in previous studies (Becker, 1976; Dehne and Schoenbeck, 1978; Schoenbeck, 1979 ). Furthermore, the indirect assay using a split rooted sprig system where G. intraradices was used to challenge R. solani and G. graminis var. graminis in separate conetainers revealed no correlation between AMF colonization and disease severity in the case of either pathogen. Based on the results of this experiment, there is no systemic defense response afforded to the St. Augusti negrass plant by AMF colonization. While neither the direct nor indirect experiments revealed a positive role for AMF in St. Augustinegrass root disease severity, the evaluations did provide valuable information about AMF that was previously unknown. Ar buscular mycorrhizal fungi do colonize St. Augustinegrass with a diversity of species, but the relationship appears to be neutral role in this species. Based on this information, the focus of future research on AMF in St. Augustinegrass sod should involve a thorough evaluation of AMF species and their individual effects on the host. Additionally, field trials designed to evaluate various sod management strategies and their effects on AMF for the purpose of manipulating the symbiosis into a mutually benefi cial relationship would be worthwhile. While no positive effects of AMF on disease severity were observed in these studies, the potential for reduced pesticide use and cost with the use of mycorrhizae justifies further evaluations.

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104 APPENDIX A SELECTIVE MEDIA RECI PES FOR ISOLATION OF G. GRAMINIS VAR. GRAMINIS AND R. SOLANI FROM PLANT TISSUE A. Semi selective media recipe for isolation of G. graminis var. graminis from plant tissue ( Goo ch, 2002). a. 500 ml deionized water in 1000 ml Erlenmeyer flask b. 4.8 g PDA (potato dextrose agar) – (Difco Laboratories, Inc., Detroit, Michigan) c. 2 g solidifying agar – Difco Laboratories, Inc. d. Autoclave for 20 min at 121 C and 15 psi e. Amend with: a. 0.01 g rifampicin b. 0.01 g streptomycin sulfate B Semi selective media recipe for isolation of R. solani from plant tissue (Adapted from Adams and Butler, 1983). a. 500 ml deionized water in 1000 ml Erlenmeyer flask b. 3.8 g granulated agar – Difco Laboratories, Inc. c. 0.5 g KH 2 PO 4 d. 1 ml MgSO 4 .7H 2 O e. A utoclave for 20 min at 121 C and 15 psi f. Amend with: a. 0.35 g neomycin sulfate b. 0.1 g casein hydrolsyate c. 1 ml Benlate d. 1 ml tannic acid e. 2 drops Ridomil (metalayxyl)

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105 APPENDIX B NUTRIENT SOLUTION (2 0 0 20) USED IN DIRECT A ND INDIRECT TRIALS DESCRIBED IN CHAPTER IV Total N = 20 % 1.97 % Nitrate N 18.03 % Urea N Soluble Potash (K 2 O) = 20 % 1. MgSO 4 7H 2 O (0.34 mg) 2. CuSO 4 5H 2 O (0.1 mg) 3. Fe EDTA (150 mg) 4. MnSO 4 H 2 O (0.05 mg) 5. ( NH 4 ) 4 Mo 7 O 24 4H 2 O (400 mg) 6. ZnSO 4 7H 2 O (0.6 mg) 7. KNO 3 (190 mg) 8. Ca (NO 3 ) 2 4H 2 O (50 mg) 9. NaCl (1.0 mg) *All contained in 1 litre of water

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106 APPENDIX C RHIZOCTONIA SOLANI AND G. GRAMINIS VAR. GRAMINIS INOCULUM PRODUCTION PROTOCOLS A. Sterile substrate inoculation with JK2 ( G graminis var. graminis ) isolate (Ada pted from Datnoff and Elliott, 1997). a. 250 ml perennial ryegrass (BrightStar II) se ed/500 ml wide mouth Erlenmeyer flask b. 125 ml deionized water/flask c. Autoclave substrate two consecutive days for 90 min/d at 121 C and 15 psi d. Aseptically chop 7 d old Petri dish of JK1 G. graminis var. graminis isolate and mix into sterile substrate in flas k with 30 ml sterile deionized water e. Incubate substrate at 25 C for four weeks with 24 h darkness f. Shake flasks twice weekly to prevent substrate packing B. Sterile substrate inoculation with PDC 7884 ( R. solani) isolate ( Adapted from Sneh et al., 1991 and Gaskill, 1968 ). a. 25 g oat seed/250 ml Erlenmeyer flask b. 25 30 ml deionized water; soak overnight c. Autoclave substrate three consecutive days for 90 min/day at 121 C and 15 psi d. Once cooled, inoculate flask with three to four 7 mm plugs of actively growing R. solani mycelium e. Incubate substrate at 25 30 C for two to three weeks f. Shake flasks to loosen seeds and prevent packing g. After three weeks incubation, pour seed into sterile Petri dishes; allow to air dry uncovered for two weeks h. Store in sterile vials at 4 C until inoculation

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107 APPENDIX D ADDITIONAL DATA ANAL YSIS RESULTS REFEREN CED IN CHAPTER IV DIRECT EXPERIMENTS y = 0.0053x + 2.2148 R 2 = 0.0005 1 2 3 4 5 6 0 5 10 15 20 Mean Percent G. intraradices Colonization Mean Disease Severity (1-6 Scale) Appendix D 1. The direct effect of G. intraradices colonization on take all root rot disease severity in ‘Floratam’ St. Augustinegrass. Values represent the mean of three trials with four replicates/trial.

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108 y = 0.0308x + 1.8769 R 2 = 0.4006 1 2 3 4 5 6 0 20 40 60 80 100 Mean Percent R. solani Colonization G. intraradices Mean Disease Severity (Scale 1-6) Appendix D 2. The relationship between R. solani colonization and brown patch disease severity in ‘Floratam’ St. Augustinegrass. Values represent the means of three trials with four r eplicates/trial.

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109 y = 0.0216x + 1.0526 R 2 = 0.2458 1 2 3 4 5 6 0 20 40 60 80 100 Mean Percent R. solani Colonization + G. intraradices Mean Disease Severity (1-6 Scale) Appendix D 3. The relationship between R. solani colonization and G. intraradices on brown patch disease severity in ‘Floratam’ St. Augustinegrass. Values represent th e means of three trials with four replicates/trial.

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110 APPENDIX E ADDITIONAL DATA ANAL YSIS RESULTS REFEREN CED IN CHAPTER IV INDIRECT EXPERIMENTS Appendix E 1. Photograph depicting a conetainer used in the indirect experiment with drilled hole and cut to allow for sprig to be inserted without tissue dam age. a

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111 y = 0.0353x + 0.8983 R 2 = 0.3205 1 2 3 4 5 6 0 20 40 60 80 100 Percent G. graminis var. graminis Colonization G. intraradices Mean Disease Severity (Scale 1-6) Appendix E 2. The indirect effect of G. graminis var. graminis on take all root rot diease severity in ‘Floratam’ St. Augustinegrass without G. intraradices Values represent the means of three trials with four replicates/trial on an adjacent split sprig system.

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112 y = -0.0661x + 1.7796 R 2 = 0.3471 1 2 3 4 5 6 0 20 40 60 80 100 Percent G. intraradices Colonization Mean Disease Severity (1-6 Scale) Appendix E 3. The effect of Glomus intraradices colonization on brown patch and take al l root rot disease severity in ‘Floratam’ St.Augustinegrass on plants in the split sprig assay. Values represent the means of three trials with four replicates/trial.

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113 y = 0.0342x + 1.2048 R 2 = 0.3393 1 2 3 4 5 6 0 20 40 60 80 100 Mean Percent R. solani Colonization + G. intraradices Mean Disease Severity (Scale 1-6) Appendix E 4. The indirect effect of R. solani on disease severity in ‘Floratam’ St. Augustinegrass with G. intraradices on an adjacent split sprig system. Values represent the means of three trials with four replicates/trial.

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114 y = 0.0271x + 1.7119 R 2 = 0.339 1 2 3 4 5 6 0 20 40 60 80 100 Mean Percent G. graminis var. graminis + G. intraradices Colonization Mean Disease Severity (Scale 1-6) Appendix E 5. The indirect effect of G. graminis var. graminis on di sease severity in ‘Floratam’ St. Augustinegrass with G. intraradices Values represent the means of three trials with four replicates/trial.

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115 APPENDIX F ANALYSIS OF VARIANCE TABLES FOR CHAPTERS 2, 3, AND 4 The SAS System 22:22 Wednesday, July 19, 2006 2 The GLM Procedure Dependent Variable: sporeden Sum of Source DF Squares Mean Square F Value Pr > F Model 41 38.55619444 0.94039499 3.94 <.0001 Error 66 15.73407222 0.23839503 Corrected Total 107 54.29026667 R Square Coeff Var Root MSE sporeden Mean 0.710186 8.054095 0.488257 6.062222 Source DF Type I SS Mean Square F Value Pr > F date 11 28.08166667 2.55287879 10.71 <.0001 location 2 0.49388889 0.24694444 1.04 0.3606 rep(location) 6 1.21259444 0.20209907 0.85 0.5379 rainfall 1 0.02743228 0.02743228 0.12 0.7355 soiltemp 1 0.00744901 0.00744 901 0.03 0.8602 date*location 20 8.73316316 0.43665816 1.83 0.0349 Source DF Type III SS Mean Square F Value Pr > F date 11 12.8810 5106 1.17100464 4.91 <.0001 location 2 0.52131593 0.26065796 1.09 0.3411 rep(location) 6 1.21259444 0.20209907 0.85 0.5379 rainfall 0 0.00000000 . soiltemp 0 0.00000000 . date*location 20 8.73316316 0.43665816 1.83 0.0349 Tests of Hypothes es Using the Type III MS for date*location as an Error Term Source DF Type III SS Mean Square F Value Pr > F date 11 12.88105106 1.17100464 2.68 0.0267

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116 The SAS System 22:22 Wednesday, July 19, 2006 3 The GLM Procedure Tukey's Studentized Range (HSD) Test for sporeden NOTE: This test controls the Type I ex perimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 66 Error Mean Square 0.238395 Critical Value of Studentized Range 4.79129 Minimum Significant Difference 0.7798 Means with the same le tter are not significantly different. Tukey Grouping Mean N date A 6.7344 9 May A A 6.66 22 9 June A A 6.5511 9 April A A 6.5100 9 Nov A B A 6.2689 9 August B A B A C 6.1467 9 Oct B A C B A C 6.0522 9 Sept B A C B A C 5.9922 9 5 Dec B C B D C 5.7244 9 March B D C B D C 5.7189 9 Feb D C D C 5.3800 9 Jan D D 5. 0056 9 4 Dec

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117 The SAS System 22:22 Wednesday, July 19, 2006 5 The GLM Procedure Dependent Variable: percolon S um of Source DF Squares Mean Square F Value Pr > F Model 41 1744.970096 42.560246 3.71 <.0001 Error 66 757.324989 11.474621 Corrected Total 107 2502.295085 R Square Coeff Var Root MSE percolon Mean 0.697348 13.89690 3.387421 24.37537 Source DF Typ e I SS Mean Square F Value Pr > F date 11 1410.787241 128.253386 11.18 <.0001 location 2 4.613424 2.306712 0.20 0.8184 rep(location) 6 23.386811 3.897802 0.34 0.9134 rainfall 1 11.152745 11.152745 0.97 0.3278 soiltemp 1 7.560355 7.560355 0.66 0.4199 date*locati on 20 287.469520 14.373476 1.25 0.2430 Source DF Type III SS Mean Square F Value Pr > F date 11 1315.392404 119.581128 10.42 <.0001 location 2 5.718640 2.859320 0.25 0.7802 rep(location) 6 23.386811 3.897802 0.34 0.9134 rainfall 0 0.000000 . soiltemp 0 0.000000 . date*location 20 287.469520 14.373476 1.25 0.2430 Tests of Hypotheses Using the Type III MS for date*location as an Error Term Source DF Type III SS Mean Square F Value Pr > F date 11 1315.392404 119.581128 8.32 <.0001

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118 The SAS System 22:22 Wednesday, July 19, 2006 6 The GLM Procedure Tukey's Studentized Range (HSD) Test for percolon NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 66 Error Mean Sq uare 11.47462 Critical Value of Studentized Range 4.79129 Minimum Significant Difference 5.41 Means with the same letter are not significantly different. Tukey Grouping Mean N date A 29.007 9 Jan A A 28.854 9 Feb A A 28.584 9 April A B A 26.731 9 May B A B A 26. 133 9 4 Dec B A B A 25.923 9 June B A B A C 24.849 9 March B C B D C 22.636 9 August B D C B D C 21.680 9 Sept D C D C 19.914 9 Nov D C D C 19.504 9 Oct D D 18.688 9 5 Dec

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119 The SAS System 22:22 Wednesday, July 19, 2006 7 The CORR Procedure 2 Variables: percolon sporeden Simple Statistics Variable N Mean Std Dev Sum Minimum Maximum percolon 108 24.37537 4.83590 2633 12.71000 38.32000 sporeden 108 6.06222 0.71231 654.72000 4.36000 7.66000 Pearson Correlation Coefficients, N = 108 Prob > |r| under H0: Rho=0 percolon sporeden perc olon 1.00000 0.00758 0.9380 sporeden 0.00758 1.00000 0.9380 The SAS System 22:22 Wednesday, July 19, 2006 8 The CORR Procedure 8 Variables: percolon soiltemp percolon rainfall sporeden soiltemp sporeden rainfall Simpl e Statistics Variable N Mean Std Dev Sum Minimum Maximum percolon 108 24.37537 4.83590 2633 12.71000 38.32000 soiltemp 108 3.74222 2.35241 404.16000 0 8.69000 percolon 108 24.37537 4.83590 2633 12.71000 38.32000 rainfall 108 25.23417 6.55045 2725 11.77000 34.54000 sporeden 108 6.0 6222 0.71231 654.72000 4.36000 7.66000 soiltemp 108 3.74222 2.35241 404.16000 0 8.69000 sporeden 108 6.06222 0.71231 654.72000 4.36000 7.66000 rainfal l 108 25.23417 6.55045 2725 11.77000 34.54000 Pearson Correlation Coefficients, N = 108 Prob > |r| under H0: Rho=0 percolon soiltemp percolo n rainfall sporeden soiltemp sporeden rainfall percolon 1.00000 0.02488 1.00000 0.14948 0.00758 0.02488 0.00758 0.14948 0.7983 0.1226 0.9380 0.7983 0.9380 0.1226 soiltemp 0.02 488 1.00000 0.02488 0.61090 0.48819 1.00000 0.48819 0.61090 0.7983 0.7983 <.0001 <.0001 <.0001 <.0001 percolon 1.00000 0.02488 1.00000 0.14948 0.00758 0.02488 0.00758 0.149 48

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120 0.7983 0.1226 0.9380 0.7983 0.9380 0.1226 rainfall 0.14948 0.61090 0.14948 1.00000 0.45921 0.61090 0.45921 1.00000 0.1226 <.0001 0.1226 <.0001 <.0 001 <.0001 sporeden 0.00758 0.48819 0.00758 0.45921 1.00000 0.48819 1.00000 0.45921 0.9380 <.0001 0.9380 <.0001 <.0001 <.0001 soiltemp 0.02488 1.00000 0.02488 0.61090 0. 48819 1.00000 0.48819 0.61090 0.7983 0.7983 <.0001 <.0001 <.0001 <.0001 sporeden 0.00758 0.48819 0.00758 0.45921 1.00000 0.48819 1.00000 0.45921 0.9380 <.0001 0.9380 <.0001 <.0001 <.0001 rainfall 0.14948 0.61090 0.14948 1.00000 0.45921 0.61090 0.45921 1.00000 0.1226 <.0001 0.1226 <.0001 <.0001 <.0001 Appendix F 1. Analysis of variance tables for spore density and percent colonization data in Chapter 2, and Pearson’s product moment correlation coefficients for attempted correlations between variables and soil chemical characteristics and soil moisture and soil temperature.

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121 The SAS System 22:13 Wednesday, July 19, 2006 2 The GLM Procedure Dependent Variable: rsolani Sum of Source DF Squares Mean Square F Value Pr > F Model 28 23746.3116 848.0826 1.98 0.0023 Error 559 240032.3874 429.3960 Corrected Total 587 263778.6990 R Square Coeff Var Root MSE rsolani Mean 0.090024 120.3404 20.72187 17.21939 Source DF Type I SS Mean Square F Value Pr > F date 6 13979.59184 2329.93197 5.43 <.0001 location 2 1001.27551 500.63776 1.17 0.3124 rep(location) 6 644.48696 107.41449 0.25 0.9592 amfcolon 1 818.37429 818.37429 1.91 0.1680 amfsd 1 132.41846 132.41846 0.31 0.5789 date*location 12 7170.16457 597.51371 1.39 0.1653 Source DF Type III SS Mean Square F Value Pr > F date 6 13183.23554 2197.20592 5.12 <.0001 location 2 634.32230 317.16115 0.74 0.4782 rep(location) 6 564.06333 94.01055 0.22 0.9707 amfcolon 1 39.58906 39.58906 0.09 0.7615 amfsd 1 3.47586 3.47586 0.01 0.9283 date*location 12 7170.16457 597.51371 1.39 0.1653 Tests of Hypotheses Using the Type III MS for date*location as an Error Term Source DF Type III SS Mean Square F Value Pr > F date 6 13183.23554 2197.20592 3.68 0.0262

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122 The SAS System 22:13 Wedn esday, July 19, 2006 3 The GLM Procedure Tukey's Studentized Range (HSD) Test for rsolani NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. Alpha 0.05 Error Degrees of Freedom 559 Error Mean Square 429.396 Critical Value of Studentized Range 4.18483 Minimum Significant Difference 9.4616 Means with the same letter are not significantly different. Tukey Grouping Mean N date A 24.405 84 29 Dec A B A 22.024 84 29 Apr B A B A C 19.940 84 19 Oct B A C B A C 17.560 84 5 Jan B C B C 14 .583 84 31 Mar C C 11.310 84 31 Aug C C 10.714 84 21 Jun Appendix F 2. Anal ysis of variance tables for Rhizoctonia solani percent colonization data in Chapter 3.

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123 The SAS System 22:07 Wednesday, July 19, 2006 1 The REG Procedure Model: MODE L1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 14.08211 14.08211 27.28 0.0005 Error 9 4.64516 0.51613 Corrected Total 10 18.72727 Root MS E 0.71842 R Square 0.7520 Dependent Mean 3.54545 Adj R Sq 0.7244 Coeff Var 20.26316 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 0.09677 0.69486 0.14 0.8923 rsolcolon 1 0.06323 0.0121 0 5.22 0.0005 The SAS System 22:07 Wednesday, July 19, 2006 3 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 7.05085 7.05085 5.19 0.0437 Error 11 14.94915 1.35901 Corrected Total 12 22.00000 Root MSE 1.16577 R Square 0.3205 Dependent Mean 3.00000 Adj R Sq 0.2587 Coeff Var 38.85892 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 0.89831 0.97771 0.92 0.3779 gggcolon 1 0.03525 0.01548 2.28 0.043 7

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124 The SAS System 22:07 Wednesday, July 19, 2006 5 The REG Procedure Model: MODEL1 Dependen t Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 0.92562 0.92562 5.32 0.0438 Error 10 1.74105 0.17410 Corrected Total 11 2.66667 Root MSE 0.41726 R Square 0.3471 Dependent Mean 1.33333 Adj R Sq 0.2818 Coeff Var 31.29439 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.77961 0.22797 7.81 <.0001 amfcolon 1 0.06612 0.02867 2.31 0.0438 The SAS System 22:07 Wednesday, July 19, 2006 7 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 5. 06124 5.06124 5.14 0.0469 Error 10 9.85542 0.98554 Corrected Total 11 14.91667 Root MSE 0.99274 R Square 0.3393 Dependent Mean 2.41667 Adj R Sq 0.2732 Coeff Var 41.07909 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.20482 0.60671 1.99 0.0751 rsolcolon 1 0.03422 0.01510 2.27 0.0469

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125 The SAS System 22:07 Wednesday, July 19, 2006 9 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 2.25989 2.25989 5.13 0.0470 Error 10 4.40678 0.44068 Corrected Total 11 6.66667 Root MSE 0.66384 R Square 0.3390 Dependent Mean 2 .33333 Adj R Sq 0.2729 Coeff Var 28.45011 Parameter Estimates Parameter Standard Variable DF Estimate Err or t Value Pr > |t| Intercept 1 1.71186 0.33472 5.11 0.0005 gggcolon 1 0.02712 0.01198 2.26 0.0470 The SAS System 2 2:07 Wednesday, July 19, 2006 12 The GLM Procedure Dependent Variable: ggg Sum of Source DF Squares Mean Square F Value Pr > F Model 24 16766.7893 698.6162 3.32 <.0001 Error 563 118364.1273 210.2382 Corrected Total 587 135130.9167 R Square Coeff Var Root MSE ggg Mean 0.124078 102.9557 14.49959 14.08333 Source DF Type I SS Mean Square F Value Pr > F date 6 12105.214 29 2017.53571 9.60 <.0001 location 2 501.73810 250.86905 1.19 0.3040 rep 2 81.93466 40.96733 0.19 0.8230 amfcolon 1 57.95802 57.95802 0.28 0.5998 amfsd 1 1.82548 1.82548 0.01 0.9258 date*location 12 4018.11879 334.84323 1.59 0.0895 Source DF Type III SS Mean Square F Value Pr > F date 6 9889.269923 1648.211654 7.84 <.0001 location 2 570.223651 285.111825 1.36 0.2585 rep 2 73.695910 36.847955 0.18 0.8393 amfcolon 1 98.114370 98.114370 0.47 0.4948 amfsd 1 174.670045 174.670045 0.83 0.3624 date*location 12 4018.118793 334.843233 1.59 0.0895

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126 The SAS System 22:07 Wednesday, July 19, 2006 13 The GLM Procedure Tukey's Studentized Range (HSD) Test for ggg NOTE: This test controls the Type I experimentwise error rate, but it generally has a higher Type II error rate than REGWQ. A lpha 0.05 Error Degrees of Freedom 563 Error Mean Square 210.2382 Critical Value of Studentized Range 4.18472 Minimum Significant Difference 6.6204 Means with the same letter are not significantly different. Tukey Grouping Mean N date A 20.012 84 31 Aug A A 19.345 84 21 Jun A B A 14.881 84 19 Oct B A B A 13.393 84 31 Mar B A B A 13.393 84 29 Apr B B 12.202 84 5 Jan C 5.357 84 29 Dec Appendix F 2. Analysis of variance tables for Gaeumannomyces graminis var. graminis percent colonization data in Chapter 3.

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127 The SAS System 22:16 Wednesday, July 19, 2006 1 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Var iance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 3.57143 3.57143 3.13 0.1075 Error 10 11.42857 1.14286 Corrected Total 11 15.00000 Root MSE 1.06904 R Square 0.2381 Dependent Mean 3.50000 Adj R Sq 0.16 19 Coeff Var 30.54414 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.71429 1.05624 1.62 0.1357 rsolcolon 1 0.02857 0.01616 1.77 0.1075 The SAS System 22:16 Wednesday, July 19, 20 06 3 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 7.07065 7.07065 4.39 0.0600 Error 11 17.69858 1.60896 Corrected Total 12 24.76923 Root MSE 1.26845 R Square 0.2855 Dependent Mean 2.69231 Adj R Sq 0.2205 Coeff Var 47.11381 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.63830 0.61365 2.67 0.0218 gggcolon 1 0.02284 0.01089 2.10 0.0600

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128 The SAS System 22:16 Wednesday, July 19, 2006 5 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 0.00526 0.00526 0.00 0.9471 Error 9 10.17656 1.13073 Corrected Total 10 10.18182 Root MSE 1.06336 R Square 0.0005 Dependent Mean 2.27273 Adj R Sq 0.1105 Coeff Var 46.78771 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 2.21483 0.90713 2.44 0.0373 amfcolon 1 0.00526 0.07714 0.07 0.9471 The SAS System 22:16 Wednesday, July 19, 2006 8 The REG Procedure Model: MODEL1 Dependent Variable: wgtai Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 0.50760 0.50760 3.02 0.1130 Error 10 1.68209 0.16821 Co rrected Total 11 2.18969 Root MSE 0.41013 R Square 0.2318 Dependent Mean 0.85583 Adj R Sq 0.1550 Coeff Var 47.92203 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.52905 0.40522 3.7 7 0.0036 rsolcolon 1 0.01077 0.00620 1.74 0.1130

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129 The SAS System 22:16 Wednesday, July 19, 2006 10 The REG Procedure Model: MODEL1 Dependent Variable: wgtai Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 0.00729 0.00729 0.03 0.8644 Error 11 2.62282 0.23844 Corrected Total 12 2.63011 Root MSE 0.48830 R Square 0.0028 Dependent Mean 1.00615 Adj R Sq 0.0879 Coeff Var 48.53145 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.04000 0.23623 4.40 0.0011 gggcolon 1 0.00073333 0.00419 0.17 0.8644 The SAS System 22:16 Wednesday, July 19, 2006 12 The REG Procedure Model: MODEL1 Dependent Variable: wgtai Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 0.00796 0.00796 0.03 0.8576 Error 9 2.10153 0.23350 Corrected Total 10 2.10949 Root MSE 0.48322 R Square 0.0038 Dependent Mean 0.95091 Adj R Sq 0.1069 Coeff Var 50.81681 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.02212 0.41223 2.48 0.0350 amfcolon 1 0.00647 0.03506 0.18 0.8576 Appendix F 4. Analysis of variance tables for the direct assay in the split sprig challenge including Gaeumannomyces graminis var. graminis and Rhizoctonia solani data in Chapter 4.

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130 The SAS System 22:14 Wednesday, July 19, 2006 1 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 14.08211 14.08211 27.28 0.0005 Error 9 4.64516 0.51613 Corrected Total 10 18.72727 Root MSE 0.71842 R Square 0.7520 Depen dent Mean 3.54545 Adj R Sq 0.7244 Coeff Var 20.26316 Parameter Estimates Parameter Standard Variable DF Est imate Error t Value Pr > |t| Intercept 1 0.09677 0.69486 0.14 0.8923 rsolcolon 1 0.06323 0.01210 5.22 0.0005 The SAS System 22:14 Wednesday, July 19, 2006 3 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 7.05085 7.05085 5.19 0.0437 Error 11 14.94915 1.35901 Corrected Total 12 22.00000 Root MSE 1.16577 R Square 0.3205 Dependent Mean 3.00 000 Adj R Sq 0.2587 Coeff Var 38.85892 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 0.89831 0.97771 0.92 0.3779 gggcolon 1 0.03525 0.01548 2.28 0.0437

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131 The SAS System 22:14 Wednesday, July 19, 2006 5 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Va riance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 0.92562 0.92562 5.32 0.0438 Error 10 1.74105 0.17410 Corrected Total 11 2.66667 Root MSE 0.41726 R Square 0.3471 Dependent Mean 1.33333 Adj R Sq 0.2 818 Coeff Var 31.29439 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.77961 0.22797 7.81 <.0001 amfcolon 1 0.06612 0.02867 2.31 0.0438 The SAS System 22:14 Wednesday, July 19, 2006 7 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 5.06124 5.06124 5.14 0.0469 Error 10 9.85542 0.98554 Corrected Total 11 14.91667 Root MSE 0.99274 R Square 0.3393 Dependent Mean 2.41667 Adj R Sq 0.2732 Coeff Var 41.07909 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Interce pt 1 1.20482 0.60671 1.99 0.0751 rsolcolon 1 0.03422 0.01510 2.27 0.0469

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132 The SAS System 22:14 Wednesday, July 19, 2006 9 The REG Procedure Model: MODEL1 Dependent Variable: dissev Analysis of Variance Sum of Mean Source DF Squares Square F Value Pr > F Model 1 2.25989 2.25989 5.13 0.0470 Error 10 4.40678 0.44068 Corrected Total 11 6.66667 Root MSE 0.66384 R Square 0.3390 Dependent Mean 2.33333 Adj R Sq 0.2729 Coeff Var 28.45011 Parameter Estimates Parameter Standard Variable DF Estimate Error t Value Pr > |t| Intercept 1 1.71186 0.33472 5.11 0.0005 gggcolon 1 0.02712 0.01198 2.26 0.0470 Appendix F 4. Analysis of variance tables for the indirect assay in the split sprig challenge including Gaeumannomyces graminis var. graminis and Rhizoctonia solani data in Chapter 4.

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150 BIOGRAPHICAL SKETCH Whitney Colleen Elmore, youngest daughter of Malcolm and Donna Elmore of Lucas, Kentucky, attended Barren County High School in Glasgow, Kentucky, and graduated in 1994. Whitney grew up with a sister, Emilee, and later two nephews, Ryan and Dustin Mosier. As an active member of FFA, Whitney served as vice president of her chapter, lettered in varsity golf and track and field, and participated in the BETA Club as well as many other clubs and activities. In 1994, she began her coll egiate career at Western Kentucky University in Bowling Green, Kentucky where she received an Associate of Science degree in turf grass management in 1997 and Bachelor of Science degree in agriculture in 1998. Finishing her undergraduate degree, she purs ued her Master of Science Degree in turfgrass science/agriculture working on hydrophobic soils with Dr. Haibo Liu. Before becoming a recipient of the Sigma Xi Award for the Outstanding Graduate Research Paper in 2001, she became a member of the Golden Key National Honor Society. Upon the completion of her master’s program in 2001, Whitney began her Ph.D. in plant pathology working on diseases of turfgrasses, at the University of Florida under the guidance of Dr. James Kimbrough. While pursuing her docto rate, she became a member of Gamma Sigma Delta, the Honor Society of Agriculture, in 2003 and received scholarships from the Florida Turfgrass Association and the Florida Nursery Growers Association. Whitney is currently teaching classes at Santa Fe Commu nity College in

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151 Gainesville, Florida, and has accepted a faculty position at Macon State College in Macon, Georgia.


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POPULATION AND IDENTIFICATION OF MYCORRHIZAL FUNGI IN ST.
AUGUSTINEGRASS IN FLORIDA AND THEIR EFFECT ON SOILBORNE
PATHOGENS














By

WHITNEY COLLEEN ELMORE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006















DEDICATION





This dissertation is dedicated to my family in the memory of my father,
Malcome Elmore.















ACKNOWLEDGMENTS

I would like to thank my parents, Malcome and Donna Elmore, for their loving

support and my sister, Emilee. I would also like to acknowledge a very special person,

LaVette Burnette, for all of the patience and caring attention she has shown me for many

years. I would also like to thank Dr. James Kimbrough and his wife, Jane, for their

support, both emotionally and spiritually. I would also like to thank Drs. Jim Graham

and Kevin Kenworthy for agreeing to serve on my graduate committee and for their

willingness to offer advice on my studies. I also owe Dr. Vertigo Moody a big "thank

you" for motivating me to finish my Ph.D. as well as for his technical support in writing.

Additionally, I would like to say a big "thank you" to Dr. Gerald Benny both for

serving on my committee and for his attention in the lab. Dr. Benny is always ready to

help with research, or simply listen to my ramblings about research and politics which I

appreciate greatly. I would like to extend a personal "thank you" to the Department of

Plant Pathology staff, Gail Harris, Lauretta Rahmes, and Donna Perry. These ladies

always have a smile ready and a helping hand for students. I would also like to thank

Eldon Philman and Herman Brown for their assistance in experimental studies at the

greenhouse complex. They seem to always have a good solution or answer to any

problem or question. Finally, I would like to extend my sincerest appreciation to the

Department of Plant Pathology, namely Dr. Gail Wisler, at the University of Florida and

to the Institute of Food and Agricultural Sciences for financial and technical support in









this endeavor. I would not have been able to fulfill my dreams without the help and

support from all of these people.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..................................................................................................iii

LIST OF TABLES ..... ......... ........ ....... ... .. .... ........vii

LIST OF FIGURES............... .. ...................... ........viii

A B STRA CT .................. ............ ......................... ................ ........ xii

CHAPTER

1 GENERAL IN TRODU CTION ...................................... ...................... ............... 1

M ycorrhizal Types and Phylogeny ......................................................................... 2
Arbuscular M ycorrhiza Physiology ................................ ...............4
A rbuscular M orphology ................................................................. ..............5
M ycorrhizal C olonization ................................................................ ............... 6
M ycorrhizal Rhizosphere Interactions.................. ............................................ .8
Effects of Abiotic Factors on Mycorrhiza ...................................12
Effects of Seasonality on M ycorrhiza.............................. ...... ......... 14
M ycorrhizas in Grasses ................... ..... .................................... ...... 16

2 POPULATION AND IDENTIFICATION OF ARBUSCULAR
MYCORRHIZAL FUNGI IN ST. AUGUSTINEGRASS .......................................24

M materials and M ethods.................................... ............... 25
Results .......................... .. .. ...... ......... .29
Discussion ................... ................. ....... ....... ........34

3 THE EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI ON
GAEUMANNOMYCES GRAMINIS VAR. GRAMINIS AND RHIZOCTONIA
SOLANI COLONIZATION OF ST. AUGUSTINEGRASS SOD IN NORTH
CEN TRAL FLORID A SOILS ....................................................... 57

M materials and M ethods.................................... ............... 63
Results and Discussion ............................................... ..... .. 65










4 EFFECT OF GLOMUS INTRARADICES ON THE EXTENT OF DISEASE
CAUSED BY GAEUMANNOMYCES GRAMINIS VAR. GRAMINIS AND
RHIZOCTONIA SOLANI IN ST. AUGUSTINEGRASS ...............................74

Material and Methods ...... .................... ......... ........77
Direct Experiments ............. ... ............... ...............77
Indirect Experiments.......... ...... ............... 82
Results .............................. ... ...... ...........84
Direct Experiments ............. ... ............... ...............84
Discussion ................................. ................86
Results .................................... ..... ...........87
Indirect Experiment ...... ......... ..............................87
D iscu ssio n .......... ..... .... ......... ....................... 8 8

5 SUMMARY AND CONCLUSIONS ........................... ...............100

APPENDIX

A SELECTIVE MEDIA RECIPES FOR ISOLATION OF G. GRAMINIS VAR.
GRAMINIS AND R. SOLANI FROM PLANT TISSUE .............. ................104

B NUTRIENT SOLUTION (20-0-20) USED IN DIRECT AND INDIRECT
TRIALS DESCRIBED IN CHAPTER 4................................................................105

C RHIZOCTONIA SOLANI AND G. GRAMINIS VAR. GRAMINIS INOCULUM
PRODUCTION PROTOCOLS ....... ................................... 106

D ADDITIONAL DATA ANALYSIS RESULTS REFERENCED IN CHAPTER 4
DIRECT EXPERIM ENTS .............................................. ............... 107

E ADDITIONAL DATA ANALYSIS RESULTS REFERENCED IN CHAPTER 4
INDIRECT EXPERIM ENTS ............................................................. ...............110

F ANALYSIS OF VARIANCE TABLES FOR CHAPTERS 2, 3, AND 4.............115

L IST O F R E F E R E N C E S ........................................................................................... 133

B IO G RA PH ICA L SK ETCH .......................................................................... .......... 150
















LIST OF TABLES


Table page

2-1. Species of AMF positively identified at each sod farm location from pot cultures
of sorghum-sudangrass within a combination of field and sterile, low P soil..........45

2-2. Evaluation of analysis of variance data for spore density data from each sod farm
location by date..................... ............... ........ 48

2-3. Pearson correlation coefficients (r) for AMF spore density and soil moisture and
temperature. .......................................................50

2-4. Evaluation of analysis of variance data for percent root length colonized from
each sod farm location................... ... ...................................... ...... 54

2-5. Chemical characteristics of soils sampled for AMF at three north central Florida
sod farm locations during January, April, August, and November 2005..............56




















LIST OF FIGURES


Figure page

2-1 A-C. 'Floratam' St. Augustinegrass sod farms located at (A) Fort McCoy
(Marion County), (B) Lake Butler (Union County), and (C) Starke (Bradford
County) in north central Florida. ............................................................ ............ 40

2-2. Sorghum-sudangrass pot cultures containing 50% (w/w) field soil combined
w ith 50% sterile, low P soil. ............................................................ 41

2-3. Spore extract from field soil following the wet sieving procedure...........................42

2-4 2-7. Stained arbuscular mycorrhizal structures observed within 'Floratam' St.
Augustinegrass. ..................................................43

2-8 2-11. Stained arbuscular morphology types found within 'Floratam'
St. Augustinegrass................... ................. ................. ..........44

2-14 2-19. Arbuscular mycorrhizal fungal spores identified at the Lake
Butler sod farm location......... .. ............................ .. .......46

2-20 2-28. Arbuscular mycorrhizal fungal spores identified at the Fort
M cCoy sod farm location............... .. ........................... .........47

2-29. Spore density with increasing soil moisture levels over a 12-month period at the
Starke sod farm location. ................................................................................... .......51

2-30. Spore density with increasing soil moisture levels over a 12-month period at the
Fort M cCoy sod farm location. .................................... ..................... 51

2-31. Spore density with increasing soil moisture levels over a 12-month period at the
Lake Butler sod farm location. ........................................ ................ 52

2-32. Spore density with increasing soil temperatures over a 12-month period at the
Starke sod farm location. ................................................................................... .......52

2-33. Spore density with increasing soil temperatures over a 12-month period at the
Fort M cCoy sod farm location. .................................... ..................... 53









2-34. Spore density with increasing soil temperatures over a 12-month period at the
Starke sod farm location. ..........................................................................................53

3-1. 'Floratam' St. Augustinegrass sod mat infected with Gaeumannomyces graminis
var. graminis. Insert in bottom right-hand comer depicts underside of a mat
with rotting roots. ....................................................68

3-2 3-3. Comparison of healthy 'Floratam' St. Augustinegrass sod mat and sod
affected by brow n patch. ................................................ ............... 69

3-4. Deeply-lobed hyphopodia isolated from Gaeumannomyces graminis var.
graminis in 'Floratam' St. Augustinegrass sod samples. Scale bar = 40 [im..........70

3-5. Medium isolation plate depicting a Gaeumannomyces graminis var. graminis
colony isolated from 'Floratam' St. Augustinegrass sod samples. Arrow points
to colony. ........................................................70

3-6. Rhizoctoniasolani hyphae isolated from 'Floratam' St. Augustinegrass sod
exhibiting diagnostic 900 branching at constriction points and characteristic
septa. Scale bar = 40 rim. Arrow points to branching pattern..............................71

3-7. Medium isolation plate depicting light brown Rhizoctonia solani colony isolated
from 'Floratam' St. Augustinegrass sod samples...........................71

3-8. Mean percent of Rhizoctonia solani colonization of 'Floratam' St.
Augustinegrass in north central Florida. ................................. ............. 72

3-9. Mean percent of Gaeumannomyces graminis var. graminis colonization of
'Floratam' St. Augustinegrass in north central Florida ................................ 73

4-1. Rhizoctoniasolani isolate (PDC 7884) colony used to prepare inoculum in direct
and indirect experim ents................................... ......... 90

4-2. Gaeumannomyces graminis var. graminis isolate (JK2) used to prepare inoculum
in direct and indirect experim ents. ............................................. 90

4-3. Conetainers filled with low P soil and 'Floratam' St. Augustinegrass sprigs
inoculated in trial 1 of the direct experiment............... ....................................91

4-4. Glomus intraradices isolate (FL 208 A) used in direct and indirect assays to
inoculate 'Floratam' St. Augustinegrass sprigs. ..................................... 91

4-5. Photo showing nylon sleeves and plastic clips used in direct and indirect
experiments to clear and stain root segments from treatment replicates................92

4-6. Photo of mycorrhizal St. Augustinegrass root with arbuscules and intraradical
hypha of Glomus intraradices stained with 0.05% trypan blue from the direct
experiment G. intraradices inoculated control sprigs. ........... ... ..........92









4-7. 'Floratam' St. Augustinegrass sprigs after inoculation with Rhizoctonia solani
depicting disease severity rating scale (1-6)............................ ............. 93

4-8. 'Floratam' St. Augustinegrass sprigs after inoculation with Gaeumannomyces
graminis var. graminis depicting disease severity rating scale (1-6).................. 94

4-9 4-10. Photo depicting re-isolation plates of the two pathogenic isolates used to
challenge Glomus intraradices in both the direct and indirect experimental trials..95

4-11. Photo of the indirect experimental trial 3 containers arranged in a randomized
complete block design with four replicates per treatment..................... ..........96

4-12. Photo showing a close-up view of the experimental units of the indirect
experimental trial 1 depicting the split-root assay................................ ..........96

4-13. Photo showing the split-root assay of the indirect experimental trial 2 after
inoculation with ryegrass seeds inoculated with Gaeumannomyces graminis var.
graminis (JK2). ....................................................97

4-14. The direct effect of G. graminis var. graminis on St. Augustinegrass take-all
root rot disease severity without G. intraradices. ...................................... 98

4-15. The direct effect of G. graminis var. graminis on St. Augustinegrass take-all
root rot disease severity with G. intraradices. ................ .................. ..........98

4-16. The indirect effect of R. solani without G. intraradices on St.
Augustinegrass brown patch disease severity in an adjacent split sprig system....99

D-1. The direct effect of G. intraradices colonization on take-all root rot disease
severity in 'Floratam' St. Augustinegrass. ....................... ............. 107

D-2. The relationship between R. solani colonization and brown patch disease
severity in 'Floratam St. Augustinegrass....................................................... 108

D-3. The relationship between R. solani colonization and G. intraradices on brown
patch disease severity in 'Floratam' St. Augustinegrass...................................... 109

E-1. Photograph depicting a container used in the indirect experiment with drilled
hole and cut to allow for sprig to be inserted without tissue damage.....................110

E-2. The indirect effect of G. graminis var. graminis on take-all root rot disease
severity in 'Floratam' St. Augustinegrass without G. intraradices.................... 111

E-3. The effect of Glomus intraradices colonization on brown patch and take-all root
rot disease severity in 'Floratam' St.Augustinegrass on plants in the split sprig
assay. ..........................................................112









E-4. The indirect effect of R. solani on disease severity in 'Floratam' St.
Augustinegrass with G. intraradices on an adj acent split sprig system............ 113

E-5. The indirect effect of G. graminis var. graminis on disease severity in
'Floratam' St. Augustinegrass with G. intraradices. .................... 114

F-1. Analysis of variance tables for spore density and percent colonization data in
Chapter 2, and Pearson's product moment correlation coefficients for attempted
correlations between variables and soil chemical characteristics and soil
m oisture and soil tem perature. ....................................................... 120

F-2. Analysis of variance tables for Rhizoctonia solani percent colonization data in
Chapter 3. ............................... ........... 122

F-2. Analysis of variance tables for Gaeumannomyces graminis var. graminis percent
colonization data in Chapter 3 ......................................... ... ..... 126

F-4. Analysis of variance tables for the direct assay in the split-sprig challenge
including Gaeumannomyces graminis var. graminis and Rhizoctonia solani data
in Chapter 4. ......................................................129

F-4. Analysis of variance tables for the indirect assay in the split-sprig challenge
including Gaeumannomyces graminis var. graminis and Rhizoctonia solani data
in Chapter 4. ...................................................... 132














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

POPULATION AND IDENTIFICATION OF MYCORRHIZAL FUNGI IN ST.
AUGUSTINEGRASS IN FLORIDA AND THEIR EFFECT ON SOILBORNE
PATHOGENS

By

Whitney Colleen Elmore

August, 2006

Chair: James W. Kimbrough
Major Department: Plant Pathology

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of more than 90% of

all land plants. Mycorrhizae are documented in many crops as positive associations with

roots of plants that help reduce disease severity soilbome pathogens and increase nutrient

and water uptake while lowering plant stress and ultimately management costs.

However, there is no information concerning the effects of AMF colonization in St.

Augustinegrass.

In Florida, St. Augustinegrass sod production contributes hundreds of millions of

dollars to the economy annually while supplying a product to homeowners and

commercial entities with great aesthetic value. The use of AMF in St. Augustinegrass

sod production has many potential benefits to the sod industry and the environment

including lowered management costs, pesticide use and pollution. In these studies, a

survey of St. Augustinegrass sod farms in north central Florida revealed a moderate level

of AMF colonization as well as a diverse population of AMF species. Direct and indirect









pathogen challenges with the ubiquitous AMF, Glomus intraradices, in St.

Augustinegrass plants suggested a limited role for AMF in lowering disease severity in

two of the more devastating diseases of St. Augustinegrass in Florida, brown patch and

take-all root rot.

While no positive correlation was observed between AMF colonized St.

Augustinegrass plants and the soilborne pathogens Rhizoctonia solani or

Gaeumannomyces graminis var. graminis, effective assays for mycorrhizal St.

Augustinegrass evaluations were developed and foundation information concerning the

association between St. Augustinegrass and AMF provided valuable data, which may

help in the development of future AMF evaluations in St. Augustinegrass field trials and

with other AMF species. These results were the first to suggest an association between

AMF and St. Augustinegrass, and to evaluate their potential effects on disease severity.














CHAPTER 1
GENERAL INTRODUCTION

"Mykorrhizen" was a term first applied by the German forest pathologist, A.B.

Frank, who described structures in plant roots as "fungus-roots" (1885). Harley (1989)

described them as a mutualistic symbiosis in which a fungus and host exist as one.

Despite minuscule differences in description, mycorrhizas are recognized by scientists as

economically important in most agricultural crops. In fact, the mutually beneficial

relationships are actually three-way associations in which the soil, plant root, and fungus

interact to produce symbiotic effects.

In 1879, de Bary defined symbiosis as "the living together of differently named

organisms," which included both parasitic and beneficial relationships. Later, Raymer

(1927), commenting on the nature of symbionts, acknowledged such partnerships, but did

not provide functional information concerning the fungi involved. However, after many

years of advanced research throughout the 1960's and 70's, the meaning of the

relationship was refined to refer to naturally beneficial relationships exclusively. Most

likely, organisms co-existing became symbiotic as a result of selection pressures exerted

over the course of time (Remy et al., 1994). In fact, it is possible that the movement of

plants from water to land could not have occurred without mycorrhizal associations

(Nicolson, 1975; Pirozynski and Malloch, 1975). It is now recognized that mycorrhizas

are the norm and not the exception within the Kingdom Planta. With ancient lineages

stretching across evolutionary history, Bryophytes, Angiosperms, Pteridophytes, and

some Gymnosperms all possess these associations









(Fitter, 1991), while members of the Brassicaceae seem to evade infection by any type of

mycorrhizal fungi (Gerdemann, 1968), even in close proximity to mycorrhizal plants.

Involved in mycorrhizal symbiosis are members of the fungal taxa Ascomycotina,

Basidiomycotina, Zygomycotina, Deuteromycotina, and Glomeromycota (Schtissler et al.,

2001; Srivastava et al., 1996). Infrequently found living as saprobes, most of these fungi

are widespread across various soil types with strong biotrophic host dependence (Smith

and Read, 1997).

Mycorrhizal Types and Phylogeny

Types of mycorrhizae are divided based on their fungal associations, extent of

root penetration, presence or lack of an external mantle and/or sheath, as well as the intra-

and intercellular structures produced inside of the host root (Srivastava et al., 1996).

Presently, seven types of mycorrhizae are recognized by taxonomists (Bagyaraj, 1991).

The types of mycorrhizae include: Ectomycorrhizae, Ectendomycorrhizae, Arbutoid,

Monotropoid, Ericoid, Orchidoid, and Endomycorrhizae or the vesicular-arbuscular

mycorrhizae (Bagyaraj, 1991). Endomycorrhizae, also known as vesicular-arbuscular

mycorrhizae or VAM, were taxonomically placed within the Order Glomales of the

Phylum Zygomycota based on morphological features of asexual spores resembling

sexual reproductive structures of the Zygomycota. Six genera are recognized within the

Glomales: Glomus, Sclerocystis, Gigaspora, Scutellospora, Acaulospora, and

Entrophospora (Morton and Benny, 1990). In 2001, Schussler et al., using information

provided by small subunit rRNA gene sequences, proposed a new Phylum, to separate

arbuscular mycorrhizal fungi from other fungal groups in a monophyletic clade.

Schussler et al. (2001) suggested that they be removed from the Zygomycota and placed

into a newly erected Phylum Glomeromycota. Small subunit rRNA gene sequencing also









placed Geosiphon pyriformis, an endocytobiotic fungus, which is a distant relative of the

arbuscular mycorrhizal fungi, within this new Phylum (Schussler et al., 2001). Within

the same article, Schussler et al. (2001) also suggested that the Glomus genus be emended

to include the termination -eraceae, with the family named Glomeraceae and the higher

taxon names reflecting this change with Glomerales. Furthermore, Schussler et al.

(2001) suggested three new orders, mostly diverged from the Ascomycetes and

Basidiomycetes, be recognized as well. These are the Archaeosporales, Diversisporales,

and the Paraglomerales. Based on a combination of molecular, ecological, and

morphological characteristics, these fungi can now be separated from other fungal

groups. The use of molecular techniques such as small subunit rRNA sequencing has led

to the recent introduction of other species within the genus Glomus. Walker et al. (2004)

and Rani et al. (2004) also used this technology to add Glomus hyderabadensis from

India, and a new genus Gerdemannia, to the growing list of arbuscular mycorrhizal fungi

collected and speciated around the world. Based on their distinct molecular differences

from the Zygomycota and placement into a new phylum, Goto and Maia (2005) recently

suggested that spores of the arbuscular mycorrhizal fungi be referred to as

glomerospores. Indeed, these spores are not chlamydospores, conidia, or azygospores, so

differentiation based on molecularly distinct features is pertinent.

Forming vesicles and arbuscules within cortical root cells, fungi of the

Glomeromycota produce aseptate hyphae without the presence of a sheath or mantle.

Gigaspora and Scutellospora produce arbuscules only within roots and vesicles only

within the soil, and, therefore, the vesicular-arbuscular mycorrhizal term has been

emended to simply read as arbuscular mycorrhizae. The name was amended simply









because arbuscules are the most basic and one of the few commonalities between the

members of the group (Morton and Benny, 1990). Taylor et al. (1995) proposed that

Glomites be included as a new fossil genus of Glomales, and two years later, Wu and Lin

(1997) added another genus, Jimtrappea. However, these two genera are not widely

accepted. Currently, there are about 150 recognized species described within the

Glomales, of which only a few have been carefully studied and recognized as endo-

mycorrhizal (Morton and Bentivenga, 1994; Morton and Benny, 1990; Morton et al.,

1992; Pirozynski and Dalpe, 1989; and Stuessy, 1992). Glomeromycota are not known to

produce sexual reproductive spores and, therefore, are characterized and classified by

their resting structures. These structures vary in wall characteristics, size, shape, and

color (Morton et al., 1992; Morton and Bentivenga, 1994; and Morton and Benny, 1990).

Arbuscular Mycorrhiza Physiology

The most widespread of the mycorrhizae, both geographically and among species,

the arbuscular mycorrhizae occur frequently in the top 15-30 cm of cultivated soil

(Bagyaraj, 1991). Arbuscular mycorrhizae-forming fungi colonize and form associations

with most agriculturally and horticulturally important plant species, from fruit and forest

trees to shrubs and grasses. Unlike other mycorrhizae, these associations do not typically

lead to noticeable external morphological changes in plant roots, and they cannot be

observed easily without staining procedures (Phillips and Hayman, 1970). In most cases,

plants which have formed associations with other types of mycorrhizal fungi, such as

basidiomycetes and ascomycetes, do not form relationships with arbuscular mycorrhizae.

From the standpoint of the fungus, host specificity exists while the opposite view would

be held about the host due to the wide host range of most of the arbuscular mycorrhizal

fungi (Gerdemann, 1955). Their limited capacity to be grown from spores, vesicles, or









hyphae from root residue has led to special methodologies in order to maintain strains

and for taxonomic evaluation. Typically, single spore types are cultivated in "pot

cultures" on plant roots so that characteristics of spores, their mode of colonization, and

effects on plant growth can be studied (Smith and Read, 1997).

Arbuscular Morphology

Named by Gallaud (1905) for the structures formed inside cortical root cells,

arbuscules are similar to branched haustoria, which form early on in the association

between plant root and the repeatingly branched fungal hyphae. Baylis (1975) and St.

John (1980) suggested that the form of the root system is a defining factor in the extent to

how plants react, nutritionally, and in growth to mycorrhizal colonization. Evolving

across phylogenetic lines many times, it appears that dicotyledons have a large incidence

of associations with fungal species which form mycorrhizal associations, with very few

being non-mycorrhizal in nature (Trappe, 1987). In comparison, the lines of

monocotyledons studied by Cronquist (1981) are heavily mycorrhizal, with arbuscular

mycorrhizas predominating except in the Orchidaceae, which have mycorrhizas formed

by Basidiomycetes. In plants forming primarily magnolioid type roots, with wide

diameters up to 1.5 mm, slow growth habits, and little root-hair development,

mycorrhizas are usually well accepted and form greatly receptive relationships. On the

other hand, roots that are primarily fine and rapidly growing with long root-hairs lack the

same responsiveness (Baylis, 1975; St. John, 1980). Mycorrhizal relationships were first

described by the type of colonization patterns, referred to as either Arum- or Paris-type

(Gallaud, 1904). In fact, there appears to be a continuum between the two forms, with

intermediate types along the way.









The Arum-type, which was considered the most common association, develops

primarily within cultivated crops and consists of intercellular hyphae and arbuscules. In

contrast, the Paris-type of symbiosis involving intercellular hyphae, arbusculate coils,

and hyphal coils, typically develops within forest trees and herbs (Dickson, 2004). In

surveys of mycorrhizal plants and trees from both natural and cultivated environments, it

appears that most plant families are dominated by only one symbiotic type (Smith and

Smith, 1997). There are, however, a few plant families that appear to possess

intermediate forms of the colonization types, including the Poaceae (Smith and Smith,

1997). In an extensive survey of various plant families and mycorrhizal fungi, eight

distinct classes of colonization types were found along a continuum ranging from the

Paris- to Arum-type (Dickson, 2004). Most researchers agree that one fungus can form

either type of arbuscular colonization with most of the specificity in structure dependent

upon the host plant (Barrett, 1958; Gerdemann, 1965). Brundett and Kendrick (1988)

commented on the presence of intercellular spaces within the host root cortex as being the

main factor influencing arbuscular type. Conversely, in tomato, Cavagnaro et al. (2001)

suggested that the colonization type was dependent on both the host and fungus involved.

Mycorrhizal Colonization

In mycorrhizal colonization, the host plasmalemma is invaginated with the

encroaching arbuscules. These are physiologically active sites for nutrient translocation,

for 4-6 days, within the roots (Bracker and Littlefield, 1973; Brundett et al., 1984).

Arbuscules are important sites for P exchange for plants under deficient conditions

(Simth and Read, 1997). The vesicles, which are small and usually dark, globular or

spherical structures, form later in the association and arise from swelling of terminal and

intercalary hyphal cells. Vesicles act as storage sites for lipids (Srivastava et al., 1997).









Transversing long distances of soil beyond nutrient depletion zones and reaching areas

untouched by growth limited root hairs, the external hyphae absorb nutrients such as P

and make it available to plants, rendering these plants more equipped to survive nutrient

competitions (Nicolson, 1967). Once the fungal hyphae and plant roots become closely

associated in space, a functionally and structurally complex symbiotic relationship is

formed between the compatible organisms.

Formed only on unsuberized root tissue, certain areas of the root are more readily

colonized even though mycorrhizae can develop on any portion of young root tissue

(Brundett and Kendrick, 1990). Based on mathematical and geometrical models, root

tissue directly behind the meristematic area is considerably more susceptible to

penetration and colonization when compared to other root segments (Garriock et al.,

1989; Bonfante-Fasolo et al., 1990). This area of discrete colonization was described

earlier as the mycorrhizal infection zone by Marks and Foster (1973), who considered the

area to be "non-static," thus growing with the root. Furthermore, Brundett and Kendrick

(1990) found that the fungus penetrates and colonizes root cells with little or no suberin

deposition, which has been shown to occur just prior to or after fungal penetration.

Usually, epidermal and outermost cortical cell colonization is minimal with the

intercellular hyphae formed in the inner cortex and the majority of the colonization is

deep within the cortex where arbuscules are formed (Srivastava et al., 1997).

With the aid of cellulolytic and pectinolytic enzymes produced by the fungus,

direct penetration of the outermost cell wall is the preferred mode of hyphal entry (Jarvis

et al., 1988). Physiochemical aspects of the epidermal cell wall seem to be the primary

reasons for preferential site penetration (Jarvis et al., 1988). After cell to cell contact









between fungus and host, the external mycelia swell to form defined appresoria (20-40

lm in length). Within these appresoria, infection hyphae are formed and penetrate host

cell walls (Garriock et al., 1989). Once penetration has occurred via mechanical and

enzymatic interactions, the host's plasmalemma appears to extend around the fungus

(Bracker and Littlefield, 1973). Arbuscule formation takes between 4-5 days after which

extramatrical hyphae occurs promoting new penetration sites (Brundett et al., 1984).

Arbuscules are major contributors to the transfer of nutrients, in particular sugars,

between the plant to fungus and inorganic materials, mainly P, from the fungus to the

plant (Smith and Gianinazzi-Pearson, 1988).

Mycorrhizal Rhizosphere Interactions

A necessary component of plant life, the macro element P, occurs as part of DNA

and RNA nuclei and as part of plant membranes as phospholipids (Griffiths and

Caldwell, 1992; Smith and Read, 1997). Present in high amounts within active

meristematic regions as part of nuclear proteins and as part of ADP, ATP, NADP, and

NAD, P is partly responsible for oxidation-reduction reactions such as respiration,

nitrogen and fat metabolism, and photosynthesis, which are necessary for life (Beever

and Burns, 1980; Munns and Mosse, 1980). Symptoms of deficiency often include

purple or red leaf pigmentation, dead and/or necrotic leaves, petioles, and fruits,

premature leaf drop, stunting, and poor vascular tissue development (Srivastava et al.,

1997). An important aspect of arbuscular mycorrhizal associations is the increase in P

uptake by the plant.

The importance of arbuscular mycorrhizal fungi for P absorption was first

suggested by Baylis (1959) and then Gerdemann (1964). Later, Baylis (1967), Daft and

Nicolson (1966), Holevas (1966), and Murdoch et al. (1967) provided advanced









information showing the close association between mycorrhizas and P nutrition of the

host. Interestingly, Mosse (1973) once remarked that more than one quarter of

mycorrhizal text is devoted to P research. In fact, Sanders and Tinker (1973) stated that

"the value of these mycorrhizas for the phosphate nutrition of plants in deficient

environments may rival that of Rhizobium in nitrogen." Obviously, such a strong

statement must be supported by an abundance of research. As mycorrhizal research

progressed during the last three decades, P research remained an important topic. For

instance, in 1986, Gianinazzi-Pearson and Gianinazzi studied the kinetic associations

between P concentration in soil solutions and its effect on root and shoot tissues, while

Young et al. (1986) evaluated the effect of arbuscular mycorrhizal fungi inoculation on

soybean yield and P utilization in tropical soils. Later, Koide (1991) determined that it is

the variation among plant species in phenological, morphological, and physiological traits

that influence P demand and supply which are directly connected to potential response of

mycorrhizal associations. Once absorbed, P is allocated for plant functions or stored for

later use (Cox and Sanders, 1974). Since P deficiency is caused by both P availability

and plant demand, mycorrhizal associations can have various effects based on the plant

species (Koide, 1991).

In low P soils, mycorrhizal plants have an advantage over non-mycorrhizal plants

with root to shoot ratios lowered and shoot fresh weight to dry weight ratios higher in

mycorrhizal plants (Tinker, 1978). The plant's growth rate is influenced by interactions

in mycorrhizal colonization such as nutritional, and non-nutritional, physiological effects,

such as pH, temperature, microbial turnover, phosphatase activity, soil and plant

moisture, and/or iron (Fe) or aluminum (Al) chelate concentration (Nye and Tinker,









1977; Rusell, 1973). In P deficient soils, studies have shown that plant species with few

root hairs are strongly mycorrhizal, providing evidence that root anatomy has a strong

correlation to mycorrhizal colonization (Crush, 1974; Baylis, 1975).

Smith and Read (1997) wrote "the focus (of current research) is on P uptake, as

well as on the uptake of other nutrients for which there is now unequivocal evidence of

mycorrhizal involvement." Furthermore, they noted that "there is excellent evidence to

demonstrate that external hyphae of VA mycorrhizal fungi absorb non-mobile nutrients

(P, Zn, Cu) from soil and translocate them rapidly to the plants, thus overcoming

problems of depletion in the rhizosphere which arise as a consequence of uptake by

roots." Throughout the 1960's, reviews of the occurrence of arbuscular mycorrhizal

colonized plants and anatomy were the norm in mycorrhizal research (Smith and Read,

1997). There had been little mention of mineral nutrition until Mosse (1957) released

details of an experiment with apple seedlings which provided evidence for increased

amounts of potassium (K), iron (Fe), and (copper) Cu in mycorrhizal plant tissue versus

noninoculated control plants. Other researchers such as Gerdemann (1964) established

that P tissue concentrations were also higher in mycorrhizal plants, although the

mechanisms were not yet clearly understood. Mosse (1973) reported a shift in

mycorrhizal research from pot experiments to study the anatomy of arbuscular

mycorrhizal fungi to that of plant growth and P uptake. Now, the mechanisms underlying

the mycorrhizal effect on P uptake are coming to light including extraradical hyphae

growing into soil not already colonized by roots; hyphae that are more effective than

roots, due to size and spatial distribution, in competing with free-living microorganisms

or mineralized or solubilized P; the kinetics of P uptake into hyphae may differ from









roots; and that mycorrhizal roots can use sources of P in soil that are not plant available

(Smith and Read, 1997).

Hyphal pathways between plants may offer links for soil-derived nutrient transfer,

as is the case with plant-derived carbon (C), which can have important roles in the inter

plant and species competition in the environment (Smith and Read, 1997). Enzymes are

not the only substances produced by arbuscular mycorrhizal fungi. An Iron-containing

glycoproteinaceous substance called glomalin, produced by these fungi, is deposited in

soils (Rilling et al., 2003). Glomalin is considered to be linked to soil Carbon storage due

to its effect on soil aggregation (Rilling et al., 2003). Consistently correlated with soil

aggregate water stability, glomalin is involved in C and N content as well as being useful

as a potential land-use change indicator (Rilling et al., 2003). After many years of

taxonomic research with proteins and soil stability, micronutrient uptake research has

increased following studies by Mosse (1957), Daft et al. (1975), and Gildon and Tinker

(1983) where uptake of Cu and zinc (Zn) were observed in apples and maize when

inoculated with arbuscular mycorrhizal fungi. The uptake of other micronutrients is not

well documented, however, Marschner and Dell (1994) observed that the uptake of

manganese (Mn) is usually reduced by mycorrhizal associations. Occasionally, instances

of increased K concentrations in plant tissues have been reported, which is to be expected

given the immobility of the K ion within the soil matrix (Srivastava et al., 1997).

Conversely, with increased P uptake as well as other nutrients in mycorrhizal plants

comes the risk of accumulating toxic elemental levels. With improved P nutrition and

plant growth, the uptake of heavy metals per plant is greatly increased as demonstrated









by El-Kherbawy et al. (1989) on alfalfa inoculated with arbuscular mycorrhizae in

various soil pH levels with and without rhizobia.

Effects of Abiotic Factors on Mycorrhiza

Many climatic and physiochemical or abiotic features of the soil influence

arbuscular mycorrhizal establishment, growth and benefit. For instance, light, which is

not directly required by mycorrhizas in some cases, is essential for the host to thrive and

translocate photosynthates to the root, which in turn provides a home for mycorrhizal

fungi. In other cases, arbuscular mycorrhizal fungi are stimulated by light to increase

root colonization and spore production as well as plant response to mycorrhizal

colonization (Furlan and Fortin, 1973; Hayman, 1974).

The rate of photosynthesis and translocation of its products are heavily influenced

by air temperature (Furlan and Fortin, 1973; Hayman, 1974). By increasing air

temperature to 260 C an increase in plant growth is typical (Hayman, 1974). Soil

temperatures also influence mycorrhizal development at all stages: spore germination,

hyphal penetration, and proliferation within cortical root cells (Schenck and Schroder,

1974; Smith and Be, 1979). Optimal temperatures vary for spore germination between

species and other stages in development. The ability of the arbuscular mycorrhizal spores

to survive following host death or harvest is also dependent on soil temperature, though

also affected by soil texture (Bowen, 1980).

Soil pH is an additional determinant factor in mycorrhizal growth and

development. The efficiency of the mycorrhizae is directly determined by its ability to

adapt to soil pH. Soil pH affects both spore germination and hyphal development (Angle

and Heckman, 1986; Green et al., 1976). The interaction of soil pH and mycorrhizal

development is difficult with soil type, plant and fungal species and P forms involved.









Typically, mycorrhizas are able to colonize and grow well in soils of pH 5.6 to 7.0, but

not in soils of pH 3.3 to 4.4, as reported by Hayman and Mosse (1971).

Generally, mycorrhizas are not found within aquatic conditions, due to a

reduction in colonization, however, some aquatic plants are commonly mycorrhizal, such

as Lobelia dortmanna L. and Eichhornia crassipes [Martius] Solms (Read et al., 1976).

Conversely, most plants found within drought are typically mycorrhizal, which aids in

their survival in harsh conditions (Sondergaard et al., 1977). Arbuscular mycorrhizal

colonization of roots affects many mechanisms in plant water determination. Root

hydraulic conductivity, leaf gas exchange and expansion, phytohormone regulation, and

leaf conductants are all affected by interactions with arbuscular mycorrhizas (Gogala,

1991; Hardie and Leyton, 1981; Koide, 1985; Nelson, 1987; Auge et al., 1986). Fungal

mycelium is involved in the transport of water especially at low soil potentials, which has

made arbuscular mycorrhizae colonization and development a hot research topic in arid

and tropical landscapes (Faber et al., 1991).

Mycorrhizal roots and organic matter content play important roles in arbuscular

mycorrhizal survival and development as well. Organic root debris may act as a reserve

for soil inocula (Warner and Mosse, 1980), while in arid areas contact between

susceptible plant roots and colonized root residue is considered by Rivas et al. (1990) to

be the most important means for mycorrhizal dissemination when little water is available

for spore transport. Soil structure, pH, water, and nutrient availability are all affected by

organic matter content, thus influencing mycorrhizal associations (Khan, 1974; Daniels

and Trappe, 1980; Johnston, 1949). For instance, Johnston (1949) suggested that organic

materials such as manures can enhance tropical soil mycorrhizas in cotton stands. And,









Sheikh et al. (1975) reported that spore population and organic matter content were

positively correlated in soils with 1-2% organic matter, but low in soils with 0.5%

organic matter or less. Organic matter and root residue are important ecologically as part

of the three-way soil, plant and fungal mycorrhizal relationship.

Effects of Seasonality on Mycorrhiza

Seasonality is another abiotic contributor to arbuscular mycorrhizal colonization.

Seasonality has been shown to affect spore production as a function of host and climate

(Hetrick, 1984), while seasonal patterns can be correlated with P availability and soil

water potential in combination with host growth stages, other biotic and abiotic factors,

and management practices such as fertilization (Cade-Menun et al., 1991; Yocums,

1985). Hayman (1975) demonstrated that fertilizers such as P and Nitrogen (N) could

potentially reduce spore number and fungal colonization with N having a more

detrimental effect than P. Despite the possibility for soil chemical treatment injury,

arbuscular mycorrhizae can be found in fertile soils, which Hayman et al. (1976)

contributed to other factors such as host species, soil type, and management practices

influencing fungal survival and development.

As previously mentioned, management practices such as pesticide applications, in

particular, fungicides, may inhibit the effect of arbuscular mycorrhizal fungal sporulation

and colonization (Nemec and O'Bannon, 1979; El-Giahmi et al., 1976). Rhodes and

Larsen (1979) examined arbuscular mycorrhizae of turfgrasses in field and greenhouse

conditions. The researchers discovered that when fungicides were applied to bentgrass,

infection averaged 9 to 17%, however, in non-treated field plots, the roots were infected

at a rate of 40-60 percent. The same observation was reported in the greenhouse

evaluations, with one fungicide, PCNB, totally eliminating mycorrhizae (Rhodes and









Larsen, 1979). Conversely, DBCP, a nematicide, has actually been reported by Bird et al.

(1974) to enhance arbuscular mycorrhizal development.

It is imperative to mention that mycorrhizal interactions lie along a continuum

from mutualistic to parasitic based on the cost to benefit ratio colonization. Obviously,

mycorrhizal associations can be mutualistic, but they can also be parasitic, commensal,

amensal, and even neutral in nature (Johnson et al., 1997). Where, along this continuum

the association will fall, depends on a complex hierarchy mediated by biotic and abiotic

factors within the rhizosphere and ecosystem being affected. No doubt, this range of

mycorrhizal associations is greatly affected by time and space. The complexity of

mycorrhizal investigations is ultimately confounded by the fact that the plant and fungal

perspective on costs to benefits differs greatly from situation to situation (Johnson et al.,

1997).

With this in mind, Ryan and Graham (2002) presented the point-of-view that

arbuscular mycorrhizal fungi do not play such a vital role in production agricultural

systems, in relation to nutrition and growth, simply because the high cost of energy from

the plant to support the fungal invader outweighs the benefits of that association. This

outcome is not beneficial in terms of crop production and may, in fact, be detrimental.

Nonetheless, those production systems not considered to be within a natural or traditional

cultivated production system, such as sod, still need much attention where mycorrhizal

symbiosis is concerned before a definitive yes or no can be applied to functional use of

mycorrhizal fungi. Conversely, in 1997, Srivastava et al. concluded that "there is little

doubt that vesicular arbuscular mycorrhizae fungi will emerge as a potential tool for

improving crop plants in the years to come." These opinions, in conjunction with the









increased concern for environmental quality and sustainable technologies warrants an

examination of more specific research reports in agricultural crops. In this review, the

concentration is on turfgrass research.

Mycorrhizas in Grasses

There has been a considerable amount of research on mycorrhizal fungi

associated with grasses (Hetrick et al., 1988, 1991; Trappe, 1981; Bethlenfalvay et al.,

1984). Though much of the work conducted on grasses was begun in the 1970's,

Nicolson (1955) examined mycotrophic nature in grasses and later (Nicolson, 1956) with

mycorrhizae in both grasses and cereals. These first studies in grasses and cereals were

mainly concentrated on the ecological aspects of mycorrhizal infection. In fact, it was

not until Nicolson (1956) showed diagrammatically that external hypha penetrate the root

hairs or epidermal cells and spread throughout the cortex of grasses. Additionally,

Nicolson noted that arbuscules form later in the inner cortical layers, which was valuable

information in the study of grasses and their mycorrhizal partners.

In experiments on fescue (Festuca ovina L.), cocksfoot (Dactylis glomerata L.),

sand fescue (Festuca rubra var. arenaria L.), and marram grass (Ammophila arenaria L.:

Link), Nicolson (1956) found that mycorrhizal infection was prevalent throughout a wide

range of different habitats and soil types, although the incidence of infection varied

greatly between habitats and communities. With a lull in ecological studies throughout

the 1960's, environmental issues surpassed many of the more basic research topics. In

1979, Rhodes and Larsen examined the effects of fungicides on mycorrhizal development

in cool-season turfgrasses. Again, Rhodes and Larsen (1981) conducted a similar study,

where the effects of fungicides on bentgrasses and the mycorrhizal fungus, Glomus

fasciculatus, were explored. Arbuscular mycorrhizas of 'Penncross' creeping bentgrass









(Agrostispalustris Huds.) were studied in greenhouse experiments to evaluate popular

fungicides, such as, chloroneb and maneb, which did not affect mycorrhizal development.

However, foliar applications of PCNB, chlorothanil, bayleton, anilazine, benomyl, and

chloroneb at various weeks after inoculation with Glomusfasciculatus resulted in

significantly reduced mycorrhizal colonization, thus limiting their beneficial effects.

Later, studies of mycorrhizas in turfgrasses seemed to swing back toward

ecological studies with the introduction of seasonal and edaphic variation of arbuscular

mycorrhizal infection (Rabatin, 1979). In a population survey, Rabatin (1979) sampled

for Glomus tenuis infection in Panicum virgatum L., Poa compressa L., Poapratensis L.,

Poapalustris L., Phleum pratense L., and Festuca etalior L., all cool-season meadow

grasses. Rabatin (1979) determined that the greatest percentage of root infection by this

fungus occurred in grass roots from dry, P deficient fields. Moreover, the percent of

infection was lowest in the cool, wet months of the spring. Thus, Rabatin (1979)

concluded that mycorrhizal infection tends to be greater in drier, P deficient soils versus

wet or flooded conditions.

Bagyaraj et al. (1980) concluded that a study of the spread of mycorrhizas from

the site of infection along the root to deeper soil layers was necessary to provide

important information for plant inoculations. This was done in grasses since the roots

grow out of the inoculated sites quickly. Researchers collected root samples from various

depths and found that roots at 3 4 and 8 9 cm were mycorrhizal at 45 days after

inoculation. However, when roots were collected from deeper layers, the roots were only

mycorrhizal after 75 days. The research lead Bagyaraj et al. (1980) to conclude that

mycorrhizal infection of warm-season grasses such as Sudangrass (Sorghum bicolor L.:









Moench), was spread to deeper layers by mycelial growth through the root, which was

helpful information when researching inoculation methodologies important in such

experiments as population surveys where pot cultures are a necessary to speciate the

fungi collected. In an attempt to determine the distribution and occurrence of

mycorrhizal fungi in Florida's agricultural crops, Schenck and Smith (1981) examined

bahiagrass (Paspalum notatum Fluigge) and digitgrass (Digitaria decumbens Stent)

among 30 Cucurbitaceae, Leguminosae, Solanaceae, and Vitaceae crops. In a population

survey, the authors found that mycorrhizal fungi in Glomus occurred most frequently in

Florida, with species of Gigaspora found regularly in central and south Florida and

Entrophospora collected only once (Schenck and Smith, 1981). Furthermore,

Acaulospora was found in the highest frequency in the grasses evaluated. In this

instance, there was no correlation among species or genera occurrence and the available

soil P or soil pH.

In another study, endomycorrhizas and bacterial populations were examined in

three cool-season grasses. Agrostis tenuis Sibth., Deschampsiaflexuosa L.: Trin., and

Festuca ovina L., were collected and examined by Lawley et al., (1982) for mycorrhizal

associations. In this case, the researchers noticed that mycorrhizal abundance was lowest

when Agrostis species were partnered with other plants and highest when partnered with

Festuca.

Finally, Sylvia and Burks (1988) began working with grasses other than those

only found in cool-season climates. Beach erosion in coastal areas became a major

economic concern in the late 1980's; beach grasses such as sea oats (Uniola paniculata

L.) were often utilized to restore southeastern beaches to slow loss of sand. It was









unclear whether or not these grasses relied on arbuscular mycorrhizal associations for

survival in the harsh climate. Sylvia and Burks (1988) found that isolates of Glomus

deserticola and G. etunicatum significantly increased the dry mass, height, and P content

of the sea oats, while other isolates had little or no effect.

In the search for a better host for inoculum production, compared to the traditional

bahiagrass, Sreenivasa and Bagyaraj (1988) evaluated seven grasses for their ability to

quickly produce large masses of mycorrhizal spores for inoculations. Grasses such as

guinea grass (Panicum maximum Jacq.) and rhodes grass (Chloris gayana Kunth) were

studied and all were found to be mycorrhizal. However, the highest root colonization

was observed in the rhodes grass, as well as the highest production of spores and

infective propagules. Studies on other warm-season grasses such as St. Augustinegrass

(Stenotaphrum secundatum [Walt.] Kunze), Centipedegrass (Eremochloa ophiuroides

[Munro] Hack.), or even bermudagrass (Cynodon dactylis L.: Pers.) have not been

identified.

In studies of the difference in responses of C3 and C4 grasses to P fertility and

mycorrhizal symbiosis, Hetrick et al. (1990) showed that warm-season grasses such as

big bluestem (Andropogon geradii Vitm.) and indian grass (Sorghastrum nutans L.:

Nash), responded positively to mycorrhizae or P fertilization, or mycorrhization in cool-

season grasses, such as perennial ryegrass (Lolium perenne L.). In warm-season grasses,

there was a positive relationship between root colonization and dry weight, with an

inverse relationship between mycorrhizal root colonization and P fertilization. The

evaluation provided evidence that the C3 and C4 grasses display profoundly different

nutrient acquisition strategies (Hetrick et al., 1990b).









The effect of mycorrhizal symbiosis on regrowth of rhizomes of big bluestem was

assessed as a function of clipping tolerance (Hetrick et al., 1990a). Mycorrhizal clipped

plants were larger than nonmycorrhizal clipped plants, but the effect diminished with

successive clippings as did mycorrhizal root colonization. This information on clipping

tolerance indicates that mycorrhizal turfgrasses respond similarly when clipped or mowed

under constant turf management.

Hetrick et al. (1991) compared the root architecture of five warm and five cool-

season grasses in an attempt to evaluate whether mycorrhizal symbiosis confers a greater

tolerance to drought, soilborne disease, vigor, and yield through direct or indirect

improved nutritional status of the host plant. The cool-season grasses had significantly

more primary and secondary roots than the warm-season grasses and the diameter of

those roots was smaller than that of the warm-season grasses. The mycorrhizas did not

affect the number or diameter of cool-season grass roots, however, the warm-season

grasses did respond to mycorrhizal inoculation. Additionally, the root length was

significantly increased in the warm-season grasses with mycorrhizal infection when

compared to the cool-season grasses. Through the aid of topological analysis of root

architecture, mycorrhizal symbiosis was shown to inhibit root branching in warm-season

grasses, but had no effect on cool-season grass rooting (Hetrick et al., 1991). The

researchers concluded that mycorrhizal-dependent warm-season grasses have unique root

architecture, allowing energy to be conserved for root development, while the less

dependent cool-season grasses do not exhibit the same benefits of mycorrhizal infection.

In studies designed to determine the dependence of warm-season grasses on

arbuscular mycorrhizae and relationships between mycorrhizae and P availability and









plant density, Brejda et al. (1993) and Hetrick et al. (1994) evaluated sand bluestem

(Andropogon geradii var. paucipilus Nash), switchgrass (Panicum virgatum L.), and

Canada wild rye (Elymus canadensis L.).

The popular cool-season grasses, creeping bentgrass (Agrostis stolonifera L.) and

Kentucky bluegrass (Poa pratensis L.) were evaluated in relation to the impact of

arbuscular mycorrhizae and P status on plant growth (Charest et al., 1997). The authors

revealed that as mycorrhizal infection increased in the grasses, root colonization

increased to more than 40% with lowered P fertilization. This information could be

particularity helpful in warm-season grasses where P may have a major impact in soils,

such as those found throughout Florida. The researchers of this study concluded that

arbuscular mycorrhizal symbiosis could be considered as a potential fertilizer reduction

agent (Charest et al., 1997).

More recently, mycorrhizal symbiosis and fertilizer relationships have dominated

arbuscular mycorrhizal research; however, the majority of this work has concerned cool

and warm-season prairie grasses. The emphasis of molecular technologies has resulted in

less applied types of research being performed with grasses and mycorrhizas. Using

terminal restriction fragment length polymorphism (T-RFLP), Vandenkoornhuyse et al.

(2003) assessed the diversity of arbuscular mycorrhizal fungi in various cool-season

grasses, which co-occurred in the same research plots. Based on a clone library, the level

of diversity was consistent with past studies; showing that mycorrhizae fungal host-plant

preference exists, even between grass species.

Obviously, there is limited information on warm-season turfgrasses when

compared to the warm-season prairie and cool-season meadow grasses. In the Southeast,









warm-season turfgrasses are highly valued for their drought resistance, aesthetic

importance and generally low maintenance on some home lawns, golf courses, soccer,

and football fields. Species such as bermuda, St. Augustinegrass, seashore paspalum

(Paspalum vaginatum Swartz), zoysia (Zoysia sp.) bahia, and centipede are used in

landscapes throughout Florida. St. Augustinegrass is dominant residential species in

Florida (Trenholm, 2004). Haydu et al., (2002) estimated that 36% of the total lawn

acreage in Florida, or 1.5 million acres, was comprised of St. Augustinegrass in 1996.

Valued for its shade tolerance, ability to adapt to various soils, and color, St.

Augustinegrass cultivars such as 'Floratam', 'bitterblue', 'Raleigh', and 'Floratine'

became popular with home owners. Chinch bug resistant 'Floratam' quickly became the

number one cultivar upon its release in the 1970's. St. Augustinegrass is a desirable

species home lawn, however problems with disease susceptibility can be devastating.

Two examples are brown patch (Rhizoctonia solani Ktuhn) and take-all root rot

(Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. graminis).

To date, research evaluating the potential benefit of mycorrhizae in St.

Augustinegrass has been neglected such as reduced fertilizer use and production cost.

The method of production of St. Augustinegrass may result in limited benefits of

mycorrhizal research. St. Augustinegrass is produced vegetatively as sod throughout the

southeast. Once or twice a year, the sod is harvested leaving "ribbons" or strips of grass

behind. These ribbons are responsible for re-growth, through stolons, of the sod field.

Harvesting cycles would make lengthy mycorrhizal studies difficult. An extensive

survey of this plant system in relation to the arbuscular mycorrhizal fungi is warranted.









The overall objective of this research is to investigate the impact of mycorrhizal

fungi on warm-season turfgrasses in Florida. A survey of the population and

identification of arbuscular mycorrhizal fungi associated with St. Augustinegrass roots in

Florida sod is provided in Chapter II. In Chapter III, a survey of root pathogens is

explored in relation to arbuscular mycorrhizal colonization in sod production fields.

Chapter IV includes studies designed to determine whether or not arbuscular mycorrhizal

fungi affect root disease caused by pathogenic isolates of R. solani and G. graminis var.

graminis, and if potential affects are direct fungal interactions or indirect systemically

acquired mechanisms of resistance. In Chapter V, a general summary and conclusions

concerning arbuscular mycorrhizal fungi in St. Augustinegrass in Florida are provided.














CHAPTER 2
POPULATION AND IDENTIFICATION OF ARBUSCULAR MYCORRHIZAL
FUNGI IN ST. AUGUSTINEGRASS

There is no information regarding arbuscular mycorrhizal fungi (AMF) in the

popular warm-season St. Augustinegrass (Stenotaphrum secundatum). In Florida, St.

Augustinegrass sod is a valuable commodity in home lawns and commercial landscapes.

'Floratam' the most common and widely adaptable cultivar is extensively used across the

state. It is also the primary cultivar grown in Florida for sod. In north central Florida,

sod production is increasing and growers are eager to increase production and lower

pesticide and fertilizer inputs. No information exists about mycorrhizas in this species.

The information is potentially useful in sod management to reduce disease severity,

chemical usage, and other production costs. In most cases, AMF populations are

decreased by agricultural practices are associated with conventional farming. St.

Augustinegrass sod production is unique in that it is not a traditional or natural plant

system. Currently, no information is available to growers to make informed decision

about inoculation with these fungi. The feasibility of inoculation studies for nutrient

acquisition, pesticide, and disease management can be performed using mycorrhizal fungi

more efficiently in the future once St. Augustinegrass is determined to be mycorrhizal.

Of current interest to mycorrhizal researchers is the ecology of mycorrhizal

populations and their benefit to both organic and more conventional cropping systems.

Information from less natural and conventional systems like St. Augustinegrass









sod is timely and could shed light on a little known ty cropping method. Mycorrhizal

systems and those interactions within it are complex and require extensive evaluation,

especially in crops not yet known to possess such associations. This evaluation may

supply valuable answers about mycorrhizal ecology. The objective of this study is to

determine if AMF colonize St. Augustinegrass, to what extent, and to identify the

colonizing fungi.

Materials and Methods

Sampling.|| 'Floratam' St. Augustinegrass plant roots and associated soil were

collected monthly from three sod farms in three counties (Marion, Bradford, and Union)

in north central Florida from December 2004 through December 2005 with the exception

of July. Each of the sod farms had been cropped with 'Floratam' St. Augustinegrass for

12 years or more (Fig. 2-1 A-C).

Ten subsamples of soil were taken from three (3 m2) plots per sod farm with a

1.27 cm diameter soil probe to a depth of approximately 15 cm as suggested by Brundrett

et al. (1995). Root samples from each plot were extracted with a small hand trowel.

Subsamples of roots and soil from each plot were pooled, resulting in three separate

composite plot samples per location. Root samples were placed into plastic ziplock bags

separate from soil samples and stored at room temperature for approximately 1 d prior to

spore extraction and root manipulation for mycorrhizal evaluation. Approximately 200 g

of field soil from each plot were combined with 200 g of a low P, low organic matter soil

mined from the UF/IFAS Plant Science Research and Education Unit in Citra, Florida.

This soil was then potted into 10 cm clay pots sown with sorghum-sudangrass hybrid

seed (Sorghum bicolor [L.] Moench x Sorghum sudanense) cv. Summergrazer III. Low P









soil was used in pot cultures to enhance sporulation of potentially cryptic species in order

to facilitate their recovery and identification (Fig. 2-2).

The cultures were incubated for 60 d at 20-25 C with 12 h artificial light

(day/night). The seed was surface-sterilized using a 10% sodium hypochlorite and

deionized water solution for 30 sec and rinsed for 1 min with sterile deionized water prior

to planting. The pot cultures received a Peter's 20-0-20 (Spectrum Group, St. Louis,

MO) nutrient solution, devoid of P, every two weeks. Approximately 90 d later, single

spores from the field soil pot cultures were selected from spore extracts (Fig. 2-3). This

process was accomplished by wet sieving, decanting (Gerdemann and Nicolson, 1963),

and 40% sucrose (v/v) centrifugation (Jenkins, 1964). These spores were used to

inoculate sterile, low P soil (Citra, Florida) and sorghum-sudangrass hybrid seed for

spore production and subsequent identification of the sporulating AMF as suggested by

Gerdemann and Trappe (1974). The soil was sterilized twice for 90 min at 121 C at 15

psi for two consecutive days. Samples of field soil were also submitted to the IFAS

Extension Soil Testing Laboratory in Gainesville, Florida on a tri-monthly basis for soil

nutrient composition and pH testing. Soil pH, from all three fields, ranged from 5.6 to

7.0 during the 12 month sampling period. Phosphorous levels ranged from 5 to 119 ppm.

Root preparation. 11 Young, healthy-appearing fibrous roots were rinsed in tap water

and separated with a scalpel from the plant crown and/or seminal roots. Selected roots

were cut into 1-2 cm long segments and cleared of cell and wall components in 10%

KOH (w/v) under pressure in an autoclave for approximately 20 min (Brundrett et al.,

1996). The root segments were cooled, then rinsed in tap water, and placed into hot

0.05% trypan blue with glycerol overnight to stain mycorrhizal structures (Bevege, 1968;









Phillips and Hayman, 1970; Kormanik and McGraw, 1982). Excess stain was rinsed

from the root segments with tap water and then mounted in water on glass slides to view

vesicles and arbuscules. Slight pressure applied to the cover slip, with occasional heating

over an alcohol burner, aided in flattening the root segments adequately for microscopic

evaluation of mycorrhizal structures in root cells.

One hundred root segments were evaluated per sample for intensity of

colonization and to identify any variations in arbuscular morphology which might exist.

Mycorrhizal structures on glass slides were viewed with a Nikon Optiphot compound

microscope at 200, 400, and 1000x magnifications, and photographs were taken with a

Nikon CoolPix 990 digital camera. In order to judge the amount of mycorrhizal root

colonization, the grid line intersect method was used to estimate the total root length

colonized by AMF (Newman, 1966; Tennant, 1975; Giovannetti and Mosse, 1980).

Spore extractions. I Mycorrhizal spores were extracted by wet sieving and decanting

by mixing 100 g of air-dried sample soil with 300 ml of tap water, blending at low speed

in a commercial Waring blender for 1 min, and then allowed to settle for 1 min. The

supernatant was then passed through a series of Tyler 250, 125, and 38 mrn mesh sieves

(Daniels and Skipper, 1982). The remaining fraction was rinsed with tap water to remove

sediment and any organic materials left behind. The fraction was decanted into 50 ml

centrifuge tubes containing a 40% sucrose/deionized water solution (w/v) (Jenkins,

1964). The tubes were centrifuged for 3 min at 2,000 rpm in a Dynac III centrifuge. The

supernatant, containing the spores, was decanted off the top of the tube into a 38 .im

mesh sieve and rinsed to remove the sucrose. The extracted spores were collected in a

9 cm Petri dish with tap water rinse and viewed with a Zeiss dissecting scope.









Mycorrhizal spore densities were enumerated by using an ocular field method described

in the International Culture Collection of (Vesicular) Arbuscular Mycorrhizal Fungi for

high spore densities (Morton, 2005).

Intact and parasite free spores were selected using a Gilson 20 .il pipetman.

These spores were used to inoculate 10 cm diameter clay pots containing the low P,

sterile soil (as described above) and planted with surface-sterilized sorghum-sudangrass

hybrid seed. The monocultures were kept at 20-25 C for approximately 60 d. At that

point, any spores that had been produced as a result of the inoculations were extracted as

previously mentioned, and used to inoculate another crop of sorghum-sudangrass in

sterilized, low P soil. The second generation of monocultures were then maintained for

60-90 d and processed for spore extraction and mycorrhizal identification.

Arbuscular mycorrhizal fungi tleutificutioo ||I Identification of the mycorrhizal fungi

associated with St. Augustinegrass was accomplished by selecting healthy, single spores

with a 20 [il Pipetman and mounting in either sterile, deionized water or (1:1 v/v) PVLG

(polyvinyl alcohol-lactic acid) + Melzer's reagent (Khalil et al., 1992). The spores were

then viewed at 200, 400, and 1000x using a Nikon compound microscope and identified.

Using arbuscular mycorrhizal descriptions by Schenck and Perez (1988), a tentative

determination to genus was made based on the average measurement of 20 similar spores

per pot. The species was determined based on taxonomic descriptions from the INVAM

Species Guide (Schenck and Perez, 1988). Identifying characteristics of the

monocultured spores, such as spore wall number and width, hyphal appendages, the

presence or absence of germ shields, approximate overall spore diameter and color in

reagents, were used as described by Schenck and Perez (1988).









Statistical analysis. |I Spore density and percent colonization data were analyzed using

the General Linear Model procedure (SAS Institute, Version 9.0, 2004) (Appendix F-l).

The survey was performed using a random model in a randomized complete block design

with multiple samplings at multiple locations. The percent root colonization data were

transformed with the arcsine square root transformation prior to an analysis of variance

due to distribution of propagules within soil being highly variable resulting in a non-

normal frequency of distribution points (St. John and Hunt, 1983; Friese and Koske,

1991). Spore density data were transformed to their natural log prior to analysis of

variance to prevent violation of the assumption of normal distribution. Significant

interactions were separated using Tukey's Studentized Range Distribution test.

Correlations between percent colonization or spore density data, with soil nutrient

composition, and percent colonization to spore density were done in SAS using Pearson

product-moment correlation coefficients. Regression analyses also were performed with

the regression procedure in SAS.

Results

Root Evaluation. || Roots, collected from sod fields evaluated in this survey revealed

the first evidence of an interaction between AMF and St. Augustinegrass. In stained

roots mounted on glass slides, AMF structures such as internal vesicles, intra and

extraradical hypha, and an assortment of arbuscular types were observed. Bulbous

appressoria (Fig. 2-4) were noted at inoculation points along the length of the root, giving

rise to carbohydrate storage vesicles of various shapes within cortical root cells (Figs. 2-

5, 2-6). Copious amounts of intra and extraradical hypha were observed within and along

the outer surface of root tissue (Fig. 2-7). Most notably, a variety of arbuscular types

were observed within the cortical root cells. Arbuscules, or haustoria-like structures,









have been categorized into two morphological types (Gallaud, 1904); Arum- and Paris-

types. These intercellular mycorrhizal structures are the presumed active fungal sites of

nutrient translocation between host and fungus (Bracker and Littlefield, 1973; Brundett et

al., 1984).

In this study, field grown plant roots were found to contain both the Arum- and

Paris- type of arbuscules along with a variety of intermediate Arum- morphologies.

Intermediate forms of the Arum- type found in cortical root cells of St. Augustinegrass

sod plants ranged from a typical "feathery" form (Fig. 2-8) extending from intracellular

hypha to a "dense-compact" form between cells of conjoined intercellular hyphae (Fig. 2-

9). A "grainy" form (Fig. 2-10) was also found in cortical root cells on several occasions.

This could be a collapsing arbuscule instead of an intermediate arbuscular form. The

Paris-type arbuscule found in St. Augustinegrass plant roots shows a typical arbusculate

coil (Fig. 2-11) in the root cell, while intermediate forms were not observed. An unusual

structure was found along intercellular hyphae that resembled a hyphal mat with a

mantle-like appearance often found in conjunction with certain types of ectomycorrhizas

(Fig. 2-12). This may be a new arbuscular form found in the Poaceae. This structure

was only observed once in St. Augustinegrass plants harvested in April 2005 at the Fort

McCoy location.

Spore density evaluation. | Further evidence supporting an interaction between AMF

and St. Augustinegrass was observed outside the root within the rhizosphere. AMF

spores clinging to epidermal tissue on roots were frequently observed in field samples

and in pot cultures using field soil from each farm location and sorghum-sudangrass as

the trap plant. The three sod farms sampled in this survey have been cropped solely in









'Floratam' St. Augustinegrass sod for more than 12 years. Weeds are heavily controlled

with herbicides at each location. The AMF spores recovered from field soil are entirely

dependent upon the St. Augustinegrass plants because they are obligate heterotrophs.

The limited availability of other plant species at each location, and the availability of

numerous spore types for pot culturing and subsequent AMF identification, provides

adequate evidence of AMF colonizing St. Augustinegrass plants in North Central Florida

soils.

Additional mycorrhizal structures such as auxiliary cells were frequently

observed in slide mounts of spores from both pot cultures and field soil (Fig. 2-13).

Selected single spores that appeared non-parasitized and viable, were chosen under light

microscopy for culturing in sterile, low P soil in order to obtain consistent spore

structures compatible with identification procedures. Spores, retrieved from pot cultures

were used as sieved soil sub-cultures to produce another generation of spores capable of

being readily identified from their morphological structures according to Schenck and

Perez (1988). Table 2-1 lists the species of AMF positively identified from sub-cultures

of soil from each location over a year-long period.

Species of Glomus were the most commonly encountered AMF in north central

Florida soils at each location. At the Lake Butler location, Glomus species included: G.

etunicatum Becker & Gerdemann (Fig. 2-14), G. intraradices Schenck & Smith (Fig. 2-

15, 2-16), G. reticulatum Bhattacharjee & Mukerji (Fig. 2-17, 2-18), and G. uggregutimln

Schenck & Smith (Fig. 2-19). Glomus species isolated at the Fort McCoy location

included: G. ambisporum Smith & Schenck (Fig. 2-20), G. formosanum Wu & Chen









(Fig. 2-21), G. macrocarpum Tulasne & Tulasne (Fig. 2-22), G. gerdemannii Rose,

Daniels & Trappe (Fig. 2-23), G. intraradices, and G. etunicatum.

Acaulospora spinosa Walker & Trappe (Fig. 2-24) and an unidentified species of

Scutellospora were isolated at Lake Butler. Additional AMF genera were found at Fort

McCoy including: Entrophospora infrequens [Hall] Ames & Schneider (Fig. 2-25), A.

denticulata Sieverding & Toro (Fig. 2-26), A. lacunosa Morton (Fig. 2-27), and

Scutellospora minute [Ferr. & Herr.] Walker & Sanders (Fig. 2-28). The Starke location

was unusual in species diversity with only 3 species isolated: Glomus etunicatum, G.

intraradices, and Scutellospora minute. One unique spore type was found at the Fort

McCoy location, but could not be grown in a pot culture successfully. The unidentified

spore type was observed on two occasions during the late spring of 2005 in very small

numbers and appeared to be either a species of Acaulospora or Entrophospora based on

morphology. Without a sufficient number of cultivated spores for microscopic

evaluation, positive identification of the species was not possible.

Sieving field soil from each location not only yielded spores for pot culturing, but

also enabled a numerical count of spore density, which is a good indicator of the

infectivity of the AMF in the soil and their level of activity in the rhizosphere. The total

spore density at the three locations ranged from 78 to 2,132 spores per 100 g of dry soil

(non-transformed data). Spore density but did not vary among or within sod farm

locations (P < 0.0001), indicating that variations in soil factors did not significantly affect

AMF spore production between locations from December 2004 through December 2005

(Table 2-2). Spore production did vary significantly (P < 0.0001) between monthly

sampling, which suggested a possible seasonal influence on spore production. Greater









spore density totals occurred in soils collected during the warmer summer and fall

months, as compared to, lowered spore production occurring in the cooler months of

winter and spring. Total spore density in December 2004 was significantly lower when

compared to December 2005. This might be explained by increased rainfall, prior to the

sampling period, in north central Florida during the 2004 hurricane season.

With spore densities varying between dates, analysis of variance for these points

showed a significant date by location interaction (P < 0.05) indicating that seasonal

effects and unknown variations in site-related effects might measurably influence the

total spore density. In this survey, rainfall and soil moisture where positively correlated

to spore density (Table 2-3).

Based on the regression equations, a quadratic response was generated in total

spore density to soil moisture at each location. Spore density at the Starke location

increased at soil moisture levels between 0 and 2 cm, but declined until soil moisture

levels reached 6 cm where another increase was observed (Fig. 2-29). Above 9 cm a

decrease in spore density occurred (r=0.73). The same general response to soil moisture

was noted at the Fort McCoy location except where soil moisture declined to

approximately 8 cm (r=0.61) (Fig. 2-30). At the Lake Butler location, spore density

increased slightly until soil moisture levels reached 7 to 8 cm when a slight decline in

spore density was observed (r=0.68) (Fig. 2-31). This lends credibility to the theory that

excessive rainfall during the hurricane season of 2004 lowered spore production in

December of that year.

A quadratic response was also produced in total spore density to temperature at

each location. Spore density at the Starke location (r= 0.60) (Fig. 2-32) decreased from









15 C until the temperature reached 20 C. Between 20 C and 28-29 C a gradual increase

in spore density was observed until the temperature reached 30 C. At that point there was

another gradual decrease in spore density, which seemed to level off near 35 C. At the

Fort McCoy location (r=0.84) a gradual increase in spore density was observed until the

temperature was approximately 28-29 C, then a decline was noted (Fig. 2-33). At the

Lake Butler location (r=-0.59) a slight increase in spore density occurred across all

temperature ranges (Fig. 2-34). Based on these data, it appears that soil temperatures

above 28-30 C have a detrimental effect on the AMF. In addition, this temperature range

might also damage host root tissue.

Percent colonization evaluation. |I Percent root length colonized by AMF yielded no

significant difference among or within location differences, but there was a significant

date interaction (P < 0.0001). Colonization was generally highest in the cooler months of

winter and spring, with lower colonization occurring in the warmer summer and fall

months except in December 2005, when colonization was the lowest. The amount of root

length colonized ranged from 13 to 39% across the sampling dates (non-transformed

data). No correlation was found between temperature and soil moisture in relation to

percent root length colonized (Table 2-4).

Discussion

Dickson (2004) suggested an Arum-Paris continuum of mycorrhizal symbioses in

a survey of 12 colonized plant families, with arbuscule formation dependent on the

fungus as well as the host plant. Most mycorrhizal angiosperms were once thought to

only produce the Arum-type of arbuscule, which consists of both intercellular hyphae and

arbuscules, while most angiosperms and bryophytes were thought to only produce the

Paris-type with intercellular hyphae and arbuscular coils (Dickson, 2004). The majority









of scientific research has been conducted on flowering plants versus trees and bryophytes

causing these fallacies to be argued as fact until Smith and Smith (1997) produced a

comprehensive list of plant families that included their arbuscule types. The list showed

that the Paris-type is in fact most common among all plant families and that,

"intermediate" or transitional arbuscular morphotypes were observed in some plant

species. One genus (Ranunculus) forms both types within the same plant (Smith and

Smith, 1997).

Experiments on maize (Zea mays) and the tuliptree (Liriodendron tulipfera),

among many others, revealed that AMF can form either type of arbusculate structure

based on the host plant (Barrett, 1958; Gerdemann, 1965). In a field experiment using

tomatoes (Lycopersicon esculentum) and other annual crops, investigators found that

arbuscule morphology is actually dependent on intercellular spaces in cortical root cells

(Brundrett and Kendrick, 1988; Cavagnaro et al., 2001). Intermediate forms of the Arum

and Paris-type arbuscules are common in certain plant families such as those described in

three cultivars of flax (Linum usitatissimum), which Dickson et al. (2003) referred to as

arbuscules "in pairs in adjacent longitudinally arranged cortical cells arising from a

single, radial intercellular hyphae."

On rare occasions, both arbuscule types (Arum and Paris) occur in the same plant

species, which Smith and Smith (1997) noted in the family Poaceae. The Paris- and

Arum- types were found in millet, ryegrass, and wheat. In addition, a series of

intermediate forms between the two main types of arbuscules were also observed. The

same can be said for St. Augustinegrass plants in relation to AMF colonization. In field

studies, environmental effects may interact to influence fungal and plant response to the









mycorrhizal interaction. Sylvia et al. (1993) suggested that even in the presence of high

amounts of soil P, water stress and pesticide applications can have extensive effects on

mycorrhizal response. Rabatin (1979) noted that soil moisture may have the greatest

effect on the degree of infection of Glomus species in field situations. Furthermore, the

stages of plant development (Saif and Khan, 1975) as well as temperature (Giovannetti,

1985; Schenck and Kinloch, 1980; Smith & Smith, 1997; Sylvia, 1986) all play a major

role in mycorrhizal activity.

In this survey, AM fungi preferred warmer months for spore production and

cooler months for colonization of St. Augustinegrass plants. In the north central region

of Florida, St. Augustinegrass does not usually go completely dormant in cooler

temperatures, and there is usually some plant activity during the winter months especially

in the roots where AMF colonization occurs. This increase in colonization during cooler

temperatures may be an effort to preserve valuable carbon and energy reserves for future

spore production. Subsequent proliferation in the warmer months, while the plant host is

most active, would provide more carbohydrates from a symbiotic interaction (Johnson et

al., 1997). It is also possible that AMF are actually acting as a parasite in the winter

months when colonization is highest while the plant is less active.

During less than optimal winter growing conditions, the St. Augustinegrass plant

is less able to defend itself against infection and colonization due to lowered metabolic

activity. Johnson et al. (1997) suggested a mycorrhizal continuum ranging from

mutualistic to parasitic in some managed habitats where humans unknowingly altered the

association through management regimes. Another possibility is environmentally









induced parasitism due to morphological, phenological, and physiological differences in

the symbionts which may influence the mycorrhizal association (Johnson et al., 1997).

Conversely, in natural habitats, mycorrhizal associations have evolved over many

years to encourage fitness in the plant and the fungus making the interaction continually

mutual (Johnson et al., 1997). St. Augustinegrass sod systems are not traditional

cropping systems needing continual management inputs from man, nor are they a natural,

non-impacted habitat. St. Augustinegrass sod could be referred to as a non-conventional

cropping system due to minimal inputs after harvesting where ribbons of grass are left

behind for re-growth. Cloned host plants are in constant supply in sod fields providing

the AMF with a dependable host, but when the plant is semi-dormant throughout the

winter months the fungi may actually pose a threat to the health of the plant because net

costs in carbon might then exceed net benefits in some situations. For example, during

instances of lowered metabolic activity in the winter, plants lower photosynthetic ability

and subsequent output and will not benefit from the added benefits of a mutual

interaction. Acquisition of nutrients and water is less important during these times, but

St. Augustinegrass may be harmed by the loss of stored carbon to AMF. Throughout the

year, there are potential times when the interaction between plant and AMF is such that

the symbiosis might actually be neutral in nature (Johnson et al., 1997).

An attempt was made in this survey to correlate spore density to the percent root

length colonized, but no correlation was found. Some researchers have reported a

correlation between the two variables (Giovannetti, 1985; Miller et al., 1979) while

others have observed no such relationship (Giovannetti and Nicolson, 1983; Hayman and

Stovold, 1979). This is most likely due to the vast variations observed in soils, plant









species and their developmental stage, and fungal specificity. Many mycorrhizal studies

suggest a significant interaction with soil P where spore production or colonization is

lowered by increasing levels of P. Correlations between soil chemical characteristics

such as P content to spore density and percent root colonization have been reported in

grasses (Brejda et al., 1993). Others suggest that mycorrhizal ecology plays less of a

role. P content in south Florida soils had no effect on AMF in tropical forage legume

pastures (Medina-Gonzalez et al., 1988), nor did potassium or pH in studies of cultivated

soils (Abbott and Robson, 1977; Hayman, 1978).

In this survey, soil samples from each location were evaluated during the months

of January, April, August, and November 2005 in an attempt to correlate soil Mg, Ca, K,

P, soil pH, and organic matter percentage to spore density and/or percent root length

colonized, but a correlation was not observed (Table 5). One theory to explain the lack of

correlation between AMF and P content, in this case, might be explained by asexual

organisms, without the cost of sexual reproduction and consequently no genetic

variability, and having scores of mutations that accumulate over a long period of time

(Helgason and Fitter, 2005). The Glomeromycota possess ancient asexual lineages

(Gandolfi et al., 2003). This apparent genetic isolation would presumably cause

mutations to allow for some adaptations such as P tolerance. In AMF the coenocytic

mycelium is multinucleate providing a set of mutations within the DNA of all nuclei

(Helgason and Fitter, 2005). Reductions in fitness due to a lack of genetic variability due

to asexual reproduction may never be noticed in AMF because mutated, non-functional

genes from one nuclear lineage might be subjugated by functional alleles on another

nucleus (Helgason and Fitter, 2005).









Arbuscular mycorrhizal fungi in these sod fields are secluded, thus reducing

genetic variability, so it is possible that the ancient fungi are capable of evolving and

adapting through mutations to tolerate large amounts of added nutrients like P. P is

widely used in large amounts in St. Augustinegrass to promote root growth and health for

winter survival and spring green-up. Through years of isolation in sod fields and large

applications of P on a frequent basis, these fungi might have evolved a mechanism

through spontaneous mutation to tolerate elevated P levels. This is speculation, but the

lack of spore density and percent colonization variable correlation to P levels could be

due to genetic mutation in the fungi within these fields leading to a significant adaptation

and evolutionary event.

Overall root colonization and spore density were low to moderate, which suggests

that the AMF populating St. Augustinegrass sod production soils are moderately active.

This situation might lend itself to field inoculation where AMF could potentially provide

a level of root disease protection, which might lower pesticide use and cost. It could also

lead to increased and more efficient P acquisition and use when combined with more

conducive management strategies. On the other hand, inoculation with AMF might be

ineffective in situations where genetic isolation combined with perennial cropping and

moderate to heavy fertilizer inputs are unavoidable for proper management.






40















Figure 2-1 A-C. 'Floratam' St. Augustinegrass sod farms located at (A) Fort McCoy
(Marion County), (B) Lake Butler (Union County), and (C) Starke (Bradford
County) in north central Florida.
































Fig. 2-2. Sorghum-sudangrass pot cultures containing 50% (w/w) field soil combined
with 50% sterile, low P soil.






42


Fig. 2-3. Spore extract from field soil following the wet sieving procedure.








































Figs. 2-4 2-7. Stained arbuscular mycorrhizal structures observed within 'Floratam' St.
Augustinegrass.

Fig. 2-4. Bulbous appressoria found originating from extraradical hypha.
Bar = 40 im.
Fig. 2-5. Circular type of AMF vesicle stained with trypan blue. Bar = 40 rim.
Fig. 2-6. Oblong type of AMF vesicle stained with trypan blue. Bar = 40 im.
Fig. 2-7. Extraradical hyphae observed with light microscopy infecting
and colonizing roots. Bar = 20 rim.











































Figs. 2-8 2-11. Stained arbuscular morphology types found within 'Floratam'
St. Augustinegrass.

Fig. 2-8. Feathery form of the Arum-type arbuscule morphology, stained
with trypan blue, within cortical root cells. Bar = 40 inm.
Fig. 2-9. Dense and compacted Arum-type arbuscule morphology stained
with trypan blue. Bar = 40 inm.
Fig. 2-10. Grainy or collapsing Arum-type arbuscule morphology stained
with trypan blue. Bar = 40 inm.
Fig. 2-11. Paris-type coiled arbuscule, stained with trypan blue, within
cortical root cells. Bar = 40 inm.
Fig. 2-12. Net-like AMF structure observed in roots across adjacent
cortical root cells. Bar = 20 inm.
Fig. 2-13. Auxiliary cells of an AMF observed in spore extracts from field
soil. Bar = 40 nm.










Table 2-1. Species of AMF positively identified at each sod farm location from pot
cultures of sorghum-sudangrass within a combination of field and sterile, low
P soil.


Location


AMF SDecies


Lake Butler
Lake Butler
Lake Butler
Lake Butler
Lake Butler
Lake Butler

Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy
Fort McCoy

Starke
Starke
Starke


Acaulospora spinosa
Glomus etunicatum
G. intraradices
G. reticulatum
G. agglegutIlIn
Scutellospora sp.

A. denticulata
A. lacunose
Entrophospora infrequens
G. ambisporum
G. etunicatum
G. formosanum
G. gerdemanii
G. intraradices
G. macrocarpum
Scutellospora minute

G. etunicatum
G. intraradices
S. minute
































1 17











19



Figs. 2-14 2-19. Arbuscular mycorrhizal fungal spores identified at the Lake
Butler sod farm location.

Fig. 2-14. A spore of Glomus etunicatum stained in Melzer's reagent. Bar = 20 [im.
Fig. 2-15. A spore of G. intraradices in deionized water. Bar = 20 [im.
Fig. 2-16. Spore wall morphology of G. intraradices spore stained in
Melzer's reagent (arrows point to cell wall layers). Bar = 40 [im.
Fig. 2-17. A spore of G. reticulatum in deionized water. Bar = 20 ism.
Fig. 2-18. Spore wall morphology of G. reticulatum in deionized water
(arrows point to cell wall layers). Bar = 40 ism.
Fig. 2-19. A broken spore of G. aggregatilni in Melzer's reagent. Bar = 20 ism.


















20 1_ 22


















26 -_ 27 28



Figs. 2-20 2-28. Arbuscular mycorrhizal fungal spores identified at the Fort
McCoy sod farm location.

Fig. 2-20. A spore of Glomus ambisporum stained in Melzer's reagent. Bar = 20 rim.
Fig. 2-21. A spore of G. formosanum stained in Melzer's reagent. Bar = 20 rim..
Fig. 2-22. A spore of G. macrocarpum stained in Melzer's reagent. Bar = 20 rim.
Fig. 2-23. A spore of G. gerdemannii stained in Melzer's reagent. Bar = 20 rim.
Fig. 2-24. A spore of Acaulospora spinosa stained in Melzer's reagent. Bar = 20 rim.
Fig. 2-25. A spore of Entrophospora infrequens stained in Melzer's reagent.
Bar = 20 rm.
Fig. 2-26. A spore of A. denticulata stained in Melzer's reagent. Bar = 20 rim.
Fig. 2-27. A spore of A. lacunosa stained in Melzer's reagent. Bar = 20 rim.
Fig. 2-28. A spore of Scutellospora minute stained in Melzer's reagent. Bar = 20 rm.









Table 2-2. Evaluation of analysis of variance data for spore density data from each sod
farm location by date.


Date Location


Dec. '04 Fort McCoy
Lake Butler
Starke


Jan '05




Feb '05


Fort McCoy
Lake Butler
Starke

Fort McCoy
Lake Butler
Starke


March '05 Fort McCoy
Lake Butler
Starke

April'05 Fort McCoy
Lake Butler
Starke

May'05 Fort McCoy
Lake Butler
Starke

June'05 Fort McCoy
Lake Butler
Starke

Aug'05 Fort McCoy
Lake Butler
Starke

Sept'05 Fort McCoy
Lake Butler
Starke


Total Spore Density
(spores/100g air-
dried soil)
5.06t
4.82
5.13
mean 5.00 d
5.42
5.17
5.54
mean 5.38 cd
5.80
5.78
5.56
mean 5.71 bcd
5.53
6.33
5.30
mean 5.72 bcd
6.48
6.84
6.32
mean 6.55 a
6.72
6.54
6.94
mean 6.73 a
6.90
6.78
6.29
mean 6.66 a
5.90
7.01
5.88
mean 6.26 ab
6.36
6.13
5.66
mean 6.05 abc









Oct '05


Fort McCoy
Lake Butler


Starke

Nov'05 Fort McCoy
Lake Butler
Starke


Dec '05


Fort McCoy
Lake Butler
Starke


6.65
6.08
5.70
mean 6.14 abc
6.22
6.54
6.76
mean 6.51 a
5.81
5.80
6.36
mean 5.99 abc


tEach value is the average of three sample plots/location (10 sub-samples/plot).
Means followed by the same letter are not significantly different according to
Tukey's (HSD) Studentized Range Test (P = 0.0001).























Table 2-3. Pearson correlation coefficients
and temperature.


Percolont
Sporeden
Rainfall


Sporedent
-0.007


(r) for AMF spore density and soil moisture


Rainfall
-0.14
0.45***


Soiltempt
0.02
0.48***
0.61***


*** Significant at P = 0.0001, respectively.
f Percolon = percent root length colonized; Sporeden = spore density;
Rainfall = amount of rainfall in month preceding sampling date;
Soiltemp = soil temperature for sampling date.














8

7

60

0
U5

S4



2


Soil Moisture (cm)

Fig. 2-29. Spore density with increasing soil moisture levels over a 12-month period at
the Starke sod farm location.


0)
C 0

G) 0


0..


Soil Moisture (cm)


Fig. 2-30. Spore density with increasing soil moisture levels over a 12-month period at
the Fort McCoy sod farm location.


y = -0.0264X4 + 0.4465x3 2.3561x2 + 4.4331x + 3.4988
2


y = -0.0067X4 + 0.1113x3 0.6217x2 + 1.5001x + 4.6121
R2 = 0.6136




































Soil Moisture (cm)

Fig. 2-31. Spore density with increasing soil moisture levels over a 12-month period at
the Lake Butler sod farm location.


y = -3E-06x6 + 0.0004X5 0.028x4 + 0.9465x3 17.252x2 + 160.12x 583.17
R = 0.6062






15 20 25 30 3!
Soil Temperature (C)


Fig. 2-32. Spore density with increasing soil temperatures over a 12-month period at the
Starke sod farm location.


y = 0.0118x4 0.195x3 + 0.9987x2 1.4413x + 5.8091
R2 = 0.6888


o

c S
.5
C( 0

00
ni )







53















y = 0.0002X4 0.0206X3 + 0.8144x2 13.691x + 88.104
R2 = 0.8455


Soil Temperature (C)


Fig. 2-33. Spore density with increasing soil temperatures over a 12-month period at the
Fort McCoy sod farm location.


Fig. 2-34. Spore density with increasing soil temperatures over a 12-month period at the
Starke sod farm location.


8

7, ,
6 0


STy = 1 E-05x4 + 0.0009x3 0.1124x2 + 3.1385x 20.969
R2 = 0.5939


2
1

0
15 20 25 30 35
Soil Temperature (C)









Table 2-4. Evaluation of analysis of variance data for percent root length colonized from
each sod farm location.


Date
Dec. '04


Location
Fort McCoy


Lake Butler
Starke

Jan'05 Fort McCoy
Lake Butler
Starke

Feb '05 Fort McCoy
Lake Butler
Starke

March'05 Fort McCoy
Lake Butler
Starke

April '05 Fort McCoy
Lake Butler
Starke

May'05 Fort McCoy
Lake Butler
Starke

June'05 Fort McCoy
Lake Butler
Starke

Aug'05 Fort McCoy
Lake Butler
Starke

Sept '05 Fort McCoy
Lake Butler
Starke

Oct '05 Fort McCoy
Lake Butler
Starke

Nov '05 Fort McCoy


%Colonization (GIM)

27.226t
25.47
25.69
mean 26.13 ab1
28.28
28.05
30.69
mean 29.01 a
24.86
29.91
31.79
mean 28.85 a
26.96
24.01
23.58
mean 24.84 abc
26.98
30.03
28.74
mean 28.58 a
24.62
28.35
27.21
mean 26.73 ab
25.25
29.54
22.97
mean 25.92 ab
23.00
22.76
22.14
mean 22.63 bcd
23.81
18.27
22.96
mean 21.68 bcd
21.13
18.87
18.51
mean 19.50 cd
18.08






55


Lake Butler 20.94
Starke 20.71
mean 19.91 cd
Dec '05 Fort McCoy 18.87
Lake Butler 19.90
Starke 17.29
mean 18.68 d

tEach value is the average of three sample plots/location (10 sub-
samples/plot).
Means followed by the same letter are not significantly different according
to Tukey's (HSD) Studentized Range Test (P = 0.0001).























Table 2-5. Chemical characteristics of soils sampled for AMF at three north central
Florida sod farm locations during January, April, August, and November
2005.


P/g soil
9
112
38
12
91
27
35
88
56
55
47
45


Soil Nutrient Levels
Ca K
883 13
455 75
306 117
830 16
418 91
359 103
260 37
1065 97
903 87
392 82
414 91
370 83


pHt
5.5
5.8
5.7
7.0
5.8
5.7
5.9
6.3
6.2
5.4
5.7
5.4


OMt
1.57
2.31
2.02
1.70
2.53
2.01
1.34
2.97
2.08
1.99
2.07
1.93


tSoil pH, nutrient level, and organic matter content based on the mean of three composite
samples/location.
OM = Organic matter content.
*FM = Fort McCoy location.
**LB = Lake Butler location.


Date
Jan '05
Jan '05
Jan '05
April '05
April '05
April '05
Aug '05
Aug '05
Aug '05
Nov '05
Nov '05
Nov '05


Location
FM*
LB**
Starke
FM
LB
Starke
FM
LB
Starke
FM
LB
Starke














CHAPTER 3
THE EFFECT OF ARBUSCULAR MYCORRHIZAL FUNGI ON
GAEUMANNOMYCES GRAMINIS VAR. GRAMINIS AND RHIZOCTONIA
SOLANI COLONIZATION OF ST. AUGUSTINEGRASS SOD IN NORTH CENTRAL
FLORIDA SOILS


Take-all root rot and brown patch are two of the more common and devastating

diseases of St. Augustinegrass sod throughout Florida. Take-all root rot, caused by

Gaeumannomyces graminis (Sacc.) Arx & D. Olivier var. graminis, is a disease of both

grasses and cereals (Nilsson, 1969; Huber and McCay-Buis, 1993). Take-all root rot was

first described in Sweden in the early 1800's infecting grasses (Mathre, 1992). It is one

of several G. graminis varieties which infect many important crops worldwide (Rovira

and Whitehead, 1983). This particular variety of the fungus infects all cultivars of St.

Augustinegrass (Elliott, 1995; Datnoff et al., 1997). In the late 1980's, large, chlorotic

patches of St. Augustinegrass were observed on sod farms in South Florida and were

confirmed as the first disease symptoms of G. graminis var. graminis infection observed

in this species (Elliott, 1993). The disease was found in St. Augustinegrass throughout

Alabama, Florida, and Texas (Fig. 3-1) and it is notably more severe in the summer and

fall months, especially during periods of increased precipitation (Elliott, 1993). Early

studies suggested that the fungus preferred alkaline or high pH soil, mild winters, thatch-

accumulation and frequent light irrigation, however the conditions that predisposed the

stand to disease or prompted disease escape are not known (Guyette, 1994).

Management recommendations included elimination of low areas where water

accumulates, watering only when needed, and the use of pH decreasing









fertilizers in the fall, as well as thatch prevention and aeration (Guyette, 1994).

Fungicides were recommended as preventative but not curative treatments, which limited

management options to growers (Guyette, 1994). The effect of systemic fungicides on G.

graminis var. graminis infection and colonization of turfgrasses was evaluated; but

results indicated that preventative and/or curative rates of fungicides did not limit take-all

root rot disease or increase turfgrass quality (Elliott, 1995). Biological controls were

explored in an attempt to decrease take-all root rot in wheat and turfgrasses. The effects

of bacterial isolates, actinomycetes, and fluorescent pseudomonads on the roots of wheat

were evaluated as antagonists against G. graminis var. tritici (Sivasithamparam and

Parker, 1978). These organisms make up a large portion of the microbial community of

soils and researchers expected their production of antibiotics or toxic metabolites would

inhibit take-all in wheat in suppressive soils. While combinations of these

microorganisms reduced disease, none were successful alone (Sivasithamparam and

Parker, 1978). To date, no effective curative or preventative controls for take-all root rot

are recognized for use in St. Augustinegrass.

In order to determine the impact of arbuscular mycorrhizal fungi (AMF) on take-

all root rot in St. Augustinegrass sod, it is necessary to accurately diagnose G. graminis

var. graminis and determine its population within the field. The diagnosis of take-all root

rot involves several characteristics and diagnostic tools for isolation and identification.

The pathogen is somewhat elusive and may be easily confused with other fungi if the

scientist is not familiar with the morphology of the fungus and patterns of infection. The

ascomycete, G. graminis var. graminis, is classified in the order Diaporthales because it

produces ascospores in black, flask-shaped, ostiolate perithecia, which are fully enclosed









and lined with hyaline periphyses (Landschoot, 1997; Walker, 1973). The perithecia are

typically 200-400 [m x 150-300 C[m in length, with the neck portion 100-400 C[m in

length and 70-100 C[m wide (Landschoot, 1997). The asci, clavate in shape, are

unitunicate, are formed in a hymenium, and range in length from 80-140 C[m and 10-15

[lm in width. The apex of the ascus, which has a refractive apical ring, is generally

yellowish en masse. Each ascospore is typically 70-110 C[m in length, 2-4 C[m in width

and they usually contain 3-8 septa, but there may be 11 or 12 septa produced. The

anamorphic state, which is rarely observed, is a Philaphora species that produces conidia

5-14 C[m in length x 2-4 C[m in width. The use of conidia as taxonomic criterion is not

recommended due to variation between isolates and their non-descript morphology. In

culture, mycelia range from short to aerial, white to gray, green to brown, or black

(Landschoot, 1997).

Dark runner hyphae are typically observed on and around the crown portion of the

plant, with extension onto the stem and stolons. The roots usually have relatively fewer

dark surface runner hyphae, compared to the foliar portion of the plant, which may

remain green. Instead of dark runner hyphae, the roots are often covered with dark

brown to black lesions and subsurface hyaline hyphae. The cortical browning of roots is

thought to be a host defense mechanism, while the discoloration of shoots is a necrotic

symptom of disease (Penrose, 1992). The name "take-all root rot," implies that the roots

are the first plant parts to be severely affected whether facilitated by feeding damage

from nematodes or mole crickets, mechanical damage from sod production, cultural

techniques, or through natural openings.









After the initial invasion, the seminal roots are colonized internally by more

hyaline and infectious, secondary hyphae usually right behind the root tip (Henson et al.,

1999; Gilligan, 1983), which is were AMF usually colonize root tissue. Pathogenic

colonization causes an occlusion of vascular tissues resulting in the characteristic gradual

decline in plant health and potential death. Dark runner hyphae may continue up the

plant in search of more juvenile and susceptible tissue while producing deeply lobed and

melanized hyphopodia.

The hyphopodia are considered by most as superficial hyphal structures (Henson

et al., 1999) since they originate from the hyphae, however they behave much in the same

way as appressoria, which develop from the germ tube of germinating fungi providing

infection pressure and anchoring the fungus to plant tissue (Agrios, 2004). Hyphopodia

cluster and develop into an infection cushion which provides the added structural stability

while helping to maintain the turgor pressure required for colonization (Henson et al.,

1999). The force of exertion of G. graminis var. graminis is associated with reduced cell

wall permeability, turgor, and wall rigidity (Bastmeyer et al., 2002). The deeply lobed

hyphopodia are unique to G. graminis var. graminis and may exist to allow the fungus to

overcome plant resistance mechanisms. Plants of St. Augustinegrass may benefit from

AMF colonization in the presence of Gaeumannomyces graminis var. graminis. But, it is

possible for AMF to have a negative impact on plants in some situations, or they may

even be neutral in nature (Johnson et al., 1997).

Brown patch or Rhizoctonia blight, caused by Rhizoctonia solani Ktuhn (Figs. 3-2,

3-3), is most active in St. Augustinegrass from November to May when temperatures

average









25 C and below (Elliott and Simone, 2001). Brown patch is typically worse in periods of

excessive rainfall or irrigation, or when grass leaves remain wet for more than 48 hours

(Elliott and Simone, 2001). In the field, small chlorotic patches of sod gradually turn

brown as infected leaf blades die, hence the name brown patch (Elliott and Simone,

2001). As patches expand, they may coalesce into large rings of yellow-brown sod with

dark and wilted margins. It is not uncommon for sod to appear green and healthy in the

center of the rings. Grass blades are killed near the crown due to restriction of water and

nutrient transport, which creates a dark rot near the base of the blade. Infected blades can

easily be pulled from the leaf sheath due to the soft rot (Elliott and Simone, 2001). Most

usually the stolons and leaves are affected more than the roots themselves. A barrage of

chemical controls, such as azoxystrobin, fluotanil, and mancozeb offer effective brown

patch control when used as preventatives. Cultural controls include irrigating only when

necessary between 2 and 8 AM and removal of mower clippings from the site. However,

the use of quick release nitrogen during periods of R. solani activity seems most

beneficial (Elliott and Simone, 2001). The use of chemicals in sod production has been

controlled in recent years and these restrictions will continue according to state and

federal regulations. Effective disease prevention strategies including the use of biological

controls, such as AMF, are essential research objectives in an industry where quality is of

utmost importance to buyers and growers.

Brown patch was first described in St. Augustinegrass in the 1980's (Hurd and

Grisham, 1983; Martin and Lucas, 1984) as an aerial type of pathogen common to a

variety of crops including corn, soybean, and rice (Sneh et al., 1991). Other pathogenic

species of Rhizoctonia affecting St. Augustinegrass include R. oryzae Ryker & Gooch









and R. zeae Voorhees which cause a sheath rot or spot, but the two species are rare

(Martin and Lucas, 1984; Haygood and Martin, 1990). The telomorph, Thanatephorus

cucumeris Frank, is assigned to the Basidiomycota (Ainsworth et al., 1973). Mycelia of

R. solani appear buff to dark brown in culture with irregularly shaped light to dark brown

sclerotia (Sneh et al., 1991). Rhizoctonia solani is identified by its characteristic right

angle (900) branching between the primary and secondary hypha (Duggar, 1915) with

branches forming acute (450) angles to main hypha (Butler and Bracker, 1970).

Identification is made easier by the presence of a septum at the branches near hyphal

constrictions at the base of right angles (Duggar, 1915). Additionally, the older, main

runner hypha of R. solani are more than 7 [im in diameter with more than two nuclei per

cell (Sneh et al., 1991).

Arbuscular mycorrhizal fungi have been associated with increased nutrient and

water acquisition in plants for many years. Mycorrhizal symbiosis often results in

increased plant vigor and the use of AMF has been studied in many crops as potential

antagonists to root pathogens (Schenck, 1987; Sylvia and Williams, 1992; Smith and

Read, 1997; Yao et al., 2002). Glomus etunicatum Becker & Gerdemann and G.

intraradices Schenck & Smith are two of the more common AMF species investigated as

potential biological controls and chemical alternatives against R. solani in crops such as

potato (Yao et al., 2002) and species of Fusarium in tomato crops and alfalfa (Caron et

al., 1986; Hwang et al., 1992). In several cases, G. intraradices provided significant

control of soilborne pathogens (Niemira et al., 1996; Khalil et al., 1994; Viyanak and

Bagyaraj, 1990). Newsham et al., (1995) reported that mycorrhizal fungi are capable of

protecting annual grasses from soilborne fungi. In other surveys, researchers found that









G. intraradices significantly reduced take-all root rot caused by G. graminis var.

graminis in cool- season bentgrasses on greens with low soil P levels (Koske et al., 1995).

Reductions in take-all disease severity in mycorrhizal wheat may be due to

increased P uptake, increased root cell wall lignification, pathogen exclusion, production

of antagonistic compounds, or altered root exudates (Graham and Menge, 1992).

However, baseline information concerning pathogen colonization and potential effects of

AMF on disease in the field is necessary before experiments concerning mechanisms of

resistance and inoculation can be undertaken.

The objective of this survey was to determine the extent of R. solani and G.

graminis var. graminis colonization in production fields of 'Floratam' St. Augustinegrass

sod in north central Florida and to determine whether populations of AMF are having any

effect on disease incidence in the field. Many researchers may feel that the effects of

AMF in turfgrass systems may be outweighed by the benefits of added nutrients,

pesticides, and irrigation. However, in St. Augustinegrass sod systems where inputs are

limited, AMF may serve a greater role in plant resistance to soilborne pathogens or soil

suppressiveness.

Materials and Methods

Root Pathogen Sampling. 'Floratam' St. Augustinegrass stolons and roots were

collected on a bimonthly basis from the three north central Florida sod farms described in

chapter 2 in January through December 2005. The roots and stolons were surveyed for

take-all root rot and brown patch. From each of the three (3 m2) plots described in

chapter 2, ten subsamples of root and stolon tissue (1-5 cm above the crown) were

randomly dissected from collected plants and cut into 100 pieces of tissue 2-5 cm in

length, in order to quantify the extent of root rot disease and to isolate and identify the









causal organisms. The pieces were washed, surface-sterilized for 1 min in a 10% sodium

hypochlorite and deionized water solution, rinsed twice for 1 min with sterile deionized

water, and blotted dry.

Pathogen Identification. Forty pieces of tissue from each of the 100 segments/plot were

randomly selected for isolation of G. graminis var. graminis and forty for isolation ofR.

solani and aseptically plated into selective agar media (Appendix- A) in 15 x 100 mm

Petri dishes. Selective media (Appendix A) were used to isolate the pathogens from

tissue and to slow growth of other soilborne fungi not associated with diseased tissue.

The Petri dishes were incubated at 24 C under a 12 h diurnal cycle. Fungal growth was

monitored by light microscopy for 5-8 d or until opportunistic fungal growth required

colony transfer to sterile media, in order to isolate the desired root pathogens. Samples of

fungal colonies suspected of being R. solani or G. graminis var. graminis were mounted

in water on glass slides and viewed with a Nikon Optiphot compound microscope to

identify fungal structures microscopically. Gaeumannomyces graminis var. graminis

colonies were readily identified in media by the presence of deeply-lobed hyphopodia

(Figs. 3-4, 3-5) within melanized mycelium (Landschoot, 1997). Rhizoctonia solani

colonies (Figs. 3-6, 3-7) were identified based on the auburn to light brown color and 900

branching of the mycelium (Sneh et al., 1991).

Pathogen Quantification and Statistical Analysis. The number of colonies of G.

graminis var. graminis and R. solani observed emerging from root or stolon pieces were

used to quantify the amount of infection of these root pathogens at each sod farm

location (Figs. 3-5, 3-6). The mean colonization data were expressed as the percentage of

sampled root or stolon pieces colonized by G. graminis var. graminis or R. solani on









selective agar media (Appendix A). The survey was performed using a random model in

a randomized complete block design with multiple samplings at multiple locations. The

percent colonization data were analysed using the Generalized Linear Model (SAS

Institute, Version 9.0, 2004) (Appendix F-2; Appendix F-3). Arbuscular mycorrhizal

fungi sampling data, as described in chapter 2, were used in this survey since root

pathogen sampling occurred simultaneously in the same plot locations as the survey of

AMF in the previous chapter. Significant interactions (P < 0.05) were separated using

Tukey's Studentized Range Distribution test, and correlations between AMF percent

colonization and spore density to percent colonization of each root pathogen were done in

SAS using Pearson product-moment correlation coefficients.

Results and Discussion

No correlation between AMF spore density or percent colonization in relation to

R. solani or G. graminis var. graminis colonization were found. Additionally, no location

effects were detected in the analysis of variance among or within the sampling months (P

< 0.001). However, pathogen colonization did vary significantly between sampling

months (P < 0.001), which suggested a seasonal influence on pathogen activity in north

central Florida soils at each sod farm location. Mean values of root colonization by R.

solani were greatest in December 2004 at 24.40% and lowest in June 2005 at 10.71

percent (Fig. 3-8). The warmer months of June and August had the lowest R. solani

colonization percentages but the values were not significantly different

from values in March, January, or October. The cooler months of December and April

had the highest percentages of R. solani, although the April mean was not significantly

different (P < 0.05) from October, January, or March (Fig. 3-8). This finding is not

surprising since R. solani has optimal growth below 26 C therefore it is typically more









active in cooler weather (Elliott and Simone, 2001). Interestingly, as noted in chapter 2,

AMF spore density (Table 2-2) was generally lowest during the cooler months of

December, January, and April and highest during warmer weather, with percent

colonization highest during the cooler months when R. solani is most active in these soils

(Table 2-4).

Mean values of root colonization by G. graminis var. graminis were highest in the

warmer months of August 2005 at 20.01% and lowest in December 2004 at 5.35 percent

(Fig. 3-9). The months of August, June, and October had the highest percentages of G.

graminis var. graminis colonization, with the lowest mean values occurring in December,

January, March, and April. However, there were no significant differences (P < 0.05)

between mean values in June and October, or October, April, March, and January.

Again, this finding is not surprising because G. graminis var. graminis is most active in

warm, markedly wet conditions where there is excessive thatch accumulation (Elliott,

1993; Guyette, 1994). During the warm, humid days of summer, St. Augustinegrass sod

is often heavily irrigated and mowed, which produces favorable growth conditions for G.

graminis var. graminis because of surplus moisture and accumulating clippings which

add to thatch layers. In this survey, the pathogen is most active during periods when

AMF percent colonization is lowest suggesting a limited role for AMF in take-all root rot

disease suppression in these soils. More controlled studies might shed light on potential

AMF effects on soilborne pathogens which may be confounded during field evaluations

due to rhizosphere variability and environmental effects. If these criteria can be

evaluated under less variable conditions, beneficial AMF effects could be evaluated and






67


perhaps manipulated for optimal disease suppression and concurrent decreases in

pesticide use.










































Fig. 3-1. 'Floratam' St. Augustinegrass sod mat infected with Gaeumannomyces
graminis var. graminis. Insert in bottom right-hand corner depicts underside
of a mat with rotting roots.











































Figs. 3-2 3-3. Comparison of healthy 'Floratam' St. Augustinegrass sod mat and sod
affected by brown patch.
Fig. 3-2. Healthy 'Floratam' St. Augustinegrass sod mat.
Fig. 3-3. 'Floratam' St. Augustinegrass sod mat infected with R. solani causing brown
patch.


.-::li:;u~; r;
































Fig. 3-4. Deeply-lobed hyphopodia isolated from Gaeumannomyces graminis var.
graminis in 'Floratam' St. Augustinegrass sod samples. Scale bar = 40 nm.
























Fig. 3-5. Medium isolation plate depicting a Gaeumannomyces graminis var. graminis
colony isolated from 'Floratam' St. Augustinegrass sod samples. Arrow
points to colony.

































Fig. 3-6. Rhizoctonia solani hyphae isolated from 'Floratam' St. Augustinegrass sod
exhibiting diagnostic 900 branching at constriction points and characteristic
septa. Scale bar = 40 inm. Arrow points to branching pattern.


Fig. 3-7. Medium isolation plate depicting light brown Rhizoctonia solani colony
isolated from 'Floratam' St. Augustinegrass sod samples. Arrows point to
colonies.
























30
S25 a








Dec Jan April March June Aug Oct
04 05 05 05 05 05 05
Date
Date


Fig. 3-8. Mean percent of Rhizoctonia solani colonization of 'Floratam' St.
Augustinegrass in north central Florida. Means followed by the same number
are not significantly different according to the Tukey's mean separation test (P
< 0.05). The percent colonization is based on the mean number of colonies
where R. solani was recovered.


























25
a a
. 020
.5 ab ab ab
E 15
e b
10


0
Dec Jan April March June Aug Oct
04 05 05 05 05 05 05
Date


Fig. 3-9. Mean percent of Gaeumannomyces graminis var. graminis colonization of
'Floratam' St. Augustinegrass in north central Florida. Means followed by the
same number are not significantly different according to the Tukey's mean
separation test (P < 0.05). The percent colonization is based on the mean
number of colonies where G. graminis var. graminis was recovered.














CHAPTER 4
EFFECT OF GLOMUS INTRARADICES ON THE EXTENT OF DISEASE CAUSED
BY GAEUMANNOMYCES GRAMINIS VAR. GRAMINIS AND RHIZOCTONIA
SOLANI IN ST. AUGUSTINEGRASS

Arbuscular mycorrhizal fungi (AMF) are widespread symbionts in the majority of

plant species; and are associated with increased plant vigor via improved nutrient uptake,

especially P, and increased water acquisition (Smith and Read, 1997). The beneficial

effects of AMF on crop yield have been thoroughly documented (Harley and Smith,

1983). There is much debate on whether or not AMF alter plant resistance to pathogens

by an indirect mechanism or simply interact directly with the pathogens themselves.

When AMF act as pathogen antagonists, there are likely one or more mechanisms

of resistance. For example, AMF may be deterring pathogen infection by increasing

plant vigor through improved nutrient acquisition, the AMF themselves may be

producing anti- microbial metabolites, or the AMF may be stimulating the plant's own

natural defense response to colonization by increasing phytoalexin production (Schenck,

1970). Previous studies have indicated that AMF symbiosis greatly improves plant

resistance to abiotic pressures such as water stress (Sylvia and Williams, 1992) and

transplant shock (Menge et al., 1978) in various crops. AMF have also been evaluated as

biological controls against biotic stresses such as bacterial pathogens (Weaver and

Wehunt, 1975), parasitic nematodes (Baltruschat et al., 1973; Schenck and Kellam,

1978), viral pathogens (Daft and Okusanya, 1973; Giannakis and Sanders, 1989), and

soilborne fungal pathogens (Jeffries, 1987; Schenck, 1987; Hooker et al., 1994;

Linderman, 1994; Azc6n-Aquilar and Barea, 1996).









The vast majority of evaluations concerning the effects of AMF on disease severity

involve fungal pathogens (Schenck and Kellam, 1978). The first report of an interaction

between mycorrhizal fungi and fungal pathogens involved soybean (Glycine max L.

Merr) and Phytophthora root rot, where the mycorrhizal plants actually had higher rates

of disease versus the nonmycorrhizal plants (Ross, 1972). In other reports, AMF had no

effect on disease at all (Ramirez, 1974; Sherinkina, 1975). Depending on the stage of

host plant development, plant and mycorrhizal fungal species, and the complexities

between biotic and abiotic rhizosphere factors, there is evidence that mycorrhizal

interactions lie along a continuum ranging from mutualistic to parasitic, commensal,

amensal, and potentially even neutral (Johnson et al., 1997). However, there are many

reports of mycorrhizal colonization reducing disease severity in many plant systems such

as pea, tomato, soybean, wheat, and peanut involving such fungal pathogens as Fusarium

solani Mart. (Sacc.), G. graminis (Sacc.) Arx & Olivier var. tritici J. Walker, Sclerotium

rolfsii (Sacc.), Pythium spp., Phytophthora parasitica Dastur, and R. solani Ktuhn

(Graham and Menge, 1992; Dehne, 1982; Krishna and Bagyaraj, 1983; Zambolim and

Schenck, 1983; Hedge and Rai, 1984; Vigo et al., 2000; Yao et al., 2002).

In fact, the effects of mycorrhizal colonization on disease severity is potentially so

important that Newsham et al. (1995) suggested that the benefits of AMF to disease

suppression may be as important as the nutritional benefits derived from the symbiosis in

some instances. For example, in temperate grasslands, the effects of a direct AMF

interaction with root pathogens reduced disease severity and increased plant vigor and

fecundity greatly (Newsham et al., 1995). Soilborne pathogen suppression by AMF

includes both physical and physiological mechanisms (Sharma et al., 1992). Physical









plant defense responses against pathogen penetration are: increased lignification (Dehne

and Schoenbeck, 1978), greater mechanical strength and nutrient flow within vascular

systems (Schoenbeck, 1979), and direct competition with the pathogen for cortical

infection courts and resources (Graham, 2001). Becker (1976) observed that pathogen

penetration of root cells was directly reduced by the presence of AMF and not indirectly

by a systemic plant resistance based on thickening cell walls. In some cases the direct

influence of AMF may be the only reason for observations of disease resistance. It is

important to establish whether or not particular plant systems benefit, suffer, or remain

unaltered by mycorrhizal colonization. If the relationship appears to be beneficial,

Gerdemann (1975) remarked that the effect of mycorrhizal fungi on disease should be

determined whether resistance is due to direct or indirect mechanisms.

The host-pathogen relationship can be greatly influenced by indirect or

physiological effects of AMF through increased P nutrition, enhanced mycorrhizal root

growth which aids in disease escape, or up-regulation of pathogenesis-related proteins

(Gianinazzi-Pearson and Gianinazzi, 1989; Blee and Anderson, 2000; Graham, 2001).

AMF may also be responsible for lowering disease severity in complex reactions

involving host physiology such as the production of rhizosphere leachates from

mycorrhizal plant roots. These leachates have been observed to substantially limit the

production of zoospores and sporangia of Phytophthora cinnamomi Ronds in sweet corn

and chrysanthemum (Meyer and Linderman, 1986).

There appears to be no information concerning the effects of AMF, if any, on

disease severity in St. Augustinegrass. If there is a direct or indirect beneficial effect of

AMF on disease severity of St. Augustinegrass in relation to brown patch or take-all root









rot, several questions will remain concerning the actual mechanism of observed

resistance. However, without basic information and techniques to differentiate between

direct and indirect effects and to determine what extent disease severity may or may not

be lowered, further evaluations would not be warranted.

The economic importance of AMF in soils of north central Florida St.

Augustinegrass sod fields may be considerable where diseases such as brown patch and

take-all root rot reduce harvestable hectares. Arbuscular mycorrhizal fungi can stimulate

plant vigor and possibly interact directly or indirectly with soilborne pathogens to limit

disease. AMF have been observed colonizing St. Augustinegrass (see Chapter 2), and

they might benefit sod production. The potential AMF benefits to sod growers include

reduced loss of sod and revenue to soilborne pathogens, and lowered management costs

through reduced fungicide use. The potential advantages of AMF inoculation or field

manipulation with specialized techniques may also benefit the environment by decreasing

soil and water pollution through reduced of fungicide use. For these reasons, it is prudent

to evaluate the potential benefits of AMF to disease resistance whether by direct or

indirect mechanisms in St. Augustinegrass sod. As part of ongoing research on the effect

of AMF on disease severity in St. Augustinegrass, the objective of this study was to

determine the effect of G. intraradices, on St. Augustinegrass in disease development by

challenging it both directly and indirectly with G. graminis var. graminis or R. solani.

Material and Methods

Direct Experiments

St. Augustinegrass Sprig Propagation and Stock Plants.- 'Floratam' St.

Augustinegrass sprigs having no apparent signs or symptoms of disease were obtained

from Hendrick's Turf Farm (Lake Butler, Florida). The sprigs were rooted in flat, plastic









nursery trays or 18 cm clay pots in a sterilized Arrodondo fine sand medium

supplemented with a nutrient solution (Appendix B) every three weeks. The sprigs were

grown and maintained in a growth chamber at 25-27 C under cool-white fluorescent

bulbs with irradiance at 25 ilE/m2/s and a 15 h photoperiod/day. Sprigs were watered

every other day throughout the experimental period with water adjusted to pH 6.0-6.5.

After approximately 6 weeks of propagation, selected sprigs, not in direct contact with

soil, were excised from the edge of the flat trays and replanted as sterile stock plantlets.

These sub-cultured plants were maintained as described above until additional sprigs, not

touching the soil and hanging from the edge of the tray, were collected for

experimentation.

R. solani Inoculum Production.- A virulent strain of R. solani (PDC 7884) (Fig. 4-1)

isolated from diseased St. Augustinegrass submitted by a homeowner in Leon County,

Florida was provided by the Plant Disease Clinic (Institute of Food and Agricultural

Sciences, University of Florida, Gainesville, Florida). The isolate was cultured at 4 C

and stored on potato dextrose agar (Difco Laboratories, Inc., Detroit, Michigan) for

approximately 2 weeks. An oat (Avena sativa L.) inoculum was prepared according to

Sneh et al. (1991) and Gaskill (1968) with modifications (Appendix C) and inoculated

with agar plugs from actively growing R. solani (PDC 7884) mycelium or with sterile

agar plugs (control). The inoculum substrate was incubated at 21 C with a 12 h

photoperiod for 4 weeks and shaken 2-3 times/week to prevent packing of the oat seeds.

The inoculated seeds were then air-dried, sealed in plastic zip-lock bags, and stored at

room temperature until use.









G. graminis var. graminis Inoculum Production.- A virulent strain of G. graminis var.

graminis (JK2) was collected and identified from diseased St. Augustinegrass (Fig. 4-2)

from the lawn of Dr. James Kimbrough (Gainesville, Florida) and isolated on selective

media amended with antibiotics (Appendix A). Actively growing G. graminis var.

graminis mycelium from a single Petri dish was chopped and combined with sterilized

ryegrass seed as described by Datnoff and Elliott (1997) with modification (Appendix C).

The inoculated flasks of sterile ryegrass seed substrate and uninoculated control flasks

were incubated in total darkness at 21 C for 4 weeks prior to use. The flasks were shaken

2-3 times/week to prevent packing of the inoculated ryegrass seed.

Mycorrhization of 'Floratam' St. Augustinegrass Sprigs.- Sprigs of 'Floratam' St.

Augustinegrass were selected from the edge of sterile stock plants in flat trays, as

previously described. Sprigs were inspected visually for any signs or symptoms of

potential pathogens or diseases, and if healthy, were selected for experimental use. The

sprigs were then planted into 6.8 cm wide by 18 cm deep containers (Steuwe and Sons,

Inc., Corvallis, Oregon) filled with a sterilized low P soil, as mentioned in Chapter 2 (Fig.

4-3). The sprigs were then placed in a controlled growth room with a 15 h photoperiod/d

at 21-25 C, watered daily with pH adjusted 6.0-6.5 deionized water, and maintained for

approximately 3 weeks to allow root development to occur and transplant shock to

subside. After the 3 week growth period, the sprigs, with approximately 8 cm of root

length, were inoculated with approximately 20 spores of G. intraradices (FL 208A) (Fig.

4-4) obtained from the INVAM Culture Collection (Morgantown, West Virginia) or

noninoculated water controls. The FL 208A isolate was selected because it was first

isolated in a citrus grove in central Florida, near Orlando, in 1978 in 7.0-7.5 pH soil,









which is similar to that of the sod fields in north central Florida. The sprigs were then

acclimatized for approximately 4 weeks in the growth room to allow the AMF time to

colonize the sprig roots, which was determined at 2 and 4 weeks in extra experimental

units.

Pathogen Inoculation.- The AMF colonized sprigs were inoculated with either R. solani

(PDC 7884) or the G. graminis var. graminis (JK2) isolate or uninoculated as controls by

gently pushing the soil aside to expose a portion of the roots near the crown of the sprig.

Approximately 3-5 infected seeds of either the R. solani inoculated oat substrate or G.

graminis var. graminis inoculated ryegrass seed substrate were placed equidistant from

the crown in each container at a 1-2 cm distance from the plant. The soil was carefully

replaced following inoculation. Inoculated sprigs were maintained in the growth room

for approximately 4 weeks with a 15 h photoperiod/d at 21-25 C. Each cone was

supplied with a nutrient solution devoid of P on two occasions at 50 ml/conetainer

(Appendix B). Plants were watered daily with 50 ml water/conetainer adjusted to 6.0-6.5

pH.

Mycorrhizal Evaluation.- Roots from the sprigs were rinsed in tap water and separated

with a scalpel from the plant crown. Selected roots were cut into 1-2 cm long segments,

put into porous nylon sleeves, inserted in small, plastic clips (Fig. 4-5), and the cell and

wall components cleared in 10% KOH (w/v) under pressure in an autoclave for

approximately 20 min at 121 C psi (Brundrett et al., 1996). The root segments were

cooled, then rinsed in tap water, and placed into 0.05% trypan blue in 25% glycerol

overnight to stain mycorrhizal structures (Bevenge, 1968; Phillips and Hayman, 1970;

Kormanik and McGraw, 1982). Excess stain was rinsed from the root segments with tap









water and then the roots were mounted in water on glass slides to view vesicles,

intraradical hyphae, and arbuscules (Fig. 4-6).

Root segments from each replicate were pooled from each treatment, and

evaluated for intensity of colonization. Mycorrhizal structures on glass slides were

viewed with a Nikon Optiphot compound microscope at 200, 400, and 1000x

magnifications, and photographs were taken with a Nikon CoolPix 990 digital camera. In

order to judge the amount of mycorrhizal root colonization, the grid line intersect method

was used to approximate the total root length colonized by AMF (Newman, 1966;

Tennant, 1975; Giovannetti and Mosse, 1980).

Direct Experiment Disease Assessment.- Disease severity (root and shoot rot) was rated

at the conclusion of a 3 week growth period on both the AMF inoculated, pathogen

inoculated, and control sprigs. Disease severity was assessed using an arbitrary disease

scale from 1 to 6 with 1 = no symptoms of disease; 2 = 1-25% disease; 3 = 26-50%

disease; 4 = 51-75% disease; 5 = 76-100% disease; and 6 = plant death (Figs. 4-7; 4-8).

The presence of either the R. solani or G. graminis var. graminis pathogens on each

infected sprig was confirmed by re-isolation of each pathogen (Figs. 4-9; 4-10) on

selective media (Appendix A). For each sprig, the percent colonization of the pathogen

and/or AMF was recorded as described in Chapters 2 and 3.

Direct Experiment Design and Statistical Analysis.- The experiment was performed using

a factorial arrangement (1 cultivar of St. Augustinegrass) x (1 AMF + uninfected

pathogen control) x (1 R. solani-infected + 1 AMF) x (1 R. solani- infected AMF) and

(1 G. graminis var. graminis- infected + 1 AMF) x (1 G. graminis var. graminis AMF)

and (uninfected pathogen control + uninoculated AMF control) in a randomized complete









block design with four replicates/treatment (Fig. 4-11). Regression analyses were

performed with the regression procedure in SAS (SAS Institute, 2004) (Appendix F-4).

All data presented are the means of four replicates. As there were no differences between

trials based on the ANOVA, all data presented were combined for the purpose of

presenting the results and discussion more easily.

Indirect Experiments

St. Augustinegrass sprigs were produced and maintained in the same manner as described

above in the Direct Experiment section as were mycorrhization and pathogen inoculum

production, inoculation, and quantification. However, in this experiment, the potential

effects of indirect AMF interactions with soilborne pathogens were evaluated instead of

the potential direct impacts of mycorrhization. Instead of a direct challenge between

AMF and pathogen in one container, indirect effects were investigated using a split-root

assay.

Indirect AMF Challenge Split-Root Assay.- Sterile, 4 week old 'Floratam' St.

Augustinegrass sprigs with approximately 8 cm of healthy root tissue were placed into

two adjacent containers with one rooted end of the sprig in one container and the other

rooted end in another container (Fig. 4-12). Holes (1 cm in diameter) were drilled 2.5

cm from the top of each 6.5 cm wide by 18 cm deep container (Steuwe and Sons, Inc.,

Corvallis, Oregon) prior to planting, on one side of the container (Appendix E- 1). A cut

was made from the top of the drilled hole to the top of each container to allow the sprig

to be inserted into the hole without tissue damage. Sprigs were planted into containers

filled with sterile low P soil as previously described and maintained in the growth room

for 3 weeks to limit transplant shock and acclimatize the sprigs. Sprigs were then

inoculated with the G. intraradices isolate (FL 208A) as described in the direct









experiment above, or a control substrate in one container, with either the G. graminis

var. graminis isolate (JK2) or R. solani isolate (PDC 7884) inoculated or an uninoculated

control substrate in the adjacent container occupied by the other rooted end of that same

sprig (Fig. 4-13). The containers were watered daily with 50 ml water/conetainer

adjusted to pH 6.0-6.5 and supplied with a nutrient solution on two occasions (Appendix

B). The sprigs were maintained for 3 weeks in the growth chamber at 21-25 C with a 15

h photoperiod. The sprigs were visually inspected every 2-3 d for the presence of

invading pathogenic mycelia along the stolon portion of the sprig to prevent cross

contamination. The presence of the pathogen used to inoculate one container was not

observed in any of the adjacent experimental units containersr) based on the lack of

recovery of the pathogen from adjacent containers by selective media isolation

(Appendix A). The stolon portion spanning the distance between the two adjacent

containers was approximately 5 cm in length. Percent G. intraradices colonization was

measured using the gridline intersect method described in the previous section.

Indirect Experiment Design and Statistical Analysis.- The experiment was performed

using a factorial arrangement (1 cultivar of St. Augustinegrass) x (1 AMF + uninfected

pathogen control) x (1 R. solani-infected + 1 AMF) x (1 R. solani- infected AMF) and

(1 G. graminis var. graminis- infected + 1 AMF) x (1 G. graminis var. graminis AMF)

and (uninfected pathogen control + uninoculated AMF control) split-root assay in a

randomized complete block design with four replicates. The entire experiment was setup

three times from January May 2006. Regression analyses were performed with the

regression procedure in SAS (SAS Institute, 2004) (Appendix F-5). All data presented









are the means of four replicates/treatment. No differences were found between trials

based on the ANOVA, therefore, data were pooled for analysis.

Results

Direct Experiments

Mycorrhizal Colonization.- In the direct experiment, mean values of root colonization by

the AMF, Glomus intraradices, were 10% for the R. solani- infect + AMF treatment,

11.3% for the AMF inoculated control treatment (no pathogen), and 11.7% for the G.

graminis var. graminis-infected + AMF treatment, respectively, after mycorrhizal

inoculation. Root colonization of AMF was not significantly affected by the direct

presence of either pathogen nor did the AMF control treatment (no pathogen) have any

direct effect, either positive or negative, on disease severity itself (Appendix D-1). In this

study, the colonization of plants by AMF, G. intraradices, apparently had a neutral effect

on the St. Augustinegrass plants without the direct presence of either pathogen nor did

the AMF affect plant growth.

Disease Development.- The direct effect of G. intraradices on brown patch (caused by R.

solani) disease severity was evaluated by first investigating the relationship of the R.

solani- infected control (no AMF) treatment (Appendix D-2) to disease severity. The

mean percent colonization of the R. solani-infected control treatment was 60%, but the

disease severity (mean = 3.8 on a scale of 1 to 6) was not significantly correlated with the

mean colonization percentage of R. solani using the regression procedure in SAS (SAS

Institute, 2004). Since there was no definitive relationship between plant disease

severity and the percentage of R. solani colonization with this treatment, there was no

need to assume that G. intraradices in the R.solani-infected + AMF treatment would have

a beneficial effect on disease severity. This was supported by the regression analysis









comparing the relationship of disease severity to percent R. solani colonization (mean

colonization = 57%) in the R. solani- infected + AMF treatment (Appendix D-3) where

disease severity (3.3 on a scale of 1 to 6) was not correlated to the mean percentage of

AMF colonization (mean colonization = 18%). In this study, the AMF treatments had no

effect on disease severity in the direct presence of R. solani regardless of the mean

colonization of the pathogen or AMF.

The direct effect of G. intraradices on disease severity was also evaluated in this

study for take-all root rot caused by G. graminis var. graminis. Based on regression, the

relationship between disease severity and the G. graminis var. graminis- infected control

(no AMF), it appears that the pathogen (mean colonization = 42.8%) had a significant

relationship (r2 = 0.65) with disease severity (2.4 on a scale of 1 to 6). This model shows

that as disease severity increases so does G. graminis var. graminis percent colonization

in a direct pathogenicity challenge (Fig. 4-14). This finding suggests that the AMF could

potentially have a direct effect on disease severity and that the relationship could be

evaluated since the percent colonization of G. graminis var. graminis had a measurable

effect on disease severity. The regression analysis of disease severity (mean = 3.3 on a

scale of 1 to 6) to the G. graminis var. gramninis- infected + AMF inoculated treatment

revealed a highly correlated relationship between the treatment and disease severity (r2 =

0.81). As disease severity increased according to this treatment, so did the percent

colonization of G. graminis var. graminis even in the direct presence of AMF (mean =

8.6%) (Fig. 4-15). There was no apparent reduction or increase in disease severity.

Therefore, the AMF have no direct beneficial effect on take-all root rot disease severity.

Additionally, the AMF treatment alone could not be correlated to a reduction in percent









G. graminis var. graminis colonization (data not shown) nor did the treatment have a

direct effect on lowering take-all root rot disease severity since the disease severity trend

did not differ from that of the G. graminis var. graminis- infected AMF treatment.

Since disease severity was not affected by G. intraradices in the G. graminis var.

graminis- infected + AMF treatment or correlated to the percent of G. graminis var.

graminis colonization in the control uninoculated with AMF, it appears that the AMF

colonization had no direct negative or positive impact on the pathogen or disease

severity. In this study, the interaction between AMF and the plant in the direct presence

of the pathogens, G. graminis var. graminis and R. solani would thus be considered

neutral in nature.

Discussion

More importantly, this study demonstrates that mycorrhization with the AMF, G.

intraradices, did not reduce development of R. solani or G. graminis var. graminis in

direct contact nor did the AMF treatment reduce or increase disease severity of brown

patch or take-all root rot in 'Floratam' St. Augustinegrass, as has been observed in other

mycorrhizal studies (Ross, 1972; St. Arnaud et al., 1994; Mark and Cassells, 1996).

Arbuscular mycorrhizal fungi have been associated with increased disease severity in

some instances with R. solani, so analysis based on this assumption was as necessary as

assuming the AMF treatment would lower disease severity (Ramirez, 1974; Sherinkina,

1975; Johnson et al., 1997; Yao et al., 2002). No beneficial effects of AMF inoculation

on take-all root rot or brown patch disease severity in St. Augustinegrass were observed.

This is perhaps due to the relatively low levels of mycorrhizal root colonization. Possibly

AMF inoculation would be more beneficial to plants with a higher level of mycorrhizal

colonization.









In summary, the results show that the purported beneficial effects of direct AVIMF

interactions with plant roots such as increased cell wall lignification or the production of

antagonistic mycorrhizal root exudates did not play a role in this study (Becker, 1976;

Dehne and Schoenbeck, 1978; Graham, 2001). Thus, inoculation with G. intraradices

will not improve disease severity or reduce disease development. The effects of such an

interaction within field trials could potentially yield contradictory results, and the

microbial and environmental variability within the rhizosphere would make such

experiments difficult at best.

Results

Indirect Experiment

In order to thoroughly evaluate the potential effects of AMF on disease severity

and/or soilborne pathogen development, another series of studies involving a more

indirect method was performed simultaneously with the direct experiment described

above. This assay was designed to isolate potential systemic resistance responses from

mycorrhization which have been documented (Gianinazzi-Pearson and Gianinazzi, 1989;

Blee and Anderson, 2000; Graham, 2001).

In this assay, the R. solani control (no AMF) treatment revealed a significant

correlation between pathogen colonization and disease severity (Fig. 4-16). In this

instance, as percent colonization of R. solani (mean = 54.9%) increased so did disease

severity (mean = 3.5 on a scale of 1 to 6; r2 = 0.75). Since there was a significant

relationship between the pathogen and disease severity, the regression procedure in SAS

was also used to analyze the indirect effects of the R. solani + G. intraradices treatment

on disease severity. The combination of this pathogen and AMF in an indirect assay,

where one container was inoculated with R. solani and the other container containing