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1 WAITEA AND RHIZOCTONIA PATHOGENS OF SEASHORE PASPALUM THE ROLE OF SALINITY IN DISEASE EXPRESSION AND CHARACTERIZATION OF A NEW WAITEA CIRCINATA VARIETY CAUSING BASAL LEAF BLIGHT By STEVEN JOSEPH KAMMERER A DISSERTATION P RESENTED 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 2011
2 2011 Steven Joseph Kammerer
3 To my wife and best friend Ros emary, my greatest sourc e of support and encouragement and to God for all his blessings and guidance in my life
4 ACKNOWLEDGMENTS I would like to thank my advisor, Phil Harmon for all his guidance and help but more importantly for accepting me as a grad uate student. I also want to thank all the members of my graduate committee, Lee Burpee, David Norman, and Kevin Kenworthy for their help and support throughout this project. I also want to thank my fellow graduate students. Todd Cooper has been a great friend, and I thank him for all his advice and help maintaining the turfgrass for these studies. Norma Flor was also a good friend and lent me assistance Other people who were helpful to me in this endeavor, Patti Rayside, Carol Stiles, Brenda Rutherfor d, and the superintendents at the golf courses I worked with at Old Palm, Boca West, Vero Beach Country Blub Parkland, Tuscany Reserve, the Plantation at Somerset, Hammock Bay and the Oaks Club, for allowing me to sample and collect data over two years. T hanks to the University of Florida and to my employer Syngenta for seeing potential in me and the knowledge I would gain upon completion to justify funding my tuition for this endeavor Lastly, I express my deepest appreciation to my wife, without her urg ing over the years, I would have never even attempted to return to school for such a daunting understanding and confidence in me for all the days and nights whe n I wa s gone in addition to my already busy schedule as a full time Syngenta employee is a lot for any family to endure During the lonely, frustrating nights away from home alone in a camper, their love, encouragement and support kept me going.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 REVIEW OF LITERATURE ................................ ................................ .................... 14 Seashore Paspalum ................................ ................................ ............................... 14 Rhizoctonia ................................ ................................ ................................ ............. 18 Rhizoctonia and Turfgrass ................................ ................................ ...................... 23 Rhizoctonia solani ................................ ................................ ............................ 23 Rhizoctonia zeae and Rhizoctonia oryzae ................................ ....................... 27 Salinity Issues ................................ ................................ ................................ ......... 30 2 SURVEY FOR RHIZOCTONIA SPECIES ISOLATED FROM SEASHORE PASPALUM GOLF COURSES IN FLORIDA ................................ .......................... 35 Introduction ................................ ................................ ................................ ............. 35 Material s and Methods ................................ ................................ ............................ 38 Turfgrass Sampling ................................ ................................ .......................... 38 Temperature, soil pH and EC determination ................................ .............. 39 Fungal isolations ................................ ................................ ........................ 40 Morphological Characterization and Groupings ................................ ................ 40 Molecular DNA methods and isolate identification ................................ ..... 41 Correlation of Edaphic Factors and Isolation Frequency of Fungi .................... 43 Results ................................ ................................ ................................ .................... 44 Isolates Recovered Phylogenetics, Morphology and Descriptions ................ 44 Means and Isolate Recovery Frequencies ................................ ....................... 46 Isolate Recovery Corre lations ................................ ................................ .......... 47 Discussion ................................ ................................ ................................ .............. 47 3 IDENTIFICATION OF A NEW WAITEA CIRCINATA VARIETY CAUSING BASAL LEAF BLIGHT OF SEASHORE PASPALUM ................................ ............. 63 Introduction ................................ ................................ ................................ ............. 63 Materials and Methods ................................ ................................ ............................ 65 Turfgrass Sampling and Isolate Maintenan ce. ................................ ................. 65
6 Colony and Fungal Characteristics. ................................ ................................ .. 66 Internal Transcribed Spacer Region DNA Sequencing. ................................ ... 66 DNA Cloning. ................................ ................................ ................................ .... 67 Temperature, Growth Studies. ................................ ................................ ......... 68 Pathogenicity Studies. ................................ ................................ ...................... 68 Results ................................ ................................ ................................ .................... 71 Colony Morphology and Characterization. ................................ ........................ 71 rDNA ITS Sequence Analysis. ................................ ................................ ......... 7 1 Turfgrass Pathogenicity Studies. ................................ ................................ ...... 72 Discussion ................................ ................................ ................................ .............. 73 4 THE INTERACTION OF CHRYSORHIZA ZEAE RHIZOC TONIA SOLANI AG 2 2LP AND SALINE WATER ON PASPALUM VAGINATUM ................................ 85 Introduction ................................ ................................ ................................ ............. 85 Materials and Methods ................................ ................................ ............................ 86 Pathogen Isolations and Identification ................................ .............................. 86 Inoculum Preparation ................................ ................................ ....................... 87 Turfgrass Establishment and Maintena nce ................................ ...................... 88 Salt Treatments ................................ ................................ ................................ 88 Chrysorhiza zeae in o culations ................................ ................................ ... 89 Rhizoctonia so lani AG 2 2LP inoculations ................................ ................. 90 Results ................................ ................................ ................................ .................... 91 Chrysorhiza zeae Salinity Experiment 1 ................................ ........................... 91 Chrysorhiza zeae Salinity Experiment 2 ................................ ........................... 92 Rhizoctonia solani AG 2 2LP Salinity Experiment 1 ................................ ......... 92 Rhizoctonia solani AG 2 2LP S alinity Experiment 2 ................................ ......... 93 Discussion ................................ ................................ ................................ .............. 94 LIST OF REFERENCES ................................ ................................ ............................. 104 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 115
7 LIST OF TABLES Table page 1 1 dissertation ................................ ................................ ................................ ......... 34 2 1 Species utilized in this study and identification from sampling effort (or otherwise noted) utilizing rDNA internal transcribed spacer (ITS) region sequences for genetic characterization ................................ .............................. 51 2 2 Isolate recovery frequencies and SP tissue location ................................ .......... 52 2 3 Mean of Rhizoctonia like fungus isolation recovered from seashore paspalum tissue, soil EC (sali nity), soil pH, soil temperature, and canopy temperatures ... 52 2 4 Mean Rhizoctonia like isolate recovery from golf course sites, EC and pH over nine sampling events ................................ ................................ .................. 53 2 5 Mean recovery frequencies of total and individual Rhizoctonia like isolates recovered, soil and canopy temperatures, EC, and pH values from SP golf courses over nine sampling events ................................ ................................ .... 54 2 6 Glimmix (General Linear Mixed model) for mean incidence of selected fungus on SP tissue vs. soil temperature, canopy temperature, EC and pH ...... 54 2 7 Pearson correlati on coefficients for mean incidence of selected fungi on SP tissue vs. soil temperature, canopy temperature, EC and pH. ............................ 55 3 1 Isolates of Waitea circinata and related fungi that were examined uti lizing rDNA internal transcribed spacer (ITS) region sequences ................................ 77 3 2 Comparison of hyphal and sclerotial characteristics of isolates from symptomatic SP in South Florida ................................ ................................ ........ 78 3 3 Mycelial growth rates of Waitea circinata isolates after 24 h on potato dextrose agar. ................................ ................................ ................................ ..... 78 3 4 Pathogenicity of Waitea circinata isolate SK PSA TM4 on S P, bermudagrass, and roughstalk bluegrass ................................ ................................ ................... 79 4 1 Trial 1 Interaction of C. zeae inoculation and saline water on 'SeaDwarf' SP .. 97 4 2 Trial 2 Interaction of C. zeae inoculation and saline water on 'SeaDwarf' SP .. 97 4 3 saline irrigation as affected b y inoculation with Rhizoctonia solani AG 2 2LP .... 97
8 4 4 Trial 1 Interaction of R. solani AG 2 2LP inoculation and saline water on 'SeaDwarf' SP ................................ ................................ ................................ ..... 98 4 5 Trial 2 Interaction of R. solani AG 2 2LP inoculation and saline water on 'SeaDwarf' SP ................................ ................................ ................................ ..... 98
9 LIST OF FIGURES Figure page 2 1 Locations and distances between golf courses utilized in this survey. ............... 56 2 2 Phylogenetic distance tree comparing nucleotide sequences from the rDNA of isolates of Thanatephorus Waitea and Ceratobasidiu m (UCAG unknown Ceratobasidium anastomsis group). ................................ .................... 57 2 3 Thanatephorus cucumeris ( Rhizoctonia solani ) 28 d culture isolates. A) Rhizoctonia solani AG 2 2IIIB. B) under 100X magnification. C) Rhizoctonia solani AG 2 2LP. D) under 100X magnification. ................................ ................ 58 2 4 Ceratobasidium spp. 28 d colony characteristics. A) Ceratobasidium sp. AG G. B) Ceratobasidium sp. AG L. C) Ceratobasidium AG Q. .D) unidentified Ceratobasidium anastomosis group. ................................ ................................ .. 59 2 5 Waitea circinata varieties. A1) W c var. prodigus ; colony, A2) 40X stained safranin O, A3) sclerotia. B1) W c var. zeae ; co lony, B2) 100X, B3) sclerotia. C1) W c var. oryzae colony, C2) 100X, C3) sclerotia. .............. 60 2 6 Average soil temperatures (6 cm depth) compared to average turf canopy temperatures on eight Sout h Florida SP fairways over a two year period. ......... 61 2 7 Number of Thanatephorus cucumeris and Waitea circinata isolates recovered compared to historical average 5 year data from the FAWN for Fort La uderdale, FL for soil temperatures at 10 cm. ................................ .......... 61 2 8 Number of Thanatephorus cucumeris and Waitea circinata isolates recovered compared to historical average 5 year data from the FAWN for Fort La uderdale, FL for rainfall. ................................ ................................ .......... 62 3 1 ................................ ................................ ...... 79 3 2 A) Characteristics of UWC isolate (SK PSA TM4) colony on PDA. B) Sclerotial characteristics left to right of W circinata var. circinata UWC, W circinata var. oryzae and W circinata var. zeae ................................ ............... 80 3 3 Comparison of mycelial growth rates (mm per day) of Waitea circinata varieties on potato dextrose agar at 15, 20, 25, 30, 35, and 40C. ..................... 81 3 4 Genetic distance matrix based on sequence data utilizing rDNA internal transcribed spacer (ITS)1, 5.8S rRNA, and ITS2 regions of the rDNA locus from isolates of Waitea circinata and related fungi. ................................ ........... 82
10 3 5 Phylogenetic d istance tree comparing nucleotide sequences from the rDNA of Waitea circinata isolates and related fungi. ................................ .................... 83 3 6 Basal leaf blight symptoms 3 d after inoculation with W. c var. prodigus : A) inoculated (left) and inoculated (right). ................................ 84 4 1 C. zeae coupled with saline water (0 to 20,000 ppm NaCl). ................................ ........... 99 4 2 % Severity chlorosis/necrosis (SEV7) and SP 7 d after inoculation with (+) C zeae coupled with saline water (0 to 20,000 ppm NaCl). ................................ ................................ ........................... 100 4 3 culated with (+) C zeae coupled with saline water (0 to 20,000 ppm NaCl). ........................ 101 4 4 SP 27 d after inoculation with (+) C z eae coupled with saline water (0 to 20,000 ppm NaCl). ................................ ................................ ........................... 102 4 5 Turf quality (TQ) and % severity chlorosis/necrosis (SEV) ratings of R. solani) coupled wi th saline water (0 to 2 0,000 ppm NaCl). Bars or boxes with same letter are statistically equivalent (t 05 ). ......................... 103
11 LIST OF ABBREVIATION S bp base pairs (of a nucleotide sequence) d days h hour or hours FAWN Florida Automated Weather Network SP Seashore paspalum UCAG Unidentified Ceratobasidium anastomosis group UWC Unidentified Waitea circinata variety
12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Part ial Fulfillment of the Requirements for the Degree of Doctor of Philosophy WAITEA AND RHIZOCTONIA PATHOGENS OF SEASHORE PASPALUM THE ROLE OF SALINITY IN DISEASE EXPRESSION AND CHARACTERIZATION OF A NEW WAITEA CIRCINATA VARIETY CAUSING BASAL LEAF BLIGHT By Steven Joseph Kammerer August 2011 Chair: Philip F. Harmon Major: Plant Pathology Seahore paspalum is growing in popularity in different regions of the world as a high quality warm season turfgrass with the added benefit of tolerance to salinity. A great diversity of Rhizoctonia like isolates were recovered from eight seashore paspalum fairways across middle to Southern Florida including but not limited to R solani AG 2 2LP the causal pathogen of large patch disease The most diverse group of is olates recovered were Waitea circinata varieties. Soil temperatures, canopy temperatures, soil pH and soil electrical conductivity (as an indicator of salinity and sodium levels) was measured in conjunction with the isolates. Through the use of statistic al models, soil and canopy temperatures and increasing salinity were significant indicators of increased recovery of R solani 2 2LP and W. c var zeae from seashore paspalum tissue. Through the seashore paspalum sampling program, a new pathogen was isolat ed and identified. Waitea circinata var. prodigus causes a basal leaf blight of seashore paspalum. In controlled environment pathogenicity s t udie s, s ymptoms progressed most rapidly on creeping bentgrass and roughstalk bluegrass.
13 symptoms of basal leaf blight progress es from a foliar necrosis on the basal leaves to progressing rapidly up the turfgrass plant. Profuse pink to yellow mycelia with small salmon to yellow aggregate sclerotia developed in culture most rapidly at temperatures of 25 to 35C. Irrigation studies with four concentrations of sodium chloride (0, 10,000, 15,000 and 20,000 ppm) coupled with inoculations of pathogenic strains of Chyrsorhiza ze ae and R. solani AG 2 compared to mock inoculated turfgrass which served as an untreated comparison. Significant decreases in turf quality and increases in turf damage resulted as salinity increased and when inoculated with C. zeae and R. solani AG 2 2LP Dry weight was not a good consistent indicator of pathogenicity with either C. zeae or R. solani but did decrease when C. zeae inoculation was coupled with salt levels of 15,000 ppm and above. Ther e were no interactions, positive or negative, between saline water and SP quality or turf damage caused by R solani or C zeae.
14 CHAPTER 1 REVIEW OF LITERATURE Seashore Paspalum Paspalum vaginatum Swartz is a diploid (2n = 20) species in the Poaceae wi th a dark green foliage forming a dense canopy (Morton, 1973; Duncan and Carrow, 2000b; Duncan, 2003) The genus Paspalum i ncludes halophytic, salt water tolerant grass es with several reported places of origin from 1759 to 1983 (Duncan and Carrow, 2000 b; Duncan 2003). Seashore paspalum (SP), Paspalum vaginatum Swartz, most probably originat es from South Africa and is found growing naturally along seashores coastal riverbanks subject to periodic flooding, as well as in brackish waters (McCarty, 2005) S eashore paspalum produces a root system with deep, dense rhizomes and stolons more robust than bermudagrass, Cynodon dactylon (L.) Pers. A warm season perennial turfgrass species, SP has a fine to medium leaf texture with folde d leaf vernation, no auricl es and pointed leaf blade tips Leaves have large, compact s heaths containing short ligules (0.5 mm long) with a pubescent, broad collar. SP stems are smooth and flattened producing long, dense rhizomes with short inter n odes. Stolon nodes are described as especially pubescent. Although seed head production can be prolific, reproduction is handicapped due to self incompatibility between the same genotypes Flowering culms range from 8 to 60 cm in height, producing two to three smooth spikelets ranging in size from 3.5 to 4.0 mm long (Beard 2002). Paspalum vaginatum is part of a diverse group of grasses, subfamily Panicoideae, and in the tribe Panicea, which include such genera as Axonopus (common and tropical carpetgrass), Digitaria (crabgrass), Panicum (torpedograss), Pennisetum (kikuyugrass) and Stenotaphrum (St. Augustinegrass) in temperate to tropical climates (Watson and
15 Dallwitz, 1992). Because of their durability and pleasing a esthetic properties, St. Augustinegrass and kikyugrass are maintained a s desirable turfgrass es in lawns, industrial parks and sometimes as borders around golf course fairways. The gen us Paspalum contains noted species Paspalum notatum ( bahiagrass ) and Paspalum distichum L. ( saltwater couch or siltgrass ) (Watson and Dallwitz 1992 ; Smiley et al., 1994) Bahiagrass is a utility turf species with poor quality due to its slow recuperative potential, abundant production of seed head s and limited climate range encompassing warm humid climate s representative of tropical and sub tropical areas ( Beard and Beard, 2005 ). Paspalum vaginatum is a warm season species which utilizes the C 4 photosynthetic pathway, dicarboxylic acid cycle for energy capture and assimilation of carbon dioxide (CO 2 ) (Hull, 1992). Cool season grasses utilize a reductive pentose phosphate cycle or C 3 pathway. The C 4 versus C 3 classification is based on the number of carbon atoms in the first stage of CO 2 assimil ation during photosynthesis. During periods of elevated temperatures coupled with high light intensity, warm season C 4 grasses are more efficient than C 3 grasses in the ability to store CO 2 within the bundle sheath cells of the chloroplasts (Nobel, 1991). C 3 grasses incur higher photorespiration rates under these conditions because, in the absence of store d CO 2 oxygen (O 2 ) competes with CO 2 for the primary enzyme binding site necessary for carbon assimilation. Thus total respiration in C 3 grasses can ex ceed photosynthesis during these periods of extended high temperatures and high light intensities leading to increased environmental stress and an overall turfgrass decline or death (Hull, 1992).
16 Following breeding efforts, SP was introduced into the gol f course and lawn care markets in 1996 as a high quality turfgrass species with new cultivars requiring fewer environmental inputs (e.g. nitrogen fertilizer and irrigation water) compared to other turfgrass species and also having fewer pest problems (Alla r, 2001; Daniel 2003 ; Duncan 1996; Duncan and Carrow 2000a ; Kuo and Fermanian, 2001 ; Williams 2002) With its robust root and rhizome system SP can survive on as little as 30 to 50 percent of the water required by bermudagrass Seashore paspalum als o has a higher tolerance to low light levels and water logged soils than bermudagrass. These factors give SP a favorable environmental profile (Huang et al., 1997 ; Duncan and Carrow 2000 b; McCarty 2005 ). The decreased water requirement of SP compared to bermudagrass is more pronounced when low quality water such as saline water or effluent water versus potable water is used to irrigate the grass (Huang 1997). Lee et al. (2004 ) reported differences between varieties of SP in their tolerances to droug ht and saline stress. Seashore paspalum was noted to be highly salt tolerant and cabable of thriving in areas damaging to other turfgrass species (Dudeck and Peacock 1985 ; Lee et al., 2004). In salt affected ecosystems where only saline or re cycled wat er is available for irrigation, SP may be the only option for high quality turfgrass Seashore paspalum is a vigorous growing turfgrass species capable of having a desirable color and good mowing strip e c haracteristics but is noted to be prone to thatch a ccumulation (McCarty, 2005) A number of cultivars based on ecotypes selected for their adaptation to various environments now exist for use in turfgrass markets. Some of these cultivar s are better adapted to higher heights of cut in fairways while oth ers can tolerate lower heights of cut on putting greens. Seashore paspalum uses can range from low
17 maintenance such as a remediation turfgrass species in high salt areas, to higher maintenance uses on golf courses. As of September 1999, it was reported that there were 16,743 golf courses in the United States, Florida having more than any other state at 1228 golf courses ( verbal discussion with Todd Lowe, USGA Agronomist personal communication 2007 ). At the time ( 20 11 ) there we re estimated to be forty or more golf courses in Florida with SP and several courses in use or being constructed with SP in the Carib b ean Due to its noted poor cold tolerance, ideal geographical range overlaps with bermudagrass in tropical or subtropical areas of the world where freezing temperatures are transient or short in duration and severity (Duncan and Carrow 2000 a; Duncan & Carrow 2000b ; Duncan 2003 ; McCarty 2005). Scaptericus vicinus the tawny mole cricket, can be extremely damaging to bermudagrass, and many g olf courses routinely apply insecticides to manage mole crickets Braman (2004) indicate d that SP was more tolerant to tawny mole crickets than was bermudagrass. At the time of the introduction of SP to the turfgrass industry turfgrass breeders speculat ed that SP would have few fungal disease problems especially when established in saline environments, because fungi have poor salt tolerance (Duncan and Carrow 2000 b ). Very little research exists pertaining to disease susceptibility of SP Because SP t olerate s salt levels that would severely damage most other grasses and broadlea f species the use of salt as a herbicide or nematicide has been suggested (Hixson et al., 2004, Wiecko 2003). Hixson et al. (2004) investigated the effects of saline water tr eatments on control of sting nematode, Belonolaimus longicaudatus a significant pest
18 of SP in addition to effects of saline water on root length. Research with the parasitic nematodes Belonolaimus longicaudatus and Hoplolaimus galeatus the lance nemat ode, indicate d SP supported similar population counts to those supported by bermudagrass, suggesting similar susceptibility between the grasses (Hixson et al., 2004). I n the amended salt water treatments there was a definitive range of 10 to 15 dS m 1 tha t resulted in increased densities of B. longicaudatus whereas reproduction and feeding decreased at levels above 25 dS m 1 Root stunting due to B. longicaudatus occurred at salinity levels of 0 to 10 dS m 1 but decreased at 25 dS m 1 Gaeumannomyces gr aminis var. graminis an ectotrophic, root infecting fungus that has been associated with warm season turfgrass es SP (Elmore et al., 2002). Dollar spot, caused by Sclerotinia hom oeocarpa F. T. Bennett, has been reported to cause up to 95% turfgrass death on bahiagrass pasture fields (Blount 2002). Dollar spot also affects SP on US golf courses and was reported in China as a disease of Paspalum vaginatum ( Smiley et al., 1994, Lv et al., 2010). T he degree of su sceptibility of Paspalum vaginatum to the various pathogenic Rhizoctonia spp. is not well known (Morton, 1973). A study by Canegallo and Martin (2006) demonstrated that Rhizoctonia solani AG 2 2LP, caused large patch disease on several cultivars of SP in coastal South Carolina. Leaf and sheath spot caused by Rhizoctonia zeae has been diagnosed by the Univ. of Florida Rapid Turf D iagnostics lab ( P.F. Harmon personal communication ). Leaf and sheath spot was among the most diagnosed disease on the samples o f turfgrass submitted in 2006. Rhizoctonia T he genus Rhizoctonia was first noted by de Candolle in 1815 to consist of imperfect fung i that produced uniform sclerotia and were associated with roots of plants
19 ( Sneh et al., 1998). The name Rhizoctonia was derived from the Greek word s r h iza meaning root and ktonos murder or destroy. Rhizoctonia is classified in the Fungi Imperfecti. Thanatephorus, the teleomorph of Rhizoctonia, is a Hymenomycete in the Basidiomycotina. Rhizoctonia species are noted to be pathogens of economic concern on both monocots and dicots encompassing a wide host range of important agronomic crops including but not limited to cereals, fruits, legumes, vegetables and many annual, perennial, herbaceous and woody orname ntal plants (Farr et al., 1989). Some Rhizoctonia species are non pathogenic and develop mycorhizal, symbiotic relationship s with tropical orchids ( Farr et al., 1989, Otero et al., 2002). Classification of Rhizoctonia species historically has been based on the number of nuclei per hypha l cell, ability of hyphae to anatamose, and characteristics such as sclerotial morphology and mycelial pigmentation (Sneh et al 1998) A number of species with binucleate hyphal cells, such as Rhizoctonia cerealis and m any diverse multinucleate species that have been subdivided into variety of designations and anastomosis groups have at one time been included in the genus (Sanders et al., 1978; Martin et al., 1983a; Martin and Lucas 1984a ; Hyakumachi et al., 199 8; Tomos a Peterson and Trevathan, 2007). Rhizoctonia hyphae are uniform in size exhibiting consistent acute and right angle branching near the distal septum of younger hyphae with constriction at the point where branching occurs. Sclerotia are survival structu res that some Rhizoctonia species form (Hyakumachi et al., 1998). Sclerotia, if produced, can vary considerably in color and size. Monilioid cells, swoll en cells similar to sclerotia, produced by many isolates of R
20 solani and binucleate Rhizoctonia spp. also can serve as survival structures in soil, organic matter or thatch (Sneh et al., 1998). The sexual state of some Rhizoctonia species has been observed and differences in the teleomorphs have led to separate and distinct teleomorphic nomenclatures ba sed on morphological differences in basidia, basidiospores and sterigmata (Sneh et al., 1998, Stalpers and Andersen 1996) The t axonomy of teleomorphs and anamorphs can be confusing because different names are assigned for perfect and imperfect states o f the same fungal species. Several taxonomic revisions have stemmed from studies where the teleomorph stages of Rh i zoctonia species were induced and characterized (Moore, 1987; Parmeter et al., 1967; Oniki et al., 1985 ). Molecular biolog ists have pionee red a new approach to identification and classification of Rhizoctonia species, varieties and subgroups (Parmeter et al., 1967 ; Hayakawa et al., 2006 ; Hyakumachi et al., 1998 ; Yokoyama et al., 1985). Martin (1987) introduced a rapid method for differenti ation of multi nucleate versus bi nucleate Rhizoctonia spp. associated with turfgrass diseases. Fungal hyphae were incubated up to 24 h on agar coated microscope slides and subsequently stained with a DNA binding probe, 4 d i amidino 2 phenylindole (DAPI ) DAPI staining causes nuclei within fungal cells to fluore sce when viewed with ultraviolet light microscopy A major scientific breakthrough involving the purification of heat stable DNA polymerase coupled with oligonucleotide synthesis led to the abil ity to amplif y specific regions of DNA through repetitive replication cycles in a process known as p olymerase c hain reaction (PCR) (Arnheim and Erlich, 1992 ; White et al., 1990 ; Smyth 1992). The use of PCR has greatly facilitated the identification and d ifferentiation of fungal species,
21 varieties and strains This major advancement came with the discovery of methods to read portions of the DNA sequence of an organism through a process known as sequencing. As an example, i nternal transcribed spacer (ITS) regions exist as non functional and variable sequences between highly conserved sequences encoding ribosomal subunits (rDNA as a whole) C omparisons between sequences published within shared information banks such as GenBank ( http://www.ncbi.nlm.nih.gov/genbank ) have led to a greater understanding of the evolutionary relationships of fungal species. Restriction Fragment Length Polymorphism (RFLP) analysis utilizes differences between homologous DNA sequen ces namely the diffe ring locations of restriction enzyme sites to identify genetic differences between isolates without the need for sequencing Through the use of specialized digestive restriction enzymes that cut or DNA at these specialized s ites, different fragment lengths result These fragments are then separated by size electoph o retically and visualized with ultraviolet light after staining with ethidium bromide. U se of specific restriction enzymes, such as Hap II (HpaII), have been used to cut DNA within specific regions to assist in the identification and differentiation of Rhizoctonia spp. into varieties with a shared teleomorph such as Waitea circinata var. circinata W. c var. zeae and W. c var. oryzae (Toda et al., 2005 ; de la Ce rda et al., 2007). Random amplification of polymorphic DNA (RAPD) analysis uses PCR in a non template specific way (Williams et al ., 1990). Short, random nucleotide sequences or primers are used and template DNA is subjected to PCR amplification. La rge template DNA is required as this increases the likelihood of amplification. This technique is
22 useful for determining new or divergent species within large populations Knowledge of the target DNA sequence is not needed with RAPD analysis. If success ful, m ultiple random fragments bands will result following gel electrophoresis. The size and location of these bands across multiple isolates are used to identify genetic diversity within and between species RAPD PCR analysis was used to group geneticall y similar Rhizoctonia isolates and to identify variability in the W circinata varieties that led to the identification of a new pathogen of bentgrass (Toda et al., 1999 ; Toda et al., 2005). T axonomic reclassifications based largely on the increased under standing of the evolutionary relationships of these similar looking fungi have resulted in the proposal of several new anamorphic genera, each associated with a distinct teleomorph. For Rhizoctonia like fungi Moore (1987) suggested the name Moniliopsis b ecause the epithet Rhizoctoni a was not published according to the International Rules of Botanical Nomenclature, and Moniliopsis predated the name Rhizoctonia However, due to extensive reference in mycological and plant pathological literature, the name Rhizoctonia has been conserved (Stalpers et al., 1998) In an effort to associate a separate and distinct anamorph with each known teleomorph, the name Rhizoctonia is limited to fungi that have a Thanatephorus teleomorph while the name Ceratorhiza has be en proposed for fungi with a Ceratobasidium teleomorph ( Moore 1987) S imilarly the name Chrysorhiza has been proposed as the anamorphic genus for Waitea teleomorphs (Stalpers and Andersen 19 96 ). Although the Rhizoctonia names are still commonly used by plant pathologists the taxonom ic epiphets and synonyms used in this dissertation are listed in Table 1 1. Interchageable use of anamorphic and teleomorphic nomenclature will be used in this dissertation.
23 Rhizoctonia and Turfgrass Rhizoctonia was first no ted to cause brown spots on creeping bentgrass putting greens in Pennsylvania by Piper and Coe (1918) The disease was described as damaged turfgrass associated with white mycelium and as not respond ing to lime or fertilizer A ing leading to sclerotia formation also was noted. Rhizoctonia spreads chiefly via the growth of mycelium from sclerotia in the soil, thatch or below ground turfgrass tissue s and causes disease in much the same way today Visual symptoms of infected tur fgrass can vary significantly depending on the causal Rhizoctonia species as well as the target turfgrass host species (Smiley et al., 1994; Couch, 1995). Symptoms may appear as yellow to brown rings or patches that may expand under an environment conduci ve to the pathogen but not necessarily ideal for the host. Rhizoctonia associated diseases of turfgrass are referred to by a number of names such as brown patch, brown ring patch, foliar blight, large patch, leaf and sheath spot, sheath blight and yell ow patch ( Aoyagi et al., 1998 ; Burpee and Martin 1992 ; Couch 1995, Martin and Lucas, 1984a, Smiley et al., 1994, Toda et al., 2005 ; Vargas 1994 ). Chrysorhiza zeae R. oryzae Ceratorhiza cerealis and six anastomosis groups ( AG ) of R. solani are cited as pathogenic on turfgrass (Burpee and Martin, 1992; Haygood and Martin 1990; Martin and Lucas, 1984a.). Rhizoctonia solani The most common and widespread pathogenic Rhizoctonia is Rhizoctonia solani Kuhn, teleomorph Thanatephorus cucumeris (A. B. Frank) Donk. Thanatephorus cucumeris can be differentiated from similar teleomorphs based on the shape and size of the sterigmata and basidiospore (Sneh et al., 1998 ). Unfortunately the sexual stage
24 of R solani is rarely observed in turfgrass and it is diffi cult to induce in culture Therefore, diagnostic differentiation via sexual structures is not practical. Rhizoctonia solani has the widest host range and has been the most studied Rhizoctonia species that infects turfgrass es Infection by R solani has been reported to occur on over twelve turfgrass species and it is unlikely that any species of turfgrass is immune (Couch 1985). On cool season turfgrass species, R solani causes the disease brown patch also known as Rhizoctonia blight (Couch 1995). Disease is most sever e in poorly drained soils and with turfgrass stimulated by nitrogen (Hearn, 1943, Couch, 1995 ). The ideal temperature range at which R solani infects cool season turfgrass was determined to be between 20 to 30 o C (Burpee and Martin 1992). R. solani AG 1 IB and AG 2 2 were observed to grow optimally in vitro at a temperature of 27 o C, while AG 4 and AG 5 grew optimally at 25 o C and 21 o C respectively. Wet conditions that persist for at least 10 h or high relative humidity conditions, g reater than 95%, coupled with night temperatures above 2 0 C were ideal for Rhizoctonia solani (AG 1) infection (Smiley et al., 1992). On warm season turfgrasses R. solani is an active pathogen from early fall to late spring in t he Southern areas of the U nited States (Couch 1995 ; Martin, 2000). R. solani AG 2 2IV was observed infecting warm season turfgrasses at temperatures below 20 C ( Burpee and Martin 1992 ). D isease symptoms are most noticeable as large patches of dead and damaged turfgrass are slo w to green up in the s pring following w inter dormancy (Ayogai et al., 1998; Ayogai et al., 1999; Canegello and Martin, 2007). Rhizoctonia solani AG 2 2IV and AG 2 2LP are associated with leaf, crown and stolon rot symptoms of large patch on warm season tu rfgrass species
25 ( Burpee and Martin, 1992; Ayogai et al., 1998 ; Martin, 2000; Blazier and Conway 2004 ; Canegallo and Martin 2007 ; Hyakumachi et al., 1998 ; Martin 2 000). Large patch disease symptoms from R. solani can be very severe on zoysiagrass St Augustinegrass, bermudagrass, centipedegrass and SP (Aoyagi et al., 1998; Canegallo and Martin, 2007; Couch, 1995; Hurd and Grisham 1983 ; Hyakumachi et al., 1998 ; Smiley et al., 1994). Isolates of Rhizoctonia solani can be separated into groups based on host pathogenicity or distinguished utilizing anast o mosis groupings (AG) (Ogoshi 1987). Determination of a nastomosis compatibility of two isolates entails the use of differential media or amended water agar to induce hyphal fusi on between the isolates an d then observing the subsequent reaction of the fused cells (Carling and Sumner, 1992 ; Sneh et al., 1998 ; Yokoyama et al., 1988 ). To test a R. solani sample, a representative nown AG group is compared to an unknown isolate. Ther e are several different methods for pairing these isolates such as o n water agar in petri dishes, on microscope slides coated with agar on cover slips, or placement of cellophane on agar media ( Carling et al., 1987 ; Hyuakumachi et al., 198 8; Ogoshi 1987, Parmeter et al., 1967 ; Sneh et al., 1988 ; Zhang and Dernoeden 1995 ) A system was proposed by Carling et al. (1988) to categorize the degrees of anasto mosis between Rhizoctonia solani contact ing fusion between hyphae and membranes without death. Perfect fusion, where hyphae from each isolate pair with vi sible passing of nuclei between the pairs occur, indicates that the unknown
26 isolate is in the same AG group (Carling et al., 1988 ; Carling and Sumner 1992 ; Sneh et al., 1998) Rhizoctonia solani has been assigned to different anastomosis groups and subgroups based on in vitro morphological differences such as mycelial color and formation of sclerotia (Burpee and Martin 1992 ; Hyakumachi et al., 1998 ; Hayawaka et al., 2006 ; Tomosa Peterson and Trevathan 2007). Zhang and Dernoe den (1995) demonstrated that culture characteristics can be reliable in identification of the different Rhizoctonia solani AG groupings. Hyakumachi et al., (1998) utilized RFLP analysis on ribosomal DNA in the nuclear coded genes to segment the previous A G 2 2 groups pathogenic on turfgrass designating the subgroup AG 2 2LP R hizoctonia solani AG 2 2 LP grew optimally in vitro at temperatures of 25 o C compared to 28 o C for AG 2 2IIIB and AG 2 2 IV The four R. solani AG groups identified as pathogen s on turfg rass include two subgroups of AG 1 (IA and IB), t hree subgroups under AG 2 (2IIIB 2IV and 2LP ), AG 4 and AG5 (Zhang and Dernoeden 1995 Burpee and Martin 1992 ; Martin and Lucas 1984a Hyakumachi et al.,1998 ). R. solani AG 4 was isolated by Martin and Lucas (198 3a ) and identified as a pathogen of perennial ryegrass ( Lolium perenne ), Tif way Cynodon dactyon (L.) Pers. X Cynodon transvaalensis (Burtt Davy)] and red fescue ( Festuca rubra ) Tomos a Peterson and Trevathan (2007) explored pathogenicity of AG 4 and AG 1 IB reporting high rates of infection on creeping bentgrass ( Agrostis palustris Huds.). C ultur e characteristics such as pigmentation, zonation, sclerotia moniloid cells and growth rates at various temperatures can help di stinguish between R. solani groups
27 that are pathogenic to turfgrass (Carling and Sumner 1992 ; Ogoshi 1987 ; Oniki, 1986). Rhizoctonia solani AG 1 IA is brown to light brown in culture with large sclerotia (2 to 5 mm) whereas R. solani AG 1 IB has mycelia darker brown in pigmentation with smaller irregular sclerotia (Sneh et al., 1998). Rhizoctonia solani AG 2 2IIIB has buff to dark brown pigmentation with distinct sclerotial formation and zonation in culture while AG 2 2IV and AG 2 2LP both exhibited dar k brown aerial mycelial growth, no zonation, and no sclerotial formation in culture (Hyakumachi et al., 1998). Rhizoctonia zeae and Rhizoctonia oryzae When investigating 42 isolates obtained in Japan from rice, corn or paddy soil, three isolates of R. z eae and R. oryzae were induced to develop the same perfect state named Waitea circinata (Oniki et al., 1985). Waitea circinata Warcup & Talbot was classified as a hymenomycete similar to Thanatephorus but differing in basidiospores that are unable to repe titively germinate on shorter horn like sterigmata (Warcup and Talbot 1962 ). Gunnell (1986) discovered that the teleomorph of R. zeae and R. oryzae was Waitea circinata but also reported variability in basidiospore morphology between the species Addit ionally Gunnell noted structural variability in the anamorphic sclerotia of each species. Thus the teleomorphs of R. zeae and R. oryzae were assigned varietal names under Waitea circinata to recognize these differences. The establishment of anastamosis g roup oryzae (WAG O) and anastamosis group zeae (WAG Z) was proposed (Zhang and Dernoeden, 1995 ; Sneh et al., 1998). It is common for researchers to use conserved DNA sequences to assign a teleomoprphic name in the absence of direct observation of the sexu al structures (de la Cerda et al., 2007; Toda et al., 2005; Toda et al., 2007). Molecular methods utilizing phylogenetics has validated separation W circinata from T cucumeris Waitea circinata and T. cucumeris
28 share the same Phylum ( Basidiomycota ) but differ in Class, Order and Family (Lawrey et al., 2008). Waitea circinata has been placed in the Class Agaricales, Order Corticiales and Family Corticiaceae whereas T cucumeris is placed in the Class Hymenomycetes, Order Ceratobasidiales and family Cera tobasideaceae (Lawrey et al., 2008). Bruns et al., 1998, placed W circinata in the Class Agarimycetes, closely related to an ectomycorhizal fungus of oak seedlings, Piloderma croceum Variety designations were proposed to recognize differences within the W circinata species as they relate to the anamorphic nomenclature (e.g. W. c. var. circinata W. c. var. oryzae and W. c var. zeae ; R. circinata var. circinata R. circinata var. oryzae and R. circinata var. zeae ) (Leiner and Carling 1994). This propo sed nomenclatur al change from R. zeae and R. oryzae to varieties R. c. var. zeae and R. c. var. oryzae has been utilized by some but ignores the designation and association of Chrysorhiza as the anamorphic genus of Waitea circinata (Piryatmojo et al., 2002 a; Toda et al., 1999 ; Toda et al., 2005 ; de la Cerda et al., 2007). Chrysorhiza zeae (Voorhees) Andersen and Stalpers was a significant pathogen of the Poaceae family whereas W. circinata was once thought to only be saprophytic (Warcup and Talbot 1962; Burpee and Martin 1992 ; Stalpers and Andersen 1996 ; Garcia et al., 2008 ). Chrysorhiza zeae Voorhees, teleomorph = Waitea circinata Warcup & Talbot, in addition to being a major pathogen of corn ( Zea ), oats ( Avena ), sunflower ( Helianthus ), soybean ( Glyci ne ), and wheat ( Triticum ) was noted as a pathogen of warm and cool season turfgrass species (Haygood and Martin 1998 ; Martin and Burpee, 1992; Martin et al., 1983b).
29 Chrysorhiza zeae is white to buff or salmon to pink in c ulture with reddish ball s haped sclerotia in artificial media (Sneh et al., 1998 ; Smiley et al., 1994). L ea f and sheath spot occurs at temperature s higher than typical ly associated with diseases caused by R. solani (Couch 1995). Leaf and sheath spot has been w by some turfgrass professionals growing temperature of 30 o C and above (Martin and Lucas, 1984a ; Burpee and Martin 1992 ; Elliottt 1999). Chrysorhiza zeae exhibits greater tolerance to the benzimidazole and dicarboximide class of fungicides than R. solani (Carling et al., 1990 ; Elliott 1999 ; Martin et al., 1984b; Royals 2002). Th is differential sensitivity to fungicides such as thiophante methyl, a benizimidazole effective against R. solani may resul t in population increases in insensitive Rhizoctonia like populations such as C zeae where these Rhizoctonia selective fungicides are used (Elliott 1999). Rhizoct onia oryzae fits th e concept of Chrysorhiza, similar to C. zeae having the Waitea circinata teleomorph, but the original name was not published according to the tenets of the International Code of Botanical Nomenclature (Andersen and Stalpers, 1994). Supporting taxonomic work regarding Latin diagnos e s and descriptions are required before R. oryz ae can be recognized as Chrysorhiza oryzae however referencing R. oryzae as W. c. var oryzae is valid. Rhizoctonia oryzae Ryker & Gooch (1938) was first noted as a foliar disease on rice in Louisiana distinctly different from C zeae Rhizoctonia oryza e grows rapidly at temperatures of 30 o C and 32 o C Rhizoctonia oryzae is white to salmon to pink in c ulture with small aggregate salmon colored sclerotia that form on the PDA agar surface ( Smiley et al., 1994; Zhang and Dernoeden, 1995; Gunnell, 1998; Sneh et al., 1998).
30 There is less information on the pathogenicity of R. oryzae on turfgrass compared to C zeae Rhizoctonia oryzae was demonstrated to cause a sheath rot and foliar lesions on centipedegrass and St. Augustinegrass progressing over five days in pathogenicity studies (Haygood and Martin, 1990). Growth of a collection of isolates (Elliott 1999) became limited at 20 C while Tomosa Peterson and Trevathan (2007) found optimal temperatures for growth to be 26 and 28 C for two isolates obtained from corn. Salinity Issues Water quality is an important issue that can ultimately dictate agronomic decisions in turfgrass management especially during periods of drought or in areas of the world where rainfall is limited. High soluble salts can have d etrimental e ffect s on the function of turfgrass roots. Salinity, whether due to proximity to bodies of salt water, indicative of coastal areas, or due to presence or accumulation of salts in irrigation water or soil is becoming a limiting factor in devel opment of golf courses around the world and in Florida (Neylan 2007). Golf courses, and the game of golf, were devised to utiliz e land unsuitable for agriculture, pasture or other uses in Scotland (Beard 2005). According to a 2003 U.S. EPA report, o ver 300 Superfund sites, formerly contaminated properties, landfills, or abandoned hazardous dump/waste sites, have been converted to golf courses, playgrounds, parks, sports fields, etc. as of 2002 With approximately 23% of g classified as saline soils, these areas have limitations for typical agriculture (Szaboles 1989). Seashore paspalum has been demonstrated to withstand heavy salt, CsCl, treatments with no deleterious effects when compared to zoysiagrass and St. Augusti negrass (Kuo 2001). The ability to utilize saline soils for production of turfgrass is especially attractive as agricultural food production is restricted due to the limited range of salt tolerant food crops. Some regions of the world
31 have limited or no access to quality irrigation water outside of recycled or reclaimed water which can be high in salts O cean waterfront areas are a magnet for tourism, and SP is noted as a turfgrass that can be planted on the banks of seaside estuaries (Allar 2001). R apid population growth is occurring in arid areas where soil and water salinity put increased demands on available potable water (Marcum 2004). Salin e soils and water are problems that increase as human populations increase. There is an increasing need for arable land for agriculture as well as for urbanization. Accordingly, SP is attractive as the most salt tolerant warm season turfgrass species (Allar 2001 ; Duncan and Carrow, 2000b ) Electrical conductivity (EC) is used to determine the total conce ntration of soluble salts in water. EC is measured in decisemens/m (dS m 1 ) or as total dissolved salts (TDS) in ppm (Marcum 2004 ). Salt water is approximately 55 dS m 1 whereas salinity of irrigation water can be classified as low, medium, high and ver y high, ranging from less than 0.25 to greater than 2.25 ds m 1 Irrigation with saline water, due to high evaporative demand, can result in quick salt accumulation in the soil profile. Reliance on medium saline irrigation water, alone, without rainfall or flushing of salts, can accumulate salts. Accounting for evaporation, a 2.5 cm irrigation even t of water with an EC of 2 dS m 1 will deposit 3 kg of salt on to a 1000 ft 2 area (Marcum 2004). The main hazardous component of high EC irrigation water is s odium, Na+, which can accumulate in the root zone displacing essential nutrients such as magnesium (Mg 2+ ) and calcium (Ca 2+ ) directly or via precipitation with bicarbonates or carbonates (Richards 1954). While tolerant to high salinity, it has been noted that continued irrigation of SP with high saline water or accumulation of salts in soil can be a stress detrimental to turf
32 quality (Duncan and Carrow 2005a ; Berndt 2005). Duncan and Carrow (2005 b ) noted that depending on the level of salinity, applica tion rates of growth regulators such as trinexapac ethyl required for maintenance of SP, will vary as water high in salinity has a growth suppressive effect on SP SeaDwarf SP resulted from tests with saline water irrigation caus ing complete stolon emergence suppression at EC w levels about 19.1 ds m 1 (Berndt study are that in order to maintain the highest quality turf irrigation of SP with the freshest water possible should be p erformed as often as possible. More importantly, it is noted that while tolerant to sal ine water SP can be stressed by too much or too long of a reliance on high salinity irrigation. While exhibiting 50% reduction in top growth when irrigated with an 18 .4 ds m 1 SP exhibited enhanced rooting (Peacock and Dudeck 1985). This effect varies depending on the variety of SP Salinity is generally regarded as a stress to most turfgrass species. Salt injury might be misdiagnos ed as disease, whereas salt and disease management should be addressed together (Yenny 1994). T he salt tolerance of SP while high in comparison to bermudagrass, varies among cultivar s and can still be damaging at certain levels ( Berndt 2005 ; Raymer an d Braman 2005). Duncan and Carrow ( 2000a) stated that SP has few pathogen problems in comparison to other warm season turfgrass species since fungi do not function optimally at high salt levels. High soluble salts, however, have been implicated as enhan cing disease symptoms from Pythium aphanidermatum and Labyrinthula terrestris on cool season turfgrass species (Camberato et al., 2005 ; Martin et al., 2002 ; Rasmussen 1988). Outside of these two pathogens, little has been published on the
33 effects of salt on pathogens of turfgrass. There is indication that use of salts may decrease pathogenicity of certain pathogens in other crops. Use of Potassium chloride, KCl, while not effective in vitro against Fusarium graminearum did reduce disease incidence of s talk rot of corn when applied to infested soil at 113 to 225 kg ha 1 ( Liu et al., 2007 ). Asparagus, a salt tolerant plant, is susceptible to Fusarium crown and root rot caused by Fusarium oxysporum and Fusarium proliferatum Studies by Elmer (1992, 2003 and 200 4 ) demonstrated that applications of NaCl at 560 to 1,120 kg ha 1 versus KCl, KNO 3 NH 4 NO 3 or Ca(NO 3 ) 2 suppressed disease development while significantly increasing fresh weight and root health of asparagus with subsequent reductions in colony for ming units of both pathogens isolated from roots. Disease suppression of Fusarium crown rot of asparagus was not due to direct fungicidal activity but due to enhanced host resistance likely caused by increases in Mn reducing bacteria. The salt tolerance of SP has been exploited to address weeds and other turfgrass problems. Various weeds such as crabgrass can be controlled fairly effectively through the use of varying concentrations of seawater mixed with potable water (Pool 2006). Early breeding work with SP has been in soils or areas not deemed high in salinity, therefore the role of salinity as it relates to virulence of fungal pathogens of SP has not been explored.
34 Table 1 1. taxonomy and nomenclature for fungal isolates in this d issertation Causal fungus (pathogen) nomenclature Anamorph name basionym Teleomorph Rhizoctonia solani 1 Moniliopsis solani Thanatephorus cuc u meris 2 Ceratorhiza cerealis Rhizoctonia cerealis Ceratobasidium cereale Rhizoctonia oryzae Monioliopsis ory zae Waitea circinata var. oryzae 3 ,4 Rhizoctonia circinata ---Waitea circinata var. circinata 7 ------Waitea circinata var. prodigus 8 1 Moore, 1987 2 Ryker and Gooch, 1938 3 Oniki et al., 1985 4 Warcup and Talbot, 1962 5 Stalpers and Andersen, 199 6 6 Stalpers, et al., 1998 7 de la Cerda and Wong, 2007 8 Kammerer et al., 2011
35 CHAPTER 2 SURVEY FOR RHIZOCTONIA SPECIES ISOLATED FRO M SEASHORE PASPALUM GOLF COURSES IN FLOR IDA Introduction In 2010, there were at least twenty five golf courses in Florida g rassed with cultivars such as Seashore paspalum (SP) requires different cultural practices compared to bermudagrass including an aggressive aerification program to manage the biomass produced over time (McCarty, 2005; Zinn, 2010) D iseases on SP and the causal pathogens have not been fully explored. In comparison to salinity and light studies, b asic pathology work with SP is minimal with few peer reviewed publications Little is known about the distribution, ecology and impact of fungal populations on established SP turf. S eashore paspalum was stated to be susceptible to a number of pathogens causing major diseases such as dollar spot and large patch (Canegallo and Martin 2007). Curvularia blight and take all root rot were cited as contributing to failure of SP greens (Duncan and Carrow 2005). Gaumannomyces graminis var. graminis is a noted pathogen of SP (Elmore et al., 2002) Zinn (2010) acknowledges that in humid environments diseases such as Pythium large patch, brown patch, fairy ring, Rhizoctonia and especially dollar spot can be more problematic to SP versus when grown in an arid environment. In 2010, dollar spot caused by Sclerotinia homoeocarpa F. T. Bennett was reported as a serious disease of SP (Lv et al., 2010). While noted for its low n itrogen requirement, increasing the n itrogen and p otassium fertility for SP can be an advantageous cultural practice to minimize dollar spot infestations (Smiley et al., 1994;
36 Couch, 1995; Duncan and Carrow, 2000b). Outside of increased dollar spot tolerance, nitrogen applications to SP resulted in increased turf quality, another fe ature highly regarded in the golf course market (Kopec et al., 2005 ). Unfortunately, increased nitrogen fertility for dollar spot can result in a more conducive environment for Rhizoctonia diseases of turfgrass (Bloom and Couch, 1960; Burpee, 1995). To date utilization of SP for golf courses around the world has been most prevalent in tropical to sub tropical climates representative of the environment in Florida. In humid environments like Florida, R solani is known to be a major pathogen of significa nce on a wide range of plants (Farr et al., 1981). In addition to n itrogen rich tur f grass, temperatures ranging from 21 to 32 C coupled with persistent wet conditions for at least 10 h are ideal for R solani infection of cool season turfgrasses (Smiley et al., 1992). O n warm season turfgrasses especially in the transition zone areas of the United States R. solani can be very problematic (Couch 1995, Martin 2000 ). R. solani AG 2 2LP has optimal growth temperatures at 25 C while R. solani AG 2 2IIIB and R. solani AG 2 2IV grow optimally at 28 C ( Hyakumachi et al., 1998). The disease susceptibility of several cultivar s of SP was limited to research efforts with Rhizoctonia solani AG 2 2LP in South Carolina (Can ega llo 2007). Couch (1995) observed i nfection of warm and cool season turfgrasses by Chrysorhiza zeae was more likely with higher temperature s daytime temperatures of 28 to 33 C compared to infection by R. solani These high temperatures are more conducive for growth of C zeae than fo r R. solani (Martin and Lucas, 1984a ; Burpee and Martin 1992 ; Elliottt 1999). L eaf and sheath spot disease caused by C. zeae can
37 result in symptoms sometimes referred to as at temperature s of 30 C Rhizoctonia ory zae grow s rapidly at temperatures of 30 C and 32 C similar to C zeae (Ryker and Gooch 1938). Information on the pathogenicity of R. oryzae in turfgrasses is limited compared to C zeae Salinity and scarcity of quality irrigation water is noted as a major limiting factor in golf course development in Florida and around the world (Neylan 2007). Evidence suggests that changes in salinity affects photosynthetic parameters, leaf temperature, osmotic and leaf water potential, rate of transpiration and relativ e leaf water content (Sultana et al., 19 99). High soluble salts can negatively affect the osmotic potential and corresponding ability of turfgrass roots to take up water. The tolerance of SP to high salinity is well documented; however, continued relianc e on irrigation with high saline water is a stress that can be detrimental to turf quality (Duncan and Carrow 2005a ; Berndt 2005). O n cool season turfgrass species diseases caused by Pythium aphanidermatum and Labyrinthula terrestris were more severe o n turf irrigated with water containing h igh soluble salts compared to low levels of salts (Camberato et al., 2005 ; Martin et al., 2002 ; Rasmussen 1988). Precipitation and displacement of essential nutrients with bicarbonate and carbonates is associated w ith high EC irrigation water containing s odium (Richards 1954). The c orrelation of the incidence of disease caused by Rhizoctonia like fungi on SP and fluctuating soil EC values is unknown. The role of soil pH in infection of turfgrass by fungal pathogen s is not fully understood. Take all disease of wheat, caused by Gaumannomyces graminis var. tritici is alleviated by acidification of alkaline soils (Ownley et al., 1991). Nutrient
38 imbalances and deficiencies can cause stress on turfgrass. Severity of brown patch, caused by R. solani was observed to be greater on creeping bentgrass ranging from a pH 5.6 to 9.0 when n itrogen levels were high (Bloom and Couch, 1960). Sharp increases in soil pH resulted in increased spring dead spot and bermudagrass dec line disease severity on bermudagrass in Alabama (Hagan, 1997). High soil pH resulted in declines in populations of beneficial bacteria found to be suppressive to G. graminis var. tritici colonization of wheat roots (Bull et al., 1991, Ownley et al., 2003 ). Some of these suppressive bacteria reduce m anganese to unavailable forms as soil pH levels exceed 7. Applications of c alcium to SP, which can increase soil pH, has been a common practice in the maintenance of turfgrasses as a means of alleviating sodi um accumulation (Duncan, 1996). The objectives of this study were to: 1. Determine the incidence and identities of Rhizoctonia like fungal species infecting SP fairways in Florida; 2. Calculate correlation coefficients for Rhizoctonia like fungus isolatio n and environmental and edaphic factors. Materials and Methods Turfgrass S ampling Eight SP golf course fairways and/ or roughs across the state of Florida were sampled nine times, approximately every two months, from August 2007 to January 2009 to obtain a two year ecological survey focusing on Rhizoctonia and Rhizoctonia like fungi. The nine sampling periods were; September 4 5, 2007 (Sept 07), October 29 30, 2007 (Oct 07), January 3 4, 2008 (Jan 08), March 3 4, 2008 (Mar 08), May 5 6, 2008 (May 08), July 8 9, 2008 (July 08), August 25 26, 2008 (Aug 08), November 3 4, 2008 (Nov 08), and January 5 6, 2009 (Jan 09). One c up cutter plug sample, approximately 15 cm in depth and 10.75 cm in diameter, w as removed from
39 each golf course fairway or rough where patches, rings, foliar blight, thinning of the foliar canopy or some degree of localized chlorosis and/or necrosis occured The samples were immediately placed in a polyethylene bag on ice. Each of the eight golf courses were located in Fl orida; two in Boca Raton, Fort Myers, two in Naples, Sarasota, Vero Beach, and West Palm Beach. F our SP cultivars were Palm Beach), Parkland Go lf Course (Boca Raton), The Plantation (Fort Myers) and Vero Beach Country Club (Vero Beach) (Figure 2 1) Temperature, soil pH and EC determination Corresponding agronomic data were collected in conjunction with each sample removed from each golf course. Soil temperature to a 6 cm depth was recorded with an analog soil thermometer, and canopy temperature was determined using a Fluke 61 Infrared thermometer (Fluke Corp., China) from the same location from which a cup cutter sample was removed In the laboratory, soil was shaken from the cup cutter sample and collected. Turf s amples were washed of any remaining soil and dead organic matter. Soil samples ( 10 cc ) w ere air dried then added to 20 mL of deionized water (~24 C) and stirred in a 100 cc cup. The slurry mixture of soil and water was allowed to settle for 15 minutes then stirred again directly prior to taking pH readings using a calibrated Corning pH mete r 120 (Corning Inc., Corning, NY ). Soil electrical conductivity, EC, values representative of sodium levels were taken using an Oakton Conductivity /TDS/OC Meter CON 11 Series ( Eutech Instruments Singapore) on a S cm 1 scale set for 23C.
40 Fungal isol ations Turf samples were transported on ice in a s tyrofoam cooler for no more than three days from collection to processing. In the laboratory, using a knife, all cup cutter turf s amples were sliced approximately 0.5 cm below the soil surface separating t he underground stolons, rhizomes, and roots from the above ground leaves sheaths and stolons. Sixteen 1.5 to 2 cm above ground and sixteen below ground tissue pieces displaying some form of chlorosis or necrosis collected from each plug, were surface st erilized in 70% ethanol for 30 seconds and rinsed twice for 30 seconds with sterilized de ionized water Using sterile tissue paper, all tissue samples were patted dry and transferred to P etri dishes containing either water agar [15 g Bacto agar ( Difco La boratories, Detroit MI )/ Liter de ionized water], one fifth strength potato dextrose agar [7.8 g PDA (Difco ),10 g Bacto agar, 150 g rifam picin (Fisher Scientific Co., Fair Lawn NJ), 0.5 g ampicillin (Sigma Chemical Co., Steinheim Germany) / Liter de ioniz ed water) ] or PDA + thiophanate methyl (Topsin M 70WP, Cerexagri Inc., King of Prussia PA) [39 g PDA, 0.1 g t hiophanate methyl, 150 g rifam picin, 0.5 g ampicillin/ Liter de ionized water]. Four pieces of above ground tissue and four pieces of below grou nd tissue from each sample were placed o n each media type. The tissue samples were incubated at 25C and observed for up to 5 d for non sporulating, Rhizoctonia like colonies and hyphae ( Sneh et al., 1991 ). Hyphal tip transfers of characteristic colonies were made to PDA + rifiampicin + ampicillin ((39 g PDA + 150 g rifamipicin + 0.5 g ampicillin)/ Liter de ionized water) to obtain pure culture s of the fungi Morphological Characterization and Groupings Colony morphology was observed and noted up to 3 w ee ks following a 5 mm mycelia plug transfer to full strength PDA. Isolates were maintained in the dark at 25C.
41 Upon colony and microscopic inspection, all isolates that closely resembled each other were grouped by growth rates, colony patterns, presence o r absence of; pigment/colors, moniloid cells, sclerotia, sclerotia shape and color, and hyphal growth characteristics (on the surface, below or above the agar), and branching patterns. One or more isolates from each group were selected for DNA sequencing. I solates were transferred to sterilized oat seed which was colonized over 2 to 3 weeks, dried in autoclaved paper bags, and stored at 15C. Molecular DNA methods and isolate identification T wenty five isolates from the paspalum sampling events five is olates from bermudagrass, one from annual bluegrass, one from perennial ryegrass and one from zoysiagrass were characterized (Table 2 1) Ribosomal DNA (rDNA) including ITS1 the 5.8S ribosomal subunit, and ITS2 of one or more fungal isolates from each di stinct morphological group was sequenced after polymerase chain reaction (PCR) amplification (White 1990). Additional sequences of related fungi were retrieved from GenBank. To extract DNA, a pproximately 50 mg of aerial mycelia was removed from each fung al colony and macerated for one minute with a 0 3200 SPM Mini Bead beater at medium speed (Biospec Products, Bartlesville, OK) in 20 L of sterile distilled de ionized water The macerated samples were processed using a QIAGEN Quick Clean Up DNA extracti on kit (Qiagen Inc., Valencia, CA) instructions Template DNA from each isolate was PCR amplified. The 50 L PCR reaction mixture had 20 p M of each oligonucleotide primer (ITS1 and ITS4), a pproximately 100 ng of template DNA and 25 L of RED Extract N Am p PCR reaction mix (Sigma
42 Aldrich, St. Louis, MO) (White et al.,1990). An Eppendorf AG 22331 thermocycler (Hamburg, Germany) was programmed for an initial 94C 3 minute cycle followed by 35 cycles of 94C for 1 minute 55 C for 1 minute and 72C for 1 minute with one last cycle of 72C for 10 minutes and a 4C indefinite holding cycle. Using 5 L of PCR product, electrophoresis was performed in a 1.5% agarose gel with 0.005% ethidium bromide. Amplification was confirmed by the presence of a 650 to 700 bp band under UV light. T he PCR product was cleaned with the QuickLyse Miniprep 250 kit (Qiagen Inc., Valencia, CA) according to manufacture Following cleaning, the amplified PCR product s for each isolate wer e inserted into Escherichia coli and cloned utilizing a Topo TA Kit (Invitrogen, Carlsbad, CA). Five transformed white bacterial clones per isolate were cultured in L uria Bertani (LB) b roth at 37C in a shaker incubator for 14 to 16 h Plasmids were ext racted from b acterial clones with a miniprep k it according to manufactures directions (Q IAGEN QuickLyse Miniprep kit Qiagen Inc., Valencia, CA) The purified plasmids were submitted to the Interdisciplinary Center for Biotechnology Research at the Unive rsity of Florida in Gainesville, FL for sequencing. Sequences were aligned using the Clustal W method in Mega Build 4 .02 (Center for Evolutionary Functional Genomics, Tempe, AZ) Consensus sequences were derived using 3 or more clone sequences for each i solate after removing the primer sequences. Using Mega 4.0 2, a phylogenetic tree was constructed using a Neighbor Joining algorithm for the Kimura Two Parameter Model (Burpee et al., 2003) Bootstrap values were determined based on 1,000 random samples of the data set. Two sequences from GenBank were included, NUK 3BG from creeping bentgrass, and a sequence for
43 Sclerotinia homeocarpa accession number GU002301. Isolates were identified based on the closest known sequences in GenBank based on Blast sear ches. Correlation of Edaphic Factors and Isolation Frequency of Fungi The golf course (site), date sampled, soil temperature, canopy temperature, soil pH, soil EC and whether an isolate was obtained from above or below ground turfgrass tissue were record ed for each isolate recovered Mean isolation frequency ( isolates per sample ) was calculated as was the mean salinity, pH, soil, and canopy temperatures associated with each sample site Data were subjected to a general linear model and means were separa Isolates were identified and grouped as Waitea circinata Thanatephorus cucumeris and Ceratobasidium spp. Random effects with significance at Pr>F at 0.01, 0.05 and 0.10 for the variables of soil temperature, canopy t emperature, soil EC and soil pH were identified. Five year averages from Sept ember 1, 2005 to November 1 2009 were obtained for soil temperatures and rainfall from archived data collected by the Florida Automated Weather Network (FAWN) for the Fort Laud erdale, FL. Correlation coefficients were assessed between t hese averages and isolate recovery, observed EC readings and soil temperatures. For environmental and edaphic correlations, data were analyzed utilizing the GLIMMIX (General Linear Mixed model) procedure, and Pearson correlation coefficients were calculated for all Rhizoctonia like isolates. S tatistical an alys e s w ere performed using statistics analysis software (SAS) for Windows version 8.02 (Cary, NC).
44 Results Isolates Recovered Phylogenetic s, Morphology and Descriptions Seventy four isolates resembling Rhizoctonia with hyphae that had right and acute angle branching were recovered from the eight SP golf courses over nine sampling events August 2007 to January 2009. Twenty five of these i solates were identified through DNA sequencing (Table 2 1 ). All sequenced isolates were within the Ceratobasidium Thanatephorus and Waitea genera. Of the 25 sequenced, approximately 43% of isolates were Thanatephorus cucumeris 28% were Waitea circinata and 24% were Ceratobasidium (Table 2 2 ). A p hylogenetic tree was constructed using the DNA sequences of the twenty five SP isolates sequences obtained from additional isolates, and sequences retrieved from GenBank from outside this study are listed in Table 2 1 ( Figure 2 2 ). Isolates recovered from SP were identified in 9 of 11 clades ; t hree associated with W circinata clades two with T. cucumeris and four with Ceratobasidium W aitea circinata var. prodigus was recovered five times over four sampli ng dates from three different golf courses. There were four distinct clades of Ceratobasidium isolates belonging to different AG groups. One of the Ceratobasidium clades was a new clade 95% related to Ceratobasidium sp. AG E. Within T cucumeris two AG groups were identified, AG 2 2LP, the causal pathogen of large patch and AG 2 2IIIB, the causal pathogen of brown patch on cool season turfgrasses The T. cucumeris AG 2 2LP isolates obtained from SP were within the same clade as isolates obtained from pe rennial ryegrass and zoysiagrass. There was less distance separation between the clades of the Ceratobasidium and Thanatephorus isolates between these genera and the Waitea circinata varieties
45 Initial growth for the T cucumeris isolates, both T. cucum eris AG 2 2LP and AG 2 2IIIB, was observed to be much slower at 25 C as compared to the W. circinata isolates on full strength PDA The young individual hyphal strands produced abundant, tight 90 angle branches After 28 d the mycelia darkened to a mil k chocolate, brown color None of the T. cucumeris isolates produced sclerotia. After the mycelia began to turn brown, moniloid hyphae w ere observ ed ( Figure 2 3 ). The Ceratobasidium isolates were variable in appearance producing light brown to gray co lored mycelia in 28 d cultures on full strength PDA (Figure 2 4 ). Some produced concentric rings of mycelia. The unknown Ceratobasidium sp. anastomosis group (UCAG) was the only C eratobasidium group to produce black sclerotia 3 to 6 mm in diameter and ir regular shaped on the agar surface It is not know n if this isolate is a turfgrass pathogen. Initial growth of the Waitea circinata varieties resulted in less consistent 90 acute angle branching, more likely to be 60 to 75 and less branching with grea ter distance between each branch compared to Thanatephorus or Ceratobasidium isolates (Figure 2 5 ) W. c. var. prodigus mycelia developed a slight yellow to pink color after filling the petri dish within 1 to 2 weeks. None of the Waitea circinata varieti es developed brown pigmentation. Both the W. c. var. zeae and W. c var oryzae isolates produced mycelia limited to the agar surface in contrast to the W. c. var. prodigus which had floccuse, prolific aerial mycelia that clumped together and attached to the underside of the petri dish lid. Isolates of all the W circinata varieties produced sclerotia embedded in the agar, including the W. c var circinata isolate (picture not shown). The W. c. var zeae isolates had uniform shaped sclerotia whereas the W c.
46 var oryzae and W. c. var prodigus isolates produced irregular sclerotia. Sclerotia of the W. c var prodigus isolates ranged from a cream yellow, orange or salmon color, irregular or spherical in shape, and were embedded in the agar in clu mped or ca tenulate arrangements. Means a nd I solate R ecovery F requencies A mean of one Rhizoctonia like isolates was recovered from samples taken with a range of isolates from 0 to 4 isolates per sample (Table 2 3 ). Mean EC values ranged from a minimum of 34 up to a maximum of 1871 S/cm. The Oaks Club location had the highest mean recovery of total Rhizoctonia like isolates. The Oaks Club and The Plantation had the highest recovery rate of T cucumeris isolates. The Oaks Club also had the highest mean recovery o f W circinata isolates (Table 2 4 ). The Oaks Club had the highest mean EC values over the nine sampling events. Vero Beach Country Club had the highest mean recovery of Ceratobasidium isolates. Boca West and Tuscany Reserve had among the highest pH leve ls. Hammock Bay and Tuscany Reserve had the lowest mean recovery of total Rhizoctonia like isolates, Hammock Bay having the lowest recovery of T cucumeris isolates. Parkland had the lowest recovery of W circinata isolates. The sampling period that deli vered the highest mean recovery of Rhizoctonia like isolates and T cucumeris isolates was Jan 08 which also subsequently had the lowest mean soil and canopy temperatures (12 and 14C respectively) of the nine sampling periods (Table 2 5 ). The sampling pe riod that resulted in the highest recovery of W circinata varieties was Sep 07 which also had among the highest soil and canopy temperatures (29 and 36C respectively). There were no differences among sampling periods for mean EC values. The soil and ca nopy temperatures were closely aligned
47 (Figure 2 6 ). Canopy temperatures were higher than soil temperatures with the Jan 08 and Jan 09 periods representing a steep drop from the previous sampling period. Looking at the 5 year average of historical soil t emperatures from the FAWN for Fort Lauderdale, FL, the highest frequency of T. cucumeris isolate recovery coincides with the lowest soil temperatures of approximately 21C (Figure 2 7 ). Recovery of W circinata isolates was not as concentrated around a sp ecific temperature range as was T cucumeris isolates. Isolate Recovery C orrelations There was a correlation between recovery of Rhizoctonia like isolates and the variables, soil temperature, canopy temperature and soil EC (Table 2 6 ) utilizing a General Linear Mixed Model (Glimmix). When the data were graphed in excel (data not shown) the trend was one of increasing total isolate recovery with decreasing soil and canopy temperatures and increasing soil salinity (increasing EC values). T. cucumeris AG 2 2LP recovery was correlated to decreasing soil and canopy temperatures. With the GLIMMIX model, W. c var. zeae and W. c. var. oryzae isolation was correlated to increasing canopy temperatures and increasing EC values respectively at P< 0.10. Waitea cir cinata var. oryzae isolation frequency correlated with increasing salinity at P< 0.05, with the Pearson model (Table 2 7 ). All correlation models indicated the strongest variable correlations being between soil and canopy temperatures ( data not shown). R ecovery of one isolate was not correlated with recovery of another isolate Discussion The recovery of three different genera of Rhizoctonia like fungi representing nine distinct phylogenetic clades from SP golf course fairways in Florida is indicative of a diverse group. While temperatures of 25 C may be ideal for in vitro growth of T
48 cucumeris AG 2 2LP (Hyakumachi et al., 1998), recovery of isolates, specifically T. cucumeris AG 2 2LP was most correlated to soil temperatures of 12 21 C and canopy temp eratures of 14 24 C for the sampled SP. Other genera, such as the W circinata varieties represent other problematic pathogens of significance in SP. There was a wider diversity of isolates in the W. circinata varieties and Ceratobasidium groups than w ith T. cucumeris Plants are defined as salt tolerant based on the EC e (electrical conductivity mean of saturated paste soil extracts from the rootzone) threshold required to cause reductions in yield below what is normally achieved under nonsaline condi tions (Carrow shoot growth increase up to 14 dS/m, before a decrease occurred ( Peacock and Dudeck, 1985 ) This was more than ten times greater than the highest EC level detect ed in the sampling program. Soil EC levels were low to moderate for the golf courses in this study (34 1871 S/cm), and there was good recovery of isolates through the sampling period. Though SP has an inherently high tolerance for salt (Duncan and Ca rrow, 2000b; Duncan, 2003; Lee et al., 2004), results in this sampling program indicate that increasing salinity may increase the likelihood for infection and subsequent disease from T. cucumeris AG 2 2LP and W. c. var. oryzae based on the correlation mode ls on recovery of these pathogens. T hanatephorus cucumeris AG 2 2IIIB, the causal pathogen of brown patch on cool season turfgrass, was identified three times, all on below ground SP tissue. This is the first known report of occurrence of T. cucumeris AG 2 2IIIB on SP. One of the T. cucumeris AG 2 2IIIB isolates recovered from the SP
49 produced brown patch symptoms when inoculum was introduced to potted creeping bentgrass (data not presented). It is not known if T. cucumeris AG 2 2IIIB is pathogenic to SP It is of interest to note that T. cucumeris AG 2 2LP, a pathogen of warm season turfgrass, was recovered from symptomatic cool season turfgrass, perennial ryegrass tissue (isolate SK SgPR). The perennial ryegrass, where this isolate was recovered, was seeded over a bermudagrass tee box. Waitea circinata varieties represent potential problematic pathogens on SP as the most widely detected variety, W. c. var. zeae has been diagnosed from seashore paspalum disease samples (Stiles et al., 2008) in additi on to being a noted disease of significance on bermudagrass (Haygood and Martin 1998 ; Martin et al., 1983b ; Martin USGA report). W. c var. oryzae and a new pathogen, W. c. var. prodigus were isolated multiple times. This new variety was most closely r elated to what was published as isolate NUK 3BG, an unknown Rhizoctonia species related to Waitea circinata recovered from creeping bentgrass and Kentucky bluegrass in Japan, and entered in GenBank as Waitea circinata var. agrostis (Toda et al., 2007). Wa itea circinata var. prodigus was demonstrated to be a new pathogen causing a basal leaf blight of SP (Kammerer et al., 2010). With the exception of W. c. var. oryzae retrieval of all the W circinata varieities were obtained from above ground tissue Fo r the Ceratobasidium isolates, the separation of total isolates from SP resulted in a range of diversity similar to the W. circinata isolates. Some Ceratobasium isolates are noted as beneficial in mycorhizal relationships (Otero et al., 2002). As contras ted to the W. circinata isolates, most of the Ceratobasidium isolations were recovered from
50 below ground tissue, roots and rhizomes. It is not known if any of these isolates were pathogenic or beneficial. In this study, i t was common for one cup cutter sa mple to yield two and sometimes more, different isolates. It is un known whether these isolates collectively translat e into increased or decreased disease symptom s but their co existence can pose challenges in regards to fungicide selection as some fungi cides control one fungus versus the other more efficiently (Royals 2002 ; Elliott 1999 ; Carling et al., 1990 ; Martin 1984b ; Blazier 2004). The canopy temperature results indicate that monitoring temperature levels may be a worthwhile indicator that turf grass managers can utilize in monitoring for large patch Increasing canopy temperatures corresponding to the optimal growing temperature of 30 C for W. c. var. zeae may indicate an increase in risk for leaf and sheath spot (Martin and Lucas, 1984a ; Bur pee and Martin 1992 ; Elliottt 1999). The EC levels, indicative of soil sodium levels versus overall salts is another environmental variable that may warrant monitoring as an additional potential stress that correlated with isolation of T. cucumeris AG 2 2LP and W. c var. oryzae from SP in this study. Golf courses in Florida and across the country are increasingly utilizing effluent, reclaimed or run off water which may contain high soluble salts. Utilizing historical data for rainfall from the FAWN, a nd looking at correlation models with the isolates recovered, recovery of T. cucumeris AG 2 2LP was highest during the drier, cooler periods of the year (Figure 2 8) Low rainfall necessitates irrigation which may result in increased salinity over time.
51 T able 2 1. Species utilized in this study and identification from sampling effort (or otherwise noted) utilizing rDNA internal transcribed spacer (ITS) region sequences for genetic characterization Isolate no. Host a Origin Date collected Identity GenBank accession no. 01 SK SMd Annual bluegrass Reston, VA 25 Apr 2008 Waitea circinata var. circinata FJ154894 05 SK BA1 Boca Raton, FL 4 Sep 2007 Waitea circinata var. zeae HM597140 09 SK 4OA3 1/5 SP Sarasota, FL 4 Sep 2007 Waitea circinata var. zeae HM597139 10 SK 5OB TM1 Sarasota, FL 6 May 2008 UCAG b 14 SK 3OA TM Sarasota, FL 30 Oct 2007 Waitea circinata var. zeae HM597141 15 SK BG UFC Gainesville, FL 7 Jan 2008 UCAG 18 SK HBB1W Naples, FL 30 Oct 2007 Ceratobasidium sp. AG L 36 SK OA W3 I Sarasota, FL 9 July 2008 Waitea circinata var. prodigus HM597147 39 SK HBA1 Naples, FL 9 July 2008 Waitea circinata var. oryzae HM597138 41 SK 4OPB 1/5/1 Palm Beach Gardens, FL 9 July 2008 Ceratobasidium sp. AG G 42 SK 0821 BG Columbia, SC 24 Aug. 2008 Waitea circinata var. oryzae HM597137 43 SK 0821 BG2 Columbia, SC 24 Aug. 2008 Thanatephorus cucumeris AG 2 2IIIB 44 SK PSA TM4 Ft. Myers, FL 4 Jan 2008 Waitea circinata var. prodigus HM597146 45 SK OA TM1 Sarasota, FL 4 Jan 2008 Waitea circinata var. prodigus HM597144 46 SK HBA W1 Naples, FL 4 Jan 2008 Waitea circinata var. prodigus HM597143 47 SK 0821 BG3 Columbia, SC 24 Aug 2008 Waitea circina ta var. oryzae 59 SK OA W1 II Sarasota, FL 4 Nov 2008 Waitea circinata var. oryzae HM597135 60 SK 4BWB W1 Boca Raton, FL 6 Jan 2009 Waitea circinata var. oryzae HM597134 61 SK PB TM1 Boca Raton, FL 6 Jan 2009 Ceratobasidium sp. HM597133 64 SK VBA2 1/5 Vero Beach, FL 21 Dec 2007 Ceratobasidium sp. AG G 66 SK PMA WA1 Ft. Myers, FL 26 Aug 2008 Waitea circinata var. prodigus HM597145 67 SK BWA W3 Boca Raton, FL 26 Aug 2008 Ceratobasidium sp AG Q 68 SK 4VBB4 W1 Vero Beach, FL 4 Sept 2007 Ceratobasidium sp. AG L 69 SK 3OB1W Sarasota, FL 30 Oct 2007 Thanatephorus cucumeris AG 2 2IIIB HM597131 70 SK OA W1 I Sarasota, FL 4 Nov 2008 Waitea circinata var. oryzae HM597136 74 SK 820 BG West Palm Bch, FL 10 July 2008 Waitea circinata var. zeae HM597142 75 SK VBB4 1/5 I 2 Vero Beach, FL 2 Mar 2008 Thanatephor us cucumeris AG 2 2LP 76 3BWA TM2 Boca Raton, FL 6 Jan 2009 Thanatephorus cucumeris AG 2 2LP HM597132 78 SK 3OA 1/5 2 Sarasota, FL 4 Nov 2008 Thanatephorus cucumeris AG 2 2LP 79 SK 4VBB1 1/5 Vero B each, FL 9 July 2008 Ceratobasidium sp. AG G 81 SK 3PMA W4 Ft. Myers, FL 4 Nov 2008 Thanatephorus cucumeris AG 2 2LP 82 SK EL 2 II Atlanta, GA 18 Sept 2008 Thanatephorus cucumeris AG 2 2LP 83 SK SgPR Perennial ryeg rass Ponte Vedra Bch, FL 7 Jan 2008 Thanatephorus cucumeris AG 2 2LP 84 GU002301 c Foshawn, China 24 Aug 2010 Sclerotinia homoeocarpa GU002301 91 NUK 3BG c Creeping bentgrass Aichi, Japan June 1999 Waitea circinata var. agrostis AB213567 a Annual bluegrass ( Poa annua L.), SP: SP ( Paspalum vaginatum Swartz), bermudagrass ( Cynodon dactylon (L.) Pers.), zoysiagrass (Zoysia japonica Steud.), perennial ryegrass (Lolium perenne L.), creeping bentgrass ( Agrostis stolonife ra L.) b UCAG = Unidentified Ceratobasidium Anastomosis Group c DNA sequence and other information were obtained from GenBank.
52 Table 2 2. Isolate recovery frequencies and SP tissue location Isolates Total recovery Above ground tissue Below ground tiss ue Thanatephorus cucumeris 32 23 9 T. cucumeris AG 2 2LP 28 23 5 T. cucumeris AG 2 2IIIB 4 0 4 Ceratobasidium spp. 18 4 14 Waitea circinata 24 21 3 W. c. var. zeae 10 10 0 W. c. var. oryzae 9 6 3 W. c. var. prodigus 5 10 0 Total 74 48 26 Table 2 3 Mean of Rhizoctonia like fung us isolat ion recovered from seashore paspalum tissue, soil EC (salinity), soil pH, soil temperature, and canopy temperatures Parameter* Mean Std Dev Median Minimum Maximum Rhizoctonia like isolates 1 1 1 0 4 EC (S/cm) 285.4 381 135 34 1871 pH 7.5 0. 4 7.5 6.6 8.3 Soil temperature (C) 23. 4 6. 2 24 7.5 34.4 Canopy temperature (C) 28. 6 10. 1 28. 4 3.9 55.6 n = 72 (eight golf courses and nine sampling events)
53 Table 2 4 Mean Rhizoctonia like isolate recov er y from golf course sites, EC and pH over nine sampling events Golf course and location a All Rhizoctonia like isolates Thanatephorus cucumeris isolates Waitea circinata isolates Ceratobasidium isolates EC (S/cm) pH The Oaks Club, Sarasota 1.8 a 0.7 a 0. 9 a 0.2 c 868.6 a 7.4 b Vero Beach C C Vero Beach 1.4 b 0.3 d 0.3 c 0.8 a 277.8 c 7.5 ab Boca West, Boca Raton 1.1 c 0.6 b 0.2 d 0.3 b 99.8 c 7.7 a The Plantation, Ft. Myers 0.9 d 0.7 a 0.2 d 0 e 143 c 7.5 ab Old Palm, West Palm 0.9 d 0.1 f 0.6 b 0.2 c 126.8 c 7.5 ab Parkland, West Palm 0.8 e 0.4 c 0 e 0.3 b 82.1 c 7.4 ab Hammock Bay, Naples 0.7 f 0.2 e 0.2 d 0.2 c 574.3 b 7.5 ab Tuscany Reserve, Naples 0.7 f 0.3 d 0.2 d 0.1 d 110.9 c 7.7 a a Means followed by the same letter are statistically equ ivalent according to GLM (t
54 Table 2 5. Mean recovery frequencies of total and individual Rhizoctonia like isolates recovered, soil and canopy temperatures, EC, and pH values from SP golf courses over nine sampling ev ents Sampling period a All Rhizoctonia like isolates Thanatephorus cucumeris isolates Waitea circinata isolates Ceratobasidium isolates Soil temp (C) Canopy temp (C) EC (S/cm) pH Sept 07 1.0 c 0 g 0.9 a 0.1 e 29 a 36 ab 247 7.6 abc Nov 07 0.9 d 0.1 f 0 .2 d 0.5 b 25 b 30 bc 388 7.7 ab Jan 08 1.9 a 1.2 a 0.4 c 0.2 d 12 e 14 d 276 7.3 cd Mar 08 0.6 e 0.5 c 0 f 0.1 e 18 d 25 c 214 7.5 bcd May 08 0.5 f 0.2 e 0.1 e 0.1 e 25 b 33 ab 228 7.2 d July 08 0.9 d 0.1 f 0.4 c 0.4 c 30 a 35 ab 226 7.6 abc Sept 08 0.9 d 0 g 0.1 e 0.7 a 30 a 38 a 197 7.2 d Nov 08 1.0 c 0.4 d 0.5 b 0.1 e 21 c 23 c 378 7.7 ab Jan 09 1.6 b 1.1 b 0.4 c 0.1 e 21 c 24 c 415 7.9 a a Means followed by the same letter are statistically equivalent according to GLM (t Table 2 6. Glimmix (General Linear Mixed model) for mean incidence of selected fung us on SP tissue vs. soil temperature, canopy temperature, EC and pH Fungal isolates soil temperature (C) canopy temperature (C) EC (S/cm) pH F value Pr> F F value Pr>F F value Pr>F F value Pr>F All Rhizoctonia like isolates 8.12 0.006** 7.24 0.009** 8.81 0.004** 0.03 0.8572 Thanatephorus cucumeris AG 2 2LP 41.04 <0.0001** 27.44 <0.0001** 2.38 0.1273 0.83 0.3658 Thanatephorus cucumeris AG 2 2IIIB 0 .20 0.6556 0.53 0.4681 0.04 0.8519 0.99 0.3221 All Ceratobasidium isolates 1.20 0.2772 0.13 0.7234 0.01 0.9267 0.53 0.4703 W. c. var. zeae 1.74 0.1918 2.90 0.0931* 2.14 0.1483 0.04 0.8332 W. c. var. oryzae 0.29 0.5901 1.09 0.2993 3.80 0.0554* 1.37 0.2446 W. c. var. prodigus 2.29 0.1349 1.24 0.2695 0.01 0.9109 3.28 0.0746* *, ** Significant at P < 0.10, 0.01 respectively.
55 Table 2 7 Pearson correlation coefficients for mean incidence of selected fungi on SP tissue vs. soil temperature canopy temperature, EC and pH. Soil temperature (C) Canopy temperature (C) EC (S/cm) pH Fungus corr. Pr>R corr. Pr>R corr. Pr>R corr. Pr>R All Rhizoctonia like isolates 0.32 *** 0.31 *** 0.33 *** 0.02 Thanatephorus cucumeris AG 2 2LP 0.61 ** 0.53 *** 0.18 0.11 Thanatephorus cucumeris AG 2 2IIIB 0.05 0.09 0.02 0.12 All Ceratobasidium isolates 0.13 0.04 0.01 0.09 W. c. var. zeae 0.16 0.20 0.17 0.02 W. c. var. oryzae 0.06 0.12 0.23 ** 0.14 W. c. var prodigus 0.18 0.13 0.01 0.21 *, **, *** Significant at P<0.10, 0.05, 0.01 respectively.
56 Figure 2 1 Locations and distances between golf courses utilized in this survey.
57 Figure 2 2 Phylogenetic distance tree comparing nucleotide sequences from the rDNA of isolates of Thanatephorus Waitea and Ceratobasidium (UCAG unknown Ceratobasidium anastomsis group) 36 SK 0A W3 I 44 SK PSA TM4 66 SK PMA WA1 46 SK HBA W1 45 SK OA TM1 Waitea circinata var. prodigus Waitea circinata var. agrostis 85 NUK 3BG 5 SK BA1 9 SK 4OA3 1/5 14 SK 3OA TM 74 SK 820 BG Waitea circinata var. zeae Waitea circinata var. circinata 1 SK SMd Poa 42 SK 0821 BG 47 SK 0821 BG3 39 SK HBA1 59 SK OA W1 II 60 SK 3OPA W2 70 SK OA W1 I Waitea circinata var. oryzae Ceratobasidium sp. AG Q 67 SK BWA W3 41 SK 4OPB 1/5 1 79 SK 4VBB1 1/5 61 SK OA W1 II 64 SK VBA2 1/5 Ceratobasidium sp. AG G 18 SK 3HB B1W 68 SK 4VBB4 W1 Ceratobasidium sp. AG L 10 SK 5OB TM1 15 SK BG UFC UCAG 43 SK 0821 BG2 69 SK 3OB1W Thanatephorus cucumeris AG 2 2IIIB 81 SK 3PMA W4 83 SK SgPR 75 SK VBB4 1/5 I 2 82 SK EL 2 II 76 SK 3BWA TM2 78 SK 3OA 1/5 2 Than atephorus cucumeris AG 2 2LP Sclerotinia hom o eocarpa 84 GU002301 98 65 64 92 100 100 24 22 99 67 99 69 66 98 80 60 87 100 100 49 53 69 96 99 100 99 0.05
58 Figure 2 3 Thanatephorus cucumeris ( Rhizoctonia s olani ) 28 d culture isolates A) Rhizoctonia solani AG 2 2IIIB B) under 100X magnification C) Rhizoctonia solani AG 2 2LP D) under 100X magnification
59 Figure 2 4 Ceratobasidium spp. 28 d colony characteristics A) Ceratobasidium sp. AG G B) Ceratobasidium sp. AG L C) Ceratobasidium AG Q .D) unidentified Ceratobasidium anastomosis group
60 Figure 2 5 Waitea circinata varieties. A1) W c var. prodigus ; colony A2 ) 40X stained safranin O A 3 ) sclerotia B1 ) W c var. zeae ; co lony B 2 ) 100X B 3 ) sclerotia C1 ) W c var. oryzae colony C 2 ) 100X C 3 ) sclerotia
61 Figure 2 6 Average soil temperatures (6 cm depth) compared to average turf canopy temperatures on eight South Florida SP fairways over a two year per iod Figure 2 7 Number of Thanatephorus cucumeris and Waitea circinata isolates recovered compared to historical average 5 year data from t he FAWN for Fort Lauderdale, FL for soil temperatures at 10 cm 18.0 20.0 22.0 24.0 26.0 28.0 30.0 0 2 4 6 8 10 12 Sept 07 Nov 07 Jan 08 Mar 08 May 08 July 08 Aug 08 Nov 08 Jan 09 soil temperure ( C) No. isolates Waitea circinata Thanatephorus cucumeris 0 5 10 15 20 25 30 35 40 45 Sept 07 Nov 07 Jan 08 Mar 08 May 08 July 08 Aug 08 Nov 08 Jan 09 AVG soil temp AVG canopy temp temperature C)
62 Figure 2 8 Number of Thanatephorus cucumeri s and Waitea circinata isolates recovered compared to historical average 5 year data from the FAWN for Fort Lauderdale, FL for rainfall 0 5 10 15 20 25 30 35 40 45 50 0 2 4 6 8 10 12 Sept 07 Nov 07 Jan 08 Mar 08 May 08 July 08 Aug 08 Nov 08 Jan 09 rainfall (cm) 5 yr avg for FLL No. isolates Waitea circinata Thanatephorus cucumeris
63 CHAPTER 3 1 IDENTIFICATION OF A NEW WAITEA CIRCINATA VARIETY CAUSING BASAL LEAF BLIGHT OF SEASHORE PASPALUM Introducti on S eashore paspalum (SP), Paspalum vaginatum Swartz is a warm season grass best adapted to tropical and subtropical climates (Morton, 1973). The species is halophytic ( saltwater tolerant ) and has been found in South Africa growing along seashores and in brackish waters but the origin of the grass is uncertain (Duncan 2003; Morton 1973). A number of synonyms has been recognized since 1759 such as Paspalum distichum P foliosum P. reptans and P reimarioides to name but a few (Duncan and Carrow, 2000 ). Seashore paspalum produces a very robust root system with deep, dense rhizomes and stolons (McCarty, 2005). Suitable uses range from low maintenance or remediation turfgrass for saline soils to higher maintenance plantings li ke lawns and golf course t urf (Duncan and Carrow, 2000 ). It is a vigorous ly growing plant capable of producing a dense, dark green canopy suitable for amenity turfgrass uses S eashore paspalum urses that requires less water and fertili zer inputs to maintain a high quality foliar canopy compar ed to other warm season turfgrass species S eashore paspalum is susceptible to a range of fungal pathogens and nematodes that affect other warm season tur fgrass species. In greenhouse studies, SP was found to be as susceptible as bermudagrass, Cynodon dactylon (L.) Pers., to the parasitic nemat odes Belonolaimus longicaudatus and Hoplolaimus galeatus ( Hixson et al., 2004 ). 1 This chapter was previously published in Plant Disease 95: 515 522 ( Kammerer, S. J., Burpee, L. L., and Harmon, P. F. 2010. Identification of a new Waitea circinata variety causing basal leaf blight of seashore paspalum. )
64 Samples of SP exhibiting dollar s pot symptoms caused by Sclerotinia homoeocarpa F. T. Bennett are routinely received in the University of Florida Extension Plant Disease Clinic ( Philip Harmon, personal c ommunication ) Dollar spot has been reported as a disease of P. vaginatum in China (L v et al., 2010). Take all root rot, caused by Gaeumannomyces graminis (Sacc.) Arx & Oliver var. graminis an ectotrophic, root infecting fungus of warm season turfgrass, also has been diagnosed on SP ( Elmore et al., 2002 ). Large patch, caused by Rhizocto nia solani AG 2 occurs frequently on SP and may require preventative fungicide applications to prevent turfgrass loss (Ayogai et al., 1999; Haygood and Martin, 1990; Martin 2000) In 2007, an unusual disease of SP was observed on three golf cours es in Florida. The disease was observed three times during the coldest period of the year for South Florida, January but also twice during the hotter periods of the year, July and September. S ymptoms were observed on SP du ring cold and hot weather, foll owing low and high fertility, and during wet and dry periods. Symptoms included irregular shaped, blighted patches of turfgrass foliage ranging from 15 to 150 cm long and 5 to 90 cm wide. A Rhizoctonia like fungus, tentatively identified as Waitea circin ata was isolated from necrotic leaves and stolons from samp les taken from these locations. Waitea circinata is recognized as the teleomorph of a number of Rhizoctonia li ke fungi (Andersen and Stalpers 1994; de la Cerda et al., 2007; Leiner and Carling, 1 994; Stalpers and Andersen, 1996; Toda et al., 2005; Toda et al., 2007; Warcup and Talbot, 1962 ). P revious work has suggested the use of variety designations for W. circinata to further differentiate the species based on biological and molecular genetic d ifferences. Varieties agrostis circinata oryzae and zeae (de la Cerda et al., 2007; Leiner and
65 Carling, 1994; Lv et al., 2010; Sharon et al., 2008; Toda et al., 2007) cause distinct diseases on graminaceous hosts but have not been reported on SP. The objective of this study was to assess the biology, pathogenicity, and genetic similarities of isolates of W. circinata sampled from SP in Florida. Materials and Methods Turfgrass S ampling and I solate M aintenance. Eight golf courses in South Florida with were visited nine times approximately every two months from August 2007 to January 2009. One s ample 10.75 cm in diameter and approximately 15 cm in depth, w as removed from an area exhibit ing foliar blight or thinning on each of the eight golf c ourse fairways or roughs at each of the nine visits (Figure 3 1 ). Each sample w as placed in a polyethylene bag and the bagged samples were placed on ice. The soil and dead organic matter were wash ed from each sample. Samples were sliced in half approximately 0.5 cm below the soil surface separating the underground stolons, rhizomes and roots from the above ground leaves and stolons Sixteen above ground and sixteen below ground necrotic tissue pieces (each approximately 1.5 to 2 cm in length) were removed from each plug, surface sterilized in 70% ethanol for 30 sec onds and rinsed twice for 30 seconds with sterilized, de ionized water. Samples were blotted dry on sterile tissue and transferred to Petri dishes containing either water agar [15 g Bacto agar ( Difco Laboratories, Detroit MI )/ Liter de ionized water], o ne fifth strength potato dextrose agar [ PDA: 7.8 g PDA (Difco ),10 g Bacto agar, 150 g rifam picin (Fisher Scientific Co., Fair Lawn NJ), 0.5 g ampicillin (Sigma Chemical Co., Steinheim Germany) / Liter de ionized water) ] or PDA +
66 thiophanate methyl (Topsin M 70WP, Cerexagri Inc., King of Prus sia PA) [39 g PDA, 0.1 g t hiophanate methyl, 150 g rifam picin, 0.5 g ampicillin/ Liter de ionized water]. Samples were incubated at 25 C and were observed for up to 5 d. N on sporulating, Rhizoctonia like colonies with hyphae that had characteristic rig ht and acute angled branching were transferred to PDA + rifampicin + ampicillin (Sneh et al., 1991). Additional hyphal tip transfers were made to obtain pure cultures. Isolates were maintained in the dark at 25C. For extended storage, isolates were transferred to steril ized oat seed that had been autoclaved for three consecutive days for 30 minutes each time. The inoculated oat seed was incubated for 2 to 3 w eeks, dried in autoclaved paper bags, and stored at 15 C Colony and F ungal C haracteristic s. Size, color, and morphology of sclerotia were noted for five uncharacterized Waitea circinata (UWC) isolates from diseased SP on four golf courses after the fungi were grown at 25 C for 28 d on PDA. To measure hyphal width and the number of nuclei per cell, isolates were transferred to sterilized microscope slides coated with 2% water agar, and incubated at 25 C in the dark for 2 d. Mycelia on the agar coated slides were stained with 6 mL 0.5% (w/v) safranin O (Fisher Scientific, Fair Lawn NJ) in dis tilled water, 3.0% (w/v) KOH, and 5 mL glycerol. Diameters of nine hyphal strands per isolate were measured, and nuclei per cell were counted (Bandoni, 1979). I nternal Transcribed Spacer R egion DNA S equencing. Ribosomal DNA, including the internal tr anscribed spacer (ITS) 1 the 5.8S ribosomal subunit, and ITS2, were sequenced from the five UWC isolates and compared to the sequences of other plant pathogenic fungi including Thanatephorus Waitea Ceratobasidium, and Sclerotinia spp (Table 3 1 ). Approx imately 50 mg aerial
67 myceli um from each isolate was removed from agar plates and macerated in 20 L sterile, distilled, de ionized water for 1 min with a 0 3200 SPM Mini Bead beater operated at medium speed (Biospec Products, Bartlesville, OK). A QIAGEN Quick Clean Up DNA extraction kit (Qiagen Inc., Valencia, CA) was used to extract DNA from Approximately 100 ng template DNA was used for each 50 L PCR reaction with 20 p M of each oligon ucleotide primer (ITS1 and ITS4) and the RED Extract N Am p PCR reaction mix (Sigma Aldrich, St. Louis, MO) (White et al., 1990) An Eppendorf AG 22331 thermocycler (Hamburg, Germany) was set for an initial 3 min cycle at 94C followed by 35 cycles of 94C for 1 min 55C for 1 min and 72C for 1 min with one final cycle of 72C for 10 min and a 4C indefinite holding cycle. Five L of PCR product w ere electrophoresed in a 1.5% agarose gel with 0.005% ethidium bromide. The presence of the expected 650 bp amplicon was visualized with UV light after ethidium bromide staining. DNA C loning. T he PCR product for each isolate of UWC was cleaned with the QuickLyse Miniprep 250 kit (Qiagen Inc., Valencia, CA) according to the manufacture T he PCR p roduct s for each isolate were inserted into E. coli and cloned with a Topo TA Kit (Invitrogen, Carlsbad, CA). Five white bacterial clones per isolate were cultured in Luria Bertani (LB) b roth in a shaker incubator at 37C for 14 to 16 h. The transformed bacterial clones were lysed and cleaned with a miniprep k it according to the IAGEN QuickLyse Miniprep kit Qiagen Inc., Valencia, CA) The purified plasmids were submitted for sequencing to the Interdisciplinary Center for Bio technology Research at the University of Florida in Gainesville, FL. All
68 sequences were aligned using the Clustal W method in Mega Build 4 .02 (Center for Evolutionary Functional Genomics, Tempe, AZ) After removing the primer sequences, consensus sequenc e s w ere de rived using three or more clone sequences for each isolate A phylogenetic tree was constructed in Mega Build 4.0 2 using a Neighbor Joining algorithm for the Kimura Two Parameter Model and a genetic distance matrix was determined (Burpee et al. 2003) Bootstrap values in Mega Build 4.0 2 were determined based on 1,000 random samples of the data set. Temperature, Growth Studies. The five UWC isolates and an isolate of W. circinata var. zeae and an isolate of W. circinata var. circinata were e valuated for colony growth rate at each of six temperatures. A mycelial plug (5 mm diameter) was removed from the edge of a colony of each isolate on PDA transferred to a 9 cm diameter Petri plate of PDA, and incubated at 15, 20, 25, 30, 35, or 40C in t he dark. Colony diameter was measured every 24 h for 4 d or until the colony filled the plate. There were three replicate plates for each isolate, and the test was performed twice. Growth rates (mm per day) per day) were calculated for the first 24 h, a nd rates were averaged across the six temperatures. Average growth rate data were analyzed with analysis of variance (ANOVA) in SAS was used to separate means by isol ate (P = 0.05). Pathogenicity Studies. Inoculations with the five UWC isolates were made onto two cultivars of SP made 3 d after inoculation, and the test was repeated t wo times. In subsequent inoculation experiments, isolate SK PSA
69 Sonesta Cynodon dactylon rough stalk bluegrass ( Poa trivialis L.). Disease severity was rated 3 d after inoculation, and the experiment was repeated. In a final inoculation experiment, the five UWC isolates Agrostis stolonifera L.). Disease severity was assessed 3 d after inoculation. Th e experiment was repeated. In all inoculation experiments, three replications per isolate and host combination were used. Data from multiple repetitions of experiments were combined where statistically appropriate and unless otherwise specified. Cool sea son grasses and bermudagrass were seeded into 7.5 cm diameter pots filled with calcine clay (Turface MVP; Profile Products LLC, Buffalo Grove, IL). The grass was grown for at least three weeks with up to one foliar trimming prior to, but not the day of, i noculation. Granular f ertilize r was applied once at 0.3 grams per pot (6 12 8 starter fertilizer SP was sprigged into either 7.5 or 10.0 cm diameter pots by placing 5 to 8 stolon pieces of SP on the surface of each pot fil led with calcine clay, and the pots were irrigated. The SP was fertilized twice with 6 12 8 fertilizer at 0.3 g per 7.5 cm diameter pot and 0.5 g per 10 cm diameter pot one month after sprigging. The SP was trimmed weekly for approximately 3 months, until the turfgrass canopy covered each pot. I noculations were made approximately 4 months after sprigging. Inoculum was produced on oat seed. Fifty grams of seed were soaked in 50 mL distilled water for 24 h in a 250 mL glass flask with a foam plug. The i mbibed seed was autoclaved for 30 min once a day for three sequential days. Sterilized seed was inoculated with 5 agar plugs transferred from the leading edge of a 1 day old PDA
70 culture of each isolate. Flasks were maintained in a dark incubator at 25C for 2 to 3 weeks. Approximately every 2 d, the flasks were vigorously shaken to mix the mycelia with the seed. The colonized oat seed was transferred to sterilized paper bags and was dried for 2 weeks in a fume hood. Inoculum was stored at 5C in steril ized 100 mL glass vials sealed with Parafilm. Turfgrass was inoculated by placing five colonized oat seeds equidistant in the foliar canopy of a pot that had been irrigated with 100 mL water. Each pot w as placed in a 30 by 50 cm polyethylene bag contain in g a moist paper towel to maintain high humidity. The bags were sealed and placed in an incubator for 3 d at 30C with 12 h light per day Pots inoculated with sterile oat seed served as a control treatment for all inoculation experiments Disease incid ence was assessed 3 d after inoculation of the SP cultivars. Disease severity was assessed on pots of turfgrass utilizing the Horsfall Barratt rating scale for inoculation experiments with SK PSA TM4 and the five UWC isolates on bentgrass (Horsfall and Ba rratt 1945). Data were subjected to logit transformation and converted to a 0 to 100% severity scale after ANOVA, and means separation was performed utilizing SAS v ersion 9.0 At the conclusion of these studies, eight pieces of host tissue from at least one replication of each inoculated turfgrass species were excised, surface sterilized with 70% ethanol, rinsed, and transferred to PDA and water agar. Isolated fungi were transferred into pure culture and identified by morpholo gical characteristics as described above.
71 Results Colony M orphology and C haracterization. A summary of the morphological characteristics of UWC isolates SK PSA TM4, SK PMA WA1, SK OA W3 I, SK OA TM1 and SK HBA W1 is presented in Table 3 2 All UWC isol ates had right and acute angle hyphal branching, and produced extremely pro fuse, light yellow to pink mycelia l growth on PDA ( Fig ure 3 2 ). The aerial myce lium of each isolate clumped, clustered, and attach ed to the underside of the P etri dish lid. No b asidiospores or other sexual ( teleomorphic ) structures were either found associated with the symptomatic turfgrass samples or in vitro at any time up to 120 d after isolation Irregularly shaped, salmon colored to brown sclerotia, 3 to >7 mm in diameter w ere observed e mbedded in the PDA after 2 to 4 weeks of incubation. Isolate SK 3 OA W3 I produced sclerotia that were light yellow, while SK HBA W1 produced light brown sclerotia. Hyphal diameter averaged 5 m with six to seven nuclei per cell for each UWC isolate. Maximum growth rates for the UWC isolates occurred at 30 C (Figure 3 3 ). When comparing rates of growth on PDA encompassing temperatures from 15 to 40 C SK PSA TM4, SK OA TM1, and SK OA W3 I had statistically similar growth rates that were s ignificantly greater than the growth rates of isolates SK PMA W1, SK HBA W1, and SK SMd (Table 3 3 ). The growth rate of isolate SK BA W1 was significantly greater than that of all the other isolates tested. The growth rate of isolate SK SMd was significa ntly less than that of all other isolates tested. rDNA ITS S equence A nalysis. Genetic pairwise distances for all ITS sequences according to the Kimura Two Param eter Model had a range of 0.000 to 1.697, isolate numbers and identities
72 described in Table 3 1 ( Figure 3 4 ). When p airing sequence s the smaller the number, the greater the similarity between the sequences and the larger the number the greater the dissimilarity between the sequences. The minimum and maximum genetic distance among the UWC isolate s was 0.000 a nd 0.0021 (100 and 99.79% similarity respectively ) with no more than 1 bp difference in the 589 bp sequenced. A 9 to 14 bp difference was detected between UWC isolates and W. circinata var. agrostis isolates (Sharon et al., 2006). The UWC isolates formed a distinct phylogenic clade with 99% support (Figure 3 5 ). The clade formed by the UWC isolates was closest to the clade formed by W. circinata var. agrostis followed by the W. circinata var. zeae clade, and the W. circinata var. circinat a single isolate clade. The W. circinata var. oryzae clade was most distant from the UWC clade. Turfgrass Pathogenicity Studies. Both cultivars of SP developed symptoms following inoculation with each of the UWC isolates. Symptoms included water soaki ng, chlorosis, and then necrosis of the lower leaf blades and sheaths 3 d after inoculation. Abundant aerial mycelium was observed in the lower leaf canopy for each isolate (Figure 3 6 ). Symptoms developed in each of the three repetitions of the experimen t, and isolates were recovered from symptomatic tissue for each isolate on each SP cultivar. No symptoms were observed in the non inoculated control treatments. UWC isolate SK PSA TM4 caused an average of 57 to 97% disease severity on Son esta roughstalk bluegrass after 3 d of incubation at 30C (Table 3 4 ) Disease severity was greatest on roughstalk bluegrass For all the turfgrass cultivars inoculated, the lower
73 leaves were blighted, and the sheaths turned yellow to brown with a water soaked appearance. Mycelium was copiously present in the lower and upper turfgrass canopy. The infected turfgrass was unifory blighted, water soaked in appearance, with little to no healthy tissue rem aining 3 d after inoculation. There were no individual or distinct leaf or sheath lesions present. Isolates used in the pathogenicity studies were recovered from necrotic foliage of the various turfgrass species inoculated The UWC isolates SK PSA TM4 S K OA TM1 SK HBA W1, SK PMA WA1, and SK OA W3 I caused (meanstandard deviation) 981.4, 927.0, 887.3, 8513, and 7815% disease severity (respectively) on creeping bentgrass 3 d after inoculation. Prolific aerial mycelium of each isolate grew through ou t the lower turfgrass canopy Water soa king and necrosis of the leaf blades and sheaths were visible by day 2 Disease symptoms with all UWC isolates on all turfgrass species began as a lower (basal) leaf blight quickly progressing to an overall crown an d foliar blight. No leaf spots, lesions, or sclerotia were observed. Discussion Rhizoctonia like fungus merits a discussion of the taxonomy of the group. Over the last 150 years, diverse groups of fungal species have been assigned to the form genus Rhizoctonia s. l. (Andersen and Stalpers 1994, de la Cerda et al., 2007, Garcia et al., 2006, Mazzola et al., 1996, Moore 1987, Priyatmojo et al., 2002a). However, taxonomic reclassifications and clarification s have resulted in the development of several new anamorphic genera, each associated with a distinct teleomorph ic genus For example, the anamorphs of Thanatephor us Ceratobasidium and Waitea are currently recognized as Rhizoctonia ( Stalpers et al., 1998 ) Ceratorhiza ( Moore, 1987), and Chrysorhiza ( Stalpers and
74 Andersen, 1996), respectively Taxonomy and nomenclature of the teleomorphs of Rhizoctonia like fungi have been based on characteristics of basidiocarps, sterigmata, basidiospores and other sexual structures (Sneh et al., 1991; Stalpers and Andersen, 1996). Teleomorphs of the Rhizoctonia like pathogens of turfgrass rarely are observed, so isolate identification tends to be based on the morphology of anamorphs, which lack asexual spores and fruitin g bodies ( Burpee and Martin, 1992 ). Traditionally, characteristics such as mycelial color, the number of nuclei per hyphal cell, and the shape, size and color of sclerotia, have been used to identify these fungi (Toda et al., 2005; Toda et al., 2007). In addition, isolates have been grouped based on the ability of hyphae to anastamose (Carling et al., 2002; Garcia et al., 2006; Kuninaga et al.; 1997, Priyatmojo et al., 2002a; Stalpers et al., 1998; Yokoyama et al., 1985). The hymenomycete Waitea circinat a Warcup and Talbot (1962) was described to include fungi similar to Thanatephorus but having shorter than Thanatephorus horn like sterigmata and basidiospores that fail to undergo repetitive germination. Mycelia of W. circinata were considered to be sap robic (Warcup and Talbot, 1962) prior to the diagnosis of what is believed to be the anamorph, Chrysorhiza zeae (Voorhees) Andersen & Stalpers [= R. zeae ] (Garcia et al., 2008; Oniki et al., 1985; Stalpers and Andersen, 1996 ) as a pathogen of several memb ers of the Poaceae (Burpee and Martin 1992, Garcia et al., 2008). Isolates of C. zeae also have been referred to as W. circinata var. zeae (de la Cerda et al., 2007; Toda et al., 2005; Toda et al., 2007). The epithet Chrysorhiza was created to accommodat e fungi with a Waitea teleomorph (Stalpers and Andersen, 1996), while Rhizoctonia anamorphs are associated with Thanatephorus (Stalpers et al., 1998).
75 Sequences from phylogenetically conserved regions of DNA such as the rDNA ITS region have been used to d istinguish varieties of Waitea circinata (de la Cerda et al., 2007, Garcia et al., 2008, Leiner and Carling 1994, Sharon et al., 2008). Isolates may be identified based on the similarity of conserved sequences such as ITS1, the 5.8S ribosomal subunit, and ITS2 when teleomorphic structures are not present (de la Cerda et al., 2007, Sharon et al., 2008 ). The UWC isolates we found associated with SP in Florida appear to be morphologically and genetically distinct from previously described species affecting t urfgrass. Morphological character istics that distinguished the SP isolates from established varieties of W circinata included abundant yellow to pink aerial mycelium on PDA and salmon to yellow brown, aggregated sclerotia measuring 3.4 to 7.3 mm in diam eter e mbedded in the agar medium after 2 to 4 weeks of incubation at 30C on full strength PDA. In addition, analyses of rDNA sequences revealed that the isolates from diseased SP formed a clade that was significantly distinct from that of isolates of W. circinata vars. agrostis circinata, oryzae, or zeae. Variability in growth rates, h ost ranges, temperature optima disease symptoms and hyphal and sclerotia color reinforce the differentiation of the UWC clade Based on morphological and sequence data, we propose that these isolates from SP comprise a new variety designated W. circinata var. prodigus. DNA sequence data indicated that isolates of W. circinata var. prodigus were only 97 to 98% similar to W. circinata var. agrostis and formed a distinct cl ade with 99% support in an ITS phylogenetic tree. Isolates of W. circinata var. agrostis were recovered from diseased cr eeping bentgrass in Japan (Sharon et al., 2006) They were not observed to produce abundant yellow to pink mycelium, and the sclerotia were only 1 to 3 mm in diameter
76 and dark brown in contrast to the sclerotia l described above as characteristic of W. circinata var. prodigus. Diagnostic differentiation among Thanatephorus spp. and Waitea spp. anamorp hs affecting SP c ould be assisted gre atly by routinely plating samples on both standard and thiophanate methyl amended (100 g/ mL ) agar media Growth of the LP strain of R. solani that causes large patch is inhibited on the amended medium. Isolates of C. zeae that cause leaf and sheath spot and W. circinata var. prodigus will grow on both media, but the prodigus isolates appear more floccose within 48 h of plating. Mis identification of these pathogens could result in unnecessary applications of ineffective fungicides. For example, symptom s caused by C zeae or W circinata var. prodigus could be confused with brown patch or large patch caused by R. solani resulting in ineffective management of the disease with applicati ons of thiophanate methyl (Elliott 1999). Accurate identification of the causal agent for fungicide selection is an important factor in turfgrass disease management The proposed common W. circinata var. prodigus, is reflective of the aggressive blighting caused by the pathoge n on the oldest, basal leaves progressing over time to form diffuse necrotic patches in turfgrass swards. The rapid colonization and blight of four turfgrass species at 30C in controlled environment al conditions in this study, and the rapid growth rate of W. circinata var. prodigus in v itro at 25 to 35C suggest this pathogen could be a potential threat to multiple species of turfgrass over a wide range of climatic conditions.
77 Table 3 1. Isolates of Waitea circinata and related fungi that were examine d utilizing rDNA internal transcribed spacer (ITS) region sequences Isolate no. Host a Origin Date collected Identity GenBank accession no. 01 SK SMd Annual bluegrass Reston, VA 25 Apr 2008 Waitea circinata var. circinata FJ154894 05 SK BA1 Boca Raton, FL 4 Sep 2007 Waitea circinata var. zeae HM597140 09 SK 4OA3 1/5 Sarasota, FL 4 Sep 2007 Waitea circinata var. zeae HM597139 14 SK 3OA TM Sarasota, FL 30 O ct 2007 Waitea circinata var. zeae HM597141 36 SK OA W3 I Sarasota, FL 9 July 2008 UWC b HM597147 39 SK HBA1 Naples, FL 9 July 2008 Waitea circinata var. oryzae HM597138 42 SK 0821 BG Colu mbia, SC 24 Aug. 2008 Waitea circinata var. oryzae HM597137 44 SK PSA TM4 Ft. Myers, FL 4 Jan 2008 UWC HM597146 45 SK OA TM1 Sarasota, FL 4 Jan 2008 UWC HM597144 46 SK HBA W1 Naples, FL 4 Jan 200 8 UWC HM597143 59 SK OA W1 II Sarasota, FL 4 Nov 2008 Waitea circinata var. oryzae HM597135 60 SK 4BWB W1 Boca Raton, FL 6 Jan 2009 Waitea circinata var. oryzae HM597134 61 SK PB TM1 Boca Raton, FL 6 Jan 2009 Ceratobasidium sp. HM597133 66 SK PMA WA1 Ft. Myers, FL 26 Aug 2008 UWC HM597145 69 SK 3OB1W Sarasota, FL 30 Oct 2007 Thanatephorus cucumeris AG2 2IIIB HM597131 70 SK OA W1 I Sarasota FL 4 Nov 2008 Waitea circinata var. oryzae HM597136 74 SK 820 BG West Palm Bch, FL 10 July 2008 Waitea circinata var. zeae HM597142 76 3BWA TM2 Boca Raton, FL 6 Jan 2009 Thanatephorus cucumeris AG2 2LP HM597132 90 ATCC MYA 4521 c Culture collection Manassas, VA Apr 2009 Sclerotinia sclerotiorum FJ810516 91 NUK 3BG c Creeping bentgrass Aichi, Japan June 1999 Waitea circinata var. agrostis AB213567 92 DAI BG c Creeping bentgrass H yoga, Japan July 1998 Waitea circinata var. agrostis AB213569 93 OHT BG c Creeping bentgrass Chiba, Japan June 1997 Waitea circinata var. agrostis AB213578 94 SHO BG c Creeping bentgrass Kanagawai, Japan July 1999 Waitea circinata var. agrostis AB213575 a Annual bluegrass ( Poa annua L.), SP: SP ( Paspalum vaginatum Swartz), bermudagrass ( Cynodon dactylon (L.) Pers.), creeping bentgrass ( Agrostis stolonifera L.) b UWC = previously uncharacterized variety of Waitea circinata c DNA sequence and other inform ation were obtained from GenBank.
78 Table 3 2 Comparison of hyphal and sclerotial characteristics of isolates from symptomatic SP in S outh Florida Colony morphology a Hyphae b Sclerotia Isolate Diameter (m) Nuclei Characteristics Size (mm) c SK P SA TM4 Yellow pink, aerial clumping, monilioid cells 5 + 0.5 7 + 1.1 Many, clumped, irregular, salmon, in agar d 5 + 1.7 SK PMA WA1 Yellow pink, aerial tufted growth 5 + 0.4 7 + 1.4 Many, irregular, yellow salmon, in agar 3 + 1.0 SK OA W3 I Yellow p ink, aerial clumping, monilioid cells 5 + 0.6 7 + 1.7 Sparse to none, yellow, spherical, in agar 5 + 1.0 SK OA TM1 Yellow, aerial clustered clumping, monilioid cells 5 + 0.3 6 + 2.5 Sparse, clumped, irregular, yellow orange, in agar 7.4 + 2.6 SK HBA W1 Yellow pink aerial clumping 5 + 0.4 7 + 1.7 Erratic, abundant, salmon cream, catenulate, scattered in agar 3.4 + 1.6 Table 3 3 Mycelial growth rates of Waitea circinata isolates after 24 h on potato dextrose agar. Isolate Growth rate (mm/day) a SK BA W1 22 a SK PSA TM4 20 b SK OA TM1 20 b SK OA W3 I 20 b SK PMA W1 19 c SK HBA W1 17 d SK SMd 11 e a Mean (n = 36) growth rate. Isolate SK BA W1 is W. circinata var. zeae and SK SMd is W. circinata. var. circinata Means followed by the same let ter are statistically similar according to
79 Table 3 4. Pathogenicity of Waitea circinata isolate SK PSA TM4 on SP bermudagrass, and roughstalk bluegrass % Disease severity a Inoculum SP b SP Sonesta bermudagrass c roughstalk bluegrass d Non inoculated 10 b 5 b 5 b 3 b SK PSA TM4 69 a 57 a 57 a 97a a Ratings were taken 3 d after inoculation and incubation at 30C. Means (n = 6) within columns f b SP ( Paspalum vaginatum Swartz) c Bermudagrass ( Cynodon dactylon (L.) Pers.) d Roughstalk bluegrass ( Poa trivialis L.) Figure 3 1. Symptoms of basal leaf blight of SP in south Florida A) aIsle B A B
80 Figure 3 2. A) Characteristics of UWC isolate (SK PSA TM4) colony on PDA B) S clerotial characteristics left to right of W circinata var. circinata UWC W circinata var. oryzae and W circinata var. zea e A B
81 Figure 3 3. Comparison of mycelial growth rates (mm per day) of Waitea circinata varieties on potato dextrose agar at 15, 20, 25, 30, 35, and 40C 0 5 10 15 20 25 30 35 40 45 15 20 25 30 35 40 Rate of growth (mm growth / 24 hrs) Temperature ( C) SK OA W 3 I SK BA W 1 SK PMA W 1 SK SMd SK PSA TM 4 SK HBA W 1
82 1* 5 9 14 36 39 42 44 45 46 59 60 61 66 69 70 74 76 90 91 92 93 94   0.038  0.038 0.000  0.040 0.004 0.004  0.053 0.040 0.040 0.045  0.053 0.054 0.054 0.056 0.076  0.048 0.054 0.054 0.057 0.076 0.009  0.055 0.042 0.042 0.047 0.002 0.079 0.078  0.053 0.040 0.040 0.045 0.000 0.076 0.076 0.002  0.053 0.040 0.040 0.045 0.000 0.076 0.076 0.002 0.000  0.048 0.054 0.054 0.057 0.076 0.009 0.000 0.078 0.076 0.076  0.048 0.054 0.054 0.057 0.076 0.009 0.000 0.078 0.076 0.076 0.000  0.937 0.895 0.895 0.904 0.878 0.935 0.923 0.883 0.878 0.878 0.923 0.923  0.053 0.040 0.040 0.045 0.000 0.076 0.076 0.002 0.000 0.000 0.076 0.076 0.878  0.912 0.883 0.883 0.892 0.852 0.895 0.892 0.857 0.852 0.852 0.892 0.892 0.163 0.852  0.048 0.054 0.054 0.057 0.076 0.009 0.000 0.078 0.076 0.076 0.000 0.000 0.923 0.076 0.892  0.035 0.009 0.009 0.009 0.042 0.056 0.056 0.045 0.042 0.042 0.056 0.056 0 .890 0.042 0.863 0.056  0.919 0.899 0.899 0.909 0.868 0.912 0.909 0.873 0.868 0.868 0.909 0.909 0.152 0.868 0.020 0.909 0.880  1.658 1.635 1.635 1.646 1.604 1.695 1.697 1.607 1.604 1.604 1.697 1.697 1.484 1.604 1.383 1.697 1.646 1.447  0. 053 0.040 0.040 0.045 0.013 0.081 0.075 0.016 0.013 0.013 0.075 0.075 0.855 0.013 0.838 0.075 0.047 0.854 1.578  0.055 0.042 0.042 0.047 0.016 0.078 0.072 0.018 0.016 0.016 0.072 0.072 0.855 0.016 0.838 0.072 0.050 0.854 1.592 0.002  0.050 0.03 7 0.037 0.042 0.020 0.072 0.067 0.023 0.020 0.020 0.067 0.067 0.869 0.020 0.852 0.067 0.045 0.868 1.607 0.007 0.004  0.048 0.035 0.035 0.040 0.023 0.070 0.064 0.025 0.023 0.023 0.064 0.064 0.869 0.023 0.852 0.064 0.042 0.868 1.607 0.009 0.007 0.002 Figure 3 4. Genetic distance matrix based on sequence data utilizing rDNA internal transcribed spacer (ITS)1, 5.8S rRNA, and ITS2 regions of the rDNA locus from isolates of Waitea circinata and related fungi
83 Figure 3 5. Phylogenetic distance tree c omparing nucleotide sequences from the rDNA of Waitea circinata isolates and related fungi
84 Figure 3 6. B a sal leaf blight symptoms 3 d after inoculation with W. c var. prodigus : A) B oa trivialis C Sonesta D E F ). inoculated (left) and inoculated (right) A B C D E F
85 CHAPTER 4 THE INTERACTION OF CHRYSORHIZA ZEAE RHIZOCTONIA SOLANI AG 2 2LP AND SALINE WATER ON PASPALUM VAGINATUM Introduction Seashore paspalum (SP) Paspalum vaginatum Swartz, is a turfgrass species widely recognized for its quality and tolerance to salts. Turfgrass roots can be negatively impacted in their ability to take up water due to h igh s oluble salts I rrigation water containing high salt levels is becoming a limiting factor in development of golf courses around the world and in Florida (Neylan 2007). S eashore paspalum is noted as a salt tolerant warm season turfgrass species (Allar 20 01, Duncan and Carrow, 2000b ). Soil s alinity can be a major environmental constraint limit ing the use of some of the other primary, high quality turf grass species for golf courses, including but not limited to hybrid bermudagrass, Cynodon dactylon (L.) Pe rs., and zoysiagrass Zoysia spp Sodium, Na + is the most hazardous component of high EC irrigation water and can accumulate in root zones resulting in precipitation of other essential nutrients with bicarbonates or carbonates or directly by displacement (Richards 1954). Continued reliance on highly saline irrigation water irrigation can adversely affect turf quality of SP if salts accumulate in the root zone (Duncan and Carrow 2005 a Berndt 2005). The salt tolerance of SP differs among varieties with significant damage at varying levels ( Berndt 2005 ; Raymer and Braman 2005). Environmental and cultural stresses are well documen ted as increasing susceptibility of turfgrass to fungal infection and subsequent disease symptom expression. High salinity soils or continued irrigation with highly saline water is an added stress to most turfgrass species. Tests with saline water irriga tion to SeaDwarf
86 SP r educed shoot growth resulting in total suppression of stolon s at EC w levels about 19.1 ds m 1 (Berndt 2005). Certain varieties of SP such as SP exhibited enhanced rooting w hile exhibiting 50% reduction in top growth in respo nse to irrigation with an 18.4 dsm 1 high salt water mixtures (Peacock and Dudeck 1985). Upon its introduction as a high quality warm season turfgrass alternative to bermudagrass, SP was promoted as having few er disease issues especially in areas of hig h soil or irrigation salt levels based on the negative effects of salt on fungi ( Duncan and Carrow 2000a) Stress due to salt injury and d isease management should be jointly investigated for interactions of one with the other (Yenny 1994). Leaf and she ath spot caused by Chrysorhiza zeae, and large patch, caused by Rhizoctonia solani AG 2 2LP, have been identified as prominent diseases of seashore paspalum (SP) (Haygood and Martin ; 1998 ; Martin et al., 1983 b ). Chrysorhiza zeae has been isolated from SP exhibiting symptoms of leaf and sheath spot (Stiles et al., 2008 ) Large patch disease on several cultivars of SP in coastal South Carolina was identified as being caused by Rhizoctonia solani AG 2 2LP (Canegallo and Martin, 2006). Leaf and sheath spot i s favored by temperatures approaching and exceeding 30 o C (Martin and Lucas, 198 4a; Burpee and Martin 1992 ; Elliottt 1999) which is higher than what is optimal for large patch development (Couch 199 5). Limited research results ha ve been published on the interaction of saline water and disease caused by known warm season turfgrass pathogens such as C zeae and R solani AG 2 2LP. Materials and Methods Pathogen Isolations and Identification Isolates of C. zeae and R. solani AG 2 2LP were obtained from a s couting program conducted every two months from August 2007 to January 2009, on eight SP
87 golf courses in South Florida Turfgrass s amples exhibiting foliar blight o r thinning of the foliar canopy were removed from golf course fairways or roughs Above g round and below ground surface sterilized chlorotic SP tissue pieces and the use of selective media were utilized to identify isolates of Rhizoctonia like fungi These fungi exhibit ed uniform hyphal diameter with right and acute angle d branching and no s pore production T he isolates were transferred via hyphal tips to obtain pure cultures in sterilized potato dextrose agar (PDA). Isolates were maintained in the dark at 25C. Rhizoctonia solani AG 2 2LP and C zeae isolates were confirmed via ITS regi on DNA sequencing of ribosomal DNA including ITS1, the 5.8S ribosomal subunit, and ITS2 utilizing polymerase chain reaction. Specifically, the PCR product from extracted DNA from the fungal isolates was cleaned with a QuickLyse Miniprep 250 kit according CA). The PCR products for each isolate were inserted into E. coli and cloned with a Topo TA Kit (Invitrogen, Carlsbad, CA). The transformed bacterial clones were lysed and cleaned with a miniprep kit a ccording to manufactures directions (QIAGEN QuickLyse Miniprep kit, Qiagen Inc., Valencia, CA). The purified plasmids were submitted for sequencing to the Interdisciplinary Center for Biotechnology Research at the University of Florida in Gainesville, FL The resulting sequences confirmed the identity of the isolates utilized in these experiments as Chrysrohiza zeae or Rhizoctonia solani AG 2 2LP. Inoculum Preparation Fifty grams of oat seed were soaked in 50 mL of distilled water for 24 h in a glass flas k with a foam plug. The imbibed oat seed were autoclaved once a day for three sequential days Using one day old PDA cultures of each C. zeae and R. solani AG 2
88 2LP isolate 5 agar plugs from the leading edge of growth were transferred to the s terilized seed T he flasks were vigorously shaken approximately every 2 days to mix the mycelia with the seed and were maintained at 25 C for 2 to 3 weeks in a dark incubator. The oat seed colonized with C. zeae and R. solani AG 2 2LP were dried for 2 weeks under a fume hood in autoclaved p aper bags. Inoculum was stored at 5 C in sterilized 100 mL glass vials sealed with parafilm. Turfgrass E stablishment and M aintenance on the surf ace of each pot filled with calcine clay (Turface MVP; Profile Products LLC, Buffalo Grove, IL) The pot s of SP were irrigated twice daily on an automated irrigation system and maintained in a greenhouse for 3 to 4 months. The SP was fertilized twice at 0.5 grams per 10 cm pot one month after sprigging (6 12 LLC Lakeland, FL). The SP was trimmed up to three times weekly until the turfgrass canopy completely closed over the surface of the calcine clay for each pot. A pprox imately four months after sprigging saline water treatments and i noculations were applied Salt T reatments Two experiments were conducted to determine the effects of saline water applications and C. zeae and R. solani AG 2 2LP inoculations on SP It was determined that 150 mL of water result ed in approximately 15% drainage of water through each pot of SP For all four experiments saline water treatments were poured topically twice daily, once in the morning and once in the afternoon for three subsequen t days to the pots of SeaDwarf SP at concentrations of 0, 10,000, 15,000 and 20,000 ppm sodium chloride (NaCl) (Morton pool salt). The salt was dissolved in
89 distilled water to the appropriate concentrations. There were three replications o f each treatment. Treatments were arranged in a completely randomized design Chrysorhiza zeae inoculations Directly f ollowing the last saline water treatment, the SP was inoculated by uniformly placing six colonized oat seeds of isolate SK BA 1 or six sterile oat seed as a mock inoculated untreated check, with in the foliar canopy of each pot Pots were placed in polyethylene bags containing a moist paper towel to maintain high humidity. For the first study, t he bags were sealed and placed in an incub ator for 3 d at 30C with 12 h of light and 12 h of darkness per 24 h cycles The SP pots w ere removed from the bags and transferred to a greenhous e for 4 additional days, 7 d after inoculation, and rated for turf quality and severity of symptoms reflecte d as chlorosis and necrosis Turf q uality ratings were made on a scale of 1 to 9, with 9 being the best possible turfgrass quality (dark green, no chlorosis or necrosis apparent). Turfgrass severity ratings were made utilizing the Horsfall Barratt scale (Horsfall and Barratt, 1945) then transformed using logit transformation to percent turf damage prior to conducting statistical analysis Following the last ratings, t he SP was removed from the pots and thoroughly flushed with water to dislodge all calcine clay from the roots and rhizomes. Four pieces of s tem and leaf tissue were excised from each treatment, surface sterilized in 70% ethanol for 30 seconds, then twice rinsed with sterile water prior to transferring to 2% water agar media and 1/5 th strength PDA + rifampicin and ampicillin for observance of the presence of Chrysorhiza mycelial growth. labeled paper bags and dried at 65 C for 7 d in a GS Blue M Electric Constant Temperature Cabinet, model OV 51O A 3 (General Signal Co., Blue Island, IL). Dried SP was weighed and dry weights recorded.
90 For the second trial, the methodology a s previously described was repeated but modified in that t he potted SP turfgrass plants were plac ed in the polyethylene bag s for 7 d at 30C then rated for turf quality and severity of chlorosis and necrosis Following the 7 d incubation period, the SP pots were removed from the bags and transferred to a greenhous e for 20 additional days, 27 d after inoculation, and rated aga in for turf quality and turf severity as described above. Following completion of the study, colonization of the SP tissue was explored and dry weights determin ed in the same manner as was performed in the first trial. All turfgrass quality, severity ra tings and dry weight data w ere subjected to variance general linear model (GLM) = 0.05 ) utilizing SAS v. 9.0 (SAS Institute Inc., Cary, NC). Rhizoctonia solani AG 2 2LP inoculations Saline water applications at the same concentrations and inoculations with six colonized oat seeds by R solani AG 2 2LP ( isolate SK VBB4 1/5 I 2 ) or six sterile oat seed were made using the same methodology as described for the C. zeae inoculation studies. Pots were placed in polyethylene bags containing a moist paper towel to maintain high humidity for 7 d at 25 C After 7 d, the potted SP was transferred to a greenhouse and rated 14 d post inoculation. Host tissue was was plated after surface sterilization and the presence of fungi was noted in the same manner as was performed for the C. zeae inoculat ion studies The first inoculation trial with isolate SK VBB4 1/5 I 2 resulted in low disease severity, and t here was no recovery of the isolate from surface sterilized tissue 14 d after inoculation. In order to enhance disease symptoms, a different R. solani AG 2 2LP isolate (isolate 07 14 recovered from at Belle Glade, FL
91 24 Apr., 2007) was used for the second saline water pathogenicity trial and the incubation period in sealed polyethylene bags at 25 C was extended from 7 to 14 d. A f t er 7 and 14 d post incubation the potted SP removed from the bags were rated for t urf q uality and for severity of symptoms using the methodology and rating scales utilized with the C. zeae inoculation and saline studies. For the second tri al, the SP treatments were rated at 7 d and immediately returned to the sealed bags in the incubator for an additional 7 d. A second set of ratings were made and the studies were terminated at 14 d post inoculation. Foliar chlorosis and necrosis rating s were transformed using logit transformation from the Horsfall Barratt scale (Horsfall and Barratt 1945) to percent turf damage before conducting statistical analysis. All data were analyzed utilizing analysis of variance and means were separated accordi ng to ) utilizing SAS v. 9.0 (SAS Institute Inc., Cary, NC). Results Chrysorhiza zeae Salinity Experiment 1 No significant differences were observed in this trial at the 95% significance level. Means are presented and general trends in the data will be discussed (Figure 4 1) Turf quality was reduced in SP inoculated with C zeae but the salt water treatments did not significantly limit or enhance turf quality in the inoculated turf. The salt water treatments as well as C zeae inocu lation had no obvious effect on the severity of foliar chlorosis and necrosis in SP (Figure 4 2). Compared to the non treated check, dry weight of SP tended to be less in SP treat ed with 15,000 ppm sodium chloride, but not when treated with 10,000 or 20,0 00 ppm salt water Inoculation with C zeae had no obvious effect on dry weight of SP alone or in the presence of any of the saline treatments.
92 Seashore paspalum inoculated with C. zeae had low turf quality (Table 4 1). Saline treatments had no effect on turf quality or foliar symptoms There was no interaction, positive or negative, associated between inoculation with C. zeae and salinity. C hrysorhiza zeae was recovered and colonization and infection confirmed from all inoculated SP treatments. Chrys orhiza zeae Salinity Experiment 2 The model was not significant at the 95% confidence level. Q uality of the SP turf was not affected by any of the saline trea tments (Figure 4 3 ) Seashore paspalum i noculat ed with C. zeae had numerically lower quality af ter incubation for 7 d w hen no saline water was applied. None of the salt water treatments of the same concentration differed in quality of the turf either 7 or 27 d after inoculation Seashore paspalum had the numerically higher chlorosis and necrosis w ith the lowest dry weight s where 20,000 pppm saline water was applied compared to all other treatments (Figure 4 4) Inoculation with C. zeae had no effect on fol iar chlorosis and necrosis or the dry weight of SP with or without saline water. C hrysorhiz a zeae was recovered and confirmation of colonization and infection was obtained from all of the inoculated treatments. Rhizoctonia solani AG 2 2LP Salinity Experiment 1 No significant differences were detected for this experiment. For the first study using R. s olani AG 2 2LP (isolate SK VBB4 1/5 I 2 ), the SP quality treated with the 20,000 ppm saline water was numerically lowest compared to the non salt water check at 7 d after inoculation (Table 4 3). SP q uality was not affected by inoculation with R solani AG 2 2LP compared to SP mock inoculated with sterile oat seed at the same saline water concentrations 14 d after inoculation
93 After incubating for 7 d chlorosis and necrosis of the SP turfgrass was numerically greatest at the 20,000 ppm of sodiu m chloride compared to the water check. There were no differences in chlorosis or necrosis when comparing the same saline treatments 14 d after inoculation Differences were not apparent with applications of salt at 14 d compared to the non saline check. The SP inoculated with R. solani AG2 2LP did not differ in chlorosis or necrosis 14 d after i nocul ation compared to the SP mock inoculated with sterile seed indicating low virulence with this isolate (Table 4 4). There was no significant interaction be tween inoculation with R. solani AG 2 2LP and salt applications R hizoctonia solani was not recovered from any of the excised, surface sterilized tissue in the study confirming the lack of colonization with this isolate Rhizoctonia solani AG 2 2LP Salin ity Experiment 2 Both inoculation and salinity variables contributed significantly to the differences in severity of turfgrass chlorosis and necrosis. L ower quality ratings resulted with the SP treated with 15,000 and 20,000 ppm sodium chloride concentrat ions compared to the non saline and 10,000 ppm sal ine water applications (Figure 4 5 ) However, f or R. solani AG 2 2LP inoculated SP quality was significantly lower and levels of chlorosis and necrosis increased compared to uninoculated controls (data not shown) There were no significant differences between treatments for dry weight. S ignificant ly lower turf quality and higher chlorosis and necrosis was apparent 14 d after inoculation with R. solani AG 2 2LP compared to mock inoculated SP and with incr easing salt water concentrations (Table 4 5). There was no significant interaction between salt treatments and inoculation with Rhizoctonia solani AG 2 2LP. Rhizoctonia
94 solani was recovered in culture from excised, surface sterilized tissue from all R. s olani AG 2 2LP inoculated treatments. Discussion Pathogenicity comparisons between C. zeae and R. solani AG 2 2LP inoculated on the same turfgrass species under similar conditions is generally lacking. Most comparisons between the se fungi are based on f ungal morphology and growth characteristics in culture (Sanders et al., 1978 ; Martin et al., 1983a ; Martin and Lucas 1984a ; Moore 1987 ; Sneh 1998 ; and Hyakumachi et al., 1997 ). The two inoculation studies with C hrysorhiza zeae and Rhizoctonia solani AG 2 2LP were designed to determine the effect of saline irrigation on disease symptoms whether reflected by turf quality, chlorosis or necrosis symptoms or reductions in dry weights There were some notable differences between the four studies. T he disease s ymptoms could not be differentiated from salt damage Experimental error was high for three of the four trials conducted and resulted in not many significant differences being observed. The sources of experimental error were recorded as variation betwee n turfgrass quality of pots of SP used for the experiments and underperformance of one R. solani and the C. zeae isolates in the chosen environmental parameters. Warm season turfgrass is difficult to grow for pot studies, and the general lack of data in t he literature reflects this fact. Disease symptoms can be confused with or exacerbated by salt injury. Symptoms in c ool season turfgrass caused by the pathogens Pythium aphanidermatum ( Pythium blight ) and Labyrinthula terrestris ( rapid blight ) is enhanced due to high soluble salts (Camberato et al., 2005 ; Martin et al., 2002 ; Rasmussen 1988).
95 In the second large patch trial, s aline water applications failed to suppress disease symptoms caused by R. solani Turf quality tended to decrease with increasing concentrations of salt water. Salt water treated SP had lower turf quality and greater levels of chlorotic and necrotic tissue at every concentration greater than 0 ppm when inoculated with R. solani AG 2 2LP over the duration of the trial. General tren ds in the data from the other trials also suggest that given more uniform plant material and more replication, similar results could be expected. Extending the incubation period to 14 d and inoculating the SP with the R. solani AG 2 2LP isolate 07 14 resu lted in more definitive differences between treatments as contrasted to the first trial. Pathogenicity of the C. zeae isolate was reflected by decreased turfgrass quality ratings corresponding to the inoculated treatments versus pots that were not inocul at ed although the differences were not statistically significant. Leaf and sheath spot is a difficult disease to diagnose and causes non uniform damage on greens and other turfgrass areas where it occurs. Symptoms in pots of inoculated grass were difficu lt to distinguish from other stresses brought on by the incubation and other factors. C hrysorhiza zeae is routinely diagnosed at a high frequency from seashore RapidTu rf diagnostic clinic utilizing selective media to differentiate it from R. solani (Harmon personal communication ). The high isolation frequency of C. zeae from turfgrass samples submitted from golf courses indicates either the symptomatic turfgrass was no t easily identified as a known disease, and/or the turfgrass was not responding to actions taken by the turf manager to encourage recovery.
96 Compared to C. zeae i t was easier to visually observe R. solani colonization of SP tissue with the naked eye in e xperiment 2, as readily visible brown cob web appearing mycelia was present in abundance on the turfgrass tissue. The C. zeae myceli um was not easily visible, even directly following removal from the 100% relative humidity environment. This may expla in the difficulty golf course superintendents experience in accurately identifying leaf and sheath spot disease Salt concentrations applied up to or below 20,000 ppm did not prevent colonization by C. zeae or R. solani AG 2 2LP when SP was inoculated foll owing saline water applications S alt water applied post infection c ould possibly enhance fungal suppression but this was not tested However, t he negative effects that resulted from twice daily saline applications for 3 sequential days on turf quality and turf damage of An important factor to consider in the environmentally controlled studies versus a field situation was that the saline water applications were made in high volumes re sult ing pot. Additionally the calcine clay that served as the rooting media is not a true reflection of a finer soil texture or higher cation exchange capacity of a native soil or constructed golf course green Th us the accumulation of salts in the root zone or soil profile of SP from continued reliance on saline irrigation may be more detrimental to the turf at concentrations lower than were tested in this trial
97 Table 4 1 Trial 1 Interaction of C. zeae inoc ulation and s aline water on 'SeaDwarf' SP Treatment (salt as sodium chloride) LSD 05 ) Results (trends) inoculated NS No differences salinity (10,000 ppm, 15,000 ppm & 20,000 ppm) NS No differences inoculation X salinity NS No significant interactio n Table 4 2 Trial 2 Interaction of C. zeae inoculation and s aline water o n 'SeaDwarf' SP Treatment (salt as sodium chloride) LSD 05 ) Results (trends) inoculated NS No differences salinity (10,000 ppm, 15,000 ppm & 20,000 ppm) NS No differences inoculation X salinity NS No significant interactio n Table 4 3. Compa saline irrigation as affected by inoculation with Rhizoctonia solani AG 2 2LP Turf Quality a Chlorosis/Necrosis (%) b Treatment c 7 d after inoc 14 d after inoc 7 d after inoc 14 d after inoc Dry weight (grams) 0 ppm salt 5.8 6.7 11.1 7.4 11.4 0 ppm salt, inoc 6.2 5.8 5.6 12.8 9.8 10,000 ppm salt 3 4.8 40 24.2 10.8 10,000 ppm salt, inoc 3.7 5 20.2 22.7 10.9 15,000 ppm salt 3.5 4.5 38.7 26.3 10.3 15,000 ppm s alt, inoc 4 4.7 26.3 20.2 10.7 20,000 ppm salt 3.8 3.7 46.2 40 10.7 20,000 ppm salt, inoc 2.8 3.5 61.3 46.2 10.3 a Turf Quality rated on 0 to 9 scale, 9 = dark, green, highest quality. b Turf damage rated utilizing Horsfall Barratt sca le, data logit transformed to % chlorosis/necrosis c All treatments noted as inoc = inoculated with C. zeae all other treatments mock inoculated with sterile oat seed.
98 Table 4 4. Trial 1 Interaction of R. solani AG 2 2 LP inoculation and sal ine water on 'SeaDwarf' SP Treatment (salt as sodium chloride) 05 ) Results (trends) inoculated NS No differences salinity (10,000 ppm, 15,000 ppm & 20,000 ppm) NS No differences inoculation X salinity NS No significant interaction Table 4 5. Trial 2 Interaction of R. solani AG 2 2 LP inoculation and saline water on 'SeaDwarf' SP Treatment (salt as sodium chloride) 05 ) Results (trends) inoculated Low turf quality, high chlorosis/necrosis salinity (10,000 ppm, 15,000 ppm & 20,000 ppm) Decreasing turf quality, increasing chlorosis/necrosis with increasing salinity inoculation X salinity NS No significant interaction
99 Figure 4 SP 7 d after inoculation with (+) C zeae coupled with saline water (0 to 20,000 ppm NaCl ). 0 1 2 3 4 5 6 0 0+ 10,000 10,000+ 15,000 15,000+ 20,000 20,000+ Turf Quality (0 9 scale, 9 = best) TQ7
100 Figure 4 2. % Severity c hlorosis/n ecrosis ( SEV 7 SP 7 d after inoculation with (+) C zeae coupled with saline water (0 to 20,000 ppm NaCl ). 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 0 0+ 10,000 10,000+ 15,000 15,000+ 20,000 20,000+ Dry Weight (grams) % Severity SEV7 DW
101 Figure 4 3 Turf Quality r with (+) C zeae coupled with saline water (0 to 20,000 ppm NaCl ). 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 0 0+ 10000 10,000+ 15000 15,000+ 20000 20,000+ Turf Quality (0 9 scale, 9 = best) TQ7 TQ27
102 Figure 4 4 % Severity c hlorosis/necrosis ( SEV 27 SP 27 d after inoculation with (+) C zeae coupled with saline water (0 to 20,000 ppm NaCl ). 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 0 0+ 10000 10,000+ 15000 15,000+ 20000 20,000+ Dry Weight (grams) % Severity SEV27 DW
103 Figure 4 5 Turf quality (TQ) and % severity chlorosis/necrosis ( SEV ) ratings of R solani ) coupled with saline water (0 to 20,000 ppm NaCl ). Bars or b oxes with same letter are statistically equivalent (t tests using Fisc 05 ) a a b b b b a a 0 10 20 30 40 50 60 70 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 10,000 15,000 20,000 % Severity Turf Quality (0 to 9 scale, 9 = best) TQ14 SEV14
104 LIST OF REFERENCES Allar, B. 2001. Worth its salt. Golfdom Jan. 57(1):42 44 Andersen, T. F., and Stalpers, J. A. 1994. A check list of Rhizoctonia epithets. Mycotaxon 51:437 457. Arnheim, N. and Erlich, H. 1992. PCR strate gy. Ann Rev. Biochem. 61: 131 156. Ayogai, T., Kageyama, K. and Hyakumachi, M. 1998. Characterization and Survival of Rhizoctonia solani AG2 2 LP Associated with Large Patch Disease of Zoysia Grass. Plant Dis. 82(8):857 863. Bandoni, R. J. 1979. Safranin O as a rapid nuclear stain for fungi. Mycologia 71: 873 874. Beard, J B. 1973. Turfgrass Science and Culture. Prentice Hall Inc., Englewood Cliffs, N. J. pp. 155, 156, 217 222, 568 & 571. Beard, J. 2002. Turf management for golf courses. Ann Arbor Press. Chelsea, MI. 793 pp. Beard, J B. 2005. The history of golf (Introductory presentation at the ITRC Wales 2005). Courses, Grounds, Lawns, Sports Fields. Mich State Univ Press. Berndt, W. L. 2005. Salinity alters growth habit of Seashore Paspalum. Golf Course Mgmt. May:101 104. Blazier, S. R. and Conway, K. E. 2004. Characterization of Rhizoctonia solani isolates associated with patch diseases on turf grass. Proc. Okla. Acad. Sci. 84:41 51. Bloom, J.R. and Couch, H.B. 1960. Influence of environment on diseases of turfgrasses. I. Effect of nutrition, pH, and soil moisture on Rhizoctonia brown patch. Phytopath 50:532 534. Blount, A. R., Dankers, H., M omol, M. T., and Kucharek, T. A. 2002. Severe dollar spot fungus on bahiagrass in Florida. Online Crop Management 10.1094/CM 2002 0927 01 RS. Braman, K. 2004. Resistant Turf: Frontline defense for insect pests. USGA Turfgrass & Environ Research Onli ne January 15. 3(2): 1 10.
105 Burpee, L.L. 1995 Interactions among mowing height, nitrogen fertility and cultivar on the severity of Rhizoctonia blight of tall fescue. Plant Disease 79, 721 726. Burpee, L. L. and Martin, S. B., Jr. 1992. Biology of Rh izoctonia species associated with turfgrasses. Plant Dis. 76:112 117. Burpee, L. L., Mims, C.W., Tredway, L.P., Bae, J. and Jung, G. 2003. Pathogenicity of a novel biotype of Limonomyces roseipellis in tall fescue. Plant Dis. 87:1031 1036. Bull, C T., Weller, D. M. and Thomashow, L.S. 1991. Relationship between root colonization and suppression of Gaeumannomyces graminis var. tritici by Pseudomonas fluroescens strain 2 79. Phytopath 81:954 959. Camberato, J. J., Peterson, P. D., and Martin, S. B. 2005. Salinity alters rapid blight disease occurrence. USGA Turfgrass & Envir Research Online Aug. 15. Vol. 4(16):1 9. Canegallo, A L and Martin, B. 2007. Looking at large patch in seashore paspalum. Carolinas Green, March April: pp. 21 25. Carling, D. E., Helm, D. J., and Leiner, R. H. 1990. In vitro sensitivity of Rhizoctonia solani and other multinucleate and binucleate Rhizoctonia to selected fungicides. Plant Dis. Vol 74(11):860 863. Carling, D E., Kuninaga, S., and Brainard K. A. 2002. Hyphal anastomosis reactions, rDNA internal transcribed spacer sequences, and virulence levels among subsets of Rhizoctonia solani anastomosis group 2 (AG 2) and AG BI. Phytopath 92:43 50. Carling, D. E., Kuninaga, S. and Leiner, R. H. 19 88. Relatedness within and intraspecific groups of Rhizoctonia solani : a comparison of among grouping by anastamosis and by DNA hybridization. Phytoparasitica 16: 209 210. Carling, D. E., Leiner, R. H. and Kebler, K. M. 1987. Characterization of a new anastamosis group (AG 9) of Rhizoctonia solani Phytopath. 77:1609 1612. Carling, D. E. and Sumner, D. R. 1992. Rhizoctonia Methods for research on soilborne phytopathogenic fungi. L. L. Singleton, J. D. Mihail, and C. M. Rush, eds. Ameri can Phytopath. Society, St. Paul, MN. pp. 157 165.
106 Carrow, R. N. and Duncan, R. R. 1998. Salt Affected Turfgrass Sites: Assessment and Management. John Wiley & Sons, Inc., Hoboken, NJ. 185 pp. Couch, H. B. 1995. Diseases of Turfgrasses. 3 rd edit ion. R. E. Kreiger Publishing Company, Malabar, Florida. 348 pp. Daniel, T. 2003. Dwarfing the competition: seashore paspalum greens offer superintendent a sense of tranquility. Golf Course Management Nov. 71(11):68 74. Danneberger, K. 2007. Se ashore Paspalum on Firm Ground. Golfdom Jan. 63(1):38. de la Cerda, K., Douhan, G., and Wong, F. 2007. Discovery and characterization of Waitea circinata var. circinata affecting annual bluegrass from the Western United States. Plant Dis. 91:791 79 7. Dudeck, A. E. and Peacock, C. H. 1985. Effects of Salinity on Seashore Paspalum Turfgrasses. Agronomy Journal Vol 77:47 50. Duncan, R. R. 1996. Seashore paspalum: The next generation turf for golf courses. Golf Course Management April:49 51. Duncan, R. R. 2003. Seashore paspalum ( Paspalum vaginatum Swartz). Turfgrass Biology, Genetics, and Breeding. M.D. Casler and R. R. Duncan, eds. John Wiley and Sons, New York, N.Y. pp 295 307 Duncan, R. R.; Carrow, R. N. 2000a. Soon on gol f courses: New seashore paspalums. Golf Course Management May. 68(5):65 67. Duncan, R. R. and Carrow, R. N. 2000b. Seashore Paspalum, The Environmental Turfgrass. Ann Arbor Press, Chelseas, MI. 281 pp. Duncan, R. R. and Carrow, R. N. 2005a. M anaging seashore paspalum greens. Golf Course Management Feb.:114 118. Duncan, R. R. and Carrow, R. N. 2005b. Preventing f ailure of seashore paspalum greens. Golf Course Management. Mar.:99 102. Elliott, M. L. 1999. Comparison of Rhizoctonia ze ae isolates from Florida and Ohio turfgrass. HortScience Apr 34(2):298 300. Elmer, W. H. 1992. Suppression of Fusarium crown and root rot of asparagus with sodium chloride. Phytopath. 82:97 104.
107 Elmer, W. H. 2003. Local and systemic effects of N aCl on root composition, R hizobacteria, and Fusarium crown and root rot of asparagus. Phytopath. 93:186 192. Elmer, W. H. 2004. Combining nonpathogenic strains of Fusarium oxysporum with sodium chloride to suppress fusarium crown rot of asparagus in replanted fields. Plant Path. 53:751 758. Elmore, W. C., Gooch, M. D., and Stiles, C. M. 2002. First Report of Gaeumannomyces graminis var graminis on seashore paspalum in United States. Plant Dis. 86(4):1405. Farr, D. F., Bills, G. F., Chamu ris, G. P. and Rossman, A. Y. 1989. Fungi of Plants and Plant Products in the United States. APS Press, St. Paul, MN. pp 932 933. Garcia, M. G., Ramos, E. R., and Ochoa, R. R. 2008. First report of Chrysorhiza zeae (Voorhes) Andersen and Stalpers c ausing necrotic lesions on Cynodon dactylon (l.) pers. in Cuba. Fitosanidad 12:143 146. Garcia, V. G., Portal Onco, M. A., and Rubio Susan, V. 2006. Review. Biology and systematics of the form genus Rhizoctonia Spanish J. Agric. Research 4:55 79. Gun nell, P. S. 1986. Characterization of the teleomorphs of Rhizoctonia oryzae sativae Rhizoctonia oryzae and Rhizoctonia zeae and the effect of cultural practices on aggregate sheath spot of rice, caused by Rhizoctonia oryzae sativae Ph.D. disserta tion. University of California, Davis. Hagan, A. 1997. Control of Spring Dead Spot and Bermudagrass decline. AL Cooperative Exten Service Pub. ANR 371. Hayawaka, T., Toda, T., Pinq, Q., Mghalu, J., Yaguchi, S. and Hyakumachi, M. 2006. A New Subg oup of Rhizoctonia AG D, AG D III, obtained from Japanese zoysiagrass exhibiting symptoms of a new disease. Plant Dis. Vol. 90(11):1389 1394. Haygood, R. A., and Martin, S. B. 1990. Characterization and pathogenicity of species of Rhizoctonia associa ted with centipedegrass and St. Augustinegrass in South Carolina. Plant Dis. 74:510 514 Hearn, J. L. Jr. 1943. Rhizoctonia solani K h n and the brown patch disease of grass. Proc.Texas Acad. Sci., pp. 26 : 41 42. Hixson, A.C., Crow, W. T., McSorley, R., and Trenholm, L. E. 2004. Host Status Belonolaimus longicaudatus and Hoplolaimus galeatus Journal of Nematology 36(4):493 498.
108 Horsfall, J. G., and Barratt, R. W. 1945. An improved grading system for measu ring plant disease. Phytopath 35:655. Huang, B., R. R. Duncan, and R. N. Carrow 1997. Drought Resistance Mechanisms of Seven Warm season Turfgrasses under Surface Soil Drying: II. Root Aspects. Crop Sci. 37:1863 1869. Hull R. J 199 2 Energ y relations and carbohydrate partitioning in turfgrass Pages 175 205 in: Turfgrass D. Waddington, R. Carrow, and R. Shearman, eds. ASA, Inc., CSA, Inc. and SSSA Inc. Madison, WI. Hurd, B., and Grisham, M. P. 1983. Rhizoctonia species associated wit h brown patch of Saint Augustinegrass. Phytopath. Vol. 73(12):1661 1665. Hyakumachi, M., Mushika, T., Ogiso, Y., Toda, T. Kageyama, K. and Tsuge T. 1998. Characterization of a new cultural type (LP) of Rhizoctonia solani AG 2 2 isolated from warm se ason turfgrasses, and its genetic differentiation from other cultural types. Plant Path. 47:1 9. Kammerer, S. J., Burpee, L. L., and Harmon, P. F. 2010. Identification of a new Waitea circinata variety causing basal leaf blight of seashore paspalum. Plant Dis. 95:515 522. Kammerer, S. J. and Harmon P. F 2008. The importance of early and accurate diagnosis of Rhizoctonia diseases: Correct identification of Rhizoctonia species is necessary to efficiently manage turf diseases. Golf Course Manage ment 76:92 98. Kopec, D. M., Walworth, J. H., Gilbert, J. J., Sower, G. M., and Pessarakli M. 2005. Sea Isle 2000 on desert greens: m owing height and nitrogen fertility Golf Course Management Nov.: 84 87. Kuninaga, S., Natsuaki, T., Takeuchi, T ., and Yokosawa, R. 1997. Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani Curr. Genet. 32:237 243. Kuo, Y. J.; Fermanian, T. W. 2001. Use of seashore paspalum on phytoremediation of heavy metal contaminated soil. IXth International Turfgrass Research Conference 9:68 69. Lee, G., Carrow, R. N and Duncan, R. R. 2004. Salinity Tolerance of Seashore Paspalum Ecotypes: Shoot Growth Responses and Criteria. HortScience 39(5):1138 1142. Lee, G, Carrow, R. N. and Duncan, R. R. 2005. Criteria for Assessing Salinity Tolerance of the Halophytic Turfgrass Seashore Paspalum. Crop Sci. 45:251 258.
109 Leiner, R. H. and Carling, D. E. 1994. Characterization of Waitea circinata ( Rhizoctonia ) iso lated from agricultural soils in Alaska. Plant Dis. 78:385 388. Liu, X., Jin, J., He, P., Liu, H., and Li, W. 2007. Relationship between potassium chloride suppression of corn stalk rot and soil microorganism characteristics. Front. Agric. China 1 (2):136 141. Lv, C., Luo, L., and Hsiang, T. 2010. First report of dollar spot of seashore Paspalum ( Paspalum vaginatum ) caused by Sclerotinia hom o eocarpa in South China. Plant Dis. 94:373 373. Marcum, K. 2004. Use of saline and non potable water in the turfgrass industry: constraints and developments. Proceedings of the 4 th Intl. Crop Science Congress, 26 Sept 1 Oct. Brisbane, Australia. 12 pp. Martin, S. B. 1987. Rapid tentative identification of Rhizoctonia spp. associated with disease d turfgrasses. Plant Dis. Vol. 71(1). Martin, S. B. 2000. Distinguishing Rhizoctonia diseases on warm season grasses. Phytopath. 90:S97. Publication no. P 2000 0037 SSA. Martin, B. 2009. Identification, Pathogenicity, and Control of Rhizoctonia L eaf and Sheath Spot of Bermudagrass Putting Greens. USGA Turfgrass Research and Environmental Online. Vol 8, No 22. 10 pp. Martin, S. B., Campbell, C. L., and Lucas, L. T. 1983a. Horizontal Distribution and characterization of Rhizoctonia spp. in tal l fescue turf. Phytopath.73: 1064 1068. Martin, S. B., and Lucas, L. T. 1983b. Pathogenicity of Rhizoctonia zeae on tall fescue and other turfgrasses. Plant Dis. Vol. 67(6):676 678. Martin, S. B. and Lucas, L. T. 1984a. Characterization and path ogenicity of Rhizoctonia spp. and Binucleate Rhizoctonia like Fungi from Turfgrasses in North Carolina. Phytopath. Vol. 74(2):170 175. Martin, S. B., Lucas, L. T., and Campbell. 1984b Comparative sensitivity of Rhizoctonia solani and Rhizoctonia li ke fungi to selected fungicides in vitro. Phytopath Vol. 74(7):778 781. Martin, S. B., Stowell, L. J., Gelernter, W. D., and Alderman, S. C. 2002. Rapid blight: A new disease of cool season turfgrasses. Phytopath 92:S52
110 Mazzola, M., Wong, O., and Cook R. 1996. Virulence of Rhizoctonia oryzae and R. solani AG 8 on wheat and detection of R. oryzae in plant tissue by PCR. Phytopath 86:354 360. McCarty, L. B. 2005. Best turfgrasses for golf courses. Pages 15, 34 37 in: Best Golf Course Manage ment Practices Second Edition. Pearson Prentice Hall, Upper Saddle River, NJ. Moore, R. T. 1987. The genera of Rhizoctonia like fungi: Asco Rhizoctonia Ceratorhiza gen. nov., Moniliopsis and Rhizoctonia Mycotaxon 29:91 99. Moore, R. T. 1996. The dolipore/parenthesome septum in modern taxonomy. Pages 13 35 in: Rhizoctonia S pecies: T axonomy, M olecular B iology, E cology, P athology and D isease C ontrol B. Sneh, S. Jabaji Hare, S. Neate, and G. Dijst, eds. Kluwer Academic, Dordrect, The Netherla nds. Morton J. F. 1973. Salt tolerant siltgrass ( Paspalum vaginatum ) Proc. FL State Hort. Soc. 86:482 490. Nobel, P. S. 1991. Physiochemical and environmental p lant physiology. Academic Press, New York. 635 pp Neylan J. 2007. Turfgrass Water Us e. Australian Turfgrass Management March/ April. 9(2):28 30. Ogoshi, A. 1987. Ecology and pathogenicity of anastamosis and intraspecific groups of Rhizoctonia solani Kuhn. Ann. Rev. Phytopathology. 25:125 143. Oniki, M. Ogoshi, A., Takao, A., S akai, R. and Sumito, Tanaka. 1985. The perfect state of Rhizoctonia oryzae and R. zeae and the anastamosis groups of Waitea circinata Trans. mycol. Soc. Japan 26:189 198. Oniki, M., Ogoshi, A., and Araki, T. 1986. Development of the perfect state of Rhizoctonia solani. Ann. Phytopath. Soc. Japan 52: 169 174. Otero, J. T., Ackerman, J. D., and Bayman, P. 2002. Diversity and host specificity of endophytic Rhizoctonia like fungi from tropical orchids. American Journal of Botany 89(11):185 2 1858. Ownley, B. H., Weller, D. M., and Thomashow, L. S. 1991. Influence of In Situ and In Vitro pH on Suppression of Gaeummanomyces graminis var. tritici by Pseudomonas fluorescens 2 79. Phytopath 82:178 184.
111 Ownley, B. H., Duffy, B. K., and Weller, D. M. 2003. Identification and manipulation of soil properties to improve the Biological control performance of phenazine producing Pseudomonas fluorscens Applied and Environ Microbiol 69:3333 3343. Parmeter, J. R., Jr., Whitney, H.S., an d Platt, W. D. 1967. Affinities of some Rhizoctonia species that resemeble mycelium of Thanatephorus cucumeris Phytopath. 59:1270 1278. Peacock, C. H. and Dudeck, A. E. 1985. Physiological and growth responses of seashore paspalum to salinity. HortScience 20(1):111 112. Piper, C. V., and Coe, H. S. 1919. Rhizoctonia in lawns and pastures. Phytopath 9:89 92. Pool, N. B. 2005. Influence of salt water on weed management in seashore paspalum. Masters Thesis Univ. of Florida. 68 pp. Pr iyatmojo, A., Yamauchi, R., Carling, D., Kageyama, K., and Hyakumachi. M. 2002a. Differentiation of three varieties of Rhizoctonia circinata ; var. circinata var. oryzae and var. zeae on the basis of cellular fatty acid composition. Journal of Phytop athology 150:1 5. Priyatmojo, A., Yamauchi, R., Kageyama, K., and Hyakumachi, M. 2002b. Whole cell fatty acid composition to characterize and differentiate isolates of Rhizoctonia species associated with turfgrass species in Japan. J. Gen. Plant Pathol 68:1 7. Rasmussen, S. L. and Stanghellini, M. E. 1988. Effect of Salinity Stress on Development of Pythium Blight in Agrostis palustris Phytopath Vol. 78(11):1495 1497. Raymer, P and Braman, K. 2005. Breeding Seashore Paspalum for recrea tional use. Turfgrass and Environmental Research Summary p 32. Richards L. A. 1954. Origin and Nature of saline and alkali soils. p. 1 6. In Richards L. A. (ed.) USDA Agriculture Handbook No. 60. Diagnosis and improvement of saline and alkali soils. United States Department of Agriculture. Washington DC. Royals, J. K., II. 2002. Development and evaluation of strategic fungicide programs for control of summer diseases in bentgrass. Masters Thesis. Clemson Univ., Clemson, SC. Ryker, T. C., and Gooch, F. C. 1938. Rhizoctonia sheath spot of rice. Phytopath. 28:233 246.
112 Sanders, P. L., Burpee, L. L., and Cole, Jr. H. 1978. Preliminary Studies on Binucleate Turfgrass Pathogens that resemble Rhizoctonia solani Phytopath. Vol 68:145 148. Sharon, M., Kuninaga, S., Hyakumachi, M., Naito, S. and Sneh, B. 2008. Classification of Rhizoctonia spp. using rDNA ITS sequence analysis supports the genetic basis of the classical anastomosis grouping. Mycoscience 49:93 114. Sharon, M., Kuninaga S., Hyakumachi, M., and Sneh, B. 2006. The advancing identification and classification of Rhizoctonia spp. using molecular and biotechnological methods compared with the classical anastomosis grouping. Mycoscience 47:299 316. Smiley, R. W., Dernoeden P. H. and Clarke, B. B. 1994. Compendium of Turfgrass Diseases Second Edition. 98 pp. Smyth, N. 1992. The polymerase chain reaction history methods, and applications. Diag Molecular Path. 1(1):58 72. Sneh, B., Burpee, L. and Ogoshi, A. 199 8. Identification of Rhizoctonia species. APS Press, St. Paul, MN. 135 pp. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98: 503 517. Stalpers, J. A. and Andersen, T. F. 1996. A synopsis of the taxonomy of teleomorphs connected with Rhizoctonia S. L. Pages 49 63 in: Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control Sneh, B., Jabaji Hare, S., Neate, S., and Dijst, G, eds. Kluwer Academic, Dordrect, The Netherlands. Stalpers, J.A., Andersen, T.F., and Gams, W. 1998. Two proposals to conserve the names Rhizoctonia and R. solani ( Hyphomycetes). Taxon 47:725 726. Stiles, C. M., Harmon, P. F., and Kenworthy, K. E. 2008. Relative Pathogenicity and Fungicide Sensitivity of Isolates of Rhizoctonia and Other Fungal Pathogens and the Disease Responses of Seashore Paspalum and Zoysiagrass Cultivars. USGA 2008 Turfgrass and Environmental Research Summary. p 6. Sultana, N., Ikeda, T. an d Itoh, R. 1999. Effect of NaCl salinity on photosynthesis and dry matter accumulation in developing rice grains. Environ and Exp Botany 42: 211 220. Szaboles, I. 1989. Salt affected soils. Ph.D.C. Sci. CRC Press, Inc., Boca Raton, FL. 247 pp.
113 Toda, T., Hyakumachi, M., Suga, H., Kageyama, K., Tanaka, A., and Tani, T. 1999. Differentiation of Rhizoctonia AG D isolates from turfgrass into subgroups I and II based on rDNA and RAPD analyses. European Journ. of Plant Pathology 105: pp. 835 846. Toda, T., Mushika, T., Hayakawa, T., Tanaka, A., Tani, T., and Hyakumachi, M. 2005. Brown ring patch: a new disease on bentgrass caused by Waitea circinata var. circinata Plant Dis. 89:536 542. Toda, T., Hyak awa, T., Mwafaida Mghalu, J., Yaguchi, S., and Hyakumachi, M. 2007. A new Rhizoctonia sp. closely related to Waitea circinata causes a new disease of creeping bentgrass. J. Gen. Plant Pathol. 73:379 387. Tomosa Peterson, M., and Trevathan, L. E. 2007. Characterization of Rhizoctonia Like Fungi isolated from agronomic crops and turfgrasses in Mississippi. Plant Dis. Vol 91(3):260 265. U.S. E.P.A. 2003. Reusing cleaned up superfund sites: golf facilities where waste is left on site. www.epa.gov/superfund EPA 540 R 03 003, OSWER 9230.0 109, PB2003 104262. 73 pp. Vargas, J. M. 1994. Management of Turfgrass Diseases. CRC Press, Boca Raton, FL. pp. 20 23. Warcup, J.H. and Talbot, P.H.B. 1962. Ecology and identity of mycelia isolated from soil. Trans. Brit. Mycol. Soc. 45:495 518. Watson, L. and Dallwitz, M. J. 1992. The grass genera of the world. Oxford. 1992. 1024 pp. White, T. Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315 322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. sninsky, and T. J. White, eds. Academic Press, San Diego, CA. Wiecko, G. 2003. Ocean water as substitute for postemergence herbicides in tropical turf. Weed Tech Vol 17:788 791. Williams, J. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., and Tingey, S. V. 1990. DNA polymorphisms amplified by arbitrary primers are u seful as genetic markers. Nucl. Acids Res., 18: 6531 6535. Williams, S. 2002. Breaking the mold; Crown Colony G&CC takes a wall to wall chance on seashore paspalum. Golf Course Mgmt. Aug. 70(8): p. 34 40.
114 Yenny, R. 1994. Salinity mana gement. USGA Section Green Record. November/December:7 10. Yokoyama, K. Hyakumachi, M. and Ogashi, A. 1985. Studies of Hyphal anastomosis of Rhizoctonia solani III. hyphal anastamosis in non self anastamosis isolates. Memoirs of the Faculty of Agr ic. Hokkaido Univ. 14 (4):416 421. Zhang, M. and Dernoeden, P. H. 1995. Facilitating anastomosis grouping of Rhizoctonia solani isolates from cool season turfgrasses. Hort Science 30(6):1260 1262. Zinn, S. 2010. Paspalum grows up. Golf Course Man agement. Nov.: 68 69.
115 BIOGRAPHICAL SKETCH Steven J. Kammerer was born to Richard and Dorothy Kammerer in Xenia, OH. Steve started working at the local orchard at the early age of 15 riding his bicycle 5+ miles back and forth from home after school and over the summer Steve developed a love for being outside and working with plants. Steve attended the Ohio State University with the intention of be com ing an engineer at the urging of his father before changing majors to the field of Horticulture a se rious deviation from all the other occupations within his family. Prior to graduating in 1986 with a B.S. degree Steve completed an internship conducting field agricultural pesticide research with Mobay Chemical Company in Howe, IN. Steve was accepted i nto the University of Georgia graduate school where he completed his M.S. degree in Plant Pathology working with peaches and nectarines under the guidance of Dr. Floyd Hendrix in 198 9 Before graduation, Steve accepted and completed another internship wit h Fermenta Plant Protection as a field technical manager. After graduation, Steve accepted a full time position with Fermenta. Steve married Rosemary in 1995 and they w ere blessed with two children, a boy and a girl, Christian and McKenna From March 1 989 Steve worked through multiple company changes to his current position with Syngenta Crop Protection as a field technical manager for turfgrass and aquatics Eighteen years after graduating from UGA Steve resumed his education at UF in the pursu it of a doctoral degree in plant pathology as an aside to his full time job with Syngenta His degree requirements were completed in August 20 11 with the approval and acceptance of this dissertation. He continues as a Syngenta employee currently seeking other positions of responsibility.