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Belonolaimus longicaudatus and Hoplolaimus galeatus on Seashore Paspalum (Paspalum vaginatum)


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Belonolaimus longicaudatus AND Hoplolaimus galeatus ON SEASHORE PASPALUM ( Paspalum vaginatum ) By ADAM C. HIXSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Adam C. Hixson

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iii ACKNOWLEDGMENTS I would especially like to thank Dr. William Crow for his scientific expertise, professional guidance, and monetary assistance while pursing my master’s degree. He taught me that learning from my mistakes is the one of the most important skills in performing research. I am also indebted to my other committee members, Dr. Robert McSorley and Dr. Laurie Trenholm. Dr. McSorley’s statistical knowledge and assistance with experimental methodology were key to the completion of my degree. Dr. Trenholm always made herself available for help with turfgrass questions and overall support. Thanks go also to Mr. Frank Woods for the words of wisdom and helping me to stay sane through all the insanity. I would also like to thank my parents for all their love and guidance throughout my life. My father planted the scientific seed within me early in life. My father taught me that knowledge is something that never can be taken away from you. Collecting knowledge can be an arduous task, but the rewards are everlasting. From the time I was a baby, my mother has always been my emotional support. She has taught me that perseverance and integrity are keys to success in your career and personal life. My brother has been a very influential person in my life. He has taught me that having fun is as important as any amount of knowledge or fulfillment of goals. Allowing for time to relax and enjoy hobbies is an opportunity to clear your mind and become more successful.

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iv Most importantly, I would like to thank my wife for her never-ending love and support. As I pursue my dream, she has never doubted me. Through all the late nights and stressful days, she has always been there for emotional support and a helping hand. I am also forever grateful to my wife for the sacrifices she has made by leaving her family to be with me as I pursue my graduate degrees and career. She is a truly special person and I look forward to maturing with her for years to come.

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v TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW....................................................1 Introduction................................................................................................................... 1 Belonolaimus longicaudatus .........................................................................................2 Taxonomy..............................................................................................................2 Distribution and Biology.......................................................................................3 Host Range............................................................................................................4 Hoplolaimus galeatus ...................................................................................................5 Taxonomy..............................................................................................................5 Distribution and Biology.......................................................................................6 Host Range............................................................................................................7 Nematodes and Turfgrass.............................................................................................8 Salinity and Nematodes................................................................................................9 Seashore Paspalum.....................................................................................................10 Current Research........................................................................................................11 2 SUSCEPTIBILITY OF SEASHORE PASPALUM TO Belonolaimus longicaudatus AND Hoplolaimus galeatus .......................................................................................12 Introduction.................................................................................................................12 Materials and Methods...............................................................................................14 Results........................................................................................................................ .20 Discussion...................................................................................................................25 3 INFLUENCE OF SALT CONCENTRATION IN IRRIGATION WATER ON PATHOGENICITY AND DEVELOPMENT OF Belonolaimus longicaudatus AND Hoplolaimus galeatus ON SEASHORE PASPALUM..............................................28 Introduction.................................................................................................................28

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vi Materials and Methods...............................................................................................30 Results........................................................................................................................ .38 Discussion...................................................................................................................43 4 SURVEY OF PLANT-PARASITIC NEMATODES ASSOCIATED WITH SEASHORE PASPALUM.........................................................................................47 Introduction.................................................................................................................47 Materials and Methods...............................................................................................48 Results........................................................................................................................ .49 Discussion...................................................................................................................52 5 SUMMARY................................................................................................................55 LIST OF REFERENCES...................................................................................................59 BIOGRAPHICAL SKETCH.............................................................................................69

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vii LIST OF TABLES Table page 2-1. Composition of experimental soil compared to the United States Golf Association root zone mix specifications.....................................................................................15 2-2. Cumulative shoot dry weight and total root lengths of 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass 60 and 120 days after initial inoculations of 107 8 (2002) and 211 10 (2003) Belonolaimus longicaudatus .......................................20 2-3. Cumulative shoot dry weight and total root lengths of 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass 60 and 120 days after initial inoculations of 100 (2002) and 199 13 (2003) Hoplolaimus galeatus .................................................23 2-4. Final population densities (nematodes/100 cm3 of soil) on 'SeaIsle1' seashore paspalum and 'Tifdwarf' bermudagrass 60 and 120 days after initial inoculations of 100 (2002) and 199 13 (2003) Hoplolaimus galeatus and 107 8 (2002) and 211 10 (2003) Belonolaimus longicaudatus ................................................................24 4-1. Frequency of occurrence and population density of plant-parasitic nematode genera in soil samples collected from seashore paspalum golf courses in Florida..............51 4-2. Frequency of occurrence and population density of plant-parasitic nematode genera in soil samples collected from seashore paspalum home lawns in Florida..............52

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viii LIST OF FIGURES Figure page 3-1. Relationship between log transformation of final population densities (Pf) of H. galeatus (nematodes/100 cm3 of soil) ( Y ) and salinity treatment ( x ) in 2002 and 2003 glasshouse experiments...................................................................................40 3-2. Relationship between log transformation of final population densities (Pf) of B. longicaudatus (nematodes/100 cm3 of soil) ( Y ) and salinity treatment ( x ) in 2002 and 2003 glasshouse experiments............................................................................41 3-3. Relationship between log transformation of J2 or adults, J3, and J4 final population densities (Pf) of B. longicaudatus (nematodes/100 cm3 of soil) ( Y ) and salinity treatment ( x ) in the 2003 glasshouse experiment.....................................................42 3-4. Effects of Belonolaimus longicaudatus on root length of 'SeaIsle 1' seashore paspalum ( Paspalum vaginatum ) at increasing salinity levels. Inoculated plants received 243 13 B. longicaudatus while uninoculated plants received no nematodes. The error bars show standard deviation of individual population means. ** Indicates difference from inoculated at P 0.01, according to the oneway ANOVA procedure...........................................................................................43

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ix Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science Belonolaimus longicaudatus AND Hoplolaimus galeatus ON SEASHORE PASPALUM ( Paspalum vaginatum ) By Adam C. Hixson December 2003 Chair: William T. Crow Major Department: Entomology and Nematology Seashore paspalum ( Paspalum vaginatum ) has great potential for use in saltaffected turfgrass sites. Seashore paspalum has a natural tolerance to drought and high salinity irrigation. Therefore, use of this grass on golf courses, athletic fields, and lawns in subtropical coastal areas may aid in conservation of fresh water resources. Plantparasitic nematodes are damaging pathogens of turfgrasses in Florida, with Belonolaimus longicaudatus and Hoplolaimus galeatus considered among the most damaging. Glasshouse experiments were performed to compare the susceptibility of 'SeaIsle 1' seashore paspalum to B. longicaudatus and H. galeatus with 'Tifdwarf' bermudagrass ( Cynodon dactylon C. transvaalensis ), and to examine the effects of increasing irrigation salinity levels on B. longicaudatus and H. galeatus 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass grown in 1,500-cm3 clay pots filled with 100% United States Golf Association (USGA) specification sand and inoculated with either nematode served as experimental units. Both nematode species reproduced well on either

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x grass, but only B. longicaudatus consistently stunted root growth. Belonolaimus longicaudatus reduced root growth by 35 to 45% at 120 days after inoculation on both grasses, but these reductions did not indicate a difference ( P > 0.05) in susceptibility between grasses. These data suggest that 'SeaIsle 1' seashore paspalum is not less susceptible to B. longicaudatus or H. galeatus than is 'Tifdwarf' bermudagrass. Final population densities of H. galeatus followed a negative linear regression ( P 0.01) with increasing salinity levels. Final population densities of B. longicaudatus were quadratically ( P 0.01) related to increasing salinity levels from 0 to 25 dS/m. An increase in population densities of B. longicaudatus was observed at moderate salinity levels (10 and 15 dS/m) compared to 0 dS/m ( P 0.05). Reproduction and feeding of B. longicaudatus and H. galeatus decreased at salinity levels of 25 dS/m and above. Root length comparisons revealed that B. longicaudatus caused root stunting at low salinity levels, 0 to 10 dS/m ( P 0.05). However, at higher salinity irrigation treatments, 15 to 25 dS/m, root lengths were not different ( P > 0.05) between inoculated and uninoculated plants. These results indicate that the ability of B. longicaudatus to feed and stunt root growth was negatively affected at salinity levels of 15 dS/m and above.

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1 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Introduction Coastal areas in the southeastern United States, including a majority of Florida, are ideal habitats for Belonolaimus longicaudatus Rau, the sting nematode (Christie, 1959; Holdeman, 1955), and Hoplolaimus galeatus (Cobb, 1913) Thorne 1935, the lance nematode (Ahmad and Chen, 1980; Williams, 1973). Both are important pathogens of turfgrass (Perry et al., 1970). New turfgrass cultivars or ecotypes are commonly introduced into the turfgrass industry, but in rare cases, a new species of turfgrass begins to become more widely utilized. Seashore paspalum ( Paspalum vaginatum Swartz) is a new turfgrass species quickly growing in popularity due to the breeding and development of cultivars with a fine leaf texture and tolerance to drought and high salinity irrigation (Duncan, 1999a; Morton, 1973). Belonolaimus longicaudatus and H. galeatus have been reported as pathogens of many bermudagrass ( Cynodon dactylon (L.) Pers and Cynodon spp. hybrids) (Di Edwardo, 1963; Giblin-Davis et al., 1992b; Johnson, 1970; Rivera-Camarena, 1963; Winchester and Burt, 1964), and St. Augustinegrass ( Stenotaphrum secundatum ) (Busey et al., 1991; Giblin-Davis et al., 1992a; Henn and Dunn, 1989; Kelsheimer and Overman, 1953) cultivars. However, no published data currently exist on the susceptibility of seashore paspalum to B. longicaudatus or H. galeatus Knowledge is also lacking on the response of B. longicaudatus and H. galeatus to high salinity irrigation. After personal communication with two turfgrass managers in south Florida, Morton (1973)

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2 hypothesized that low nematode counts may be associated with seashore paspalum irrigated with saline water. Further investigation is necessary to confirm this hypothesis. Typically, susceptibility of a plant to a pathogen is based on yield reduction caused by the pathogen, but yield is difficult to determine in turfgrass. Therefore, root length data and the reproductive capability of the nematode are major determining factors of susceptibility in turfgrasses. When levels of susceptibility are determined, soil and root nematode assays may be useful in predicting a need for treatment. This information will help avoid unnecessary post-plant nematicide treatments or failure to apply a needed one. Belonolaimus longicaudatus Taxonomy The first reported nematode in the genus Belonolaimus was the species B. gracilis collected from the rhizosphere of a pine tree ( Pinus spp.) near Ocala, FL (Steiner, 1949). Subsequently, B. gracilis was reported to damage peanut ( Arachis hypogaea ) in Virginia (Owens, 1951), strawberry ( Trifolium fragiferum ), celery ( Apium graveolens ), and corn ( Zea mays ) in Florida (Christie et al., 1952), and cotton ( Gossypium spp.), soybean ( Glycine max ), and cowpea ( Vigna unguiculata ) in South Carolina (Graham and Holdeman, 1953). Later, Rau (1958) described B. longicaudatus which soon became accepted as the more common sting nematode damaging agricultural crops and turfgrasses in the southeastern United States (Perry and Rhoades, 1982; Smart and Nguyen, 1991). No published data exist on B. gracilis except for the original description in 1949. Tail and stylet lengths can separate the two species. Specimens of B. longicaudatus have a shorter tail and a longer stylet than those of B. gracilis (Rau 1958). Rau (1963) continued his work with the genus Belonolaimus describing three more species: B. euthychilus, B. maritimus, and B. nortoni Presently, the genus includes 9

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3 species, the five mentioned previously, and B. anama, B. jara, B. lineatus, and B. lolii (Fortuner and Luc, 1987). Distribution and Biology Belonolaimus longicaudatus is found primarily in deep sandy soils in the lower coastal plains of the southeastern United States from Virginia to Florida (Christie, 1959; Holdeman, 1955), but has been reported in New Jersey (Myers, 1979), Kansas (Dickerson et al., 1972), Arkansas (Riggs, 1961), Oklahoma (Russell and Sturgeon, 1969), Texas (Christie, 1959; Wheeler and Starr, 1987), and recently in California (Mundo-Ocampo et al., 1994). Holdeman (1955) reported confirmed findings of B. longicaudatus in Florida, Georgia, South Carolina, North Carolina, and Virginia, with possible findings in Alabama, Mississippi, and Louisiana. Belonolaimus longicaudatus is most commonly found in Florida because it is well adapted to soils with > 80% sand content and < 10% clay content (Miller, 1972; Robbins and Barker, 1974). Populations of B. longicaudatus have been discovered at golf courses located outside the native habitat of the nematode. These areas in the Bahamas, Bermuda, Puerto Rico, Costa Rico, and California are ideal for B. longicaudatus reproduction because the soil profiles are at least 90% sand (Lopez, 1978; Mundo-Ocampo et al., 1994; Perry and Rhoades, 1982). Imported sod from Florida and Georgia appear to be the cause of the infestations in the Bahamas, Bermuda, and Puerto Rico (Perry and Rhoades, 1982). Belonolaimus longicaudatus is recognized as the most pathogenic ectoparastic plant nematode to turfgrasses in Florida. Acceptable visual quality often is not possible without treatment of the nematode infestation. Root injury typically consists of greatly reduced root systems, with short stubby roots exhibiting dark lesions along the root axis

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4 and along the tip. Aboveground symptoms usually consist of severe stunting, incipient wilting, leaf chlorosis, or death (Perry and Rhoades, 1982). Reproduction of B longicaudatus is exclusively accomplished through amphimixis, requiring both males and females to be present (Huang and Becker, 1999). Females lay eggs as long as food is readily available (Huang and Becker, 1999). Eggs are laid two at a time with one coming from each opposed ovary of the amphidelphic reproductive system (Han, 2001). The entire lifecycle was achieved in 24 days at 28 C on excised corn roots grown in vitro (Huang and Becker, 1997; 1999). Belonolaimus longicaudatus is a migratory ectoparasitic nematode that inserts its stylet deep into the root tip and withdraws the cellular contents. The presence of a host is necessary for survival and reproduction, because there is no known survival mechanism such as anabiosis or diapause (Huang and Becker, 1997). Temperature can have a dramatic effect on the survivability and reproduction of B. longicaudatus (Boyd and Perry, 1971; Robbins and Barker, 1974). Seasonal temperature variation greatly influenced the population levels and distribution of the sting nematode. April and May temperatures in Gainesville, FL are typically favorable for high populations, while June and July are least favorable (Boyd and Perry, 1971). Reproduction of B. longicaudatus was greatest at 25 to 30 C, with one population achieving some reproduction at 35 C while the other population declined. Both populations had minimal reproduction at 20 C (Robbins and Barker, 1974). Host Range Belonolaimus longicaudatus has an extremely wide host range, including many small grains, forage crops, fruits, vegetables, ornamentals, trees, and turfgrasses (Bekal

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5 and Becker, 2000; Holdeman and Graham, 1953; Owens, 1951; Perry and Rhoades, 1982; Robbins and Barker, 1973). Holdeman (1955) reported watermelon ( Citrullus lanatus ) and tobacco ( Nicotiana tabacum ) to be non-hosts. Additionally, asparagus ( Asparagus officinalis ), buckhorn plantain ( Plantago lanceolata ), okra ( Abelmoschus esculentus ), and pokeweed ( Phytolacca americana ) were reported to be non-hosts (Robbins and Barker, 1973). Later, a California population also was found to be unable to reproduce on okra, watermelon, and tobacco (Bekal and Becker, 2000). Physiological races have been suggested numerous times by many researchers due to host preferences and reproductive isolation, but none are widely recognized (Abu-Gharbieh and Perry, 1970; Owens, 1951; Robbins and Barker, 1973; Robbins and Hirschmann, 1974). Robbins and Barker (1973) reported that some poor or non-hosts for three populations of B. longicaudatus from North Carolina were actually good to excellent hosts for one population from Georgia. Also, when populations with different morphological characteristics and host preferences were allowed to mate with each other, infertile individuals were produced (Robbins and Hirschmann, 1974). Morphological differences and reproductive potential suggest that the populations from North Carolina and Georgia may be different species (Robbins and Hirschmann, 1974). Hoplolaimus galeatus Taxonomy Lance nematodes belong to the extremely diverse subfamily of Hoplolaiminae that includes hundreds of species of plant-parasitic nematodes. Hoplolaimus galeatus was originally described as Nemonchus galeatus (Cobb, 1913), but later Thorne (1935) determined this nematode to be identical in general size and shape to Hoplolaimus coronatus Hoplolaimus galeatus and H. coronatus were later found to be conspecific,

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6 with H. galeatus having date priority (Sher, 1961). The nematode was thereafter referred to as Hoplolaimus galeatus Identifying morphological characteristics on the female include an offset cephalic region, usually with 5 annules, 2 phasmids (one anterior to the vulva and the other posterior), and a rounded tail with 10 to 16 annules. Hoplolaimus galeatus reproduces by amphimixis with an equal ratio of females to males (Hirschmann, 1959; Smart, 1965; Wen and Chen, 1972). Distribution and Biology Hoplolaimus galeatus has a wider distribution than B. longicaudatus but is most numerous in the southeastern United States. Woody or graminaceous plants appear to be especially favorable hosts (Williams, 1973). Hoplolaimus galeatus is usually a seemingly unmanageable problem because of its widespread distribution and the frequent failure of nematicides to reduce their numbers while controlling other plant parasitic nematodes (Perry et al., 1970). Krusberg and Sasser (1956) found that cotton plants growing under drought conditions that were stunted, yellowing, and almost completely defoliated returned much higher numbers of H. galeatus in the soil and roots near the rhizosphere than did healthy plants. The nematodes primarily fed endoparasitically on the cotton roots, causing considerable damage to the root cortex during penetration. Disruption of the vascular tissue occurred during feeding due to the preference for phloem (Krusberg and Sasser, 1956). Slash ( Pinus elliottii ) and Loblolly pine ( Pinus taeda ) seedlings suffered 50% mortality in North Carolina due to infestation by H. galeatus Glasshouse experiments confirmed these findings, showing roots with black lesions and most of the root cortex destroyed (Ruehle and Sasser, 1962). Experiments with bermudagrass determined that total plant weight was reduced by 50% over 6 months by H. galeatus (Di Edwardo, 1963).

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7 Host Range Lance nematodes ( H. galeatus ) are pathogenic on a variety of agronomic crops and turfgrasses. The rate of reproduction of H. galeatus was higher on rye ( Secale cereale ), barley ( Hordeum vulgare ), wheat ( Triticum aestivum ), oats ( Avena sativa ), soybean, corn, cabbage ( Brassica oleracea ), and bean ( Phaseolus vulgaris ) than on pepper ( Capsicum spp.), eggplant ( Solanum melongena ), lettuce ( Lactuca sativa ), tobacco, and pea (Ahmad and Chen, 1980). Hoplolaimus galeatus can cause serious damage to cotton (Krusberg and Sasser, 1956), pine (Ruehle and Sasser, 1962), oak (Viggars and Tarjan, 1949), wheat (Ahmad and Chen, 1980), and turfgrasses (Di Edwardo, 1963; Perry et al., 1970). In a study involving H. coronatus on cotton, host symptoms were more prominent in dry conditions, but plants supplied with adequate moisture could tolerate high populations (Krusberg and Sasser, 1956). When cotton followed bahiagrass ( Paspalum notatum ), the H. galeatus population dropped to below damaging levels (Rodriguez-Kabana and Pearson, 1972). Hoplolaimus galeatus was also found to feed endo and ectoparasitically on alfalfa ( Medicago sativa ) and bermudagrass (Ng and Chen, 1980). Hoplolaimus galeatus caused reductions in magnesium and water uptake, shoot and root growth, and number of root hairs on tall fescue ( Festuca arundinacea ) (Rodriguez-Kabana et al., 1978). Lance nematodes ( Hoplolaimus spp.) were found to be associated with unhealthy St. Augustinegrass lawns in the Tampa, FL area (Kelsheimer and Overman, 1953). Other surveys specifically associated H. galeatus with damaged lawns in Florida (Good et al., 1956; Perry and Good, 1968; Whitton, 1956). Hoplolaimus galeatus reproduced well on seven different cultivars of St. Augustinegrass, indicating similar host suitability among these cultivars (Henn and Dunn, 1989). Time course experiments with 'Floratam' and

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8 'FX-313' St. Augustinegrass indicated that H. galeatus had no effect on plant growth, even though soil counts of H. galeatus exceeded the proposed action threshold of 40 nematodes/100 cm3 of soil (Crow et al., 2003) for 84 days during the course of the experiment (Giblin-Davis et al., 1995). Alyceclover ( Alysicarpus vaginalis ), corn, and some vegetables supported high populations of H. galeatus but measurable yield suppression was not detected in any of these crops (Mashela et al., 1992b; Norton and Hinz, 1976; Rhoades, 1987). Nematodes and Turfgrass In 1951, plant-parasitic nematodes were reported to be associated with the 'yellow tuft' disease of bentgrass ( Agrostis palustris ) in Virginia (Tarjan and Ferguson, 1951). A subsequent survey in Rhode Island by Troll and Tarjan (1954) indicated that several species of nematodes might be causing serious damage on turfgrass as either primary or secondary plant pathogens. Nematodes were not recognized as pathogens of turfgrass in Florida until 1953, when V. G. Perry applied three nematicides on a thinning bermudagrass golf course green at Sanford, FL. One month after treatment, the turfgrass began to grow and later completely covered the putting surface. This indicated that the death of plant-parasitic nematodes allowed the grass to recover from root damage (Perry and Rhoades, 1982). Christie et al. (1954) stated that sting and lance nematodes were responsible for most of the nematode damage to turfgrass in Florida. In Florida, B. longicaudatus became recognized as a pathogen of significant importance to turfgrasses (Christie et al., 1954; Perry et al., 1970; Rhoades, 1962). Perry and Rhoades (1982) estimated that over 80% of some 700 Florida golf courses used nematicides routinely.

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9 Many hybrid bermudagrasses ( Cynodon dactylon (L.) Pers C. transvaalensis Burtt-Davis) have been determined to be suitable and susceptible hosts to B. longicaudatus (Giblin-Davis et al., 1992b). 'Ormond' bermudagrass was demonstrated to be extremely susceptible to B. longicaudatus with severe yellowing, stunting of top growth, and stunting of the roots (Winchester and Burt, 1964). St. Augustinegrass also was found to be a suitable and susceptible host to B. longicaudatus with root reductions occurring when compared to uninoculated plants (Giblin-Davis et. al, 1992a; Rhoades, 1962). Rhoades (1962; 1965) also reported that Trichodorus christiei and T. proximus reduced St. Augustinegrass root weight when compared to uninoculated plants. Sting and lance nematodes were extracted from soil samples taken from declining areas of St. Augustinegrass in the Tampa Bay area (Kelsheimer and Overman, 1953). Sting and lance nematodes were both found to be pathogenic on 'Tifton 328' bermudagrass in a susceptibility experiment. Sting nematode populations increased from 250 to approximately 3,700 per pot, while lance nematode populations increased from 250 to approximately 2,100 per pot (Rivera-Camarena, 1963). Salinity and Nematodes High-salinity irrigation has been shown to negatively affect some root-knot nematodes. Meloidogyne incognita egg hatching decreased by 96% when exposed to a 28.8 dS/m solution (Lal and Yadav, 1975). Salinity has demonstrated negative effects on the hatching and infectivity of M. javanica and M. arenaria juveniles (Dropkin et. al, 1958; Maqbool et al., 1987). Khan and Khan (1990) found that after only 7 days of salinity exposure, M. incognita and M. javanica had significant reductions in hatching and increases in mortality. Population levels of Aphelenchus avenae, Pratylenchus

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10 thornei, Helicotylenchus spp., and Rotylenchulus reniformis were lower at increased salt concentrations over nonsaline treatments. No differences were observed in populations of Heterodera avenae and Tylenchorhynchus mashhoodi at increased salt concentrations when compared to controls (Lal and Yadav, 1976). Some species of root-knot nematodes ( M. spartinae ) and sting nematodes ( B. maritimus ) may be well adapted to high-salinity conditions (Rau, 1963; Rau and Fassuliotis, 1965). Seashore Paspalum Seashore paspalum ( Paspalum vaginatum Swartz) is a warm season, perennial grass, with both rhizomotous and stoloniferous growth, and a V-shaped inflorescence (Duncan and Carrow, 2000; Morton 1973). Seashore paspalum is indigenous to South Africa and South America, and has been found in Argentina, Brazil, Australia, the Caribbean Islands, and Pacific islands. Within North America, it has been found throughout the southern latitudes of the United States. Morton (1973) indicates that St. Simons Island, GA, is the origin of seashore paspalum in North America. The grass is best adapted to coastal areas (brackish areas, sand dunes, and beaches) with tropical and sub-tropical temperatures. Regions from 30 to 35 N and S latitudes are most suitable (Duncan and Carrow, 2000; Morton, 1973; Skerman and Riveros, 1990). Seashore paspalum encompasses a wide range of ecotypes from fine to coarse leaf textures, and can be used in a multitude of turfgrass applications (Duncan and Carrow, 2000). Several seashore paspalum ecotypes have been researched for their multiple stress resistance characteristics. In particular, advantageous characteristics include salinity tolerance (Carrow and Duncan, 1998; Marcum and Murdoch, 1994; Peacock and Dudeck, 1985), drought tolerance, (Huang et al. 1997a; 1997b), wear and traffic tolerance

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11 (Trenholm et al. 1999; 2001a; 2001b), water logging tolerance (Malcolm, 1977), and tolerance of other stresses including pH, diseases, insects, and weeds (Duncan, 1999b; Johnson and Duncan, 1997; 1998; Wiseman and Duncan, 1996). Fertility requirements are much lower for seashore paspalum than many similarly textured grasses (Beard et al., 1982; 1991). Suggested nitrogen rates for fairways, tees, and sports fields range from 0.98 to 2.94 kg/100 m2 annually depending on geographical location (Duncan, 1999b). In Florida, recommended nitrogen rates are slightly higher, ranging from 1.96 to 5.88 kg/100 m2 annually (Trenholm and Unruh, 2003). Seashore paspalum greens ecotypes were reported to have higher quality ratings at 3 to 4 mm mowing height when compared to 'Tifgreen' bermudagrass (Duncan, 1999b). Current Research The research reported hereafter was performed in an environmentally controlled glasshouse at the University of Florida Envirotron Turfgrass Research Laboratory in Gainesville, FL. Experiments were performed in consecutive summers in an optimal environment for both nematodes and turfgrasses. The objectives of the research were to: 1. Determine susceptibility of 'SeaIsle 1' seashore paspalum to a population of B. longicaudatus and a population of H. galeatus 2. Compare 'SeaIsle 1' seashore paspalum to 'Tifdwarf' bermudagrass for differences in susceptibility to a population of B. longicaudatus and a population of H. galeatus 3. Determine how high salinity irrigation can affect the reproduction of a population of B. longicaudatus and a population of H. galeatus 4. Conduct a survey to determine which plant-parasitic nematodes are associated with established seashore paspalum golf courses and home lawns in Florida.

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12 CHAPTER 2 SUSCEPTIBILITY OF SEASHORE PASPALUM TO Belonolaimus longicaudatus AND Hoplolaimus galeatus Introduction Seashore paspalum ( Paspalum vaginatum Swartz) is a warm-season turfgrass quickly expanding in popularity. Due to the fine leaf texture and natural tolerance to drought and high salinity, seashore paspalum has become more prevalent in coastal saltaffected turfgrass sites (Duncan, 1999a; Morton, 1973). One major limitation of cultivating turfgrasses in the sandy soils of the southeastern United States is the destruction of roots by phytoparasitic nematodes (Perry and Rhoades, 1982). The sting nematode ( Belonolaimus longicaudatus Rau) and the lance nematode ( Hoplolaimus galeatus (Cobb) Thorne) are destructive pathogens on a variety of agricultural crops, including turfgrasses (Ahmad and Chen, 1980; Holdeman and Graham, 1953; Perry and Rhoades, 1982; Smart and Nguyen, 1991). While B. longicaudatus is found primarily in the coastal plains of the southeastern United States (Christie, 1959; Holdeman, 1955), H. galeatus has a much wider distribution (Williams, 1973). Belonolaimus longicaudatus damages lateral roots as soon as they are formed, causing a stunting of root growth, decreased water and nutrient uptake, and decreased rates of evapotranspiration (Busey et al., 1991; Johnson, 1970; Perry and Rhoades, 1982). Hoplolaimus galeatus enters the root tissue as a migratory endoparasite and is thought to damage roots by feeding and physical tunneling through the root cortex cell walls (Henn and Dunn, 1989; Krusberg and Sasser, 1956; Lewis and Fassuliotis, 1982; Perry et al., 1970; Williams, 1973).

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13 Both nematodes have been reported as economically important pathogens of turfgrasses in the southeastern United States (Christie et al., 1954; Kelsheimer and Overman, 1953; Perry and Rhoades, 1982). Belonolaimus longicaudatus has been reported as a pathogen of many bermudagrass ( Cynodon dactylon (L.) Pers. and Cynodon spp. hybrids), and St. Augustinegrass ( Stenotaphrum secundatum (Walt.) Kuntze) cultivars (Busey et al., 1991; Giblin-Davis et al., 1992a; 1992b; Johnson, 1970). Seven cultivars of St. Augustinegrass were discovered to have similar host suitability for H. galeatus in the glasshouse and in microplots (Henn and Dunn, 1989). However, root and shoot growth of 'FX-313' and 'Floratam' St. Augustinegrass were not affected by H. galeatus in a population dynamics and pathogenicity study (Giblin-Davis et al., 1995). Two populations of B. longicaudatus readily reproduced on 'Tifdwarf' bermudagrass ( Cynodon dactylon C. transvaalensis Burtt-Davis), and caused extensive root damage (Giblin-Davis et al., 1992b; Johnson, 1970). A forage grass study determined that there was differential host suitability and susceptibility to B. longicaudatus in Digitaria spp., Paspalum spp., and Chloris spp. introductions (Boyd and Perry, 1969). At present, no published data exist on the susceptibility of seashore paspalum to H. galeatus or B. longicaudatus The objectives of this study were to: i) determine the susceptibility and host suitability of 'SeaIsle 1' seashore paspalum to a population of B longicaudatus and a population of H galeatus and ii) compare 'SeaIsle 1' seashore paspalum to 'Tifdwarf' bermudagrass for differences in susceptibility to a population of B. longicaudatus and a population of H. galeatus

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14 Materials and Methods Experiments were performed to compare the relative susceptibility of 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass to B longicaudatus and H galeatus Experiments were conducted during the spring and summer of 2002 and 2003 at the University of Florida Turfgrass Envirotron Glasshouse in Gainesville, FL. Commercially available cultivars of Paspalum vaginatum and Cynodon dactylon C. transvaalensis were evaluated in this experiment. A sod square of 'SeaIsle 1' seashore paspalum was obtained from R. R. Duncan at the University of Georgia and 'Tifdwarf' bermudagrass was available from previous experiments. Nematode-free plugs of each grass were obtained by rooting aerial cuttings of stolons from each grass in tapered RLC-7 (UV Stabilized) Super "Stubby" Cells (cell depth = 14 cm; diam. = 3.8 cm; volume = 115 ml) (Ray Leach Single Cell Cone-tainer, Stuewe & Sons, Inc., Corvallis, OR) filled with 140 g of uninfested soil. Soil used for growth media consisted of 100% United States Golf Association (USGA) specification sand. The soil texture was analyzed using the sieving method for testing a USGA root zone mix (USGA Green Section Staff, 1993) (Table 2-1). An absorbent cotton ball was placed at the bottom of each cell to prevent soil from escaping through the drain holes. The soil was then thoroughly wetted to allow for settling. A depression was made in each cell and two aerial stolons were planted on opposing sides of the depression. Stolons (5 to 8 cm long) were terminal cuttings with two or three nodes. During the fall and winter months, the cells were placed on a glasshouse bench 1.25 m below an enclosed high bay 1,000-watt metal halide growth lamp (Hi-Tek Series, Lithonia Lighting, Conyers, GA) set on a 12hour cycle to simulate the longer day-lengths required for optimal growth. The grass was

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15 fertilized biweekly using a fertilizer solution (20%-20%-20% (N-P2O5-K2O) plus trace elements) (Peters Professional All-Purpose Plant Food, Spectrum Brands, St. Louis, MO) at a rate equivalent to 49.0 kg N/ha/month, 21.6 kg P/ha/month, and 40.7 kg K/ha/month. Both grasses were allowed to develop a substantial root system for four weeks. Table 2-1. Composition of experimental soil compared to the United States Golf Association root zone mix specifications Sand Type Particle Size Experimental Soil USGA specifications Fine Gravel 2.0 to 3.4 mm 0.1% Very Coarse Sand 1.0 to 2.0 mm 3.7% Not more than 10% of the total particles in this range, including a maximum of 3% fine gravel (preferably none) Coarse Sand 0.5 to 1.0 mm 30.8% Medium Sand 0.25 to 0.50 mm 53.4% Minimum of 60% of the particles must fall in this range Fine Sand 0.15 to 0.25 mm 10.3% Not more than 20% Very Fine Sand 0.05 to 0.15 mm 1.7% Not more than 5% Data are means of five replicates Plugs of seashore paspalum and bermudagrass obtained from the cells were transferred into 14.5 16-cm-diam. clay pots (1,500 cm3) filled with 100% USGA specification sand (Table 2.1). Insect screening (7 lines 5 lines/cm2) was cut into 5 5cm squares and placed at the bottom of each pot to prevent soil from escaping through the drain hole. Roots were washed free of soil and trimmed to approximately 5 cm below the crown to promote fresh root growth. Two depressions were made in each pot on opposite sides and two plugs of turfgrass were planted per pot. These experimental units were placed on a screened bench in an environmentally controlled glasshouse and irrigated as needed for 14 days to allow for adjustment to the new environment.

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16 A population of B. longicaudatus obtained from R. M. Giblin-Davis that originated from unmanaged field soil in the Sanford, FL area and were allowed to reproduce on 'FX 313' St. Augustinegrass was used in this experiment. A population of H. galeatus was obtained from a 'Floradwarf' bermudagrass putting green at the G. C. Horn Turfgrass Field Laboratory in Gainesville, FL. Inocula were extracted from soil using a modified Baermann funnel method (McSorley and Federick, 1991). In 2002, the source of the H. galeatus population was contaminated with other plant-parasitic nematodes, so 100 individual H. galeatus of mixed life stages were handpicked to inoculate into 16 pots of each grass. Since the B. longicaudatus population was not contaminated, handpicking was not necessary. A solution of B. longicaudatus at various life stages in tap water was calibrated by counting nematodes from 1-ml aliquots on a grided counting slide (Hawksley and Sons Limited, Lancing, Sussex, United Kingdom) replicated ten times. Approximate numbers of nematodes were measured with a pipette from water suspensions of inocula. Belonolaimus longicaudatus (107 8) was inoculated into 16 pots of each grass. In 2003, H. galeatus and B. longicaudatus were obtained from the previous year's experiment and inocula for both species were obtained using a modified Baermann funnel method (McSorley and Federick, 1991). Suspensions were made for each nematode, and standardized to deliver approximately 200 nematodes per pot. Hoplolaimus galeatus (199 13) and B longicaudatus (211 10) were inoculated into 20 pots of each grass, respectively. A higher level of nematodes was used in the second year to achieve higher reproduction. In both years, nematodes were suspended in 50 ml of tap water and equally distributed into four cavities formed in the soil near the base of the plant. Uninoculated plants received 50 ml of tap water. After

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17 inoculation, the cavities were then pinched closed with surrounding soil and tap water was applied as needed to avoid wilting. Experimental units were arranged in a randomized complete block design in the glasshouse on screened benches. In 2002, separate experiments were performed for each nematode consisting of a total of 64 pots each. Each experiment had eight treatments with eight replications. In 2003, the two nematode species were placed into the same experiment, allowing for fewer control pots and a higher number of replications. The second experiment had 12 treatments with 10 replications, for a total of 120 pots. For both experiments, at 60 days after inoculation, one pot of each treatment was removed from each block and analyzed, and 120 days after inoculation the remaining pots were analyzed. Treatments were seashore paspalum and bermudagrass inoculated with the specified number of H. galeatus or B. longicaudatus allowed to reproduce for 60 or 120 days, and uninoculated controls. Throughout the course of the 2002 experiments, which lasted from 21 April 2002 to 10 September 2002, average monthly high and low temperatures in the glasshouse ranged from 31 to 34 C, and 23 to 26 C, respectively. In 2003, experiments began 28 February 2003 and ended 13 July 2003. Average monthly high and low temperatures ranged from 27 to 32 C and 19 to 24 C, respectively. Using a 1.18-liter pump sprayer, an insecticide/miticide (Mavrik Aquaflow, Wellmark International, Schaumburg, IL) was applied at the labeled rate twice during the course of 2003 experiments for control of bermudagrass mites ( Eriophyes cynodoniensis Sayed) and rhodesgrass mealybugs ( Antonina graminis Maskell). 'Tifdwarf' bermudagrass was fertilized on a biweekly basis with 40 ml of a solution consisting of 5,100 mg NH4NO3 (34% N), 3,177 mg KCl, 252

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18 mg Ca(H2PO4)2, 435 mg CaSO4, 246 mg MgSO4, 1.55 mg H3BO3, 0.34 mg MnSO4, 0.58 mg ZnSO4, 0.13 mg CuSO4, and 3.5 mg FeSO4 per 1 liter of deionized water. A separate fertilizer solution was made for 'SeaIsle 1' seashore paspalum. The KCl was doubled to compensate for the different potassium requirements of seashore paspalum (Duncan and Carrow, 2000). Total N for both grass species was 624 mg/pot for 120 days and 346 mg/pot for 60 days. Using fabric scissors (Fiskars Brand Inc., Madison, WI), the grass was trimmed biweekly to approximately 2.5 cm above the soil surface. Tissue clippings were collected in 15 cm 23 cm mailing envelopes (Quality Park Products, St. Paul, MN) using a spouted 2.84-liter sample pan (40.6 cm 30.5 cm 5 cm) (Seedburo, Chicago, IL), and dried at 75 C for 48 hours to obtain cumulative shoot dry weight. After 60 and 120 days, randomly-selected pots of each treatment from each block were brought into the laboratory for destructive analysis. This provided a total of 32 pots for each nematode at each sampling date in 2002 and a total of 60 pots at each sampling date in 2003. Shoots were trimmed as close to the soil as possible and saved for cumulative dry weight analysis. Then, using a stainless steel T-sampling tool, a root core (approximately 4-cm-diam. 14 cm deep) was taken from the center of each pot to serve as a representative root sample. The remaining contents of each pot were emptied into their individually labeled plastic bags and thoroughly hand mixed. A 100-cm3 soil sample was taken from each bag and nematodes were extracted using a modified centrifugal-sugar flotation technique (Jenkins, 1964). Nematodes in the entire soil sample were counted using an inverted light microscope at 32 magnification. Root cores were washed free of soil on a sieve with 1.7-mm pore openings nested within a sieve with 75-m pore openings. Roots were removed from any aboveground

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19 growth and placed in a 50-ml disposable plastic centrifuge tube containing 30 ml of tap water. The 75-m-pore sieve was then submerged in 5 cm of tap water to allow the finer roots to float out and separate from the soil. These fine roots were collected using laboratory forceps and placed into the 50-ml plastic centrifuge tubes. Five drops (0.25 ml) of a 1% methylene blue mixture was added to the 30 ml of tap water to stain the roots. After a minimum of 24 hours in the solution, the roots were removed, placed on a 75-m-pore sieve, and washed free of excess dye. Stained roots were placed into a glassbottom tray and scanned using a HP ScanJet 2cx desktop scanner (Hewlett Packard, Boise, ID) to create a black and white bitmap image of the roots (Kaspar and Ewing, 1997; Pan and Bolton, 1991). The GSRoot (Louisiana State University, Baton Rouge, LA) software program was used to analyze the bitmap images. This program measures root lengths and surface areas from scanned images. Root length data were recorded for seven diameter ranges (< 0.05 mm, 0.05 to 0.10 mm, 0.10 to 0.20 mm, 0.20 to 0.30 mm, 0.30 to 0.40 mm, 0.40 to 0.50 mm, and > 0.50 mm). The resulting values were summed to determine the total root length of each root sample. In 2003 at the final sampling date, H. galeatus in roots were staged and counted to determine number of nematodes/gram of root in fresh-weighed samples using a modified acid-fuchsin staining-destaining procedure (Byrd et al., 1983). Total root lengths and cumulative shoot dry weights were compared for both evaluation periods. The one-way analysis of variance (ANOVA) procedure performed using SAS software (SAS Institute, Cary, NC) was used to analyze these two responses for differences between inoculated and uninoculated treatments within each grass species.

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20 Data for B. longicaudatus and H. galeatus were analyzed separately, even when the two experiments shared controls (2003 experiment). The one-way ANOVA procedure also was used to determine differences in susceptibility between grass species by comparing final nematode population densities, total root length percent reduction, and cumulative shoot dry weight percent reduction. Total root length percent reduction was calculated by [100 (total root length of inoculated – total root length of uninoculated) / total root length of uninoculated] and cumulative shoot dry weight percent reduction was calculated by [100 (cumulative shoot dry weight of inoculated – cumulative shoot dry weight of uninoculated) / cumulative shoot dry weight of uninoculated]. Again, data for B. longicaudatus and H. galeatus were analyzed separately, even when the two experiments shared controls (2003 experiment). A difference in the susceptibility of grasses to the nematodes was determined to be significant when P 0.05. Results The population of B. longicaudatus had a high degree of association with root reduction in both turfgrasses while the population of H. galeatus caused root reduction at only one sampling date for each grass (Tables 2-2, 2-3). In the 2002 trial, the B. longicaudatus population caused a reduction in total root growth in both seashore paspalum and bermudagrass ( P 0.05) at 120 days after inoculation, but only in seashore paspalum at the 60-day sampling (Table 2-2). In 2003, B. longicaudatus extensively stunted total root growth ( P 0.05) in seashore paspalum and bermudagrass at 60 and 120 days after inoculation (Table 2-2). In both years, lengths of small diameter roots (< 0.02 mm-diam.) on plants inoculated with B. longicaudatus were less than ( P 0.01)

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21 Table 2-2. Cumulative shoot dry weight and total root lengths of 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass 60 and 120 days after initial inoculations of 107 8 (2002) and 211 10 (2003) Belonolaimus longicaudatus Cultivar DAI Treatment Dry shoot weight (g) Root length (mm) 2002 trial 'Tifdwarf' 60 U 6.2 3.4 951 212 I 6.6 2.5 762 234 120 U 13.2 3.8 1,597 312* I 11.7 3.0 947 589 'SeaIsle 1' 60 U 13.9 3.2 1,489 287* I 11.6 3.0 1,150 281 120 U 24.4 6.7 1,647 152*** I 21.5 4.2 1,044 337 2003 trial 'Tifdwarf' 60 U 4.6 1.2 1,525 192* I 4.3 1.9 1,107 386 120 U 13.3 4.0 2,385 468*** I 13.4 3.2 1,380 354 'SeaIsle 1' 60 U 7.9 2.3 1,602 301** I 5.3 1.3 984 456 120 U 21.6 4.5 2,217 315*** I 20.6 4.3 1,185 467 Data are means of 8 (2002) or 10 (2003) replicates standard deviations. DAI = Days after inoculation. I = inoculated and U = uninoculated. *, **, *** Indicate uninoculated different from inoculated significant at P 0.05, P 0.01, and P 0.001 respectively, according to the one-way analysis of variance. uninoculated plants for both grasses and sampling dates. Lengths of small diameter roots on seashore paspalum inoculated with B. longicaudatus ranged from 488.96 217.26 to 574.26 227.91 mm compared to uninoculated plants having lengths ranging from 669.82 103.53 to 1037.26 126.31 mm. Bermudagrass followed a similar trend, with lengths of small diameter roots ranging from 469.58 108.99 to 740.81 146.93 mm and 623.60 133.24 to 1218.87 175.42 mm for inoculated and uninoculated plants, respectively. Cumulative shoot dry weight was not different ( P > 0.05) from controls when either turfgrass was inoculated with B. longicaudatus Belonolaimus longicaudatus

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22 reproduced with ease on both grasses, indicating they were suitable hosts. In both trials, B. longicaudatus reproduction data were variable among replicates, with bermudagrass supporting higher populations than seashore paspalum at all sampling dates ( P 0.05), except the 120-day evaluation period in 2002 (Table 2-4). Throughout the majority of sampling times in both experiments, the population of H. galeatus was not associated with root reduction in either grass (Table 2-3). However, in the second year of the study, H. galeatus did reduce root growth ( P 0.05) in seashore paspalum and bermudagrass after 60 and 120 days of exposure, respectively (Table 2-3). Cumulative shoot dry weight was not different from controls when either turfgrass was inoculated with H. galeatus Both grasses were suitable hosts for H. galeatus with moderate reproduction occurring both years. Bermudagrass was a more suitable host at two sampling dates, supporting higher soil populations than seashore paspalum ( P 0.05) after 60 (2003) and 120 (2002) days of feeding and reproducing (Table 2-4). Root extraction of H. galeatus by Baermann tray extraction returned low recovery in 2002. However, in 2003, roots were stained through a modified acid-fuschin staining process (Byrd et al., 1983), revealing that H. galeatus readily entered the roots of both grasses. Nematodes were exclusively found within the root cortex and tended to aggregate in random, nonspecific areas of the roots. Final root population means were 63 69 and 88 55 per gram of root fresh weight for bermudagrass and seashore paspalum respectively. Populations of H. galeatus entering the roots did not indicate a difference ( P > 0.05) in host suitability between the two grasses.

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23 Table 2-3. Cumulative shoot dry weight and total root lengths of 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass 60 and 120 days after initial inoculations of 100 (2002) and 199 13 (2003) Hoplolaimus galeatus Cultivar DAI Treatment Dry shoot weight (g) Root length (mm) 2002 trial 'Tifdwarf' 60 U 6.0 1.9 927 130 I 7.2 2.2 920 203 120 U 14.1 3.0 936 344 I 11.9 1.9 993 192 'SeaIsle 1' 60 U 11.1 2.0 1,244 272 I 11.5 2.0 1,216 257 120 U 22.8 2.9 1,170 223 I 23.0 3.8 1,164 275 2003 trial 'Tifdwarf' 60 U 4.6 1.2 1,525 192 I 4.6 1.9 1,697 490 120 U 13.3 4.0 2,385 468* I 11.9 1.8 2,050 425 'SeaIsle 1' 60 U 7.9 2.3 1,602 301* I 5.9 1.6 1,276 350 120 U 21.6 4.5 2,217 315 I 21.0 4.0 2,098 410 Data are means of 8 (2002) or 10 (2003) replicates standard deviations. DAI = Days after inoculation. I = inoculated and U = uninoculated. Indicate uninoculated different from inoculated significant at P 0.05 according to the one-way analysis of variance. Root length percent reductions and cumulative shoot dry weight reductions calculated from root and shoot growth data were used to compare the effects of B. longicaudatus or H. galeatus between 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass. Differences in cumulative shoot dry weight percent reductions between grasses were not found ( P > 0.05) in either year for B. longicaudatus or H.galeatus Hoplolaimus galeatus did not cause reductions in root growth in the 2002 trial. Therefore root length percent reductions showed no differences ( P > 0.05) between the two grasses. In the 2003 trial, H. galeatus did stunt seashore paspalum root growth even

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24 though soil populations were below reported threshold levels for bermudagrass (Crow et al., 2003). Root length percent reductions demonstrated a difference ( P 0.05) between the two grass species 60 days after inoculation, with seashore paspalum showing a 19.4% root length reduction, and bermudagrass showing a 14% increase in root length compared to uninoculated plants. Belonolaimus longicaudatus reduced root growth in both grasses, but root length percent reductions did not indicate a difference ( P > 0.05) in susceptibility between 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass. In 2002, root length percent reduction averaged 13.7% for bermudagrass, and 20.2% for seashore paspalum at the 60day sampling. At 120 days after inoculation, root length percent reduction averaged between 35% and 40% for both grasses. In the second year of the study, root length percent reduction after 60 days was 26.7% and 38.4% for bermudagrass and seashore paspalum respectively. At the final sampling date, root reductions continued on the same trend with root length percent reductions above 39% for both grasses. Table 2-4. Final population densities (nematodes/100 cm3 of soil) on 'SeaIsle1' seashore paspalum and 'Tifdwarf' bermudagrass 60 and 120 days after initial inoculations of 100 (2002) and 199 13 (2003) Hoplolaimus galeatus and 107 8 (2002) and 211 10 (2003) Belonolaimus longicaudatus 2002 2003 Cultivar 60 DAI 120 DAI 60 DAI 120 DAI B. longicaudatus 'Tifdwarf' 445 330*495 530 273 220* 1,236 714* 'SeaIsle 1' 107 86 148 81 90 97 559 366 H. galeat us 'Tifdwarf' 30 16 312 188* 58 28** 171 134 'SeaIsle 1' 22 13 80 47 15 8 116 33 Data are means of 8 (2002) or 10 (2003) replicates standard deviations. DAI = Days after inoculation. *, ** Indicate 'Tifdwarf' different from 'SeaIsle 1' significant at P 0.05 and P 0.01 respectively, according to the one-way analysis of variance.

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25 Discussion Results indicate slight differences in host suitability, but no major differences in susceptibility between the two grasses. 'Tifdwarf' bermudagrass tended to be a more suitable host for B. longicaudatus and H. galeatus throughout the experiment. Lower final population densities with root length percent reductions equal to 'Tifdwarf' bermudagrass suggest that 'SeaIsle 1' seashore paspalum could be a more susceptible or less tolerant host of B. longicaudatus Since H. galeatus was not found to consistently cause root stunting in either grass, seashore paspalum cannot be assumed more susceptible than bermudagrass. Population densities of B. longicaudatus after 120 days were very high in 2003 as opposed to 2002. Higher inoculation levels and the earlier starting date in 2003 could have resulted in enhanced reproduction. Furthermore, populations in the glasshouse may peak during the late spring and early summer months, and thereafter decline as late summer and early fall temperatures become the norm. Robbins and Barker (1974) reported that reproduction of two populations of B. longicaudatus were greatest at 25 to 30 C, and minimal reproduction occurred at 20 C. Neither grass had a reduction in cumulative top growth even though root growth was stunted by B. longicaudatus Johnson (1970) and Giblin-Davis et al. (1992b) reported similar results for top growth tissue weights for 'Tifdwarf' bermudagrass. The proposed hypothesis is that root stunting caused by B. longicaudatus could stimulate the production of more photosynthetic material to overcome the damaged root system (Giblin-Davis et al., 1992b). In these experiments, mowing frequency and height did not simulate typical golf course mowing practices. Golf courses tend to have intense aboveground defoliation, causing an

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26 immense amount of stress on the root system. This only amplifies the root problems that B. longicaudatus is already causing. Both grass species were suitable hosts for the nematodes. The lack of root stunting caused by H. galeatus is very different from the reaction of the two grasses to the more virulent nematode, B. longicaudatus. Hoplolaimus galeatus caused a slight root reduction in both grasses in the second year of the study, even though reproduction appeared to be relatively low. In February and March 2003, during the 60-day experimental time period, temperatures and day-lengths were not optimal for warmseason turfgrass growth. These environmental factors may have slowed root growth and allowed the higher inoculum levels of H. galeatus to reduce root lengths compared to uninoculated controls. In 2003, statistical analysis showed that there was a blocking effect ( P 0.05) in the H. galeatus 120-day bermudagrass root length data. A significant blocking effect means the randomized complete block design was useful and accounted for confounding factors in the glasshouse. Time course experiments with 'Floratam' and 'FX-313' St. Augustinegrass indicated that H. galeatus had no effect on plant growth even though soil counts of H. galeatus exceeded 40 nematodes/100 cm3 of soil, the proposed action threshold (Crow et al., 2003), for 84 days within the course of the experiment (Giblin-Davis et al., 1995). Tarjan and Busey (1985) reported that 'Tifdwarf' and 'Tifgreen' bermudagrass inoculated with a mixed sample of phytoparasitic nematodes (including B. longicaudatus and H. galeatus ) caused 36% to 39% root dry weight reductions. These root reductions appeared to correlate with increases in H. galeatus population levels In our experiments, B. longicaudatus alone caused approximately the same amount of root reduction, while

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27 H. galeatus failed to consistently affect root growth. However, H. galeatus was able to freely enter the roots of both grasses. In field situations, many other pathogens and climatic stresses can enhance the pathogenic effects of nematodes on turfgrasses. Glasshouse and laboratory work with soybean suggested that greater plant damage resulted from inoculation with both H. galeatus and fungi in the genera Rhizoctonia Fusarium and Macrophomina than with either pathogen alone (McGawley et al., 1984). Further investigation is needed to determine if H. galeatus may be having a synergistic effect with other pathogens and/or nematodes in the soil environment in causing damage to turfgrass roots. In conclusion, our research suggests that the susceptibility of 'SeaIsle 1' seashore paspalum to B. longicaudatus and H. galeatus is not less than 'Tifdwarf' bermudagrass. There are limitations in extrapolating damaging thresholds from glasshouse pot experiments to field situations, consequently action thresholds cannot be predicted from our data. Glasshouse pot experiments eliminate the ability of climatic stresses and pathogens other than the plant-parasitic nematodes to affect plant growth. Exposing the plant to one pathogen and/or stress at a time allows us to determine damage potential exclusive of all other negative effects on plant growth. Further experimentation is needed to determine the effects of B. longicaudatus and H. galeatus on seashore paspalum growth in field conditions.

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28 CHAPTER 3 INFLUENCE OF SALT CONCENTRATION IN IRRIGATION WATER ON PATHOGENICITY AND DEVELOPMENT OF Belonolaimus longicaudatus AND Hoplolaimus galeatus ON SEASHORE PASPALUM Introduction Salt tolerant turfgrasses are becoming essential in many areas because of salt accumulation in soil, restrictions on groundwater use, and salt-water intrusion into groundwater (Carrow and Duncan, 1998; Parker, 1975). Seashore paspalum ( Paspalum vaginatum Swartz) is a warm-season turfgrass adapted for saline conditions (Malcolm and Laing, 1969; Morton, 1973). Breeding for cultivars with fine leaf texture, and tolerance to drought and high salinity irrigation has allowed seashore paspalum to become frequently used in highly managed turfgrass sites (Dudeck and Peacock, 1985; Duncan, 1999a). One major limitation of cultivating turfgrasses in the sandy soils of the southeastern United States is the destruction of roots by phytoparasitic nematodes (Perry and Rhoades, 1982). The sting nematode ( Belonolaimus longicaudatus Rau) and the lance nematode ( Hoplolaimus galeatus (Cobb) Thorne) are destructive pathogens on a variety of agronomic crops and turfgrasses (Ahmad and Chen, 1980; Perry and Rhoades, 1982; Perry et al., 1970; Smart and Nguyen, 1991). While B. longicaudatus is usually limited to the coastal plains of the southeastern United States (Christie, 1959; Holdeman, 1955; Robbins and Barker, 1974), H. galeatus has a much wider distribution (Williams, 1973). Belonolaimus longicaudatus damages lateral roots as soon as they are formed, causing stunted root growth, decreased water and nutrient uptake, and decreased rates of evapotranspiration (Busey et al., 1991; Johnson,

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29 1970; Perry and Rhoades, 1982). Hoplolaimus galeatus enters the root cortex and may damage the roots by feeding and physical tunneling through the cell walls (Krusberg and Sasser, 1956; Williams, 1973). Both nematodes have been reported as important pathogens of turfgrasses in the southeastern United States (Christie et al., 1954; Kelsheimer and Overman, 1953; Perry and Rhoades, 1982). Belonolaimus longicaudatus and H. galeatus have been reported as pathogens of many bermudagrass ( Cynodon dactylon (L.) Pers. and Cynodon spp. hybrids) and St. Augustinegrass ( Stenotaphrum secundatum (Walt.) Kuntze) cultivars (Busey et al., 1991; Giblin-Davis et al., 1992a; 1992b; Perry et al., 1970). In more recent studies, root and shoot growth of diploid and polyploid St. Augustinegrasses were not affected by H. galeatus even though the plants supported high population levels (Giblin-Davis et al., 1995; Henn and Dunn, 1989). Two populations of B. longicaudatus readily reproduced on 'Tifdwarf' bermudagrass, and caused extensive root damage (Giblin-Davis et al., 1992a, Johnson, 1970). A forage grass study determined that there is differential host suitability and susceptibility to B. longicaudatus in Digitaria spp., Paspalum spp., and Chloris spp. introductions (Boyd and Perry, 1969). Post-plant nematicides labeled for turfgrass are becoming limited and alternatives must be found to replace recently discontinued nematicides. Salinity had a detrimental effect on population densities of nematodes on some annual crops (Edongali et al., 1982; Heald and Heilman, 1971). Soil salinity has demonstrated negative effects on the hatching and infectivity of Meloidogyne incognita Kofoid and White, M javanica (Treub) Chitwood, and M arenaria (Neal) Chitwood juveniles (Bird, 1977; Dropkin et. al, 1958; Lal and Yadav, 1975; Maqbool et al., 1987). Khan and Khan (1990) found that

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30 after only 7 days of salinity exposure, M. incognita and M. javanica had reductions in hatching and increased mortality. Population levels of Aphelenchus avenae Bastain, Pratylenchus thornei Sher and Allen, Helicotylenchus spp., and Rotylenchulus reniformis Linford and Oliveira were found to be lower at increased salt concentrations compared to nonsaline treatments (Lal and Yadav, 1976). Some species of root-knot nematodes ( M spartinae Rau and Fassuliotis) and sting nematodes ( B maritimus Rau) may be well adapted to high-salinity conditions (Rau, 1963; Rau and Fassuliotis, 1965). After personal communication with two turfgrass managers in south Florida, Morton (1973) hypothesized that low nematode counts may be associated with seashore paspalum irrigated with high saline water. Further investigation is necessary to confirm this hypothesis. Seashore paspalum is known to be tolerant of high salinity irrigation, but the reaction of nematodes parasitizing seashore paspalum to the salinity is unknown. Therefore, an experiment was performed to examine the effects of salinity irrigation on B. longicaudatus and H. galeatus Objectives of the experiment were to: i) determine the effects of high salinity irrigation on the reproduction of a population of B longicaudatus and a population of H galeatus, ii) establish simple models relating irrigation salinity levels to population levels of B longicaudatus and to population levels of H galeatus iii) compare the effects of high salinity irrigation alone to the combined effects of high salinity irrigation and B longicaudatus on shoot and root growth. Materials and Methods Four separate experiments were performed, one with B. longicaudatus and one with H. galeatus in each of two years (2002, 2003). Experiments were conducted in 2002

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31 from April to September and repeated in the spring and summer of 2003 at the University of Florida Turfgrass Envirotron Glasshouse in Gainesville, FL. In preparation for these experiments, 'SeaIsle 1' seashore paspalum, a commercially available cultivar of Paspalum vaginatum was obtained from R. R. Duncan at the University of Georgia. Nematode-free plugs of grass were obtained by rooting aerial cuttings of stolons from each grass in tapered RLC-7 (UV Stabilized) Super "Stubby" Cells (cell depth = 14 cm; diam. = 3.8 cm; volume = 115 ml) (Ray Leach Single Cell Cone-tainer, Oregon) filled with 140 g of uninfested soil. Soil used for growth media consisted of 100% United States Golf Association (USGA) sand. The soil texture was analyzed using the sieving method for testing a USGA root zone mix (USGA Green Section Staff, 1993) (Table 2-1). An absorbent cotton ball was placed at the bottom of each cell to prevent soil from escaping through the drain holes. The soil was then thoroughly wetted to allow for settling. A depression was made in each cell and two aerial stolons were planted on opposing sides of the depression. Stolons (5 to 8 cm long) were terminal cuttings with two or three nodes. During the fall and winter months, the cells were placed on a glasshouse bench 1.25 m below an enclosed high bay 1,000-watt metal halide growth lamp (Hi-Tek Series, Lithonia Lighting, Conyers, GA) set on a 12-hour cycle to simulate the longer daylengths required for optimal growth. The grass was fertilized using a fertilizer solution (20%-20%-20% (N-P2O5-K2O) plus trace elements) (Peters Professional All-Purpose Plant Food, Spectrum Brands, St. Louis, MO) at a rate equivalent to 49.0 kg N/ha/month, 21.6 kg P/ha/month, and 40.7 kg K/ha/month. A substantial root system was allowed to develop for four weeks.

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32 Plugs of seashore paspalum obtained from the cells were transferred into 14.5 16-cm-diam. clay pots (1,500 cm3) filled with 100% USGA specification sand (Table 2.1). Insect screening (7 5 lines/cm2) was cut into 5 5-cm squares and placed at the bottom of each pot to prevent soil from escaping through the drain hole. Roots were washed free of soil and trimmed to approximately 5 cm below the crown to promote fresh root growth. Two depressions were made in each pot on opposite sides and two plugs of turfgrass were planted per pot. These experimental units were placed in an environmentally controlled glasshouse and irrigated with tap water as needed for 14 days to allow for adjustment to the new environment. A population of B. longicaudatus originally from unmanaged field soil in the Sanford, FL area was obtained from R. M. Giblin-Davis at the Fort Lauderdale, FL Research and Education Center and allowed to reproduce on 'FX 313' St. Augustinegrass. A population of H. galeatus was obtained from a 'Floradwarf' bermudagrass putting green at the G. C. Horn Turfgrass Field Laboratory in Gainesville, FL. Inocula were extracted from soil using a modified Baermann funnel method (McSorley and Federick, 1991). In 2002, the H. galeatus population was contaminated with other plant-parasitic nematodes, therefore handpicking was necessary to obtain an uncontaminated population. One hundred H. galeatus of mixed life stages were inoculated into each pot of seashore paspalum. Since the B. longicaudatus population was free of other plant-parasitic nematodes, handpicking was not necessary. A solution of B. longicaudatus at various life stages and tap water was calibrated by counting nematodes from 1-ml aliquots on a grided counting slide (Hawksley and Sons Limited, Lancing, Sussex, United Kingdom) replicated ten times. Approximate numbers of nematodes were measured with a pipette

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33 from water suspensions of inocula. A total of 111 16 B. longicaudatus were added to each of the inoculated pots. In 2003, H. galeatus and B. longicaudatus were obtained from the previous year's experiment and again, inocula was obtained using a modified Baermann funnel method (McSorley and Federick, 1991). Solutions were made for each nematode and standardized to deliver approximately 250 nematodes per pot. A total of 243 13 B. longicaudatus per pot and 238 8 H. galeatus per pot were inoculated for two separate experiments. A higher level of nematodes was used in the second year to achieve higher reproduction. In both years, nematodes were suspended in 50 ml of tap water and equally distributed into four cavities formed in the soil near the base of the plant. After inoculation, the cavities were then pinched closed with surrounding soil. Uninoculated controls received 50 ml of tap water. Tap water was applied as needed for two weeks to allow nematodes to adjust to their new environment before experimental treatments were initiated. The 30 pots inoculated with each nematode were then separated into five randomized blocks and treated identically except for salt concentration in irrigation water. Separate experiments were performed for each nematode with each experiment having six treatments with five replications. Salinity irrigation treatments were formulated using Instant Ocean Synthetic Sea Salt (Aquarium Systems, Inc., Mentor, OH). Ionic composition of Instant Ocean is primarily Na+ and Cland designed to mimic closely that of seawater (Atkinson and Bingman, 1998; He and Cramer, 1992). Six 50-liter carboys were used to mix salinity treatments. A 3-liter plastic laboratory pitcher filled with 2 liters of deionized water, and a 6-cm stir bar was used with a magnetic stirrer to get the artificial sea salt into solution. After adding the concentrated solution to each carboy, they were diluted to 35 liters with

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34 deionized water. An electrical conductivity meter (YSI Incorporated, Yellow Springs, OH) equipped with a 14.6 1.3-cm-diam. dip-type plastic cell was used to test the accuracy of each treatment. The electrical conductivity meter was calibrated using a 10 dS/m standard solution and temperature chart. More salt or deionized water was added to adjust the electrical conductivity to the proper treatment level. In 2002, irrigation treatments were deionized water concentrated to five salinity levels, (5, 10, 25, 40, and 55 dS/m) and deionized water (0 dS/m) to serve as a control. Irrigation treatments were adjusted in 2003 reflecting the results of the 2002 trials and were formulated by concentrating deionized water to five salinity levels, (5, 10, 15, 20, and 25 dS/m) and deionized water to serve as a control. Each day, 150 ml of each irrigation treatment was applied to the appropriate pots, excluding days before and after leaching events. The experimental units were leached on a weekly (2002) or biweekly (2003) basis to prevent build up of salts and to deliver fertilizer without changing treatment levels. 'SeaIsle 1' seashore paspalum was fertilized on a weekly basis with 20 ml of a solution consisting of 5,100 mg NH4NO3 (34% N), 6,354 mg KCl, 252 mg Ca(H2PO4)2, 435 mg CaSO4, 246 mg MgSO4, 1.55 mg H3BO3, 0.34 mg MnSO4, 0.58 mg ZnSO4, 0.13 CuSO4, and 3.5 mg FeSO4 per 1 liter of deionized water. In 2003, 40 ml of the fertilizer solution was applied on a biweekly basis. Since salinity levels were lower in the 2003 experiment, leaching was done every two weeks as opposed to every week. Total N applied for the duration of the experiment was 624 mg/pot. Liquid fertilizer was added to 580 ml or 560 ml of deionized water and varying amounts of artificial sea salt was added to concentrate the solution to each irrigation treatment. All 600 ml of solution was poured into each pot to flush out residual salts, and replace with fertilizer solution.

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35 During the course of the 2002 experiments, which lasted from 30 May 2002 to 10 October 2002, average monthly high and low air temperatures in the glasshouse ranged from 29 to 34 C, and 21 to 26 C, respectively. In 2003, the B. longicaudatus experiment began 8 April 2003 and ended 6 August 2003 and the H. galeatus experiment began 23 May 2003 and ended 20 September 2003. Average monthly high and low air temperatures ranged from 28 to 34 C and 22 to 27 C, respectively. Using a 1.18-liter pump sprayer, an insecticide/miticide (Mavrik Aquaflow, Wellmark International, Schaumburg, IL) was applied at the labeled rate twice during the course of 2003 experiments for control of bermudagrass mites ( Eriophyes cynodoniensis Sayed). After 120 days, experimental units were brought into the laboratory for analysis. The contents of each pot were emptied into their individually labeled plastic bags and thoroughly hand mixed. A 100-cm3 soil sample was taken from each bag and processed using a modified centrifugal-sugar flotation technique (Jenkins, 1964). Nematodes were counted from the entire sub-sample using an inverted light microscope at 32 magnification. For the 2003 B. longicaudatus trial, second-stage juveniles were counted separately from the other life stages. The two categories were summed to determine the total population of each soil sample. In addition to soil extraction, H. galeatus in roots from the 2003 experiment were staged and counted to determine number of nematodes/gram of root in fresh-weighed samples using a modified acid-fuchsin stainingdestaining procedure (Byrd et al., 1983). Effects of salinity were evaluated by regressing log transformations of final nematode population densities on salinity irrigation levels. In 2003, the sting nematode portion of the experiment was adjusted because B. longicaudatus was determined to stunt root growth in seashore paspalum (Chapter 2).

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36 Effects of high salinity irrigation alone were compared to the combined effect of high salinity irrigation and B. longicaudatus on shoot and root growth. Thirty additional experimental units were irrigated with salinity treatments and left uninoculated to serve as controls. Using fabric scissors (Fiskars Brand Inc., Madison, WI), the grass was trimmed biweekly to approximately 2 cm above the soil surface for all experiments. In 2003, tissue clippings were only collected from the sting nematode experiment. Tissue was placed in 15 cm 23 cm catalog envelopes (Quality Park products, St. Paul, MN) using a spouted 2.84-liter sample pan (40.6 cm 30.5 cm 5 cm) (Seedburo, Chicago, IL), and dried at 75 C for 48 hours to obtain cumulative shoot dry weight. Final destructive analysis also was adjusted from the previous years' experiments, with the inclusion of uninoculated treatments in the B. longicaudatus experiment. Shoots were trimmed as close to the soil as possible and saved for cumulative shoot dry weight analysis. Using a stainless steel T-sampling tool, a root core (approximately 4-cm-diam. 14 cm deep) was taken from the center of each pot to compare root lengths at each salinity level with inoculated treatments. Root cores were washed free of soil on a sieve with 1.7-mm pore openings nested within a sieve with 75-m pore openings. Roots were removed from any aboveground growth and placed into 50-ml disposable plastic centrifuge tubes containing 30 ml of tap water. The 75-m-pore sieve was then submerged in 5 cm of tap water to allow the finer roots to float out and separate from the soil. These fine roots were collected using laboratory forceps and placed into the 50-ml plastic centrifuge tubes. Five drops (0.25 ml) of a 1% methylene blue mixture was added to the 30 ml of tap water to stain the roots. After a minimum of 24 hours in the solution, the roots were removed, placed on a 75-m-pore sieve, and washed free of excess dye.

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37 Stained roots were placed into a glass-bottom tray and scanned using a HP ScanJet 2cx desktop scanner (Hewlett Packard, Boise, ID) to create a black and white bitmap image of the roots (Kaspar and Ewing, 1997; Pan and Bolton, 1991). The GSRoot (Louisiana State University, Baton Rouge, LA) software program was used to analyze the bitmap images. This program measures root lengths and surface areas from scanned images. Root length data were recorded for seven diameter ranges (< 0.05 mm, 0.05 to 0.10 mm, 0.10 to 0.20 mm, 0.20 to 0.30 mm, 0.30 to 0.40 mm, 0.40 to 0.50 mm, and > 0.50 mm). The resulting values were summed to determine the total root length of each root sample. In 2003, total root length measurements and cumulative shoot dry weights from the sting nematode experiment were compared at each salinity level using a one-way analysis of variance (ANOVA) procedure. Inoculated plants were compared to uninoculated at each salinity level to determine if root and/or shoot growth reduction occurred. Orthogonal contrasts were used to compare groups of final population means or individual means for selected sets of treatments. In our experiments, the highest three salinity treatments were compared to the lower three treatments, and selected individual treatments (10 dS/m and 15 dS/m) were compared to the 0 dS/m. Transformation by log10 x was performed to normalize nematode-count data and achieve a better trendline fit (Proctor and Marks, 1975). Using SAS software, (SAS Institute, Cary, NC) the quadratic least squares procedure was used to fit a linear or quadratic model to the data relating logtransformations of final nematode population levels to salinity irrigation treatment levels. Linear and quadratic regressions were drawn using Excel software (Microsoft Corporation, Redmond, WA). One-way ANOVA procedure, quadratic least squares procedure, and orthogonal contrasts were performed using SAS software.

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38 Results Reproduction of H. galeatus and B. longicaudatus was affected by increasing salinity levels (Figs. 3.1, 3.2). Final populations of H. galeatus decreased linearly ( P 0.01) with increasing salinity irrigation treatments (Fig. 3-1). The high R2-values, 0.92 (2002) and 0.83 (2003), indicate that the data followed the linear regression lines very closely. Lower salinity treatments, 0 and 5 dS/m, resulted in higher nematode reproduction than the higher salinity irrigation treatments (Fig. 3-1). Final population densities were very low at the treatment level of 25 dS/m in both years. Treatment levels of 40 and 55 dS/m were included in the first year of the study and resulted in population levels at or near zero. Final population means of H. galeatus were 1 1.4 and 2 1.4/100 cm3 of soil for the 40 and 55 dS/m treatments respectively. The ability of H. galeatus to enter the root cortex as migratory endoparasites also decreased as salinity levels increased. Final population densities within the root cortex were lower ( P 0.05) in the 15, 20, and 25 dS/m treatments (19 13.6 H. galeatus /gram of root fresh weight) when compared to the 0, 5, and 10 dS/m treatments (167 146.9 H. galeatus /gram of root fresh weight). The relationship between final population densities of B. longicaudatus and salinity treatment levels demonstrated a quadratic regression curve in both years of the experiment (Fig 3-2). The quadratic regression was clearly defined when final population data were transformed using log10 x (Fig. 3-2). Non-transformed data also showed a quadratic regression (Data not shown), but log transformation improved R2values and goodness of fit. In 2002, final populations of B. longicaudatus demonstrated a quadratic relationship from 0 to 25 dS/m, with the 25 dS/m treatment supporting a lower

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39 population level than the 5 and 10 dS/m treatments ( P 0.01). In both years, the 10 dS/m treatment supported a higher population of B. longicaudatus than the 0 dS/m treatment ( P 0.05) causing the regression lines to be quadratic (Figure 3.2). Treatment levels of 40 and 55 dS/m were included in the first year of the study and again resulted in population levels at or near zero. Belonolaimus longicaudatus final population means were 3 3.7 and < 1/100 cm3 of soil for the 40 and 55 dS/m treatments respectively. In each trial, the orthogonal contrast test revealed that B. longicaudatus final population levels in the 25 dS/m treatment were lower ( P 0.01) than those in the less saline treatments. Final B. longicaudatus population densities were higher at 10 and 15 dS/m than 0 dS/m ( P 0.05) in the second year of the study (Fig. 3.2). At 15 and 20 dS/m, secondstage juveniles (J2) were the predominant portions of the overall population, making up an average of 68 and 60% of the total population, respectively (Fig. 3.3). At lower salinity levels (0, 5, and 10 dS/m), adults, third-stage juveniles (J3), and fourth-stage juveniles (J4), were the larger portions of the final population densities (Fig. 3.3). Shoot growth was not affected ( P > 0.05) by inoculation with B. longicaudatus Root length comparisons revealed that B. longicaudatus caused root reduction ( P 0.05) at low salinity levels, 0 to 10 dS/m. However at higher salinity irrigation treatments, 15 to 25 dS/m, root lengths were not different ( P > 0.05) between inoculated and uninoculated plants (Fig. 3-4). These results indicate that the ability of B. longicaudatus to stunt root growth was negatively affected at salinity levels of 15 dS/m and above.

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40 0 0.5 1 1.5 2 2.5 3 3.5 0510152025dS/mLog10 Pf Fig. 3-1. Relationship between log transformation of final population densities (Pf) of H. galeatus (nematodes/100 cm3 of soil) ( Y ) and salinity treatment ( x ) in 2002 and 2003 glasshouse experiments. P 0.01 Y = -0.0746 x + 2.3414 R 2 = 0.92 P 0.01 Y = -0.0452 x + 2.823 R 2 = 0.832003 2002

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41 0 0.5 1 1.5 2 2.5 3 3.5 0510152025dS/mLog10 Pf Fig. 3-2. Relationship between log transformation of final population densities (Pf) of B. longicaudatus (nematodes/100 cm3 of soil) ( Y ) and salinity treatment ( x ) in 2002 and 2003 glasshouse experiments. 2003 2002 Y = -0.0058 x 2 + 0.1027 x + 2.3801 Y = -0.0068 x 2 + 0.1248 x + 1.7994 R = 0.78 P 0.01 R = 0.72 P 0.01 22

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42 0 0.5 1 1.5 2 2.5 3 3.5 0510152025dS/mLog10 Pf Fig. 3-3. Relationship between log transformation of J2 or adults, J3, and J4 final population densities (Pf) of B. longicaudatus (nematodes/100 cm3 of soil) ( Y ) and salinity treatment ( x ) in the 2003 glasshouse experiment. Y = -0.0041 x 2 + 0.0496 x + 2.3675 R 2 = 0.76 Y = -0.0086 x 2 + 0.1936 x + 1.4314 R 2 = 0.74 J2 Adults, J3, J4 P 0.01 P 0.01

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43 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 0510152025dS/mRoot Length (mm) Inoculated Uninoculated Fig. 3-4. Effects of Belonolaimus longicaudatus on root length of 'SeaIsle 1' seashore paspalum ( Paspalum vaginatum ) at increasing salinity levels. Inoculated plants received 243 13 B. longicaudatus while uninoculated plants received no nematodes. The error bars show standard deviation of individual population means. ** Indicates difference from inoculated at P 0.01, according to the one-way ANOVA procedure. Discussion Irrigation with poor quality water is becoming more common as increasing water restrictions are forcing turfgrass managers to obtain water from alternative sources. In our experiments, high salinity irrigation affected these particular populations of the two nematodes differently. Lower salinity levels (5, 10, and 15 dS/m) caused an increase in B. longicaudatus reproduction, while the population of H. galeatus steadily decreased as ** ** **

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44 salinity levels increased. Irrigation with low salinity levels (5 and 10 dS/m) resulted in denser, more vigorous root and shoot growth than the 0 dS/m treatment. Irrigating with deionized water may have caused nutrient deficiencies and elevated evapotranspiration rates. With increased root growth, more feeding sites were available for B. longicaudatus In 2003, second-stage juveniles (J2) of B. longicaudatus comprised a majority of the population in the 15 and 20 dS/m treatments. The abundance of J2 at moderate salinity levels resulted in elevated total population numbers compared to lower salinity levels, causing the regression line to be quadratic (Fig. 3.2, 3.3). The nematodes counted as J2 had a clear body cavity indicating they were probably unable to feed. Usually, J2 can easily be separated from other life stages by their dark color and stout body shape (Han, 2001). Root length data in 2003 support this hypothesis, with root reduction not occurring at the 15, 20, or 25 dS/m treatments as opposed to the lower salinity treatment levels (Fig. 3.4). Reproduction and maturation of the nematodes at higher salinity treatments probably occurred early in the experiment before the salinity was able to build up in the soil. Han (2001) stated that it is unknown whether or not feeding is necessary for J2 to develop into J3. However, J2 began molting into J3 3 to 5 days after hatching (Han, 2001). Shoot growth was not affected by inoculations with B. longicaudatus but at salinity levels above 10 dS/m, shoot growth became less vigorous. In a previous study, shoot growth began to decline in response to soil electrical conductivity levels 8 dS/m, with shoot growth being reduced by 25% and 50% at 14 and 34 dS/m, respectively (Lee, 2000). Cumulative shoot dry weight data collected from previous susceptibility

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45 experiments also showed that inoculation with B. longicaudatus did not affect aboveground plant growth (Chapter 2). Salinity treatment levels equaling 25 dS/m and above reduced B. longicaudatus populations to extremely low levels in 2002, but these high salinity levels had detrimental effect on the growth of the grass. In 2003, inoculum levels were much higher and the same quadratic regression occurred, with 25 dS/m reducing reproductions compared to treatment levels ranging from 0 to 15 dS/m ( P 0.05). Hoplolaimus galeatus are classified as migratory endoparasites on turfgrasses. Their ability to enter the roots gives them the capability to escape the effects of most nematicides (Giblin-Davis et al., 1995). In our experiment, both soil and root populations were reduced as salinity irrigation levels increased from 0 to 25 dS/m. The nematodes were probably not able to escape the effects of the salinity by entering the roots because the root cortex tissue does not exclude the elevated ion concentrations associated with saline water. Published action thresholds that justify post-plant nematicide treatment for H. galeatus on bermudagrass are 40/100 cm3 of soil (Crow et al., 2003). Soil populations more than exceeded these numbers for both experiments at 15 dS/m and less. In previous experiments, H galeatus had no effect on seashore paspalum growth even though soil counts exceeded 40/100 cm3 of soil throughout the experiments (Chapter 2). Time course experiments (Giblin-Davis et al., 1995) with 'Floratam' and 'FX-313' St. Augustinegrass also indicated that H. galeatus had no effect on plant growth. In 2002, treatment levels of 40 and 55 dS/m caused near complete mortality for both nematodes, but the shoot growth of the grass was stunted and yellowed. Even though B. longicaudatus and H. galeatus were effectively controlled, the turfgrass was

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46 not visually acceptable. Seashore paspalum can be irrigated with seawater (54 dS/m) in the field when soil conditions allow for sufficient leaching to occur and turfgrass managers fertilize, amend, and cultivate the soil properly (Carrow and Duncan, 1998). In our experiments, prolonged exposure to these high salinity levels was detrimental to turfgrass quality. In the glasshouse, we were unable to provide sufficient leaching, proper amendments, and cultivation of soil necessary for seashore paspalum survival at salinity levels near that of seawater. Results from glasshouse experiments are difficult to extrapolate to field conditions, but we can conclude that salinity irrigation had an affect on B. longicaudatus and H. galeatus nematode reproduction. The treatment salinity levels were at a continuous level throughout the experiment, and never allowed the nematodes to recover from salinity stress. A discontinuous high salinity irrigation situation would probably be more similar to irrigation with poor quality water under field conditions where rainfall can leach salt from the soil profile (Mashela et al., 1992a). Irrigation with pure seawater or with seawater as a high percentage of the blended irrigation water may have potential as an effective option for control of B. longicaudatus and H. galeatus This information may be vital to turfgrass managers currently maintaining seashore paspalum known to have a nematode problem. Further investigation is necessary to determine if frequency and timing of high salinity irrigation, in addition to amount of salinity, can have an effect on nematode reproduction and feeding.

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47 CHAPTER 4 SURVEY OF PLANT-PARASITIC NEMATODES ASSOCIATED WITH SEASHORE PASPALUM Introduction New turfgrass cultivars or ecotypes are commonly introduced into the turfgrass industry, but in rare cases, a new species of turfgrass begins to become more widely utilized. Seashore paspalum ( Paspalum vaginatum Swartz) is a warm season grass adapted for saline conditions (Malcolm and Laing, 1969; Morton, 1973). Breeding for fine leaf textured cultivars, and its natural tolerance to drought and high salinity irrigation has allowed seashore paspalum to become frequently used in highly managed turfgrass sites (Dudeck and Peacock, 1985; Duncan, 1999a). One major limitation of cultivating turfgrasses in the sandy soils of the southeastern United States is the destruction of roots by plant-parasitic nematodes (Perry and Rhoades, 1982). Surveys are necessary to determine the distribution, frequency, and abundance of phytoparasitic nematodes associated with particular plants. These surveys provide indispensable information on the probability and severity of crop losses due to plantparasitic nematode damage. Results of surveys can help to determine more confident action thresholds allowing the grower to use soil and root nematode assays to formulate a management strategy. This will help avoid unnecessary post-plant nematicide treatments or failure to apply a needed one. High population levels of some plant-parasitic nematode genera can cause significant damage to turfgrass areas (Di Edwardo, 1963; Johnson, 1970; Lucas et al., 1974; Winchester and Burt, 1964; Rhoades, 1962; 1965;

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48 Kelsheimer and Overman, 1953, Christie et al., 1954). Therefore, a survey was conducted for golf courses and home lawns with established seashore paspalum in Florida. This survey was performed to determine the plant-parasitic nematodes commonly associated with seashore paspalum. Materials and Methods A majority of the survey soil samples were taken during the spring and summer months of 2002 and 2003 from coastal areas in central and south Florida. Soil samples were collected from golf courses in or near Fort Myers, Miami Beach, Delray Beach, Vero Beach, and Naples, FL. Home lawn samples were collected from many areas in central and south Florida, primarily in the Tampa, Miami, Fort Myers, Naples, and Daytona Beach areas. Seashore paspalum is well adapted to these areas because of its tolerance to high salinity irrigation and saltwater intrusion (Malcolm and Laing, 1969; Morton, 1973). Areas sampled were in close proximity to each coast and all samples were from sandy soils. A total of 20 home lawns and 8 golf courses were sampled. These sample sizes may appear low for a survey, but a high percentage of golf courses and home lawns growing seashore paspalum in Florida were sampled. According to a United States Golf Association Florida Regional Agronomist, 12 to 15 golf courses in Florida are currently growing seashore paspalum on > 90% of their maintained areas (Lowe, pers. comm.). A non-biased approach was practiced when selecting the home lawns and golf courses to be sampled. At each golf course, four greens and four fairways were individually sampled for plant-parasitic nematodes. Two golf courses were not growing seashore paspalum over the entire course, thus fewer samples were collected. Home lawns were sampled separately for plant-parasitic nematodes. Two to seven samples were taken at each lawn. More samples were collected from larger lawns and

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49 lawns with noticeable aboveground damage. A 2.5-cm-diam. cone-shaped soil sampler was used to collect 20 soil cores (2.5-cm-diam 10 cm deep) at approximately equal intervals in a zig-zag pattern across the sampling area. If yellowing or declining areas were noticed, then separate samples were taken from these areas. Each sample was emptied into individually labeled plastic bags and thoroughly hand mixed. A 100-cm3 soil sample was removed from each bag and nematodes were extracted using a modified centrifugal-sugar flotation technique (Jenkins, 1964). Plantparasitic nematodes were identified to genus and counted using an inverted light microscope at 32 magnification. After counting, the nematodes were preserved in a 5% formalin solution. Using a compound microscope at 40 and 100 magnifications, some preserved nematodes were slide mounted and identified to the species level with reasonable confidence. Morphology keys and original nematode descriptions were used for species identification (Esser, 1971; 1973; Esser and Vovlas, 1989; Golden and Taylor, 1956; Handoo and Golden, 1992; Loof and Luc, 1990; Rau, 1958; Sher, 1961; 1963; 1966; Sledge and Golden, 1964). Results Plant-parasitic nematodes associated with seashore paspalum were the typical range of genera found parasitizing other warm-season turfgrasses. Ten genera of plant-parasitic nematodes were extracted from 60 seashore paspalum soil samples from eight golf courses (Table 4-1). In addition to the same ten genera found on seashore paspalum golf courses, two more plant-parasitic nematode genera, a total of 12, were extracted from 51 soil samples from 20 home lawns (Table 4-2). Nematodes in the genera Hemicriconemoides and Helicotylenchus are not frequently discovered in such high

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50 populations on turfgrasses in Florida as were found on seashore paspalum (Crow, pers. comm.). As with a majority of turfgrass samples in Florida, Hoplolaimus spp. was commonly found in seashore paspalum samples. The genera most frequently found were Hoplolaimus Mesocriconema, Hemicriconemoides and Helicotylenchus which were found on 100, 100, 88, and 88% of the golf courses surveyed and on 75, 95, 70, and 85% of the home lawns sampled, respectively (Table 4-1, 4-2). Xiphinema Pratylenchus and Tylenchorhynchus were found on less than 30% of the golf courses and less than 45% of the home lawns sampled (Table 4-1, 4-2). Peltamigratus and Hemicycliophora were not found in any of the golf course soil samples and were only found associated with a low percentage of the home lawns (Table 4-2). Various morphological measurements, identifying cuticular markings, and body shape were used for species identification. Species identified from soil samples taken from the rhizosphere of seashore paspalum were Belonolaimus longicaudatus Rau, Hoplolaimus galeatus (Cobb) Thorne, Helicotylenchus pseudorobustus (Steiner) Golden, Meloidogyne graminis (Sledge and Golden) Whitehead, Hemicriconemoides annulatus Pinochet and Raski, Trichodorus proximus Allen, and Peltamigratus christiei (Golden and Taylor) Sher. Previous surveys indicate that a variety of nematodes are associated with turfgrasses in the southeastern United States (Lucas et al., 1974; Parris, 1957; Sikora et al., 2001). Even though these surveys were primarily for nematodes associated with bermudagrass and bentgrass ( Agrostis spp.), many of the nematodes we found were from the same genera. Mesocriconema Hoplolaimus and Helicotylenchus were all common in our survey, which is similar to observations throughout the southeast. The relatively

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51 high frequency of B. longicaudatus found in soil samples from golf courses (50%) and home lawns (40%) was expected because of the sandy soils associated with coastal areas in Florida. Belonolaimus longicaudatus is most commonly found in Florida because it is well adapted to soils with > 80% sand content and < 10% clay content (Robbins and Barker, 1974). Belonolaimus longicaudatus is recognized as the most pathogenic ectoparastic plant nematode to turfgrasses in Florida. Acceptable visual quality is often not possible without treatment of the B. longicaudatus infestation. Table 4-1. Frequency of occurrence and population density of plant-parasitic nematode genera in soil samples collected from seashore paspalum golf courses in Florida. Nematodes/100 cm3 of soil Nematode genus Golf courses with nematode (%)a Meanb Maximumc Hoplolaimus 100 162 643 Mesocriconema 100 69 563 Hemicriconemoides 88 97 1,014 Helicotylenchus 88 469 2,808 Trichodorus 63 30 150 Tylenchorhynchus 63 7 16 Meloidogyne 63 39 123 Belonolaimus 50 16 114 Pratylenchus 25 76 167 Xiphinema 25 79 253 aPercentage of golf courses with at least one infested green or fairway. Percentage based on eight golf courses with one to four fairways and zero to four greens sampled on each golf course for a total of 60 soil samples. bMean population levels of nematodes recovered from samples in which the nematode was found. cMaximum population levels found from individual soil samples.

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52 Table 4-2. Frequency of occurrence and population density of plant-parasitic nematode genera in soil samples collected from seashore paspalum home lawns in Florida. Nematodes/100 cm3 of soil Nematode genus Home lawns with nematode (%)a Meanb Maximumc Mesocriconema 95 123 552 Helicotylenchus 85 175 743 Hoplolaimus 75 73 253 Hemicriconemoides 70 92 428 Trichodorus 60 49 234 Tylenchorhynchus 40 42 388 Belonolaimus 40 11 72 Meloidogyne 28 14 45 Pratylenchus 25 5 15 Hemicycliophora 25 22 91 Peltamigratus 5 29 57 Xiphinema 5 7 7 aPercentage of home lawns with at least one infested soil sample. Percentage based on 20 home lawns with one to five samples taken at each lawn for a total of 51 soil samples. bMean population levels of nematodes recovered from samples in which the nematode was found. cMaximum population levels found from individual soil samples Discussion Action threshold levels have not yet been established for seashore paspalum, but according to levels established for bermudagrass (Crow et al., 2003), 26 of the 51 home lawn samples and 34 of the 60 golf course samples had nematode populations above action threshold levels. Belonolaimus Hoplolaimus Helicotylenchus Trichodorus Hemicriconemoides and Mesocriconema were the genera found above action threshold levels for bermudagrass (Crow et al., 2003). Hoplolaimus galeatus B. longicaudatus and T. proximus were associated with damaged areas on golf courses and home lawns in the current survey. Drought stress during the summer was amplified when these nematodes were present. Seashore paspalum was slow to recover from dieback

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53 associated with drought. Damaged areas were more common on home lawns than on golf courses. Golf courses are usually well maintained through proper fertilization and adequate irrigation, possibly masking root damage caused by high nematode populations. Declining seashore paspalum was found when populations of T. proximus exceeded 60 nematodes/100 cm3 of soil, H. galeatus exceeded 50 nematodes/100 cm3 of soil, and when B. longicaudatus numbers were above 10 nematodes/100 cm3 of soil. High populations of Helicotylenchus spp. (> 500 nematodes/100 cm3 of soil) were often found associated with seashore paspalum. Damaged areas were not noticed in areas where high population levels of Helicotylenchus spp. were recovered from soil samples. In Florida, Helicotylenchus spp. are rarely found in turfgrass soil samples at these high population levels (Crow, pers. comm.). Their ability to damage turfgrass in Florida is not widely known. A wide range of plant-parasitic nematodes was found associated with seashore paspalum golf courses and home lawns in Florida. Some nematode genera tended to have population levels higher than those usually found associated with bermudagrass ( Cynodon spp.) in Florida. The high population levels of Helicotylenchus spp. and Hemicriconemoides spp. found in this survey suggest seashore paspalum is an excellent host for these nematodes. Approximately half of the seashore paspalum samples had nematode populations at damaging levels according to bermudagrass threshold levels. These threshold levels are approximate and can fluctuate due to host suitability, soil temperature, soil texture, soil moisture, and a variety of other soil conditions. Threshold levels used for bermudagrass seem reasonable for diagnosis of samples with B. longicaudatus H. galeatus and T. proximus but adjustments may be necessary for

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54 Helicotylenchus and Hemicriconemoides As seashore paspalum becomes more widely grown, a better understanding of its relationship with plant-parasitic nematodes can be ascertained.

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55 CHAPTER 5 SUMMARY With more than 1,300 public and private golf courses and over three million acres of residential lawns (Haydu and Hodges, 2002; Hodges et al., 1994), Florida is not only a prime location for highly managed turfgrass areas, but also an ideal habitat for Belonolaimus longicaudatus and Hoplolaimus galeatus The data reported herein indicates that this population of B longicaudatus is detrimental to the growth of 'SeaIsle 1' seashore paspalum in the southeastern United States. The reaction of seashore paspalum to the population of H galeatus was very different with only a limited effect on plant growth detected in our research. Our data show that 'SeaIsle 1' seashore paspalum is not any less susceptible to B. longicaudatus or H. galeatus than is 'Tifdwarf' bermudagrass. The population of B. longicaudatus had a high degree of association with root growth stunting ( P 0.05) in 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass while H. galeatus failed to reduce root or shoot growth in either grass ( P > 0.05) (Chapter 2). Results indicate that there are slight differences in host suitability, but no differences in root length percent reduction between the two grasses. 'Tifdwarf' bermudagrass tended to support higher populations of B. longicaudatus and H. galeatus during the experiments ( P 0.05) (Chapter 2). Shoot growth was not affected ( P > 0.05) by either nematode on either grass. 'SeaIsle 1' seashore paspalum and 'Tifdwarf' bermudagrass root growth was reduced by 35 to 45% when exposed to B. longicaudatus for 120 days. The population of H galeatus was associated with a slight abbreviation of root development in seashore

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56 paspalum and bermudagrass during the second year of the study (Chapter 2). Even though final population densities of H. galeatus more than exceeded the reported action threshold of 40 nematodes/100 cm3 of soil for bermudagrass (Crow et al., 2003), neither seashore paspalum nor bermudagrass had a consistent reduction in root growth, although H. galeatus were found within the root cortex of both grasses (Chapter 2). These results indicate that there is no reason to believe that the susceptibility of 'SeaIsle 1' seashore paspalum to H. galeatus and B. longicaudatus is lower than 'Tifdwarf' bermudagrass. The two nematodes were affected differently by high salinity irrigation (Chapter 3). Moderate salinity levels (10 and 15 dS/m) caused an increase in B. longicaudatus reproduction when compared to 0 dS/m ( P 0.05). Visual inspection revealed that irrigation with these low salinity levels resulted in a denser, more vigorous root and shoot growth than 0 dS/m. With increased root growth, more feeding sites were available for B longicaudatus At high salinity levels of 25 dS/m and higher, B. longicaudatus populations were reduced to non-damaging levels. In 2003, root reductions were not detected ( P > 0.05) at salinity levels of 15 dS/m and higher when plants inoculated with B. longicaudatus were compared to uninoculated plants with equivalent irrigation treatments (Chapter 3). A high percentage of final populations at these salinity levels consisted of second-stage juveniles (J2). Further research must be performed to determine if salinity interferes with host locating receptors of B. longicaudatus Moderate salinity levels may also be stimulating egg hatch resulting in high populations of J2. In our experiments, soil and root populations of H. galeatus were reduced as salinity irrigation levels increased from 0 to 25 dS/m (Chapter 3). Final population

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57 densities of H. galeatus demonstrated a negative sloping linear regression ( P 0.01) when compared to increasing salinity irrigation treatments. Lower salinity treatments, 0 and 5 dS/m, resulted in higher reproduction capabilities than the higher salinity irrigation treatments ( P 0.05) (Chapter 3). Final populations were very low at the 25 dS/m treatment level and above. The R2-values were 0.92 (2002) and 0.83 (2003), showing that the data followed a linear regression very closely. High salinity irrigation may be effective in controlling H. galeatus in the soil, but these nematodes are also able to enter the root cortex as migratory endoparasites. Our results indicate that the ability of H. galeatus to completely enter and tunnel within the root cortex does not provide a barrier from the effects of the high salinity irrigation (Chapter 3). The survey of nematodes associated with established seashore paspalum revealed a majority of genera typically associated with maintained turfgrass areas (Chapter 4). Hoplolaimus spp., Helicotylenchus spp., Mesocriconema spp., and Hemicriconemoides spp. were common in home lawns and golf courses. Some genera ( Hemicriconemoides and Helicotylenchus ) tended to have population levels higher than those usually found associated with bermudagrass in Florida. The relatively high frequency of B. longicaudatus at golf courses (50%) and home lawns (40%) should have been expected because of the sandy soils associated with coastal areas in Florida. Damaged areas could be found associated with parasitism by B. longicaudatus H. galeatus and Trichodorus proximus during the summer months when the grass was under the most environmental stress (Chapter 4). Nematode management in turfgrass areas is becoming increasingly difficult because of the elimination of a majority of post-plant nematicides. Alternatives in the

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58 form of different chemical formulations, biological control, or resistant plant cultivars must be found to be able to maintain acceptable turfgrass quality in many areas. With the development of salt-tolerant grasses such as seashore paspalum, high salinity irrigation could be implemented as part of a management regiment for nematodes if irrigation periods were timed properly. Water use for landscape irrigation is becoming a critical issue in Florida. Maintaining turfgrass of acceptable quality is increasingly difficult when forced to reduce water consumption or switch to alternative water sources. In Florida, recycled water was the primary source for almost half of all golf courses in 2000 and has increased from 8% in 1974 to 21% in 1994 to 49% in 2000 (Haydu and Hodges, 2002). Seashore paspalum is well adapted to irrigation from these alternative water sources and could become more prevalent as water restrictions increase. Irrigation with water containing high levels of dissolved salts reduces shoot growth of seashore paspalum (Lee, 2000), but since high yield is not essential for turfgrass managers this could be an effective nematode management technique. Further research must be performed to determine the timing, frequency, and duration of high salinity irrigation events.

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59 LIST OF REFERENCES Abu-Gharbieh, W. I., and V. G. Perry. 1970. Host differences among Florida populations of Belonolaimus longicaudatus Rau. Journal of Nematology 2: 209-216. Ahmad, M., and T. A. Chen. 1980. Effect of certain environmental factors and host plants on reproduction of Hoplolaimus galeatus Plant Disease 64: 479-480. Atkinson, M. J., and C. Bingman. 1998. Elemental composition of commercial seasalts. Journal of Aquariculture and Aquatic Sciences 8: 39-43 Beard, J. B., S. M. Batten, S. R. Reed, K. S. Kim, and S. D. Griggs. 1982. A preliminary assessment of Adalayd Paspalum vaginatum for turfgrass characteristics and adaptation to Texas conditions. Texas Turfgrass Research PR-4039, pp. 33-34, 43. Beard, J. B., S. I. Sifers, and M. H. Hall. 1991. Cutting height and nitrogen fertility requirements of Adalayd seashore paspalum ( Paspalum vaginatum ). Texas Turfgrass Research. PR-4921, pp. 107-109. Bekal, S., and J. O. Becker. 2000. Host range of a California sting nematode population. Hortscience 35: 1276-1278. Bird, A. F. 1977. The effect of various concentrations of sodium chloride on the hostparasite relationship of the root-knot nematode ( Meloidogyne javanica ) and ( Glycine max var. Lee). Marcellia 40: 167-175. Boyd, F. T., and V. G. Perry. 1969. The effect of sting nematodes on the establishment, yield, and growth of forage grasses on Florida sandy soils. Soil and Crop Science Society of Florida Proceedings 29: 288-300. Boyd, F. T., and V. G. Perry. 1971. Effects of seasonal temperatures and certain cultural treatments on sting nematodes in forage grass. Soil and Crop Science Society of Florida Proceedings 30: 360-365. Busey, P., R. M. Giblin-Davis, C. W. Riger, and E. I. Zaenker. 1991. Susceptibility of diploid St. Augustinegrasses to Belonolaimus longicaudatus Supplement to the Journal of Nematology 23: 604-610. Byrd, J., D. W., T. Kirkpatrik, and K. R. Barker. 1983. An improved technique for clearing and staining plant tissues for detection of nematodes. Journal of Nematology 15: 142-143.

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60 Carrow, R. N., and R. R. Duncan. 1998. Salt-affected turfgrass sites: assessment and management. Ann Arbor Press, Chelsea, MI. 185 pp. Christie, J. R. 1959. The sting and awl nematode. Pp. 126-135 in J. R. Christie, ed. Plant nematodes, their bionomics and control. Gainesville, FL: University of Florida Agricultural Experiment Station. Christie, J. R., A. N. Brooks, and V. G. Perry. 1952. The sting nematode Belonolaimus gracilis a parasite of major importance on strawberries, celery, and sweet corn in Florida. Phytopathology 42: 173-176. Christie, J. R., J. M. Good, and G. C. Nutter. 1954. Nematodes associated with injury to turf ( Belonolaimus gracilis ). Proceedings of the Soil and Crop Science Society of Florida 14: 167-169. Cobb, N. A. 1913. New nematode genera found inhabiting fresh water and non-brackish soils. Journal of the Washington Academy of Science 3: 438. Crow, W. T., J. W. Noling, J. R. Rich, R. A. Kinloch, and R. A. Dunn. 2003. Florida nematode management guide. University of Florida, Institute of Food and Agricultural Sciences. Gainesville, FL: University of Florida. Di Edwardo, A. A. 1963. Pathogenicity and host-parasite relationships of nematodes on turf in Florida. Florida Agricultural Experiment Station Annual Report for 1963. Gainesville, FL: University of Florida. Dickerson, O. J., W. G. Willis, F. J. Dainello, and J. C. Pair. 1972. The sting nematode, Belonolaimus longicaudatus in Kansas. Plant Disease Reporter 56: 957. Dropkin, V. H., G. C. Martin, and R. W. Johnson. 1958. Effect of osmotic concentration on hatching of some plant parasitic nematodes. Nematologica 3: 115-126. Dudeck, A. E., and C. H. Peacock. 1985. Effects of salinity on seashore paspalum turfgrasses. Agronomy Journal 77: 47-50. Duncan, R. R. 1999a. Environmental compatibility of seashore paspalum (saltwater couch) for golf courses and other recreational uses. I. breeding and genetics. International Turfgrass Society Research Journal 8: 1208-1215. Duncan, R. R. 1999b. Environmental compatibility of seashore paspalum (saltwater couch) for golf courses and other recreational uses. II. management protocols. International Turfgrass Society Research Journal 8: 1216-1230. Duncan, R. R., and R. N. Carrow. 2000. Seashore Paspalum: The Environmental Turfgrass. Ann Arbor Press, Chelsea, MI. 281 pp.

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61 Edongali, E. A., L. Duncan, and H. Ferris. 1982. Influence of salt concentration on infectivity and development of Meloidogyne incognita on tomato. Revue de Nematologie 5: 111-117. Esser, R. P. 1971. A compendium of the genus Trichodorus (Dorylaimoidea: Diphtherophoridae). Soil and Crop Science Society of Florida Proceedings 31: 244-253 Esser, R. P. 1973. A diagnostic species compendium of the genus Xiphinema Cobb 1913. Soil and Crop Science Society of Florida Proceedings 33: 88-92. Esser, R. P., N. Vovlas. 1990. A diagnostic compendium to the genus Hemicriconemoides (Tylenchida: Criconematidae). Soil and Crop Science Society of Florida Proceedings 49: 211-219. Fortuner, R., and M. Luc. 1987. A reappraisal of Tylenchina (Nemata). 6. The family Belonolaimidae Whitehead, 1960. Revue de Nematologie 10: 183-202. Giblin-Davis, R. M., P. Busey, and B. J. Center. 1992a. Dynamics of Belonolaimus longicaudatus parasitism on a susceptible St. Augustinegrass host. Journal of Nematology 24: 432-437. Giblin-Davis, R. M., P. Busey, and B. J. Center. 1995. Parasitism of Hoplolaimus galeatus on diploid and polyploid St. Augustinegrasses. Journal of Nematology 27: 472477. Giblin-Davis, R. M., J. L. Cisar, F. G. Blitz, and K. E. Williams. 1992b. Host status of different bermudagrasses ( Cynodon spp.) for the sting nematode, Belonolaimus longicaudatus Journal of Nematology 24: 749-756. Golden, A. M., and A. L. Taylor. 1956. Rotylenchus christiei n. sp., a new spiral nematode species associated with roots of turf. Proceedings of the Helminthological Society of Washington 23: 109-112. Good, J. M., J. R. Christie, and G. C. Nutter. 1956. Identification and distribution of plant parasitic nematodes in Florida and Georgia. Phytopathology 46: 13 (Abstr.). Graham, T. W., and Q. L. Holdeman. 1953. The sting nematode Belonolaimus gracilis Steiner: A parasite on cotton and other crops in South Carolina. Phytopathology 43: 434439. Han, H. R. 2001. Characterization of intraspecific variations of Belonolaimus longicaudatus by morphology, developmental biology, host specificity, and sequence analysis of ITS-1 rDNA. Ph.D. dissertation. University of Florida. Gainesville, FL.

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62 Handoo, Z. A., and A. M. Golden. 1992. A key and diagnostic compendium to the species of the genus Hoplolaimus Daday, 1905 (Nematoda: Hoplolaimidae). Journal of Nematology 24: 45-53 Haydu, J. J., and A. W. Hodges. 2002. Economic impacts of the Florida golf course industry. University of Florida, Institute of Food and Agricultural Sciences, Economic Information Report EIR 02-4. Gainesville, FL: University of Florida. He, T., and G. R. Cramer. 1992. Growth and mineral nutrition of six rapid-cycling Brassica species in response to seawater salinity. Plant and Soil 139: 285-294. Heald, C. M., and M. D. Heilman. 1971. Interaction of Rotylenchulus reniformis soil salinity, and cotton. Journal of Nematology 3: 179-182. Henn, R. A., and R. A. Dunn. 1989. Reproduction of Hoplolaimus galeatus and growth of seven St. Augustinegrass ( Stenotaphrum secundatum ) cultivars. Nematropica 19: 8187. Hirschmann, H. 1959. Histological studies on the anteriour region of Heterodera glycines and Hoplolaimus tylenchiformis (Nematoda, Tylenchida). Proceedings of the Helminthological Society of Washington 26: 73-90. Hodges, A. W., J. J. Haydu, van Blokland, and A. P. Bell. 1994. Contribution of the turfgrass industry to Florida's economy, 1991-92: a value-added approach. University of Florida, Institute of Food and Agricultural Sciences, Economic Information Report EIR 94-1. Gainesville, FL: University of Florida. Holdeman, Q. L. 1955. The present known distribution of the sting nematode, Belonolaimus gracilis in the coastal plain of the southeastern United States. Plant Disease Reporter 39: 5-8. Holdeman, Q. L., and T. W. Graham. 1953. The effect of different plant species on the population trends of the sting nematode. Plant Disease Reporter 37: 497-500. Huang, X., and J. O. Becker. 1997. In vitro culture and feeding behavior of Belonolaimus longicaudatus on excised Zea mays roots. Journal of Nematology 29: 411-415. Huang, X., and J. O. Becker. 1999. Life cycle and mating behavior of Belonolaimus longicaudatus in Gnotobiotic culture. Journal of Nematology 31: 70-74. Huang, B., R. R. Duncan, and R. N. Carrow. 1997a. Drought-resistance mechanisms of seven warm-season turfgrasses under surface soil drying: I. Shoot response. Crop Science 37: 1858-1863.

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63 Huang, B., R. R. Duncan, and R. N. Carrow. 1997b. Drought-resistance mechanisms of seven warm-season turfgrasses under surface soil drying: II. Root aspects. Crop Science 37: 1863-1869. Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48: 692. Johnson, A. W. 1970. Pathogenicity and interactions of three nematode species on six bermudagrasses. Journal of Nematology 2: 36-41. Johnson, B. J., and R. R. Duncan. 1997. Tolerance of four seashore paspalum ( Paspalum vaginatum ) cultivars to post-emergence herbicides. Weed Technology 11: 689-692. Johnson, B. J., and R. R. Duncan. 1998. Tolerance of seashore paspalum cultivars to preemergence herbicides. Journal of Environmental Horticulture 16: 76-78. Kaspar, T. C., and R. P. Ewing. 1997. ROOTEDGE: Software for measuring root length from desktop scanner images. Agronomy Journal 89: 932-940. Kelsheimer, E. G., and A. J. Overman. 1953. Notes on some ectoparasitic nematodes found attacking lawns in the Tampa Bay area. Proceedings of the Florida State Horticultural Society 66: 301-303. Khan, A. A., and M. W. Khan. 1990. Influence of salinity stresses on hatching and juvenile mortality of root-knot nematodes, Meloidogyne incognita (Race 2) and Meloidogyne javanica Pakistan Journal of Nematology 8: 107-111. Krusberg, L. R., and J. N. Sasser. 1956. Host-parasite relationship of the lance nematode in cotton roots. Phytopathology 46: 505-510. Lal, A., and B. S. Yadav. 1975. Effect of leachates from saline soils on hatching of eggs of Meloidogyne incognita Indian Journal of Nematology 5: 228-229. Lal, A., and B. S. Yadav. 1976. Effect of soil salinity on the occurrence of phytoparasitic nematodes. Indian Journal of Mycology and Plant Pathology 6: 82-83. Lee, G. 2000. Comparative salinity tolerance and salt tolerance mechanisms of seashore paspalum. PhD Dissertation. University of Georgia. 264 pp. Lewis, S. A., and G. Fassuliotis. 1982. Lance nematodes, Hoplolaimus spp., in the southern United States. Pp. 127-138 in R. D. Riggs, ed. Nematology in the southern region of the United States, Southern Cooperative Series Bulletin 276. Fayetteville, AR: Arkansas Agricultural Experiment Station, University of Arkansas.

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64 Loof, P. A. A., and M. Luc. 1990. A revised polytomous key for the identification of species of the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) with exclusion of the X. americanum -group. Systematic Parasitology 16: 35-66. Lopez, R. 1978. Belonolaimus un Nuevo integrante de la nematofauna de Costa Rica. Agronomia Costarricense 2: 83-85. Lucas, L. T., C. T. Blake, and K. R. Barker. 1974. Nematodes associated with bentgrass and bermudagrass golf greens in North Carolina. Plant Disease Reporter 58: 822-824. Malcolm, C. V. 1977. Saltland and what to do about it. Journal of Agriculture, Western Australia 18: 127-133. Malcolm, C. V., and I. A. F. Laing. 1969. Paspalum vaginatum for salty seepages and lawns. Journal of Agriculture, Western Australia 11: 474-475. Maqbool, M. A., P. Ghazala, and S. Begum. 1987. Influence of salts on the hatchability and infectivity of Meloidogyne javanica juveniles on tomato. Pakistan Journal of Nematology 5: 99-102. Marcum, B. M., and C. L. Murdoch. 1994. Salinity tolerance mechanisms of six C4 turfgrasses. Journal of the American Society for Horticultural Science 119: 779-784. Mashela, P., L. W. Duncan, J. H. Graham, and R. McSorley. 1992a. Leaching soluble salts increases population densities of Tylenchulus semipenetrans Journal of Nematology 24: 103-108. Mashela, P., R. McSorley, and L. W. Duncan. 1992b. Damage potential and reproduction of Belonolaimus longicaudatus and Hoplolaimus galeatus on alyceclover. Journal of Nematology 24: 438-441. McGawley, E. C., K. L. Winchell, and G. T. Berggren. 1984. Possible involvement of Hoplolaimus galeatus in a disease complex of 'Centennial' soybean. Phytopathology 74: 831 (Abstr.). McSorley, R., and J. J. Frederick. 1991. Extraction efficiency of Belonolaimus longicaudatus from sandy soil. Journal of Nematology 23: 511-518. Miller, L. I. 1972. The influence of soil texture on the survival of Belonolaimus longicaudatus Phytopathology 62: 670-671. Morton, J. 1973. Salt-tolerant silt grass ( Paspalum vaginatum Swartz). Proceedings of the Florida State Horticultural Society 86: 482-490. Mundo-Ocampo, M., J. O. Becker, and J. G. Baldwin. 1994. Occurrence of Belonolaimus longicaudatus on bermudagrass in the Coachella Valley. Plant Disease 78: 529.

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65 Myers, R. F. 1979. The sting nematode, Belonolaimus longicaudatus from New Jersey. Plant Disease Reporter 63: 756-757. Ng, O. C., and T. A. Chen. 1980. Histopathological study of alfalfa root infected by Hoplolaimus galeatus Phytopathology 70: 466-467. Norton, D. C., and P. Hinz. 1976. Relationship of Hoplolaimus galeatus and Pratylenchus hexincisus to reduction of corn yields in sandy soils in Iowa. Plant Disease Reporter 60: 197-200. Owens, J. V. 1951. The pathological effects of Belonolaimus gracilis on peanuts in Virginia. Phytopathology 41: 29 (Abstr.). Pan, W. L., and R. P. Bolton. 1991. Root quantification by edge discrimination using a desktop scanner. Agronomy Journal 83: 1047-1052. Parker, G. G. 1975. The hydrogeology and problems of peninsular Florida's water resources. Proceedings Florida Turfgrass Management Conference 22: 13-36. Parris, G. K. 1957. Screening Mississippi soils for plant parasitic nematodes. Plant Disease Reporter 41: 705-706 Peacock, C. H., and A. E. Dudeck. 1985. Physiological and growth responses of seashore paspalum to salinity. Hortscience 20: 111-112. Perry, V. G., and J. M. Good. 1968. The nematode problems of turfgrasses. Proceedings Florida Turfgrass Management Conference 16: 22-25. Perry, V. G., and H. Rhoades. 1982. The genus Belonolaimus Pp. 144-149 in R. D. Riggs, ed. Nematology in the southern region of the United States, Southern Cooperative Series Bulletin 276. Fayetteville, AR: Arkansas Agricultural Experiment Station, University of Arkansas. Perry, V. G., G. C. Smart, and G. C. Horn. 1970. Nematode problems of turfgrasses in Florida and their control. Proceedings of the Florida State Horticultural Society 83: 489492. Proctor, J. R, and C. F. Marks. 1975. The determination of normalizing transformations for nematode count data from soil samples and of efficient sampling schemes. Nematologica 20: 395-406. Rau, G. J. 1958. A new species of sting nematode. Proceedings of the Helminthological Society of Washington 25: 95-98.

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66 Rau, G. J. 1963. Three new species of Belonolaimus (Nematoda: Tylenchida) with additional data on B. longicaudatus and B. gracilis Proceedings of the Helminthological Society of Washington 30: 119-128. Rau, G. J., and G. Fassuliotis. 1965. Hysoperine spartinae n. sp., a gall-forming nematode on the roots of smooth cordgrass. Proceedings of the Helminthological Society of Washington 32: 159-162. Rhoades, H. L. 1962. Effects of sting and stubby-root nematodes on St. Augustine grass. Plant Disease Reporter 46: 424-427. Rhoades, H. L. 1965. Parasitism and pathogenicity of Trichodorus proximus to St. Augustine grass. Plant Disease Reporter 49: 259-262. Rhoades, H. L. 1987. Effects of Hoplolaimus galeatus on ten vegetable crops in Florida. Nematropica 17: 213-218. Riggs, R. D. 1961. Sting nematode in Arkansas. Plant Disease Reporter 45: 392. Rivera-Camarena, J. E. 1963. Pathogenic and biological aspects of sting and lance nematodes. Ph.D. dissertation. University of Florida. Gainesville, FL. Robbins, R. T., and K. R. Barker. 1973. Comparisons of host range and reproduction among populations of Belonolaimus longicaudatus from North Carolina and Georgia. Plant Disease Reporter 57: 750-754. Robbins, R. T., and K. R. Barker. 1974. The effects of soil type, particle size, temperature, and moisture on reproduction of Belonolaimus longicaudatus Journal of Nematology 6: 1-6. Robbins, R. T., and H. Hirschmann. 1974. Variation among populations of Belonolaimus longicaudatus Journal of Nematology 6: 87-94. Rodriguez-Kabana, R., R. L. Haaland, C. B. Elkins, and C. S. Hoveland. 1978. Relationship of root diameter and number of roots to damage of tall fescue by lance nematodes. Journal of Nematology 10: 297. Rodriguez-Kabana, R., and R. W. Pearson. 1972. Changes in populations of lance and stunt nematodes in cotton following a Bahiagrass sod. Journal of Nematology 4: 233 (Abstr.). Ruehle, J. L., and J. N. Sasser. 1962. The role of plant-parasitic nematodes in stunting of pines in southern plantations. Phytopathology 52: 56-68. Russell, C. C., and R. V. Sturgeon. 1969. Occurrence of Belonolaimus longicaudatus and Ditylenchus dipsaci in Oklahoma. Phytopathology 59: 118 (Abstr.).

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67 Sher, S. A. 1961. Revision of the Hoplolaiminae (Nematoda) I classification of nominal genera and nominal species. Nematologica 6: 155-164. Sher, S. A. 1963. Revision of the Hoplolaiminae (Nematoda) IV. Peltamigratus n. gen. Nematologica 9: 455-467. Sher, S. A. 1966. Revision of the Hoplolaiminae (Nematoda) VI. Helicotylenchus Steiner, 1945. Nematologica 12: 1-56. Sikora, E. J., E. A. Guertal, and K. L. Bowen. 2001. Plant-parasitic nematodes associated with hybrid bermudagrass and creeping bentgrass putting greens in Alabama. Nematropica 31: 301-306. Skerman, P. J., and F. Riveros. 1990. Tropical grasses. Food and agricultural organization of the United Nations. 119, 128, 130, 565-568 pp. Sledge, E. B., and A. M. Golden. 1964. Hysoperine graminis (Nematoda: Heteroderidae), a new genus and species of plant-parasitic nematode. Proceedings of the Helminthological Society of Washington 31: 83-88. Smart, G. C., Jr. 1965. The aperture and lumen of the spear in three members of Tylenchoidea (Nemata). Nematologica 11: 45 (Abstr.). Smart, G. C., and K. B. Nguyen. 1991. Sting and awl nematodes: Belonolaimus spp. and Dolichodorus spp. Pp. 627-667 in W. R. Nickle, ed. Manual of agricultural nematology. New York: Marcel Dekker. Steiner, G. 1949. Plant nematodes the grower should know. Soil Science Society of Florida Proceedings 4: 72-117. Tarjan, A. C. and P. Busey. 1985. Genotypic variability in bermudagrass damage by ectoparasitic nematodes. Hortscience 20: 675-676 Tarjan, A. C. and M. H. Ferguson. 1951. Observation of nematodes in yellow turf of bentgrass. USGA Journal and Turf Management 4: 28-30. Thorne, G. 1935. Notes on free-living and plant-parasitic nematodes II. Proceedings of the Helminthological Society of Washington 2: 96-98. Trenholm, L. E., R. N. Carrow, and R. R. Duncan. 2001a. Wear tolerance, growth, and quality of seashore paspalum in response to nitrogen and potassium. Hortscience 36: 780783. Trenholm, L. E., R. R. Duncan, and R. N. Carrow. 1999. Wear tolerance, shoot performance, and spectral reflectance of seashore paspalum and bermudagrass. Crop Science 39: 1147-1152.

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68 Trenholm, L. E., R. R. Duncan, R. N. Carrow, and G. H. Snyder. 2001b. Influence of silica on growth, quality, and wear tolerance of seashore paspalum. Journal of Plant Nutrition 24: 245-259. Trenholm, L. E., and B. Unruh. 2003. Seashore paspalum management for home lawn use in Florida, University of Florida, Institute of Food and Agricultural Sciences, Extension Data Information Source, ENH 897. Gainesville, FL: University of Florida. Troll, J. S. and A. C. Tarjan. 1954. Widespread appearance of plant parasitic nematodes on golf greens in Rhode Island. Plant Disease Reporter 38: 342-344 USGA Green Section Staff. 1993. USGA recommendation for a method of putting green construction: the 1993 revision. USGA Green Section Record 31: 1-3. Viggars, R. M., and A. C. Tarjan. 1949. A new root disease of pin oaks possible caused by the nematode Hoplolaimus coronatus Cobb. Plant Disease Reporter 33: 132-133. Wen, G. Y., and T. A. Chen. 1972. Fine structures of the cuticle of Hoplolaimus galeatus Journal of Nematology 4: 236-237 (Abstr.). Wheeler, T. A., and J. L. Starr. 1987. Incidence and economic importance of plant parasitic nematodes in Texas (USA). Peanut Science 14: 94-96. Whitton, G. M., Jr. 1956. Aspects of the nematode problem. Proceedings University of Florida Turf Management Conference 4: 99-102. Williams, K. J. O. 1973. Hoplolaimus galeatus Commonwealth Institute of Helminthology descriptions of plant-parasitic nematodes 2: 1-2. Winchester, J. A., and E. O. Burt. 1964. The effect and control of sting nematode on Ormond bermudagrass. Plant Disease Reporter 48: 625-628. Wiseman, B. R., and R. R. Duncan. 1996. Resistance of Paspalum spp. to Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) larvae. Journal of Turfgrass Management 1: 23-26.

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69 BIOGRAPHICAL SKETCH Adam C. Hixson was born 25 April 1978 in Waco, TX, and grew up in China Spring, TX. He graduated from China Spring High School in 1996, and began studies at Texas A&M University, College Station, TX in the fall of 1996. He was a member of the Corps of Cadets for four years while pursing his Bachelor of Science degree in entomology. After graduation from Texas A&M University in 2000, he began studies for his Master of Science degree at University of Florida, Gainesville, Florida. The title of his thesis is Belonolaimus longicaudatus and Hoplolaimus galeatus on Seashore Paspalum ( Paspalum vaginatum )."


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Copyright Date: 2008

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Material Information

Title: Belonolaimus longicaudatus and Hoplolaimus galeatus on Seashore Paspalum (Paspalum vaginatum)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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Belonolaimus longicaudatus AND Hoplolaimus galeatus ON SEASHORE PASPALUM
(Paspalum vaginatum)















By

ADAM C. HIXSON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003

































Copyright 2003

by

Adam C. Hixson















ACKNOWLEDGMENTS

I would especially like to thank Dr. William Crow for his scientific expertise,

professional guidance, and monetary assistance while pursing my master's degree. He

taught me that learning from my mistakes is the one of the most important skills in

performing research. I am also indebted to my other committee members, Dr. Robert

McSorley and Dr. Laurie Trenholm. Dr. McSorley's statistical knowledge and assistance

with experimental methodology were key to the completion of my degree. Dr. Trenholm

always made herself available for help with turfgrass questions and overall support.

Thanks go also to Mr. Frank Woods for the words of wisdom and helping me to stay sane

through all the insanity.

I would also like to thank my parents for all their love and guidance throughout my

life. My father planted the scientific seed within me early in life. My father taught me

that knowledge is something that never can be taken away from you. Collecting

knowledge can be an arduous task, but the rewards are everlasting. From the time I was a

baby, my mother has always been my emotional support. She has taught me that

perseverance and integrity are keys to success in your career and personal life.

My brother has been a very influential person in my life. He has taught me that

having fun is as important as any amount of knowledge or fulfillment of goals. Allowing

for time to relax and enjoy hobbies is an opportunity to clear your mind and become more

successful.









Most importantly, I would like to thank my wife for her never-ending love and

support. As I pursue my dream, she has never doubted me. Through all the late nights

and stressful days, she has always been there for emotional support and a helping hand. I

am also forever grateful to my wife for the sacrifices she has made by leaving her family

to be with me as I pursue my graduate degrees and career. She is a truly special person

and I look forward to maturing with her for years to come.
















TABLE OF CONTENTS
Page

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

LIST O F TA BLE S ........... .. ......... ....... ................ ............. vii

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

A B S T R A C T ............... ................................................................................. ............... ..... ix

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ....................................................1

In tro d u ctio n .................................................................................. 1
B elonolaim us longicaudatus............................................................................. 2
Taxonomy ............ 2............ ................. 2
D distribution and B iology ............................................... ............................ 3
H ost Range .................................. ....... ... ........ ................4.
Hoplolaimus galeatus ................. .. ........ ........ ... .........5
Taxonomy ............ 5............ ................. 5
D distribution and B iology ............................................... ............................ 6
H o st R an g e ....................................................... 7
N em atodes and Turfgrass ............................................................. ....................... 8
Salinity and N em atodes ..................... .. .......................... .. ........ .......... ...... .
Seashore Paspalum .......................... .............. ................. .... ....... 10
Current Research ............ .. .......... .............................. ........ 11

2 SUSCEPTIBILITY OF SEASHORE PASPALUM TO Belonolaimus longicaudatus
AND H oplolaim us galeatus ............................................... ............................ 12

Introduction .................................................. 12
M materials and M methods ....................................................................... .................. 14
R e su lts .........................................................................................................2 0
Discussion ........................ ..........................25

3 INFLUENCE OF SALT CONCENTRATION IN IRRIGATION WATER ON
PATHOGENICITY AND DEVELOPMENT OF Belonolaimus longicaudatus AND
Hoplolaimus galeatus ON SEASHORE PASPALUM ................... ................. 28

In tro d u ctio n ...................................... ................................................ 2 8









M materials and M methods ....................................................................... ...................30
R e su lts .................................................................................................................... 3 8
Discussion .............................. .. ..................... 43

4 SURVEY OF PLANT-PARASITIC NEMATODES ASSOCIATED WITH
SEASHORE PASPALUM .................................................................... 47

Introdu action .............. .................................................................................... 4 7
M materials and M methods ........................................................................ ..................4 8
R results ........... .... ..... ... ........ ..................... ............... 49
D iscu ssion ......... ...... ............ .................................... ............................52

5 S U M M A R Y .............. ..... ............ ................. ...............................................5 5

LIST OF REFERENCES .......................... ..................59

BIOGRAPHICAL SKETCH ............. .. ......... .................69















LIST OF TABLES


Table pge

2-1. Composition of experimental soil compared to the United States Golf Association
root zone m ix specifications........................................................ ............. 15

2-2. Cumulative shoot dry weight and total root lengths of 'SeaIsle 1' seashore paspalum
and 'Tifdwarf bermudagrass 60 and 120 days after initial inoculations of 107 8
(2002) and 211 + 10 (2003) Belonolaimus longicaudatus.............. .............. 20

2-3. Cumulative shoot dry weight and total root lengths of 'Sealsle 1' seashore paspalum
and 'Tifdwarf bermudagrass 60 and 120 days after initial inoculations of 100
(2002) and 199 13 (2003) Hoplolaimus galeatus. ..............................................23

2-4. Final population densities (nematodes/100 cm3 of soil) on 'Sealslel' seashore
paspalum and 'Tifdwarf bermudagrass 60 and 120 days after initial inoculations of
100 (2002) and 199 13 (2003) Hoplolaimus galeatus and 107 8 (2002) and 211
+ 10 (2003) Belonolaimus longicaudatus. .................................... .................24

4-1. Frequency of occurrence and population density of plant-parasitic nematode genera
in soil samples collected from seashore paspalum golf courses in Florida .............51

4-2. Frequency of occurrence and population density of plant-parasitic nematode genera
in soil samples collected from seashore paspalum home lawns in Florida ............52















LIST OF FIGURES


Figure page

3-1. Relationship between log transformation of final population densities (Pf) ofH.
galeatus (nematodes/100 cm3 of soil) (Y) and salinity treatment (x) in 2002 and
2003 glasshouse experim ents. ..... ........................................................ ...............40

3-2. Relationship between log transformation of final population densities (Pf) of B.
longicaudatus (nematodes/100 cm3 of soil) (Y) and salinity treatment (x) in 2002
and 2003 glasshouse experim ents. ........................................ ....................... 41

3-3. Relationship between log transformation of J2 or adults, J3, and J4 final population
densities (Pf) ofB. longicaudatus (nematodes/100 cm3 of soil) (Y) and salinity
treatment (x) in the 2003 glasshouse experiment. .............. ................................... 42

3-4. Effects ofBelonolaimus longicaudatus on root length of'SeaIsle 1' seashore
paspalum (Paspalum vaginatum) at increasing salinity levels. Inoculated plants
received 243 + 13 B. longicaudatus, while uninoculated plants received no
nematodes. The error bars show standard deviation of individual population
means. ** Indicates difference from inoculated at P < 0.01, according to the one-
w ay A N O V A procedure ................................................. ...................................43















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

Belonolaimus longicaudatus AND Hoplolaimus galeatus ON SEASHORE PASPALUM
(Paspalum vaginatum)

By

Adam C. Hixson

December 2003

Chair: William T. Crow
Major Department: Entomology and Nematology

Seashore paspalum (Paspalum vaginatum) has great potential for use in salt-

affected turfgrass sites. Seashore paspalum has a natural tolerance to drought and high

salinity irrigation. Therefore, use of this grass on golf courses, athletic fields, and lawns

in subtropical coastal areas may aid in conservation of fresh water resources. Plant-

parasitic nematodes are damaging pathogens of turfgrasses in Florida, with Belonolaimus

longicaudatus and Hoplolaimus galeatus considered among the most damaging.

Glasshouse experiments were performed to compare the susceptibility of 'SeaIsle 1'

seashore paspalum to B. longicaudatus and H. galeatus with 'Tifdwarf bermudagrass

(Cynodon dactylon x C. transvaalensis), and to examine the effects of increasing

irrigation salinity levels on B. longicaudatus and H. galeatus. 'SeaIsle 1' seashore

paspalum and 'Tifdwarf bermudagrass grown in 1,500-cm3 clay pots filled with 100%

United States Golf Association (USGA) specification sand and inoculated with either

nematode served as experimental units. Both nematode species reproduced well on either









grass, but only B. longicaudatus consistently stunted root growth. Belonolaimus

longicaudatus reduced root growth by 35 to 45% at 120 days after inoculation on both

grasses, but these reductions did not indicate a difference (P > 0.05) in susceptibility

between grasses. These data suggest that 'SeaIsle 1' seashore paspalum is not less

susceptible to B. longicaudatus or H. galeatus than is 'Tifdwarf bermudagrass. Final

population densities ofH. galeatus followed a negative linear regression (P < 0.01) with

increasing salinity levels. Final population densities of B. longicaudatus were

quadratically (P < 0.01) related to increasing salinity levels from 0 to 25 dS/m. An

increase in population densities of B. longicaudatus was observed at moderate salinity

levels (10 and 15 dS/m) compared to 0 dS/m (P < 0.05). Reproduction and feeding of B.

longicaudatus and H. galeatus decreased at salinity levels of 25 dS/m and above. Root

length comparisons revealed that B. longicaudatus caused root stunting at low salinity

levels, 0 to 10 dS/m (P < 0.05). However, at higher salinity irrigation treatments, 15 to

25 dS/m, root lengths were not different (P > 0.05) between inoculated and uninoculated

plants. These results indicate that the ability ofB. longicaudatus to feed and stunt root

growth was negatively affected at salinity levels of 15 dS/m and above.














CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

Introduction

Coastal areas in the southeastern United States, including a majority of Florida, are

ideal habitats for Belonolaimus longicaudatus Rau, the sting nematode (Christie, 1959;

Holdeman, 1955), and Hoplolaimus galeatus (Cobb, 1913) Thorne 1935, the lance

nematode (Ahmad and Chen, 1980; Williams, 1973). Both are important pathogens of

turfgrass (Perry et al., 1970). New turfgrass cultivars or ecotypes are commonly

introduced into the turfgrass industry, but in rare cases, a new species of turfgrass begins

to become more widely utilized. Seashore paspalum (Paspalum vaginatum Swartz) is a

new turfgrass species quickly growing in popularity due to the breeding and development

of cultivars with a fine leaf texture and tolerance to drought and high salinity irrigation

(Duncan, 1999a; Morton, 1973).

Belonolaimus longicaudatus and H. galeatus have been reported as pathogens of

many bermudagrass (Cynodon dactylon (L.) Pers and Cynodon spp. hybrids) (Di

Edwardo, 1963; Giblin-Davis et al., 1992b; Johnson, 1970; Rivera-Camarena, 1963;

Winchester and Burt, 1964), and St. Augustinegrass (Stenotaphrum secundatum) (Busey

et al., 1991; Giblin-Davis et al., 1992a; Henn and Dunn, 1989; Kelsheimer and Overman,

1953) cultivars. However, no published data currently exist on the susceptibility of

seashore paspalum to B. longicaudatus or H. galeatus. Knowledge is also lacking on the

response of B. longicaudatus and H. galeatus to high salinity irrigation. After personal

communication with two turfgrass managers in south Florida, Morton (1973)









hypothesized that low nematode counts may be associated with seashore paspalum

irrigated with saline water. Further investigation is necessary to confirm this hypothesis.

Typically, susceptibility of a plant to a pathogen is based on yield reduction caused

by the pathogen, but yield is difficult to determine in turfgrass. Therefore, root length

data and the reproductive capability of the nematode are major determining factors of

susceptibility in turfgrasses. When levels of susceptibility are determined, soil and root

nematode assays may be useful in predicting a need for treatment. This information will

help avoid unnecessary post-plant nematicide treatments or failure to apply a needed one.

Belonolaimus longicaudatus

Taxonomy

The first reported nematode in the genus Belonolaimus was the species B. gracilis

collected from the rhizosphere of a pine tree (Pinus spp.) near Ocala, FL (Steiner, 1949).

Subsequently, B. gracilis was reported to damage peanut (Arachis hypogaea) in Virginia

(Owens, 1951), strawberry (Trifoliumfragiferum), celery (Apium graveolens), and corn

(Zea mays) in Florida (Christie et al., 1952), and cotton (Gossypium spp.), soybean

(Glycine max), and cowpea (Vigna unguiculata) in South Carolina (Graham and

Holdeman, 1953). Later, Rau (1958) described B. longicaudatus, which soon became

accepted as the more common sting nematode damaging agricultural crops and

turfgrasses in the southeastern United States (Perry and Rhoades, 1982; Smart and

Nguyen, 1991). No published data exist on B. gracilis except for the original description

in 1949. Tail and stylet lengths can separate the two species. Specimens of B.

longicaudatus have a shorter tail and a longer stylet than those of B. gracilis (Rau 1958).

Rau (1963) continued his work with the genus Belonolaimus, describing three more

species: B. euthychilus, B. maritimus, andB. nortoni. Presently, the genus includes 9









species, the five mentioned previously, and B. anama, B. jara, B. lineatus, and B. lolii

(Fortuner and Luc, 1987).

Distribution and Biology

Belonolaimus longicaudatus is found primarily in deep sandy soils in the lower

coastal plains of the southeastern United States from Virginia to Florida (Christie, 1959;

Holdeman, 1955), but has been reported in New Jersey (Myers, 1979), Kansas

(Dickerson et al., 1972), Arkansas (Riggs, 1961), Oklahoma (Russell and Sturgeon,

1969), Texas (Christie, 1959; Wheeler and Starr, 1987), and recently in California

(Mundo-Ocampo et al., 1994). Holdeman (1955) reported confirmed findings of B.

longicaudatus in Florida, Georgia, South Carolina, North Carolina, and Virginia, with

possible findings in Alabama, Mississippi, and Louisiana. Belonolaimus longicaudatus is

most commonly found in Florida because it is well adapted to soils with > 80% sand

content and < 10% clay content (Miller, 1972; Robbins and Barker, 1974). Populations

ofB. longicaudatus have been discovered at golf courses located outside the native

habitat of the nematode. These areas in the Bahamas, Bermuda, Puerto Rico, Costa Rico,

and California are ideal for B. longicaudatus reproduction because the soil profiles are at

least 90% sand (Lopez, 1978; Mundo-Ocampo et al., 1994; Perry and Rhoades, 1982).

Imported sod from Florida and Georgia appear to be the cause of the infestations in the

Bahamas, Bermuda, and Puerto Rico (Perry and Rhoades, 1982).

Belonolaimus longicaudatus is recognized as the most pathogenic ectoparastic

plant nematode to turfgrasses in Florida. Acceptable visual quality often is not possible

without treatment of the nematode infestation. Root injury typically consists of greatly

reduced root systems, with short stubby roots exhibiting dark lesions along the root axis









and along the tip. Aboveground symptoms usually consist of severe stunting, incipient

wilting, leaf chlorosis, or death (Perry and Rhoades, 1982).

Reproduction of B. longicaudatus is exclusively accomplished through

amphimixis, requiring both males and females to be present (Huang and Becker, 1999).

Females lay eggs as long as food is readily available (Huang and Becker, 1999). Eggs

are laid two at a time with one coming from each opposed ovary of the amphidelphic

reproductive system (Han, 2001). The entire lifecycle was achieved in 24 days at 28 C

on excised corn roots grown in vitro (Huang and Becker, 1997; 1999).

Belonolaimus longicaudatus is a migratory ectoparasitic nematode that inserts its

stylet deep into the root tip and withdraws the cellular contents. The presence of a host is

necessary for survival and reproduction, because there is no known survival mechanism

such as anabiosis or diapause (Huang and Becker, 1997).

Temperature can have a dramatic effect on the survivability and reproduction of

B. longicaudatus (Boyd and Perry, 1971; Robbins and Barker, 1974). Seasonal

temperature variation greatly influenced the population levels and distribution of the sting

nematode. April and May temperatures in Gainesville, FL are typically favorable for

high populations, while June and July are least favorable (Boyd and Perry, 1971).

Reproduction of B. longicaudatus was greatest at 25 to 30 OC, with one population

achieving some reproduction at 35 OC while the other population declined. Both

populations had minimal reproduction at 20 OC (Robbins and Barker, 1974).

Host Range

Belonolaimus longicaudatus has an extremely wide host range, including many

small grains, forage crops, fruits, vegetables, ornamentals, trees, and turfgrasses (Bekal









and Becker, 2000; Holdeman and Graham, 1953; Owens, 1951; Perry and Rhoades,

1982; Robbins and Barker, 1973). Holdeman (1955) reported watermelon (Citrullus

lanatus) and tobacco (Nicotiana tabacum) to be non-hosts. Additionally, asparagus

(Asparagus officinalis), buckhorn plantain (Plantago lanceolata), okra (Abelmoschus

esculentus), and pokeweed (Phytolacca americana) were reported to be non-hosts

(Robbins and Barker, 1973). Later, a California population also was found to be unable

to reproduce on okra, watermelon, and tobacco (Bekal and Becker, 2000). Physiological

races have been suggested numerous times by many researchers due to host preferences

and reproductive isolation, but none are widely recognized (Abu-Gharbieh and Perry,

1970; Owens, 1951; Robbins and Barker, 1973; Robbins and Hirschmann, 1974).

Robbins and Barker (1973) reported that some poor or non-hosts for three populations of

B. longicaudatus from North Carolina were actually good to excellent hosts for one

population from Georgia. Also, when populations with different morphological

characteristics and host preferences were allowed to mate with each other, infertile

individuals were produced (Robbins and Hirschmann, 1974). Morphological differences

and reproductive potential suggest that the populations from North Carolina and Georgia

may be different species (Robbins and Hirschmann, 1974).

Hoplolaimus galeatus

Taxonomy

Lance nematodes belong to the extremely diverse subfamily of Hoplolaiminae that

includes hundreds of species of plant-parasitic nematodes. Hoplolaimus galeatus was

originally described as Nemonchus galeatus (Cobb, 1913), but later Thorne (1935)

determined this nematode to be identical in general size and shape to Hoplolaimus

coronatus. Hoplolaimus galeatus and H. coronatus were later found to be conspecific,









with H. galeatus having date priority (Sher, 1961). The nematode was thereafter referred

to as Hoplolaimus galeatus. Identifying morphological characteristics on the female

include an offset cephalic region, usually with 5 annules, 2 phasmids (one anterior to the

vulva and the other posterior), and a rounded tail with 10 to 16 annules. Hoplolaimus

galeatus reproduces by amphimixis with an equal ratio of females to males (Hirschmann,

1959; Smart, 1965; Wen and Chen, 1972).

Distribution and Biology

Hoplolaimus galeatus has a wider distribution than B. longicaudatus, but is most

numerous in the southeastern United States. Woody or graminaceous plants appear to be

especially favorable hosts (Williams, 1973). Hoplolaimus galeatus is usually a

seemingly unmanageable problem because of its widespread distribution and the frequent

failure of nematicides to reduce their numbers while controlling other plant parasitic

nematodes (Perry et al., 1970). Krusberg and Sasser (1956) found that cotton plants

growing under drought conditions that were stunted, yellowing, and almost completely

defoliated returned much higher numbers ofH. galeatus in the soil and roots near the

rhizosphere than did healthy plants. The nematodes primarily fed endoparasitically on

the cotton roots, causing considerable damage to the root cortex during penetration.

Disruption of the vascular tissue occurred during feeding due to the preference for

phloem (Krusberg and Sasser, 1956). Slash (Pinus elliottii) and Loblolly pine (Pinus

taeda) seedlings suffered 50% mortality in North Carolina due to infestation by H.

galeatus. Glasshouse experiments confirmed these findings, showing roots with black

lesions and most of the root cortex destroyed (Ruehle and Sasser, 1962). Experiments

with bermudagrass determined that total plant weight was reduced by 50% over 6 months

by H. galeatus (Di Edwardo, 1963).









Host Range

Lance nematodes (H. galeatus) are pathogenic on a variety of agronomic crops

and turfgrasses. The rate of reproduction ofH. galeatus was higher on rye (Secale

cereale), barley (Hordeum vulgare), wheat (Triticum aestivum), oats (Avena sativa),

soybean, corn, cabbage (Brassica oleracea), and bean (Phaseolus vulgaris) than on

pepper (Capsicum spp.), eggplant (Solanum melongena), lettuce (Lactuca sativa),

tobacco, and pea (Ahmad and Chen, 1980). Hoplolaimus galeatus can cause serious

damage to cotton (Krusberg and Sasser, 1956), pine (Ruehle and Sasser, 1962), oak

(Viggars and Tarjan, 1949), wheat (Ahmad and Chen, 1980), and turfgrasses (Di

Edwardo, 1963; Perry et al., 1970). In a study involving H. coronatus on cotton, host

symptoms were more prominent in dry conditions, but plants supplied with adequate

moisture could tolerate high populations (Krusberg and Sasser, 1956). When cotton

followed bahiagrass (Paspalum notatum), the H. galeatus population dropped to below

damaging levels (Rodriguez-Kabana and Pearson, 1972). Hoplolaimus galeatus was also

found to feed endo and ectoparasitically on alfalfa (Medicago sativa) and bermudagrass

(Ng and Chen, 1980). Hoplolaimus galeatus caused reductions in magnesium and water

uptake, shoot and root growth, and number of root hairs on tall fescue (Festuca

arundinacea) (Rodriguez-Kabana et al., 1978).

Lance nematodes (Hoplolaimus spp.) were found to be associated with unhealthy

St. Augustinegrass lawns in the Tampa, FL area (Kelsheimer and Overman, 1953). Other

surveys specifically associated H. galeatus with damaged lawns in Florida (Good et al.,

1956; Perry and Good, 1968; Whitton, 1956). Hoplolaimus galeatus reproduced well on

seven different cultivars of St. Augustinegrass, indicating similar host suitability among

these cultivars (Henn and Dunn, 1989). Time course experiments with 'Floratam' and









'FX-313' St. Augustinegrass indicated that H. galeatus had no effect on plant growth,

even though soil counts of H galeatus exceeded the proposed action threshold of 40

nematodes/100 cm3 of soil (Crow et al., 2003) for 84 days during the course of the

experiment (Giblin-Davis et al., 1995). Alyceclover (Alysicarpus vaginalis), corn, and

some vegetables supported high populations ofH. galeatus, but measurable yield

suppression was not detected in any of these crops (Mashela et al., 1992b; Norton and

Hinz, 1976; Rhoades, 1987).

Nematodes and Turfgrass

In 1951, plant-parasitic nematodes were reported to be associated with the 'yellow

tuft' disease of bentgrass (Agrostispalustris) in Virginia (Tarjan and Ferguson, 1951). A

subsequent survey in Rhode Island by Troll and Tarjan (1954) indicated that several

species of nematodes might be causing serious damage on turfgrass as either primary or

secondary plant pathogens. Nematodes were not recognized as pathogens of turfgrass in

Florida until 1953, when V. G. Perry applied three nematicides on a thinning

bermudagrass golf course green at Sanford, FL. One month after treatment, the turfgrass

began to grow and later completely covered the putting surface. This indicated that the

death of plant-parasitic nematodes allowed the grass to recover from root damage (Perry

and Rhoades, 1982). Christie et al. (1954) stated that sting and lance nematodes were

responsible for most of the nematode damage to turfgrass in Florida. In Florida, B.

longicaudatus became recognized as a pathogen of significant importance to turfgrasses

(Christie et al., 1954; Perry et al., 1970; Rhoades, 1962). Perry and Rhoades (1982)

estimated that over 80% of some 700 Florida golf courses used nematicides routinely.









Many hybrid bermudagrasses (Cynodon dactylon (L.) Pers x C. transvaalensis

Burtt-Davis) have been determined to be suitable and susceptible hosts to B.

longicaudatus (Giblin-Davis et al., 1992b). 'Ormond' bermudagrass was demonstrated to

be extremely susceptible to B. longicaudatus, with severe yellowing, stunting of top

growth, and stunting of the roots (Winchester and Burt, 1964). St. Augustinegrass also

was found to be a suitable and susceptible host to B. longicaudatus, with root reductions

occurring when compared to uninoculated plants (Giblin-Davis et. al, 1992a; Rhoades,

1962). Rhoades (1962; 1965) also reported that Trichodorus christiei and T proximus

reduced St. Augustinegrass root weight when compared to uninoculated plants. Sting and

lance nematodes were extracted from soil samples taken from declining areas of St.

Augustinegrass in the Tampa Bay area (Kelsheimer and Overman, 1953). Sting and

lance nematodes were both found to be pathogenic on 'Tifton 328' bermudagrass in a

susceptibility experiment. Sting nematode populations increased from 250 to

approximately 3,700 per pot, while lance nematode populations increased from 250 to

approximately 2,100 per pot (Rivera-Camarena, 1963).

Salinity and Nematodes

High-salinity irrigation has been shown to negatively affect some root-knot

nematodes. Meloidogyne incognita egg hatching decreased by 96% when exposed to a

28.8 dS/m solution (Lal and Yadav, 1975). Salinity has demonstrated negative effects on

the hatching and infectivity ofM. javanica and M arenaria juveniles (Dropkin et. al,

1958; Maqbool et al., 1987). Khan and Khan (1990) found that after only 7 days of

salinity exposure, M. incognita and M. javanica had significant reductions in hatching

and increases in mortality. Population levels ofAphelenchus avenae, Pratylenchus









thornei, Helicotylenchus spp., and Rotylenchulus reniformis were lower at increased salt

concentrations over nonsaline treatments. No differences were observed in populations

ofHeterodera avenae and Tylenchorhynchus mashhoodi at increased salt concentrations

when compared to controls (Lal and Yadav, 1976). Some species of root-knot nematodes

(M spartinae) and sting nematodes (B. maritimus) may be well adapted to high-salinity

conditions (Rau, 1963; Rau and Fassuliotis, 1965).

Seashore Paspalum

Seashore paspalum (Paspalum vaginatum Swartz) is a warm season, perennial

grass, with both rhizomotous and stoloniferous growth, and a V-shaped inflorescence

(Duncan and Carrow, 2000; Morton 1973). Seashore paspalum is indigenous to South

Africa and South America, and has been found in Argentina, Brazil, Australia, the

Caribbean Islands, and Pacific islands. Within North America, it has been found

throughout the southern latitudes of the United States. Morton (1973) indicates that St.

Simons Island, GA, is the origin of seashore paspalum in North America. The grass is

best adapted to coastal areas (brackish areas, sand dunes, and beaches) with tropical and

sub-tropical temperatures. Regions from 30 to 350 N and S latitudes are most suitable

(Duncan and Carrow, 2000; Morton, 1973; Skerman and Riveros, 1990). Seashore

paspalum encompasses a wide range of ecotypes from fine to coarse leaf textures, and

can be used in a multitude of turfgrass applications (Duncan and Carrow, 2000).

Several seashore paspalum ecotypes have been researched for their multiple stress

resistance characteristics. In particular, advantageous characteristics include salinity

tolerance (Carrow and Duncan, 1998; Marcum and Murdoch, 1994; Peacock and Dudeck,

1985), drought tolerance, (Huang et al. 1997a; 1997b), wear and traffic tolerance









(Trenholm et al. 1999; 2001a; 2001b), water logging tolerance (Malcolm, 1977), and

tolerance of other stresses including pH, diseases, insects, and weeds (Duncan, 1999b;

Johnson and Duncan, 1997; 1998; Wiseman and Duncan, 1996). Fertility requirements

are much lower for seashore paspalum than many similarly textured grasses (Beard et al.,

1982; 1991). Suggested nitrogen rates for fairways, tees, and sports fields range from

0.98 to 2.94 kg/100 m2 annually depending on geographical location (Duncan, 1999b).

In Florida, recommended nitrogen rates are slightly higher, ranging from 1.96 to 5.88

kg/100 m2 annually (Trenholm and Unruh, 2003). Seashore paspalum greens ecotypes

were reported to have higher quality ratings at 3 to 4 mm mowing height when compared

to 'Tifgreen' bermudagrass (Duncan, 1999b).

Current Research

The research reported hereafter was performed in an environmentally controlled

glasshouse at the University of Florida Envirotron Turfgrass Research Laboratory in

Gainesville, FL. Experiments were performed in consecutive summers in an optimal

environment for both nematodes and turfgrasses. The objectives of the research were to:

1. Determine susceptibility of 'Sealsle 1' seashore paspalum to a population of B.
longicaudatus and a population of H. galeatus.

2. Compare 'Sealsle 1' seashore paspalum to 'Tifdwarf bermudagrass for differences
in susceptibility to a population of B. longicaudatus and a population of H.
galeatus.

3. Determine how high salinity irrigation can affect the reproduction of a population
of B. longicaudatus and a population of H. galeatus.

4. Conduct a survey to determine which plant-parasitic nematodes are associated with
established seashore paspalum golf courses and home lawns in Florida.














CHAPTER 2
SUSCEPTIBILITY OF SEASHORE PASPALUM TO Belonolaimus longicaudatus
AND Hoplolaimus galeatus

Introduction

Seashore paspalum (Paspalum vaginatum Swartz) is a warm-season turfgrass

quickly expanding in popularity. Due to the fine leaf texture and natural tolerance to

drought and high salinity, seashore paspalum has become more prevalent in coastal salt-

affected turfgrass sites (Duncan, 1999a; Morton, 1973). One major limitation of

cultivating turfgrasses in the sandy soils of the southeastern United States is the

destruction of roots by phytoparasitic nematodes (Perry and Rhoades, 1982). The sting

nematode (Belonolaimus longicaudatus Rau) and the lance nematode (Hoplolaimus

galeatus (Cobb) Thorne) are destructive pathogens on a variety of agricultural crops,

including turfgrasses (Ahmad and Chen, 1980; Holdeman and Graham, 1953; Perry and

Rhoades, 1982; Smart and Nguyen, 1991). While B. longicaudatus is found primarily in

the coastal plains of the southeastern United States (Christie, 1959; Holdeman, 1955), H.

galeatus has a much wider distribution (Williams, 1973). Belonolaimus longicaudatus

damages lateral roots as soon as they are formed, causing a stunting of root growth,

decreased water and nutrient uptake, and decreased rates of evapotranspiration (Busey et

al., 1991; Johnson, 1970; Perry and Rhoades, 1982). Hoplolaimus galeatus enters the

root tissue as a migratory endoparasite and is thought to damage roots by feeding and

physical tunneling through the root cortex cell walls (Henn and Dunn, 1989; Krusberg

and Sasser, 1956; Lewis and Fassuliotis, 1982; Perry et al., 1970; Williams, 1973).









Both nematodes have been reported as economically important pathogens of

turfgrasses in the southeastern United States (Christie et al., 1954; Kelsheimer and

Overman, 1953; Perry and Rhoades, 1982). Belonolaimus longicaudatus has been

reported as a pathogen of many bermudagrass (Cynodon dactylon (L.) Pers. and Cynodon

spp. hybrids), and St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntze)

cultivars (Busey et al., 1991; Giblin-Davis et al., 1992a; 1992b; Johnson, 1970). Seven

cultivars of St. Augustinegrass were discovered to have similar host suitability for H.

galeatus in the glasshouse and in microplots (Henn and Dunn, 1989). However, root and

shoot growth of 'FX-313' and 'Floratam' St. Augustinegrass were not affected by H.

galeatus in a population dynamics and pathogenicity study (Giblin-Davis et al., 1995).

Two populations ofB. longicaudatus readily reproduced on 'Tifdwarf bermudagrass

(Cynodon dactylon x C. transvaalensis Burtt-Davis), and caused extensive root damage

(Giblin-Davis et al., 1992b; Johnson, 1970). A forage grass study determined that there

was differential host suitability and susceptibility to B. longicaudatus in Digitaria spp.,

Paspalum spp., and Chloris spp. introductions (Boyd and Perry, 1969).

At present, no published data exist on the susceptibility of seashore paspalum to

H. galeatus or B. longicaudatus. The objectives of this study were to: i) determine the

susceptibility and host suitability of 'SeaIsle 1' seashore paspalum to a population of B.

longicaudatus and a population of H. galeatus, and ii) compare 'SeaIsle 1' seashore

paspalum to 'Tifdwarf bermudagrass for differences in susceptibility to a population of B.

longicaudatus and a population ofH. galeatus.









Materials and Methods

Experiments were performed to compare the relative susceptibility of 'SeaIsle 1'

seashore paspalum and 'Tifdwarf bermudagrass to B. longicaudatus and H. galeatus.

Experiments were conducted during the spring and summer of 2002 and 2003 at the

University of Florida Turfgrass Envirotron Glasshouse in Gainesville, FL.

Commercially available cultivars of Paspalum vaginatum and Cynodon dactylon

x C. transvaalensis were evaluated in this experiment. A sod square of 'Sealsle 1'

seashore paspalum was obtained from R. R. Duncan at the University of Georgia and

'Tifdwarf bermudagrass was available from previous experiments. Nematode-free plugs

of each grass were obtained by rooting aerial cuttings of stolons from each grass in

tapered RLC-7 (UV Stabilized) Super "Stubby" Cells (cell depth = 14 cm; diam. = 3.8

cm; volume = 115 ml) (Ray Leach Single Cell Cone-tainer, Stuewe & Sons, Inc.,

Corvallis, OR) filled with 140 g ofuninfested soil. Soil used for growth media consisted

of 100% United States Golf Association (USGA) specification sand. The soil texture was

analyzed using the sieving method for testing a USGA root zone mix (USGA Green

Section Staff, 1993) (Table 2-1). An absorbent cotton ball was placed at the bottom of

each cell to prevent soil from escaping through the drain holes. The soil was then

thoroughly wetted to allow for settling. A depression was made in each cell and two

aerial stolons were planted on opposing sides of the depression. Stolons (5 to 8 cm long)

were terminal cuttings with two or three nodes. During the fall and winter months, the

cells were placed on a glasshouse bench 1.25 m below an enclosed high bay 1,000-watt

metal halide growth lamp (Hi-Tek Series, Lithonia Lighting, Conyers, GA) set on a 12-

hour cycle to simulate the longer day-lengths required for optimal growth. The grass was









fertilized biweekly using a fertilizer solution (20%-20%-20% (N-P205-K20) plus trace

elements) (Peters Professional All-Purpose Plant Food, Spectrum Brands, St. Louis, MO)

at a rate equivalent to 49.0 kg N/ha/month, 21.6 kg P/ha/month, and 40.7 kg K/ha/month.

Both grasses were allowed to develop a substantial root system for four weeks.

Table 2-1. Composition of experimental soil compared to the United States Golf
Association root zone mix specifications

Sand Type Particle Size Experimental Soil USGA specifications
Fine Gravel 2.0 to 3.4 mm 0.1% Not more than 10% of
the total particles in
this range, including a
Very Coarse Sand 1.0 to 2.0 mm 3.7% maximum of 3% fine
gravel (preferably
none)

Coarse Sand 0.5 to 1.0 mm 30.8% Minimum of 60% of
Medium Sand 0.25 to 0.50 mm 53.4% the particles must fall
in this range

Fine Sand 0.15 to 0.25 mm 10.3% Not more than 20%

Very Fine Sand 0.05 to 0.15 mm 1.7% Not more than 5%
Data are means of five replicates

Plugs of seashore paspalum and bermudagrass obtained from the cells were

transferred into 14.5 x 16-cm-diam. clay pots (1,500 cm3) filled with 100% USGA

specification sand (Table 2.1). Insect screening (7 lines x 5 lines/cm2) was cut into 5 x 5-

cm squares and placed at the bottom of each pot to prevent soil from escaping through the

drain hole. Roots were washed free of soil and trimmed to approximately 5 cm below the

crown to promote fresh root growth. Two depressions were made in each pot on opposite

sides and two plugs of turfgrass were planted per pot. These experimental units were

placed on a screened bench in an environmentally controlled glasshouse and irrigated as

needed for 14 days to allow for adjustment to the new environment.









A population ofB. longicaudatus obtained from R. M. Giblin-Davis that

originated from unmanaged field soil in the Sanford, FL area and were allowed to

reproduce on 'FX 313' St. Augustinegrass was used in this experiment. A population of

H. galeatus was obtained from a 'Floradwarf bermudagrass putting green at the G. C.

Horn Turfgrass Field Laboratory in Gainesville, FL. Inocula were extracted from soil

using a modified Baermann funnel method (McSorley and Federick, 1991). In 2002, the

source of the H. galeatus population was contaminated with other plant-parasitic

nematodes, so 100 individual H. galeatus of mixed life stages were handpicked to

inoculate into 16 pots of each grass. Since the B. longicaudatus population was not

contaminated, handpicking was not necessary. A solution of B. longicaudatus at various

life stages in tap water was calibrated by counting nematodes from 1-ml aliquots on a

grided counting slide (Hawksley and Sons Limited, Lancing, Sussex, United Kingdom)

replicated ten times. Approximate numbers of nematodes were measured with a pipette

from water suspensions of inocula. Belonolaimus longicaudatus (107 8) was

inoculated into 16 pots of each grass. In 2003, H. galeatus and B. longicaudatus were

obtained from the previous year's experiment and inocula for both species were obtained

using a modified Baermann funnel method (McSorley and Federick, 1991). Suspensions

were made for each nematode, and standardized to deliver approximately 200 nematodes

per pot. Hoplolaimus galeatus (199 + 13) and B. longicaudatus (211 + 10) were

inoculated into 20 pots of each grass, respectively. A higher level of nematodes was used

in the second year to achieve higher reproduction. In both years, nematodes were

suspended in 50 ml of tap water and equally distributed into four cavities formed in the

soil near the base of the plant. Uninoculated plants received 50 ml of tap water. After









inoculation, the cavities were then pinched closed with surrounding soil and tap water

was applied as needed to avoid wilting.

Experimental units were arranged in a randomized complete block design in the

glasshouse on screened benches. In 2002, separate experiments were performed for each

nematode consisting of a total of 64 pots each. Each experiment had eight treatments

with eight replications. In 2003, the two nematode species were placed into the same

experiment, allowing for fewer control pots and a higher number of replications. The

second experiment had 12 treatments with 10 replications, for a total of 120 pots. For

both experiments, at 60 days after inoculation, one pot of each treatment was removed

from each block and analyzed, and 120 days after inoculation the remaining pots were

analyzed. Treatments were seashore paspalum and bermudagrass inoculated with the

specified number ofH. galeatus or B. longicaudatus allowed to reproduce for 60 or 120

days, and uninoculated controls.

Throughout the course of the 2002 experiments, which lasted from 21 April 2002

to 10 September 2002, average monthly high and low temperatures in the glasshouse

ranged from 31 to 34 C, and 23 to 26 C, respectively. In 2003, experiments began 28

February 2003 and ended 13 July 2003. Average monthly high and low temperatures

ranged from 27 to 32 C and 19 to 24 C, respectively. Using a 1.18-liter pump sprayer,

an insecticide/miticide (Mavrik Aquaflow, Wellmark International, Schaumburg, IL) was

applied at the labeled rate twice during the course of 2003 experiments for control of

bermudagrass mites (Eriophyes cynodoniensis Sayed) and rhodesgrass mealybugs

(Antonina graminis Maskell). 'Tifdwarf bermudagrass was fertilized on a biweekly basis

with 40 ml of a solution consisting of 5,100 mg NH4NO3 (34% N), 3,177 mg KC1, 252









mg Ca(H2P04)2, 435 mg CaSO4, 246 mg MgSO4, 1.55 mg H3BO3, 0.34 mg MnSO4, 0.58

mg ZnSO4, 0.13 mg CuSO4, and 3.5 mg FeSO4 per 1 liter of deionized water. A separate

fertilizer solution was made for 'Sealsle 1' seashore paspalum. The KC1 was doubled to

compensate for the different potassium requirements of seashore paspalum (Duncan and

Carrow, 2000). Total N for both grass species was 624 mg/pot for 120 days and 346

mg/pot for 60 days. Using fabric scissors (Fiskars Brand Inc., Madison, WI), the grass

was trimmed biweekly to approximately 2.5 cm above the soil surface. Tissue clippings

were collected in 15 cm x 23 cm mailing envelopes (Quality Park Products, St. Paul,

MN) using a spouted 2.84-liter sample pan (40.6 cm x 30.5 cm x 5 cm) (Seedburo,

Chicago, IL), and dried at 75 C for 48 hours to obtain cumulative shoot dry weight.

After 60 and 120 days, randomly-selected pots of each treatment from each block

were brought into the laboratory for destructive analysis. This provided a total of 32 pots

for each nematode at each sampling date in 2002 and a total of 60 pots at each sampling

date in 2003. Shoots were trimmed as close to the soil as possible and saved for

cumulative dry weight analysis. Then, using a stainless steel T-sampling tool, a root core

(approximately 4-cm-diam. x 14 cm deep) was taken from the center of each pot to serve

as a representative root sample. The remaining contents of each pot were emptied into

their individually labeled plastic bags and thoroughly hand mixed. A 100-cm3 soil

sample was taken from each bag and nematodes were extracted using a modified

centrifugal-sugar flotation technique (Jenkins, 1964). Nematodes in the entire soil

sample were counted using an inverted light microscope at x 32 magnification.

Root cores were washed free of soil on a sieve with 1.7-mm pore openings nested

within a sieve with 75-pm pore openings. Roots were removed from any aboveground









growth and placed in a 50-ml disposable plastic centrifuge tube containing 30 ml of tap

water. The 75-.m-pore sieve was then submerged in 5 cm of tap water to allow the finer

roots to float out and separate from the soil. These fine roots were collected using

laboratory forceps and placed into the 50-ml plastic centrifuge tubes. Five drops (0.25

ml) of a 1% methylene blue mixture was added to the 30 ml of tap water to stain the

roots. After a minimum of 24 hours in the solution, the roots were removed, placed on a

75-mm-pore sieve, and washed free of excess dye. Stained roots were placed into a glass-

bottom tray and scanned using a HP ScanJet 2cx desktop scanner (Hewlett Packard,

Boise, ID) to create a black and white bitmap image of the roots (Kaspar and Ewing,

1997; Pan and Bolton, 1991). The GSRoot (Louisiana State University, Baton Rouge,

LA) software program was used to analyze the bitmap images. This program measures

root lengths and surface areas from scanned images. Root length data were recorded for

seven diameter ranges (< 0.05 mm, 0.05 to 0.10 mm, 0.10 to 0.20 mm, 0.20 to 0.30 mm,

0.30 to 0.40 mm, 0.40 to 0.50 mm, and > 0.50 mm). The resulting values were summed

to determine the total root length of each root sample. In 2003 at the final sampling date,

H. galeatus in roots were staged and counted to determine number of nematodes/gram of

root in fresh-weighed samples using a modified acid-fuchsin staining-destaining

procedure (Byrd et al., 1983).

Total root lengths and cumulative shoot dry weights were compared for both

evaluation periods. The one-way analysis of variance (ANOVA) procedure performed

using SAS software (SAS Institute, Cary, NC) was used to analyze these two responses

for differences between inoculated and uninoculated treatments within each grass species.









Data for B. longicaudatus and H. galeatus were analyzed separately, even when the two

experiments shared controls (2003 experiment).

The one-way ANOVA procedure also was used to determine differences in

susceptibility between grass species by comparing final nematode population densities,

total root length percent reduction, and cumulative shoot dry weight percent reduction.

Total root length percent reduction was calculated by [100 x (total root length of

inoculated total root length of uninoculated) / total root length of uninoculated] and

cumulative shoot dry weight percent reduction was calculated by [100 x (cumulative

shoot dry weight of inoculated cumulative shoot dry weight of uninoculated) /

cumulative shoot dry weight of uninoculated]. Again, data for B. longicaudatus and H.

galeatus were analyzed separately, even when the two experiments shared controls (2003

experiment). A difference in the susceptibility of grasses to the nematodes was

determined to be significant when P < 0.05.

Results

The population of B. longicaudatus had a high degree of association with root

reduction in both turfgrasses while the population ofH. galeatus caused root reduction at

only one sampling date for each grass (Tables 2-2, 2-3). In the 2002 trial, the B.

longicaudatus population caused a reduction in total root growth in both seashore

paspalum and bermudagrass (P < 0.05) at 120 days after inoculation, but only in seashore

paspalum at the 60-day sampling (Table 2-2). In 2003, B. longicaudatus extensively

stunted total root growth (P < 0.05) in seashore paspalum and bermudagrass at 60 and

120 days after inoculation (Table 2-2). In both years, lengths of small diameter roots (<

0.02 mm-diam.) on plants inoculated with B. longicaudatus were less than (P < 0.01)









Table 2-2. Cumulative shoot dry weight and total root lengths of 'SeaIsle 1' seashore
paspalum and 'Tifdwarf bermudagrass 60 and 120 days after initial
inoculations of 107 8 (2002) and 211 + 10 (2003) Belonolaimus
longicaudatus.

Cultivar DAI Treatment Dry shoot weight (g) Root length (mm)
2002 trial
'Tifdwarf 60 U 6.2 3.4 951 212
I 6.6 2.5 762 234
120 U 13.2 3.8 1,597 312*
I 11.7 3.0 947 589
'Sealsle 1' 60 U 13.9 + 3.2 1,489 + 287*
I 11.6 3.0 1,150 281
120 U 24.4 6.7 1,647 152***
I 21.5 4.2 1,044 337

2003 trial
'Tifdwarf 60 U 4.6 + 1.2 1,525 192*
I 4.3 1.9 1,107 386
120 U 13.3 4.0 2,385 468***
I 13.4 3.2 1,380 354
'Sealsle 1' 60 U 7.9 2.3 1,602 301**
I 5.3 1.3 984 456
120 U 21.6 4.5 2,217 315***
I 20.6 4.3 1,185 467
Data are means of 8 (2002) or 10 (2003) replicates + standard deviations.
DAI = Days after inoculation.
I = inoculated and U = uninoculated.
*, **, *** Indicate uninoculated different from inoculated significant at P < 0.05,
P < 0.01, and P < 0.001 respectively, according to the one-way analysis of variance.

uninoculated plants for both grasses and sampling dates. Lengths of small diameter roots

on seashore paspalum inoculated with B. longicaudatus ranged from 488.96 217.26 to

574.26 227.91 mm compared to uninoculated plants having lengths ranging from

669.82 103.53 to 1037.26 126.31 mm. Bermudagrass followed a similar trend, with

lengths of small diameter roots ranging from 469.58 108.99 to 740.81 + 146.93 mm

and 623.60 133.24 to 1218.87 175.42 mm for inoculated and uninoculated plants,

respectively. Cumulative shoot dry weight was not different (P > 0.05) from controls

when either turfgrass was inoculated with B. longicaudatus. Belonolaimus longicaudatus









reproduced with ease on both grasses, indicating they were suitable hosts. In both trials,

B. longicaudatus reproduction data were variable among replicates, with bermudagrass

supporting higher populations than seashore paspalum at all sampling dates (P < 0.05),

except the 120-day evaluation period in 2002 (Table 2-4).

Throughout the majority of sampling times in both experiments, the population of

H. galeatus was not associated with root reduction in either grass (Table 2-3). However,

in the second year of the study, H. galeatus did reduce root growth (P < 0.05) in seashore

paspalum and bermudagrass after 60 and 120 days of exposure, respectively (Table 2-3).

Cumulative shoot dry weight was not different from controls when either turfgrass was

inoculated with H. galeatus. Both grasses were suitable hosts for H. galeatus with

moderate reproduction occurring both years. Bermudagrass was a more suitable host at

two sampling dates, supporting higher soil populations than seashore paspalum (P < 0.05)

after 60 (2003) and 120 (2002) days of feeding and reproducing (Table 2-4). Root

extraction ofH. galeatus by Baermann tray extraction returned low recovery in 2002.

However, in 2003, roots were stained through a modified acid-fuschin staining process

(Byrd et al., 1983), revealing that H. galeatus readily entered the roots of both grasses.

Nematodes were exclusively found within the root cortex and tended to aggregate in

random, nonspecific areas of the roots. Final root population means were 63 69 and 88

55 per gram of root fresh weight for bermudagrass and seashore paspalum respectively.

Populations ofH. galeatus entering the roots did not indicate a difference (P > 0.05) in

host suitability between the two grasses.









Table 2-3. Cumulative shoot dry weight and total root lengths of'SeaIsle 1' seashore
paspalum and 'Tifdwarf bermudagrass 60 and 120 days after initial
inoculations of 100 (2002) and 199 13 (2003) Hoplolaimus galeatus.

Cultivar DAI Treatment Dry shoot weight (g) Root length (mm)
2002 trial
'Tifdwarf 60 U 6.0 + 1.9 927 + 130
I 7.2 + 2.2 920 203
120 U 14.1 3.0 936 344
I 11.9 1.9 993 192
'Sealsle 1' 60 U 11.1 2.0 1,244 272
I 11.5 2.0 1,216 257
120 U 22.8 2.9 1,170 223
I 23.0 3.8 1,164 275

2003 trial
'Tifdwarf 60 U 4.6 + 1.2 1,525 192
I 4.6 + 1.9 1,697 490
120 U 13.3 4.0 2,385 468*
I 11.9 1.8 2,050 425
'Sealsle 1' 60 U 7.9 2.3 1,602 301*
I 5.9 1.6 1,276 350
120 U 21.6 4.5 2,217 315
I 21.0 4.0 2,098 410
Data are means of 8 (2002) or 10 (2003) replicates + standard deviations.
DAI = Days after inoculation.
I = inoculated and U = uninoculated.
Indicate uninoculated different from inoculated significant at P < 0.05 according
to the one-way analysis of variance.

Root length percent reductions and cumulative shoot dry weight reductions

calculated from root and shoot growth data were used to compare the effects of B.

longicaudatus or H. galeatus between 'Sealsle 1' seashore paspalum and 'Tifdwarf

bermudagrass. Differences in cumulative shoot dry weight percent reductions between

grasses were not found (P > 0.05) in either year for B. longicaudatus or H.galeatus.

Hoplolaimus galeatus did not cause reductions in root growth in the 2002 trial.

Therefore root length percent reductions showed no differences (P > 0.05) between the

two grasses. In the 2003 trial, H. galeatus did stunt seashore paspalum root growth even









though soil populations were below reported threshold levels for bermudagrass (Crow et

al., 2003). Root length percent reductions demonstrated a difference (P < 0.05) between

the two grass species 60 days after inoculation, with seashore paspalum showing a 19.4%

root length reduction, and bermudagrass showing a 14% increase in root length compared

to uninoculated plants.

Belonolaimus longicaudatus reduced root growth in both grasses, but root length

percent reductions did not indicate a difference (P > 0.05) in susceptibility between

'Sealsle 1' seashore paspalum and 'Tifdwarf bermudagrass. In 2002, root length percent

reduction averaged 13.7% for bermudagrass, and 20.2% for seashore paspalum at the 60-

day sampling. At 120 days after inoculation, root length percent reduction averaged

between 35% and 40% for both grasses. In the second year of the study, root length

percent reduction after 60 days was 26.7% and 38.4% for bermudagrass and seashore

paspalum respectively. At the final sampling date, root reductions continued on the same

trend with root length percent reductions above 39% for both grasses.

Table 2-4. Final population densities (nematodes/100 cm3 of soil) on 'Sealslel' seashore
paspalum and 'Tifdwarf bermudagrass 60 and 120 days after initial
inoculations of 100 (2002) and 199 13 (2003) Hoplolaimus galeatus and
107 8 (2002) and 211 + 10 (2003) Belonolaimus longicaudatus.

2002 2003
Cultivar 60 DAI 120 DAI 60 DAI 120 DAI
B. longicaudatus
'Tifdwarf 445 330* 495 530 273 220* 1,236 714*
'SeaIsle 1' 107 86 148 81 90+ 97 559 366
H. galeatus
'Tifdwarf 30 16 312 188* 58 28** 171 + 134
'SeaIsle 1' 22 13 80 47 15 8 116 33
Data are means of 8 (2002) or 10 (2003) replicates standard deviations.
DAI = Days after inoculation.
*, ** Indicate 'Tifdwarf different from 'SeaIsle 1' significant at P 0.05 and P
0.01 respectively, according to the one-way analysis of variance.









Discussion

Results indicate slight differences in host suitability, but no major differences in

susceptibility between the two grasses. 'Tifdwarf bermudagrass tended to be a more

suitable host for B. longicaudatus and H. galeatus throughout the experiment. Lower

final population densities with root length percent reductions equal to 'Tifdwarf

bermudagrass suggest that 'Sealsle 1' seashore paspalum could be a more susceptible or

less tolerant host ofB. longicaudatus. Since H. galeatus was not found to consistently

cause root stunting in either grass, seashore paspalum cannot be assumed more

susceptible than bermudagrass.

Population densities of B. longicaudatus after 120 days were very high in 2003 as

opposed to 2002. Higher inoculation levels and the earlier starting date in 2003 could

have resulted in enhanced reproduction. Furthermore, populations in the glasshouse may

peak during the late spring and early summer months, and thereafter decline as late

summer and early fall temperatures become the norm. Robbins and Barker (1974)

reported that reproduction of two populations of B. longicaudatus were greatest at 25 to

30 C, and minimal reproduction occurred at 20 OC. Neither grass had a reduction in

cumulative top growth even though root growth was stunted by B. longicaudatus.

Johnson (1970) and Giblin-Davis et al. (1992b) reported similar results for top growth

tissue weights for 'Tifdwarf bermudagrass. The proposed hypothesis is that root stunting

caused by B. longicaudatus could stimulate the production of more photosynthetic

material to overcome the damaged root system (Giblin-Davis et al., 1992b). In these

experiments, mowing frequency and height did not simulate typical golf course mowing

practices. Golf courses tend to have intense aboveground defoliation, causing an









immense amount of stress on the root system. This only amplifies the root problems that

B. longicaudatus is already causing.

Both grass species were suitable hosts for the nematodes. The lack of root

stunting caused by H. galeatus is very different from the reaction of the two grasses to

the more virulent nematode, B. longicaudatus. Hoplolaimus galeatus caused a slight root

reduction in both grasses in the second year of the study, even though reproduction

appeared to be relatively low. In February and March 2003, during the 60-day

experimental time period, temperatures and day-lengths were not optimal for warm-

season turfgrass growth. These environmental factors may have slowed root growth and

allowed the higher inoculum levels ofH. galeatus to reduce root lengths compared to

uninoculated controls. In 2003, statistical analysis showed that there was a blocking

effect (P < 0.05) in the H. galeatus 120-day bermudagrass root length data. A significant

blocking effect means the randomized complete block design was useful and accounted

for confounding factors in the glasshouse. Time course experiments with 'Floratam' and

'FX-313' St. Augustinegrass indicated that H. galeatus had no effect on plant growth even

though soil counts ofH. galeatus exceeded 40 nematodes/100 cm3 of soil, the proposed

action threshold (Crow et al., 2003), for 84 days within the course of the experiment

(Giblin-Davis et al., 1995).

Tarjan and Busey (1985) reported that 'Tifdwarf and 'Tifgreen' bermudagrass

inoculated with a mixed sample of phytoparasitic nematodes (including B. longicaudatus

and H. galeatus) caused 36% to 39% root dry weight reductions. These root reductions

appeared to correlate with increases in H. galeatus population levels. In our experiments,

B. longicaudatus alone caused approximately the same amount of root reduction, while









H. galeatus failed to consistently affect root growth. However, H. galeatus was able to

freely enter the roots of both grasses. In field situations, many other pathogens and

climatic stresses can enhance the pathogenic effects of nematodes on turfgrasses.

Glasshouse and laboratory work with soybean suggested that greater plant damage

resulted from inoculation with both H. galeatus and fungi in the genera Rhizoctonia,

Fusarium, and Macrophomina than with either pathogen alone (McGawley et al., 1984).

Further investigation is needed to determine if H galeatus may be having a synergistic

effect with other pathogens and/or nematodes in the soil environment in causing damage

to turfgrass roots.

In conclusion, our research suggests that the susceptibility of'SeaIsle 1' seashore

paspalum to B. longicaudatus and H. galeatus is not less than 'Tifdwarf bermudagrass.

There are limitations in extrapolating damaging thresholds from glasshouse pot

experiments to field situations, consequently action thresholds cannot be predicted from

our data. Glasshouse pot experiments eliminate the ability of climatic stresses and

pathogens other than the plant-parasitic nematodes to affect plant growth. Exposing the

plant to one pathogen and/or stress at a time allows us to determine damage potential

exclusive of all other negative effects on plant growth. Further experimentation is needed

to determine the effects of B. longicaudatus and H. galeatus on seashore paspalum

growth in field conditions.














CHAPTER 3
INFLUENCE OF SALT CONCENTRATION IN IRRIGATION WATER ON
PATHOGENICITY AND DEVELOPMENT OF Belonolaimus longicaudatus AND
Hoplolaimus galeatus ON SEASHORE PASPALUM

Introduction

Salt tolerant turfgrasses are becoming essential in many areas because of salt

accumulation in soil, restrictions on groundwater use, and salt-water intrusion into

groundwater (Carrow and Duncan, 1998; Parker, 1975). Seashore paspalum (Paspalum

vaginatum Swartz) is a warm-season turfgrass adapted for saline conditions (Malcolm

and Laing, 1969; Morton, 1973). Breeding for cultivars with fine leaf texture, and

tolerance to drought and high salinity irrigation has allowed seashore paspalum to

become frequently used in highly managed turfgrass sites (Dudeck and Peacock, 1985;

Duncan, 1999a). One major limitation of cultivating turfgrasses in the sandy soils of the

southeastern United States is the destruction of roots by phytoparasitic nematodes (Perry

and Rhoades, 1982). The sting nematode (Belonolaimus longicaudatus Rau) and the

lance nematode (Hoplolaimus galeatus (Cobb) Thorne) are destructive pathogens on a

variety of agronomic crops and turfgrasses (Ahmad and Chen, 1980; Perry and Rhoades,

1982; Perry et al., 1970; Smart and Nguyen, 1991).

While B. longicaudatus is usually limited to the coastal plains of the southeastern

United States (Christie, 1959; Holdeman, 1955; Robbins and Barker, 1974), H. galeatus

has a much wider distribution (Williams, 1973). Belonolaimus longicaudatus damages

lateral roots as soon as they are formed, causing stunted root growth, decreased water and

nutrient uptake, and decreased rates of evapotranspiration (Busey et al., 1991; Johnson,









1970; Perry and Rhoades, 1982). Hoplolaimus galeatus enters the root cortex and may

damage the roots by feeding and physical tunneling through the cell walls (Krusberg and

Sasser, 1956; Williams, 1973). Both nematodes have been reported as important

pathogens of turfgrasses in the southeastern United States (Christie et al., 1954;

Kelsheimer and Overman, 1953; Perry and Rhoades, 1982). Belonolaimus longicaudatus

and H. galeatus have been reported as pathogens of many bermudagrass (Cynodon

dactylon (L.) Pers. and Cynodon spp. hybrids) and St. Augustinegrass (Stenotaphrum

secundatum (Walt.) Kuntze) cultivars (Busey et al., 1991; Giblin-Davis et al., 1992a;

1992b; Perry et al., 1970). In more recent studies, root and shoot growth of diploid and

polyploid St. Augustinegrasses were not affected by H. galeatus, even though the plants

supported high population levels (Giblin-Davis et al., 1995; Henn and Dunn, 1989). Two

populations ofB. longicaudatus readily reproduced on 'Tifdwarf bermudagrass, and

caused extensive root damage (Giblin-Davis et al., 1992a, Johnson, 1970). A forage

grass study determined that there is differential host suitability and susceptibility to B.

longicaudatus in Digitaria spp., Paspalum spp., and Chloris spp. introductions (Boyd

and Perry, 1969).

Post-plant nematicides labeled for turfgrass are becoming limited and alternatives

must be found to replace recently discontinued nematicides. Salinity had a detrimental

effect on population densities of nematodes on some annual crops (Edongali et al., 1982;

Heald and Heilman, 1971). Soil salinity has demonstrated negative effects on the

hatching and infectivity ofMeloidogyne incognita Kofoid and White, M.javanica

(Treub) Chitwood, and M. arenaria (Neal) Chitwood juveniles (Bird, 1977; Dropkin et.

al, 1958; Lal and Yadav, 1975; Maqbool et al., 1987). Khan and Khan (1990) found that









after only 7 days of salinity exposure, M. incognita and M. javanica had reductions in

hatching and increased mortality. Population levels ofAphelenchus avenae Bastain,

Pratylenchus th,,niei Sher and Allen, Helicotylenchus spp., and Rotylenchulus reniformis

Linford and Oliveira were found to be lower at increased salt concentrations compared to

nonsaline treatments (Lal and Yadav, 1976). Some species of root-knot nematodes (M.

spartinae Rau and Fassuliotis) and sting nematodes (B. maritimus Rau) may be well

adapted to high-salinity conditions (Rau, 1963; Rau and Fassuliotis, 1965). After

personal communication with two turfgrass managers in south Florida, Morton (1973)

hypothesized that low nematode counts may be associated with seashore paspalum

irrigated with high saline water. Further investigation is necessary to confirm this

hypothesis.

Seashore paspalum is known to be tolerant of high salinity irrigation, but the

reaction of nematodes parasitizing seashore paspalum to the salinity is unknown.

Therefore, an experiment was performed to examine the effects of salinity irrigation on

B. longicaudatus and H. galeatus. Objectives of the experiment were to: i) determine the

effects of high salinity irrigation on the reproduction of a population of B. longicaudatus

and a population ofH. galeatus, ii) establish simple models relating irrigation salinity

levels to population levels of B. longicaudatus and to population levels ofH. galeatus,

iii) compare the effects of high salinity irrigation alone to the combined effects of high

salinity irrigation and B. longicaudatus on shoot and root growth.

Materials and Methods

Four separate experiments were performed, one with B. longicaudatus and one

with H. galeatus in each of two years (2002, 2003). Experiments were conducted in 2002









from April to September and repeated in the spring and summer of 2003 at the University

of Florida Turfgrass Envirotron Glasshouse in Gainesville, FL.

In preparation for these experiments, 'SeaIsle 1' seashore paspalum, a

commercially available cultivar ofPaspalum vaginatum, was obtained from R. R.

Duncan at the University of Georgia. Nematode-free plugs of grass were obtained by

rooting aerial cuttings of stolons from each grass in tapered RLC-7 (UV Stabilized) Super

"Stubby" Cells (cell depth = 14 cm; diam. = 3.8 cm; volume = 115 ml) (Ray Leach Single

Cell Cone-tainer, Oregon) filled with 140 g ofuninfested soil. Soil used for growth

media consisted of 100% United States Golf Association (USGA) sand. The soil texture

was analyzed using the sieving method for testing a USGA root zone mix (USGA Green

Section Staff, 1993) (Table 2-1). An absorbent cotton ball was placed at the bottom of

each cell to prevent soil from escaping through the drain holes. The soil was then

thoroughly wetted to allow for settling.

A depression was made in each cell and two aerial stolons were planted on

opposing sides of the depression. Stolons (5 to 8 cm long) were terminal cuttings with

two or three nodes. During the fall and winter months, the cells were placed on a

glasshouse bench 1.25 m below an enclosed high bay 1,000-watt metal halide growth

lamp (Hi-Tek Series, Lithonia Lighting, Conyers, GA) set on a 12-hour cycle to simulate

the longer daylengths required for optimal growth. The grass was fertilized using a

fertilizer solution (20%-20%-20% (N-P205-K20) plus trace elements) (Peters

Professional All-Purpose Plant Food, Spectrum Brands, St. Louis, MO) at a rate

equivalent to 49.0 kg N/ha/month, 21.6 kg P/ha/month, and 40.7 kg K/ha/month. A

substantial root system was allowed to develop for four weeks.









Plugs of seashore paspalum obtained from the cells were transferred into 14.5 x

16-cm-diam. clay pots (1,500 cm3) filled with 100% USGA specification sand (Table

2.1). Insect screening (7 x 5 lines/cm2) was cut into 5 x 5-cm squares and placed at the

bottom of each pot to prevent soil from escaping through the drain hole. Roots were

washed free of soil and trimmed to approximately 5 cm below the crown to promote fresh

root growth. Two depressions were made in each pot on opposite sides and two plugs of

turfgrass were planted per pot. These experimental units were placed in an

environmentally controlled glasshouse and irrigated with tap water as needed for 14 days

to allow for adjustment to the new environment.

A population of B. longicaudatus originally from unmanaged field soil in the

Sanford, FL area was obtained from R. M. Giblin-Davis at the Fort Lauderdale, FL

Research and Education Center and allowed to reproduce on 'FX 313' St. Augustinegrass.

A population ofH. galeatus was obtained from a 'Floradwarf bermudagrass putting green

at the G. C. Horn Turfgrass Field Laboratory in Gainesville, FL. Inocula were extracted

from soil using a modified Baermann funnel method (McSorley and Federick, 1991). In

2002, the H. galeatus population was contaminated with other plant-parasitic nematodes,

therefore handpicking was necessary to obtain an uncontaminated population. One

hundred H. galeatus of mixed life stages were inoculated into each pot of seashore

paspalum. Since the B. longicaudatus population was free of other plant-parasitic

nematodes, handpicking was not necessary. A solution of B. longicaudatus at various life

stages and tap water was calibrated by counting nematodes from 1-ml aliquots on a

grided counting slide (Hawksley and Sons Limited, Lancing, Sussex, United Kingdom)

replicated ten times. Approximate numbers of nematodes were measured with a pipette









from water suspensions ofinocula. A total of 111 + 16 B. longicaudatus were added to

each of the inoculated pots. In 2003, H. galeatus and B. longicaudatus were obtained

from the previous year's experiment and again, inocula was obtained using a modified

Baermann funnel method (McSorley and Federick, 1991). Solutions were made for each

nematode and standardized to deliver approximately 250 nematodes per pot. A total of

243 + 13 B. longicaudatus per pot and 238 8 H. galeatus per pot were inoculated for

two separate experiments. A higher level of nematodes was used in the second year to

achieve higher reproduction. In both years, nematodes were suspended in 50 ml of tap

water and equally distributed into four cavities formed in the soil near the base of the

plant. After inoculation, the cavities were then pinched closed with surrounding soil.

Uninoculated controls received 50 ml of tap water. Tap water was applied as needed for

two weeks to allow nematodes to adjust to their new environment before experimental

treatments were initiated. The 30 pots inoculated with each nematode were then

separated into five randomized blocks and treated identically except for salt concentration

in irrigation water. Separate experiments were performed for each nematode with each

experiment having six treatments with five replications.

Salinity irrigation treatments were formulated using Instant Ocean Synthetic Sea

Salt (Aquarium Systems, Inc., Mentor, OH). Ionic composition of Instant Ocean is

primarily Na+ and C1 and designed to mimic closely that of seawater (Atkinson and

Bingman, 1998; He and Cramer, 1992). Six 50-liter carboys were used to mix salinity

treatments. A 3-liter plastic laboratory pitcher filled with 2 liters of deionized water, and

a 6-cm stir bar was used with a magnetic stirrer to get the artificial sea salt into solution.

After adding the concentrated solution to each carboy, they were diluted to 35 liters with









deionized water. An electrical conductivity meter (YSI Incorporated, Yellow Springs,

OH) equipped with a 14.6 x 1.3-cm-diam. dip-type plastic cell was used to test the

accuracy of each treatment. The electrical conductivity meter was calibrated using a 10

dS/m standard solution and temperature chart. More salt or deionized water was added to

adjust the electrical conductivity to the proper treatment level.

In 2002, irrigation treatments were deionized water concentrated to five salinity

levels, (5, 10, 25, 40, and 55 dS/m) and deionized water (0 dS/m) to serve as a control.

Irrigation treatments were adjusted in 2003 reflecting the results of the 2002 trials and

were formulated by concentrating deionized water to five salinity levels, (5, 10, 15, 20,

and 25 dS/m) and deionized water to serve as a control. Each day, 150 ml of each

irrigation treatment was applied to the appropriate pots, excluding days before and after

leaching events. The experimental units were leached on a weekly (2002) or biweekly

(2003) basis to prevent build up of salts and to deliver fertilizer without changing

treatment levels. 'SeaIsle 1' seashore paspalum was fertilized on a weekly basis with 20

ml of a solution consisting of 5,100 mg NH4NO3 (34% N), 6,354 mg KC1, 252 mg

Ca(H2PO4)2, 435 mg CaSO4, 246 mg MgSO4, 1.55 mg H3BO3, 0.34 mg MnSO4, 0.58 mg

ZnSO4, 0.13 CuSO4, and 3.5 mg FeSO4 per 1 liter of deionized water. In 2003, 40 ml of

the fertilizer solution was applied on a biweekly basis. Since salinity levels were lower in

the 2003 experiment, leaching was done every two weeks as opposed to every week.

Total N applied for the duration of the experiment was 624 mg/pot. Liquid fertilizer was

added to 580 ml or 560 ml of deionized water and varying amounts of artificial sea salt

was added to concentrate the solution to each irrigation treatment. All 600 ml of solution

was poured into each pot to flush out residual salts, and replace with fertilizer solution.









During the course of the 2002 experiments, which lasted from 30 May 2002 to 10

October 2002, average monthly high and low air temperatures in the glasshouse ranged

from 29 to 34 C, and 21 to 26 C, respectively. In 2003, the B. longicaudatus

experiment began 8 April 2003 and ended 6 August 2003 and the H. galeatus experiment

began 23 May 2003 and ended 20 September 2003. Average monthly high and low air

temperatures ranged from 28 to 34 C and 22 to 27 C, respectively. Using a 1.18-liter

pump sprayer, an insecticide/miticide (Mavrik Aquaflow, Wellmark International,

Schaumburg, IL) was applied at the labeled rate twice during the course of 2003

experiments for control of bermudagrass mites (Eriophyes cynodoniensis Sayed).

After 120 days, experimental units were brought into the laboratory for analysis.

The contents of each pot were emptied into their individually labeled plastic bags and

thoroughly hand mixed. A 100-cm3 soil sample was taken from each bag and processed

using a modified centrifugal-sugar flotation technique (Jenkins, 1964). Nematodes were

counted from the entire sub-sample using an inverted light microscope at x 32

magnification. For the 2003 B. longicaudatus trial, second-stage juveniles were counted

separately from the other life stages. The two categories were summed to determine the

total population of each soil sample. In addition to soil extraction, H. galeatus in roots

from the 2003 experiment were staged and counted to determine number of

nematodes/gram of root in fresh-weighed samples using a modified acid-fuchsin staining-

destaining procedure (Byrd et al., 1983). Effects of salinity were evaluated by regressing

log transformations of final nematode population densities on salinity irrigation levels.

In 2003, the sting nematode portion of the experiment was adjusted because B.

longicaudatus was determined to stunt root growth in seashore paspalum (Chapter 2).









Effects of high salinity irrigation alone were compared to the combined effect of high

salinity irrigation and B. longicaudatus on shoot and root growth. Thirty additional

experimental units were irrigated with salinity treatments and left uninoculated to serve

as controls. Using fabric scissors (Fiskars Brand Inc., Madison, WI), the grass was

trimmed biweekly to approximately 2 cm above the soil surface for all experiments. In

2003, tissue clippings were only collected from the sting nematode experiment. Tissue

was placed in 15 cm x 23 cm catalog envelopes (Quality Park products, St. Paul, MN)

using a spouted 2.84-liter sample pan (40.6 cm x 30.5 cm x 5 cm) (Seedburo, Chicago,

IL), and dried at 75 C for 48 hours to obtain cumulative shoot dry weight.

Final destructive analysis also was adjusted from the previous years' experiments,

with the inclusion of uninoculated treatments in the B. longicaudatus experiment. Shoots

were trimmed as close to the soil as possible and saved for cumulative shoot dry weight

analysis. Using a stainless steel T-sampling tool, a root core (approximately 4-cm-diam.

x 14 cm deep) was taken from the center of each pot to compare root lengths at each

salinity level with inoculated treatments. Root cores were washed free of soil on a sieve

with 1.7-mm pore openings nested within a sieve with 75-pm pore openings. Roots were

removed from any aboveground growth and placed into 50-ml disposable plastic

centrifuge tubes containing 30 ml of tap water. The 75-tm-pore sieve was then

submerged in 5 cm of tap water to allow the finer roots to float out and separate from the

soil. These fine roots were collected using laboratory forceps and placed into the 50-ml

plastic centrifuge tubes. Five drops (0.25 ml) of a 1% methylene blue mixture was added

to the 30 ml of tap water to stain the roots. After a minimum of 24 hours in the solution,

the roots were removed, placed on a 75-[tm-pore sieve, and washed free of excess dye.









Stained roots were placed into a glass-bottom tray and scanned using a HP ScanJet 2cx

desktop scanner (Hewlett Packard, Boise, ID) to create a black and white bitmap image

of the roots (Kaspar and Ewing, 1997; Pan and Bolton, 1991). The GSRoot (Louisiana

State University, Baton Rouge, LA) software program was used to analyze the bitmap

images. This program measures root lengths and surface areas from scanned images.

Root length data were recorded for seven diameter ranges (< 0.05 mm, 0.05 to 0.10 mm,

0.10 to 0.20 mm, 0.20 to 0.30 mm, 0.30 to 0.40 mm, 0.40 to 0.50 mm, and > 0.50 mm).

The resulting values were summed to determine the total root length of each root sample.

In 2003, total root length measurements and cumulative shoot dry weights from the

sting nematode experiment were compared at each salinity level using a one-way analysis

of variance (ANOVA) procedure. Inoculated plants were compared to uninoculated at

each salinity level to determine if root and/or shoot growth reduction occurred.

Orthogonal contrasts were used to compare groups of final population means or

individual means for selected sets of treatments. In our experiments, the highest three

salinity treatments were compared to the lower three treatments, and selected individual

treatments (10 dS/m and 15 dS/m) were compared to the 0 dS/m. Transformation by

logo1 x was performed to normalize nematode-count data and achieve a better trendline fit

(Proctor and Marks, 1975). Using SAS software, (SAS Institute, Cary, NC) the quadratic

least squares procedure was used to fit a linear or quadratic model to the data relating log-

transformations of final nematode population levels to salinity irrigation treatment levels.

Linear and quadratic regressions were drawn using Excel software (Microsoft

Corporation, Redmond, WA). One-way ANOVA procedure, quadratic least squares

procedure, and orthogonal contrasts were performed using SAS software.









Results

Reproduction ofH. galeatus and B. longicaudatus was affected by increasing

salinity levels (Figs. 3.1, 3.2). Final populations of H galeatus decreased linearly (P <

0.01) with increasing salinity irrigation treatments (Fig. 3-1). The high R2-values, 0.92

(2002) and 0.83 (2003), indicate that the data followed the linear regression lines very

closely. Lower salinity treatments, 0 and 5 dS/m, resulted in higher nematode

reproduction than the higher salinity irrigation treatments (Fig. 3-1). Final population

densities were very low at the treatment level of 25 dS/m in both years. Treatment levels

of 40 and 55 dS/m were included in the first year of the study and resulted in population

levels at or near zero. Final population means ofH. galeatus were 1 + 1.4 and 2

1.4/100 cm3 of soil for the 40 and 55 dS/m treatments respectively. The ability of H.

galeatus to enter the root cortex as migratory endoparasites also decreased as salinity

levels increased. Final population densities within the root cortex were lower (P < 0.05)

in the 15, 20, and 25 dS/m treatments (19 13.6 H. galeatus/gram of root fresh weight)

when compared to the 0, 5, and 10 dS/m treatments (167 146.9 H. galeatus/gram of

root fresh weight).

The relationship between final population densities ofB. longicaudatus and salinity

treatment levels demonstrated a quadratic regression curve in both years of the

experiment (Fig 3-2). The quadratic regression was clearly defined when final

population data were transformed using log10 x (Fig. 3-2). Non-transformed data also

showed a quadratic regression (Data not shown), but log transformation improved R -

values and goodness of fit. In 2002, final populations ofB. longicaudatus demonstrated a

quadratic relationship from 0 to 25 dS/m, with the 25 dS/m treatment supporting a lower









population level than the 5 and 10 dS/m treatments (P < 0.01). In both years, the 10

dS/m treatment supported a higher population of B. longicaudatus than the 0 dS/m

treatment (P < 0.05) causing the regression lines to be quadratic (Figure 3.2). Treatment

levels of 40 and 55 dS/m were included in the first year of the study and again resulted in

population levels at or near zero. Belonolaimus longicaudatus final population means

were 3 + 3.7 and < 1/100 cm3 of soil for the 40 and 55 dS/m treatments respectively. In

each trial, the orthogonal contrast test revealed that B. longicaudatus final population

levels in the 25 dS/m treatment were lower (P < 0.01) than those in the less saline

treatments.

Final B. longicaudatus population densities were higher at 10 and 15 dS/m than 0

dS/m (P < 0.05) in the second year of the study (Fig. 3.2). At 15 and 20 dS/m, second-

stage juveniles (J2) were the predominant portions of the overall population, making up

an average of 68 and 60% of the total population, respectively (Fig. 3.3). At lower

salinity levels (0, 5, and 10 dS/m), adults, third-stage juveniles (J3), and fourth-stage

juveniles (J4), were the larger portions of the final population densities (Fig. 3.3). Shoot

growth was not affected (P > 0.05) by inoculation with B. longicaudatus. Root length

comparisons revealed that B. longicaudatus caused root reduction (P < 0.05) at low

salinity levels, 0 to 10 dS/m. However at higher salinity irrigation treatments, 15 to 25

dS/m, root lengths were not different (P > 0.05) between inoculated and uninoculated

plants (Fig. 3-4). These results indicate that the ability of B. longicaudatus to stunt root

growth was negatively affected at salinity levels of 15 dS/m and above.










---- 2003
Y= -0.0452x + 2.823
R = 0.83
P < 0.01


3.5



3



2.5



L 2



- 1.5



1



0.5



0


--A-- 2002
Y= -0.0746x + 2.3414
2
R = 0.92
P < 0.01







8
0


A
I %.


A
-. A


5 10 15 20


dS/m


Fig. 3-1. Relationship between log transformation of final population densities (Pf) of H.
galeatus (nematodes/100 cm3 of soil) (Y) and salinity treatment (x) in 2002
and 2003 glasshouse experiments.


0
SA


A A










2003
Y= -0.0058x2 + 0.1027x + 2.3801
2
R = 0.78
P < 0.01


S8---
--r


A


--A-- 2002
Y= -0.0068x2 + 0.1248x + 1.7994
2
R = 0.72
P < 0.01


A


5 10 15 20


dS/m


Fig. 3-2. Relationship between log transformation of final population densities (Pf) of B.
longicaudatus (nematodes/100 cm3 of soil) (Y) and salinity treatment (x) in
2002 and 2003 glasshouse experiments.


3.5


3


2.5


2


1.5


0
o-


1


0.5


0


\ i










- J2


Y= -0.0086x2 + 0.1936x + 1.4314 Y
2
R = 0.74
P < 0.01


3.5



3



2.5


0 5 10 15


dS/m


Fig. 3-3. Relationship between log transformation of J2 or adults, J3, and J4 final
population densities (Pf) of B. longicaudatus (nematodes/100 cm3 of soil) (Y)
and salinity treatment (x) in the 2003 glasshouse experiment.


--A- -Adults, J3, J4

:-0.0041x2 + 0.0496x + 2.3675
R 2 = 0.76
P < 0.01
POPO1
0
A
8
0




A
0
A V
A A


5i,
0(


1



0.5



0










4,000 -
4,000 Inoculated

3,500 I Uninoculated

3,000

2,500 -

!2,000

1,500

S1,000 -


500










no nematodes. The error bars show standard deviation of individual
population means. ** Indicates difference from inoculated at P 0.01,
according to the one-way ANOVA procedure.
Discussion

Irrigation with poor quality water is becoming more common as increasing water

restrictions are forcing turfgrass managers to obtain water from alternative sources. In

our experiments, high salinity irrigation affected these particular populations of the two

nematodes differently. Lower salinity levels (5, 10, and 15 dS/m) caused an increase in

B. longicaudatus reproduction, while the population ofH. galeatus steadily decreased as









salinity levels increased. Irrigation with low salinity levels (5 and 10 dS/m) resulted in

denser, more vigorous root and shoot growth than the 0 dS/m treatment. Irrigating with

deionized water may have caused nutrient deficiencies and elevated evapotranspiration

rates. With increased root growth, more feeding sites were available for B.

longicaudatus.

In 2003, second-stage juveniles (J2) of B. longicaudatus comprised a majority of

the population in the 15 and 20 dS/m treatments. The abundance of J2 at moderate

salinity levels resulted in elevated total population numbers compared to lower salinity

levels, causing the regression line to be quadratic (Fig. 3.2, 3.3). The nematodes counted

as J2 had a clear body cavity indicating they were probably unable to feed. Usually, J2

can easily be separated from other life stages by their dark color and stout body shape

(Han, 2001). Root length data in 2003 support this hypothesis, with root reduction not

occurring at the 15, 20, or 25 dS/m treatments as opposed to the lower salinity treatment

levels (Fig. 3.4). Reproduction and maturation of the nematodes at higher salinity

treatments probably occurred early in the experiment before the salinity was able to build

up in the soil. Han (2001) stated that it is unknown whether or not feeding is necessary

for J2 to develop into J3. However, J2 began molting into J3 3 to 5 days after hatching

(Han, 2001).

Shoot growth was not affected by inoculations with B. longicaudatus, but at

salinity levels above 10 dS/m, shoot growth became less vigorous. In a previous study,

shoot growth began to decline in response to soil electrical conductivity levels > 8 dS/m,

with shoot growth being reduced by 25% and 50% at 14 and 34 dS/m, respectively (Lee,

2000). Cumulative shoot dry weight data collected from previous susceptibility









experiments also showed that inoculation with B. longicaudatus did not affect

aboveground plant growth (Chapter 2). Salinity treatment levels equaling 25 dS/m and

above reduced B. longicaudatus populations to extremely low levels in 2002, but these

high salinity levels had detrimental effect on the growth of the grass. In 2003, inoculum

levels were much higher and the same quadratic regression occurred, with 25 dS/m

reducing reproductions compared to treatment levels ranging from 0 to 15 dS/m (P <

0.05).

Hoplolaimus galeatus are classified as migratory endoparasites on turfgrasses.

Their ability to enter the roots gives them the capability to escape the effects of most

nematicides (Giblin-Davis et al., 1995). In our experiment, both soil and root populations

were reduced as salinity irrigation levels increased from 0 to 25 dS/m. The nematodes

were probably not able to escape the effects of the salinity by entering the roots because

the root cortex tissue does not exclude the elevated ion concentrations associated with

saline water. Published action thresholds that justify post-plant nematicide treatment for

H. galeatus on bermudagrass are 40/100 cm3 of soil (Crow et al., 2003). Soil populations

more than exceeded these numbers for both experiments at 15 dS/m and less. In previous

experiments, H. galeatus had no effect on seashore paspalum growth even though soil

counts exceeded 40/100 cm3 of soil throughout the experiments (Chapter 2). Time course

experiments (Giblin-Davis et al., 1995) with 'Floratam' and 'FX-313' St. Augustinegrass

also indicated that H. galeatus had no effect on plant growth.

In 2002, treatment levels of 40 and 55 dS/m caused near complete mortality for

both nematodes, but the shoot growth of the grass was stunted and yellowed. Even

though B. longicaudatus and H. galeatus were effectively controlled, the turfgrass was









not visually acceptable. Seashore paspalum can be irrigated with seawater (54 dS/m) in

the field when soil conditions allow for sufficient leaching to occur and turfgrass

managers fertilize, amend, and cultivate the soil properly (Carrow and Duncan, 1998). In

our experiments, prolonged exposure to these high salinity levels was detrimental to

turfgrass quality. In the glasshouse, we were unable to provide sufficient leaching,

proper amendments, and cultivation of soil necessary for seashore paspalum survival at

salinity levels near that of seawater.

Results from glasshouse experiments are difficult to extrapolate to field conditions,

but we can conclude that salinity irrigation had an affect on B. longicaudatus and H.

galeatus nematode reproduction. The treatment salinity levels were at a continuous level

throughout the experiment, and never allowed the nematodes to recover from salinity

stress. A discontinuous high salinity irrigation situation would probably be more similar

to irrigation with poor quality water under field conditions where rainfall can leach salt

from the soil profile (Mashela et al., 1992a). Irrigation with pure seawater or with

seawater as a high percentage of the blended irrigation water may have potential as an

effective option for control ofB. longicaudatus and H. galeatus. This information may

be vital to turfgrass managers currently maintaining seashore paspalum known to have a

nematode problem. Further investigation is necessary to determine if frequency and

timing of high salinity irrigation, in addition to amount of salinity, can have an effect on

nematode reproduction and feeding.














CHAPTER 4
SURVEY OF PLANT-PARASITIC NEMATODES ASSOCIATED WITH SEASHORE
PASPALUM

Introduction

New turfgrass cultivars or ecotypes are commonly introduced into the turfgrass

industry, but in rare cases, a new species of turfgrass begins to become more widely

utilized. Seashore paspalum (Paspalum vaginatum Swartz) is a warm season grass

adapted for saline conditions (Malcolm and Laing, 1969; Morton, 1973). Breeding for

fine leaf textured cultivars, and its natural tolerance to drought and high salinity irrigation

has allowed seashore paspalum to become frequently used in highly managed turfgrass

sites (Dudeck and Peacock, 1985; Duncan, 1999a). One major limitation of cultivating

turfgrasses in the sandy soils of the southeastern United States is the destruction of roots

by plant-parasitic nematodes (Perry and Rhoades, 1982).

Surveys are necessary to determine the distribution, frequency, and abundance of

phytoparasitic nematodes associated with particular plants. These surveys provide

indispensable information on the probability and severity of crop losses due to plant-

parasitic nematode damage. Results of surveys can help to determine more confident

action thresholds allowing the grower to use soil and root nematode assays to formulate a

management strategy. This will help avoid unnecessary post-plant nematicide treatments

or failure to apply a needed one. High population levels of some plant-parasitic

nematode genera can cause significant damage to turfgrass areas (Di Edwardo, 1963;

Johnson, 1970; Lucas et al., 1974; Winchester and Burt, 1964; Rhoades, 1962; 1965;









Kelsheimer and Overman, 1953, Christie et al., 1954). Therefore, a survey was

conducted for golf courses and home lawns with established seashore paspalum in

Florida. This survey was performed to determine the plant-parasitic nematodes

commonly associated with seashore paspalum.

Materials and Methods

A majority of the survey soil samples were taken during the spring and summer

months of 2002 and 2003 from coastal areas in central and south Florida. Soil samples

were collected from golf courses in or near Fort Myers, Miami Beach, Delray Beach,

Vero Beach, and Naples, FL. Home lawn samples were collected from many areas in

central and south Florida, primarily in the Tampa, Miami, Fort Myers, Naples, and

Daytona Beach areas. Seashore paspalum is well adapted to these areas because of its

tolerance to high salinity irrigation and saltwater intrusion (Malcolm and Laing, 1969;

Morton, 1973). Areas sampled were in close proximity to each coast and all samples

were from sandy soils. A total of 20 home lawns and 8 golf courses were sampled.

These sample sizes may appear low for a survey, but a high percentage of golf courses

and home lawns growing seashore paspalum in Florida were sampled. According to a

United States Golf Association Florida Regional Agronomist, 12 to 15 golf courses in

Florida are currently growing seashore paspalum on > 90% of their maintained areas

(Lowe, pers. comm.). A non-biased approach was practiced when selecting the home

lawns and golf courses to be sampled. At each golf course, four greens and four fairways

were individually sampled for plant-parasitic nematodes. Two golf courses were not

growing seashore paspalum over the entire course, thus fewer samples were collected.

Home lawns were sampled separately for plant-parasitic nematodes. Two to seven

samples were taken at each lawn. More samples were collected from larger lawns and









lawns with noticeable aboveground damage. A 2.5-cm-diam. cone-shaped soil sampler

was used to collect 20 soil cores (2.5-cm-diam x 10 cm deep) at approximately equal

intervals in a zig-zag pattern across the sampling area. If yellowing or declining areas

were noticed, then separate samples were taken from these areas.

Each sample was emptied into individually labeled plastic bags and thoroughly

hand mixed. A 100-cm3 soil sample was removed from each bag and nematodes were

extracted using a modified centrifugal-sugar flotation technique (Jenkins, 1964). Plant-

parasitic nematodes were identified to genus and counted using an inverted light

microscope at x 32 magnification. After counting, the nematodes were preserved in a 5%

formalin solution. Using a compound microscope at x 40 and x 100 magnifications,

some preserved nematodes were slide mounted and identified to the species level with

reasonable confidence. Morphology keys and original nematode descriptions were used

for species identification (Esser, 1971; 1973; Esser and Vovlas, 1989; Golden and Taylor,

1956; Handoo and Golden, 1992; Loof and Luc, 1990; Rau, 1958; Sher, 1961; 1963;

1966; Sledge and Golden, 1964).

Results

Plant-parasitic nematodes associated with seashore paspalum were the typical range

of genera found parasitizing other warm-season turfgrasses. Ten genera of plant-parasitic

nematodes were extracted from 60 seashore paspalum soil samples from eight golf

courses (Table 4-1). In addition to the same ten genera found on seashore paspalum golf

courses, two more plant-parasitic nematode genera, a total of 12, were extracted from 51

soil samples from 20 home lawns (Table 4-2). Nematodes in the genera

Hemicriconemoides and Helicotylenchus are not frequently discovered in such high









populations on turfgrasses in Florida as were found on seashore paspalum (Crow, pers.

comm.). As with a majority ofturfgrass samples in Florida, Hoplolaimus spp. was

commonly found in seashore paspalum samples. The genera most frequently found were

Hoplolaimus, Mesocriconema, Hemicriconemoides, and Helicotylenchus, which were

found on 100, 100, 88, and 88% of the golf courses surveyed and on 75, 95, 70, and 85%

of the home lawns sampled, respectively (Table 4-1, 4-2). Xiphinema, Pratylenchus, and

Tylenchorhynchus, were found on less than 30% of the golf courses and less than 45% of

the home lawns sampled (Table 4-1, 4-2). Peltamigratus and Hemicycliophora were not

found in any of the golf course soil samples and were only found associated with a low

percentage of the home lawns (Table 4-2).

Various morphological measurements, identifying cuticular markings, and body

shape were used for species identification. Species identified from soil samples taken

from the rhizosphere of seashore paspalum were Belonolaimus longicaudatus Rau,

Hoplolaimus galeatus (Cobb) Thorne, Helicotylenchuspseudorobustus (Steiner) Golden,

Meloidogyne graminis (Sledge and Golden) Whitehead, Hemicriconemoides annulatus

Pinochet and Raski, Trichodorusproximus Allen, and Peltamigratus christiei (Golden

and Taylor) Sher.

Previous surveys indicate that a variety of nematodes are associated with

turfgrasses in the southeastern United States (Lucas et al., 1974; Parris, 1957; Sikora et

al., 2001). Even though these surveys were primarily for nematodes associated with

bermudagrass and bentgrass (Agrostis spp.), many of the nematodes we found were from

the same genera. Mesocriconema, Hoplolaimus, and Helicotylenchus were all common

in our survey, which is similar to observations throughout the southeast. The relatively









high frequency of B. longicaudatus found in soil samples from golf courses (50%) and

home lawns (40%) was expected because of the sandy soils associated with coastal areas

in Florida. Belonolaimus longicaudatus is most commonly found in Florida because it is

well adapted to soils with > 80% sand content and < 10% clay content (Robbins and

Barker, 1974). Belonolaimus longicaudatus is recognized as the most pathogenic

ectoparastic plant nematode to turfgrasses in Florida. Acceptable visual quality is often

not possible without treatment of the B. longicaudatus infestation.

Table 4-1. Frequency of occurrence and population density of plant-parasitic nematode
genera in soil samples collected from seashore paspalum golf courses in
Florida.

Nematodes/100 cm of soil
Golf courses with
Nematode genus nematode (%)a Meanb Maximum'
Hoplolaimus 100 162 643
Mesocriconema 100 69 563
Hemicriconemoides 88 97 1,014
Helicotylenchus 88 469 2,808
Trichodorus 63 30 150
Tylenchorhynchus 63 7 16
Meloidogyne 63 39 123
Belonolaimus 50 16 114
Pratylenchus 25 76 167
Xiphinema 25 79 253
aPercentage of golf courses with at least one infested green or fairway.
Percentage based on eight golf courses with one to four fairways and zero to four greens
sampled on each golf course for a total of 60 soil samples.
bMean population levels of nematodes recovered from samples in which the
nematode was found.
Maximum population levels found from individual soil samples.









Table 4-2. Frequency of occurrence and population density of plant-parasitic nematode
genera in soil samples collected from seashore paspalum home lawns in
Florida.

Nematodes/100 cm3 of soil
Home lawns with
Nematode genus nematode (%)a Meanb Maximum'
Mesocriconema 95 123 552
Helicotylenchus 85 175 743
Hoplolaimus 75 73 253
Hemicriconemoides 70 92 428
Trichodorus 60 49 234
Tylenchorhynchus 40 42 388
Belonolaimus 40 11 72
Meloidogyne 28 14 45
Pratylenchus 25 5 15
Hemicycliophora 25 22 91
Peltamigratus 5 29 57
Xiphinema 5 7 7
aPercentage of home lawns with at least one infested soil sample. Percentage
based on 20 home lawns with one to five samples taken at each lawn for a total of 51 soil
samples.
bMean population levels of nematodes recovered from samples in which the
nematode was found.
Maximum population levels found from individual soil samples

Discussion

Action threshold levels have not yet been established for seashore paspalum, but

according to levels established for bermudagrass (Crow et al., 2003), 26 of the 51 home

lawn samples and 34 of the 60 golf course samples had nematode populations above

action threshold levels. Belonolaimus, Hoplolaimus, Helicotylenchus, Trichodorus,

Hemicriconemoides, and Mesocriconema were the genera found above action threshold

levels for bermudagrass (Crow et al., 2003). Hoplolaimus galeatus, B. longicaudatus,

and T proximus were associated with damaged areas on golf courses and home lawns in

the current survey. Drought stress during the summer was amplified when these

nematodes were present. Seashore paspalum was slow to recover from dieback









associated with drought. Damaged areas were more common on home lawns than on

golf courses. Golf courses are usually well maintained through proper fertilization and

adequate irrigation, possibly masking root damage caused by high nematode populations.

Declining seashore paspalum was found when populations of T proximus exceeded 60

nematodes/100 cm3 of soil, H. galeatus exceeded 50 nematodes/100 cm3 of soil, and

when B. longicaudatus numbers were above 10 nematodes/100 cm3 of soil. High

populations ofHelicotylenchus spp. (> 500 nematodes/100 cm3 of soil) were often found

associated with seashore paspalum. Damaged areas were not noticed in areas where high

population levels of Helicotylenchus spp. were recovered from soil samples. In Florida,

Helicotylenchus spp. are rarely found in turfgrass soil samples at these high population

levels (Crow, pers. comm.). Their ability to damage turfgrass in Florida is not widely

known.

A wide range of plant-parasitic nematodes was found associated with seashore

paspalum golf courses and home lawns in Florida. Some nematode genera tended to

have population levels higher than those usually found associated with bermudagrass

(Cynodon spp.) in Florida. The high population levels of Helicotylenchus spp. and

Hemicriconemoides spp. found in this survey suggest seashore paspalum is an excellent

host for these nematodes. Approximately half of the seashore paspalum samples had

nematode populations at damaging levels according to bermudagrass threshold levels.

These threshold levels are approximate and can fluctuate due to host suitability, soil

temperature, soil texture, soil moisture, and a variety of other soil conditions. Threshold

levels used for bermudagrass seem reasonable for diagnosis of samples with B.

longicaudatus, H. galeatus, and T proximus, but adjustments may be necessary for






54


Helicotylenchus and Hemicriconemoides. As seashore paspalum becomes more widely

grown, a better understanding of its relationship with plant-parasitic nematodes can be

ascertained.














CHAPTER 5
SUMMARY

With more than 1,300 public and private golf courses and over three million acres

of residential lawns (Haydu and Hodges, 2002; Hodges et al., 1994), Florida is not only a

prime location for highly managed turfgrass areas, but also an ideal habitat for

Belonolaimus longicaudatus and Hoplolaimus galeatus. The data reported herein

indicates that this population ofB. longicaudatus is detrimental to the growth of 'SeaIsle

1' seashore paspalum in the southeastern United States. The reaction of seashore

paspalum to the population ofH. galeatus was very different with only a limited effect on

plant growth detected in our research. Our data show that 'Sealsle 1' seashore paspalum

is not any less susceptible to B. longicaudatus or H. galeatus than is 'Tifdwarf

bermudagrass.

The population of B. longicaudatus had a high degree of association with root

growth stunting (P < 0.05) in 'SeaIsle 1' seashore paspalum and 'Tifdwarf bermudagrass

while H. galeatus failed to reduce root or shoot growth in either grass (P > 0.05) (Chapter

2). Results indicate that there are slight differences in host suitability, but no differences

in root length percent reduction between the two grasses. 'Tifdwarf bermudagrass tended

to support higher populations of B. longicaudatus and H. galeatus during the experiments

(P < 0.05) (Chapter 2). Shoot growth was not affected (P > 0.05) by either nematode on

either grass. 'Sealsle 1' seashore paspalum and 'Tifdwarf bermudagrass root growth was

reduced by 35 to 45% when exposed to B. longicaudatus for 120 days. The population of

H. galeatus was associated with a slight abbreviation of root development in seashore









paspalum and bermudagrass during the second year of the study (Chapter 2). Even

though final population densities of H galeatus more than exceeded the reported action

threshold of 40 nematodes/100 cm3 of soil for bermudagrass (Crow et al., 2003), neither

seashore paspalum nor bermudagrass had a consistent reduction in root growth, although

H. galeatus were found within the root cortex of both grasses (Chapter 2). These results

indicate that there is no reason to believe that the susceptibility of 'Sealsle 1' seashore

paspalum to H. galeatus and B. longicaudatus is lower than 'Tifdwarf bermudagrass.

The two nematodes were affected differently by high salinity irrigation (Chapter 3).

Moderate salinity levels (10 and 15 dS/m) caused an increase in B. longicaudatus

reproduction when compared to 0 dS/m (P < 0.05). Visual inspection revealed that

irrigation with these low salinity levels resulted in a denser, more vigorous root and shoot

growth than 0 dS/m. With increased root growth, more feeding sites were available for

B. longicaudatus. At high salinity levels of 25 dS/m and higher, B. longicaudatus

populations were reduced to non-damaging levels. In 2003, root reductions were not

detected (P > 0.05) at salinity levels of 15 dS/m and higher when plants inoculated with

B. longicaudatus were compared to uninoculated plants with equivalent irrigation

treatments (Chapter 3). A high percentage of final populations at these salinity levels

consisted of second-stage juveniles (J2). Further research must be performed to

determine if salinity interferes with host locating receptors ofB. longicaudatus.

Moderate salinity levels may also be stimulating egg hatch resulting in high populations

of J2.

In our experiments, soil and root populations of H galeatus were reduced as

salinity irrigation levels increased from 0 to 25 dS/m (Chapter 3). Final population









densities of H. galeatus demonstrated a negative sloping linear regression (P < 0.01)

when compared to increasing salinity irrigation treatments. Lower salinity treatments, 0

and 5 dS/m, resulted in higher reproduction capabilities than the higher salinity irrigation

treatments (P < 0.05) (Chapter 3). Final populations were very low at the 25 dS/m

treatment level and above. The R -values were 0.92 (2002) and 0.83 (2003), showing

that the data followed a linear regression very closely. High salinity irrigation may be

effective in controlling H. galeatus in the soil, but these nematodes are also able to enter

the root cortex as migratory endoparasites. Our results indicate that the ability of H.

galeatus to completely enter and tunnel within the root cortex does not provide a barrier

from the effects of the high salinity irrigation (Chapter 3).

The survey of nematodes associated with established seashore paspalum revealed a

majority of genera typically associated with maintained turfgrass areas (Chapter 4).

Hoplolaimus spp., Helicotylenchus spp., Mesocriconema spp., and Hemicriconemoides

spp. were common in home lawns and golf courses. Some genera (Hemicriconemoides

and Helicotylenchus) tended to have population levels higher than those usually found

associated with bermudagrass in Florida. The relatively high frequency of B.

longicaudatus at golf courses (50%) and home lawns (40%) should have been expected

because of the sandy soils associated with coastal areas in Florida. Damaged areas could

be found associated with parasitism by B. longicaudatus, H. galeatus, and Trichodorus

proximus during the summer months when the grass was under the most environmental

stress (Chapter 4).

Nematode management in turfgrass areas is becoming increasingly difficult

because of the elimination of a majority of post-plant nematicides. Alternatives in the









form of different chemical formulations, biological control, or resistant plant cultivars

must be found to be able to maintain acceptable turfgrass quality in many areas. With the

development of salt-tolerant grasses such as seashore paspalum, high salinity irrigation

could be implemented as part of a management regiment for nematodes if irrigation

periods were timed properly. Water use for landscape irrigation is becoming a critical

issue in Florida. Maintaining turfgrass of acceptable quality is increasingly difficult

when forced to reduce water consumption or switch to alternative water sources. In

Florida, recycled water was the primary source for almost half of all golf courses in 2000

and has increased from 8% in 1974 to 21% in 1994 to 49% in 2000 (Haydu and Hodges,

2002). Seashore paspalum is well adapted to irrigation from these alternative water

sources and could become more prevalent as water restrictions increase. Irrigation with

water containing high levels of dissolved salts reduces shoot growth of seashore

paspalum (Lee, 2000), but since high yield is not essential for turfgrass managers this

could be an effective nematode management technique. Further research must be

performed to determine the timing, frequency, and duration of high salinity irrigation

events.
















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BIOGRAPHICAL SKETCH

Adam C. Hixson was born 25 April 1978 in Waco, TX, and grew up in China

Spring, TX. He graduated from China Spring High School in 1996, and began studies at

Texas A&M University, College Station, TX in the fall of 1996. He was a member of the

Corps of Cadets for four years while pursing his Bachelor of Science degree in

entomology. After graduation from Texas A&M University in 2000, he began studies for

his Master of Science degree at University of Florida, Gainesville, Florida. The title of

his thesis is "Belonolaimus longicaudatus and Hoplolaimus galeatus on Seashore

Paspalum (Paspalum vaginatum)."